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PHYSIOLOGICAL REVIEWS
Vol. 81, No. 2, April 2001
Printed in U.S.A.
Mammalian Mitogen-Activated Protein Kinase Signal
Transduction Pathways Activated by Stress and Inflammation
JOHN M. KYRIAKIS AND JOSEPH AVRUCH
Diabetes Research Laboratory, Medical Services, and Department of Molecular Biology,
Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts
http://physrev.physiology.org
0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society
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I. Introduction
II. The Stress-Activated Protein Kinase/c-Jun NH2-Terminal Kinase, p38 and ERK5/Big MitogenActivated Protein Kinase-1 Pathways: The Three Major Mammalian Mitogen-Activated Protein
Kinase Signaling Pathways Activated by Environmental Stress and Inflammatory Cytokines:
Core Pathways and Their Targets
A. General considerations
B. The stress-activated protein kinases/c-Jun NH2-terminal kinases, a family of MAPKs activated
by environmental stresses and inflammatory cytokines of the TNF superfamily
C. The p38 MAPKs, a second stress-activated MAPK group
D. ERK5/big MAP kinase-1 (BMK1), a third class of stress-activated MAPK
E. SAPK, p38, and ERK5 substrates
F. MEKs upstream of the SAPKs, p38s, and ERK5
G. Several divergent families of MAP3Ks are upstream of the SAPKs, p38s, and ERK5
III. Putative Elements Upstream of Stress-Activated Mitogen-Activated Protein Kinase Core Pathways
A. Regulation of the SAPKs and p38s by Rho family GTPases
B. Regulation of the SAPKs and p38s by heterotrimeric G proteins: implications
for cardiovascular diseases
C. Regulation of the SAPKs by scaffold proteins
D. Regulation of the SAPKs by the GCKs
E. Regulation of the SAPKs and p38s by adapter proteins that couple to TNF family receptors
IV. Molecular Mechanisms Coupling Stress-Activated MAP3Ks to Upstream Signals
A. General considerations
B. MEKK1 may lie downstream of Ras and Rho family GTPases, GCK family kinases, and TRAFs
and may be targeted to an apoptotic pathway upon caspase cleavage
C. GCKs may collaborate with TRAFs to activate MAP3Ks including MEKK1
D. ASK1 is an effector for TRAFs, specifically, a TNF effector recruited by TRAF2: role of cellular
redox in ASK1 regulation
E. TAK1 is a target for TGF-␤ and IL-1 through its association with TAB1
F. MEKK4 is a putative effector for Rho family GTPases and may be activated by DNA damage
through a direct interaction with GADD45 homologs
G. Regulation of MLK3 by group I GCKs and Cdc42Hs: role of dimerization through
the Leu zippers
V. Biological Functions of the Stress-Activated Protein Kinase and p38 Pathways in the Stress
Response, Cell Cycle Control Embryogenesis, and Immune System Maturation
A. The SAPKs can participate in regulating apoptosis in response to environmental stress through
AP-1-regulated transcriptional induction of FasL
B. The p38s can trigger cell cycle arrest at G1/S as part of a signaling pathway in late G1 mediated
by Cdc42Hs and/or MEKK3
C. The SAPKs and brain development
D. The SAPKs and p38s are required for the progression of cardiomyocyte hypertrophy in response
to pressure overload and vasoactive peptides
E. The SAPKs, p38s, and ischemic/ischemic-reperfusion injury
F. MEKK3 and cardiovascular development
G. The SAPKs are key regulators of T-cell maturation, activation, and protection
from FasL apoptosis
H. The SAPKs and p38s promote the stabilization and enhanced translation of mRNAs encoding
proinflammatory proteins
VI. Concluding Remarks
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
A. Oligomerization, adapter/G protein binding, membrane translocation, and phosphorylation
as themes in MAP3K regulation
B. MAPK pathway biology
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I. INTRODUCTION
Mitogen-activated protein kinase (MAPK) signal
transduction pathways are among the most widespread
mechanisms of eukaryotic cell regulation. All eukaryotic
cells possess multiple MAPK pathways, each of which is
preferentially recruited by distinct sets of stimuli, thereby
allowing the cell to respond coordinately to multiple divergent inputs. Mammalian MAPK pathways can be activated by a wide variety of different stimuli acting through
diverse receptor families, including hormones and growth
factors that act through receptor tyrosine kinases [e.g.,
insulin, epidermal growth factor (EGF), platelet-derived
growth factor (PDGF), and fibroblast growth factor
(FGF)] or cytokine receptors (e.g., growth hormone) to
vasoactive peptides acting through G protein-coupled,
seven-transmembrane receptors (e.g., ANG II, endothelin), transforming growth factor (TGF)-␤-related polypeptides, acting through Ser-Thr kinase receptors, inflammatory cytokines of the tumor necrosis factor (TNF) family
and environmental stresses such as osmotic shock, ionizing radiation and ischemic injury. MAPK pathways, in
turn, coordinate activation of gene transcription, protein
synthesis, cell cycle machinery, cell death, and differentiation. Accordingly, these pathways exert a profound
effect on cell physiology (120, 165, 195).
All MAPK pathways include central three-tiered
“core signaling modules” (Fig. 1) in which MAPKs are
activated by concomitant Tyr and Thr phosphorylation
within a conserved Thr-X-Tyr motif in the activation loop
of the kinase domain subdomain VIII. MAPK phosphorylation and activation are catalyzed by a family of dual
specificity kinases referred to as MAPK/extracellular signal-regulated kinase (ERK)-kinases (MEKs or MKKs).
MEKs, in turn, are regulated by Ser/Thr phosphorylation,
also within a conserved motif in kinase domain subdomain VIII, catalyzed by any of several protein kinase
families collectively referred to as MAPK-kinase-kinases
(MAP3Ks). MAPK core signaling modules are themselves
regulated by a wide variety of upstream activators and
inhibitors (120, 165, 195).
The notion of multiple parallel MAPK signaling cascades was first appreciated from studies of simple eukaryotes such as the budding yeast Saccharomyces cerevisiae. To date, six S. cerevisiae MAPK signaling pathways have been identified. These have been reviewed
FIG. 1. The mitogen-activated protein kinase (MAPK) core signaling
module. Divergent inputs feed into core MAPK-kinase-kinase (MAP3K) 3
MAPK-kinase (MEK) 3 MAPK core pathways which then recruit appropriate responses.
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Kyriakis, John M., and Joseph Avruch. Mammalian Mitogen-Activated Protein Kinase Signal Transduction
Pathways Activated by Stress and Inflammation. Physiol Rev 81: 807– 869, 2001.—The molecular details of mammalian stress-activated signal transduction pathways have only begun to be dissected. This, despite the fact that the
impact of these pathways on the pathology of chronic inflammation, heart disease, stroke, the debilitating effects of
diabetes mellitus, and the side effects of cancer therapy, not to mention embryonic development, innate and
acquired immunity, is profound. Cardiovascular disease and diabetes alone represent the most significant health care
problems in the developed world. Thus it is not surprising that understanding these pathways has attracted wide
interest, and in the past 10 years, dramatic progress has been made. Accordingly, it is now becoming possible to
envisage the transition of these findings to the development of novel treatment strategies. This review focuses on the
biochemical components and regulation of mammalian stress-regulated mitogen-activated protein kinase (MAPK)
pathways. The nuclear factor-␬B pathway, a second stress signaling paradigm, has been the subject of several
excellent recent reviews (258, 260).
April 2001
MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
3) Pathway organization is mediated by scaffolding
proteins. Given the extraordinary complexity and diversity of MAPK regulation and function, it is critical that the
efficiency and selectivity of MAPK pathways be preserved. Scaffold proteins bind and sequester select MAPK
pathway components, and thereby help to maintain pathway integrity and to permit the coordinated and efficient
activation and function of MAPK components in response
to specific types of stimuli (234). Some yeast signaling
pathways include distinct scaffolding proteins that bind
and segregate groups of signaling components. Alternatively, in some MAPK pathways, the signaling components themselves possess intrinsic scaffolding properties
(see Fig. 7). Thus Ste5p of the yeast mating pheromone
pathway is a scaffolding protein that selectively binds a
MAP3K (Ste11p), a MEK (Ste7p), and a MAPK (Fus3p)
and couples them to upstream activators. Thus, although
Ste11p can function in both the mating and osmosensing
pathways, it selectively activates different MEKs in each
pathway: Ste7p for the mating pathway and Pbs2p for the
osmosensing pathway, due in part to the fact that Ste5p
maintains signaling pathway specificity by binding selectively Ste7p and not Pbs2p (120, 242).
Conversely, Pbs2p, in addition to serving as a MEK,
has intrinsic scaffold properties, selectively binding
Ste11p and the osmosensing MAPK Hog1p. Pbs2p does
not bind Fus3p, and thus Pbs2p maintains signaling pathway integrity by interacting specifically with Hog1p and
not with Fus3p or Kss1p (3, 11). Likewise, the mammalian
scaffold proteins c-Jun NH2-terminal kinase (JNK) interacting proteins (JIPs)-1 and -2 and JNK/stress-activated
protein kinase (SAPK)-associated protein-1 (JSAP1/JIP3),
like Ste5p, couple elements of different SAPK core signaling modules, while mammalian stress-activated protein
kinase (SAPK)/ERK-kinase-1 (SEK1), like Pbs2p, possess
both the properties of a protein kinase and a scaffold
protein (72, 136, 155, 336, 342, 355).
4) MAP3K regulation is by membrane recruitment,
oligomerization, and phosphorylation. MAP3K regulation
represents the entry point to MAPK pathways and is
accordingly complex. In addition to scaffold proteins
mentioned above, actual activation of MAP3Ks has been
shown to involve three key phenomena: recruitment to
the membrane, typically mediated by an upstream activating protein; homoligomerization, often within a multiprotein complex containing additional regulatory components; and phosphorylation by upstream kinases. The
mitogenic MAP3K Raf is illustrative of all three properties. Thus it is recruited to the membrane by Ras (195),
where it undergoes regulatory phosphorylation catalyzed
in part by p21-activated kinase-3 (160) and oligomerization (190). All three events are necessary for activation
(see sect. IIIA1). Stress-regulated MAP3Ks are likely similarly regulated.
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elsewhere (120). The features of yeast MAPK pathways as
well as early biochemical studies have revealed several
common principles shared by all MAPK pathways.
1) MAPKs are proline-directed kinases; however,
substrate selectivity is often conferred by specific MAPK
docking sites present on physiological substrates. The
proline-directed substrate specificity of the MAP kinases
was established using peptide substrates corresponding
to the sequences surrounding Thr 669 of the EGF receptor
(Glu-Leu-Val-Glu-Pro-Leu-Thr-Pro-Ser-Gly-Glu-Ala-ProAsn-Gln-Ala-Leu-Leu-Arg) and the site on myelin basic
protein (Ala-Pro-Arg-Thr-Pro-Gly-Gly-Arg) phosphorylated in vitro by the 42-kDa insulin- and mitogen-stimulated MAPK ERK-2. Systematic variation in the sequences
surrounding the single Thr phosphorylation site in the
EGFR peptide and the single Thr in the MBP peptide,
verified the essential role of the proline at immediately
COOH terminal to the phosphoacceptor site (50, 55, 84).
However, this proline directedness is not sufficient to
account for the high degree of substrate selectivity manifested by different MAPK subgroups. The Michaelis constant (Km) for native protein substrates is usually several
orders of magnitude lower than the Km for synthetic
peptides corresponding in amino acid sequence to the
region immediately surrounding the phosphorylation site
on these substrates. It has become apparent that most, if
not all, of the physiological substrates of the MAPKs
possess specific binding sites, often at considerable distance from the phosphorylation site in the primary sequence, that allow for a strong interaction with select
MAPK subfamilies to the exclusion of others (64, 144, 301,
351). In turn, MAPKs themselves possess complementary
docking sites that interact with the MAPK binding domains on substrate proteins (143, 301). This confers
a striking substrate specificity on a family of protein
kinases with an otherwise apparently broad in vitro
substrate profile, certainly as portrayed by synthetic
peptides.
2) There are signaling components with more than
one biological function/signaling components under multiple forms of regulation. Within MAPK core signaling
modules, there are instances wherein individual elements
can function promiscuously in several pathways. Conversely, MAPK pathway components are often subject to
regulation by multiple inputs. For example, the S. cerevisiae MAP3K Ste11p functions as part of the mating pheromone response pathway and the osmosensing pathway
(20, 242), while the mammalian MAP3K tumor progression locus-2 (Tpl-2) can activate the mitogenic ERK and
three stress-activated MAPK pathways (264). Conversely,
the yeast osmosensing MEK Pbs2p can be regulated by
three MAP3Ks: Ssk2p, Ssk22p, and Ste11p while the mammalian stress-regulated MEK stress-activated protein kinase/ERK-kinase-1 (SEK1)/MAPK-kinase-4 (MKK4) is a
putative substrate for at least 10 known MAP3Ks.
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
II. THE STRESS-ACTIVATED PROTEIN KINASE/
c-JUN NH2-TERMINAL KINASE, p38 AND
ERK5/BIG MITOGEN-ACTIVATED PROTEIN
KINASE-1 PATHWAYS: THE THREE MAJOR
MAMMALIAN MITOGEN-ACTIVATED
PROTEIN KINASE SIGNALING PATHWAYS
ACTIVATED BY ENVIRONMENTAL STRESS
AND INFLAMMATORY CYTOKINES: CORE
PATHWAYS AND THEIR TARGETS
A. General Considerations
B. The Stress-Activated Protein Kinases/c-Jun
NH2-Terminal Kinases, a Family of MAPKs
Activated by Environmental Stresses and
Inflammatory Cytokines of the TNF Superfamily
Indications that mammalian cells possessed several
MAPK pathways came shortly after the identification of
the ERKs. The protein synthesis inhibitor cycloheximide,
when administrated to rats, can elicit the in vivo activation of ribosomal S6 phosphorylation, and, in fact, this
strategy was used to activate p70 S6 kinase in vivo before
purification (10). It was originally suggested that p70 S6
kinase, like MAPK-activated protein kinase-1 (MAPKAPK1)/ribosomal S6 kinase (Rsk), a known ERK substrate
with which p70 S6 kinases shares some superficial enzymological similarities (85, 254, 292), would be activated
by ERKs. However, the ERKs were unable to activate p70
S6 kinase in vitro, suggesting that cycloheximide activated novel kinases including proline-directed protein kinases (164). This idea was supported by the demonstration that injection of cycloheximide into rats activated a
novel Ser/Thr kinase activity that could phosphorylate
microtubule-associated protein-2 (MAP2). Purification of
this kinase revealed a 54-kDa polypeptide, initially named
p54-MAP2 kinase, or p54 (164, 165). p54 could be inactivated with Tyr or Ser/Thr phosphatases, indicating that,
like the ERKs, the p54 kinase required concomitant Tyr
and Ser/Thr phosphorylation for activity (167, 165); moreover, studies with synthetic peptide substrates showed
that p54 was proline directed, but phosphorylated Ser/
Thr-Pro motifs with a specificity distinct for that of the
p42/p44 MAPKs (220).
The specificity of p54 toward native protein substrates clearly differed from that of the ERKs. In particular, p54 was unable to activate MAPKAP-K1/Rsk in vitro
under conditions wherein ERK-mediated activation of
MAPKAP-K1/Rsk was observed (164). Of note, p54 was
also unable to activate p70 S6 kinase in vitro, although
both p54 and the ERKs could phosphorylate p70 S6 kinase
in an autoinhibitory segment. p70 S6 kinase activation
was subsequently shown to require additional phosphatidylinositol (PI) 3-kinase-dependent steps (10). Most importantly, p54 was able to phosphorylate the c-Jun transcription factor at two sites (Ser-63 and Ser-73) implicated
in regulation of c-Jun and AP-1 trans-activation function
(244), at a substantially higher rate than was observed for
the p42/p44 MAPKs.
The SAPKs were cloned independently by two
groups: Kyriakis et al. (166) used the amino acid sequence
of tryptic peptides derived from purified p54 to design
specific PCR primers, whereas Dérijard et al. (68) used a
pure PCR strategy employing degenerate primers derived
from regions conserved among known MAPKs. The generation of specific antibodies enabled an analysis of the
regulation of endogenous p54 by extracellular stimuli.
Assay of p54 kinase activity immunoprecipitated from
extracts of cells subjected to various treatments revealed
that, in contrast to the p42/p44 MAPKs, p54 was not
strongly activated in most cells by mitogens such as insulin, EGF, PDGF, or FGF. In contrast, p54 was vigorously activated by a variety of noxious treatments such as
heat shock, ionizing radiation, oxidant stress, DNA damaging chemicals (topoisomerase inhibitors and alkylating
agents), reperfusion injury, mechanical shear stress, and,
of course, protein synthesis inhibitors (cycloheximide
and anisomycin) (68, 165, 166, 237).
In rationalizing the significance of p54 activation by
environmental stresses, the potent activation of p54 by
tunicamycin was conceptually important; tunicamycin inhibits N-linked protein glycosylation and leads to the
accumulation of misfolded proteins exclusively within the
lumen of the endoplasmic reticulum (ER). The ability of
an ER-localized perturbation to activate the cytosolic p54
kinase was one of the first clear-cut examples of stressactivated signal transduction through a protein kinase
(166). This formulation suggested that stress-activated
cytokines were likely regulators of p54 activity, a surmise
rapidly borne out by the demonstration that p54 is
strongly activated by all the inflammatory cytokines of the
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The insulin/mitogen-regulated extracellular signalregulated kinase (ERK) pathway was the first mammalian
MAPK pathway to be identified. This pathway is largely
regulated by the monomeric GTPase Ras which recruits
MAP3Ks of the Raf family to activate two MEKs: MEK1
and MEK2. These, in turn, activate the ERKs. The biochemistry, biology, and regulation of the ERKs have been
reviewed exhaustively elsewhere (10, 11, 195).
In recent years, it has become clear that, as with
yeast, multiple parallel mammalian MAPK pathways exist
and that most of these, in conjunction with the nuclear
factor-␬B (NF-␬B) pathway, are pivotal to stress and inflammatory responses rather than to mitogen responses.
As mammalian stress-activated signaling pathways are
elucidated, it is becoming evident that these pathways
will be important targets for novel anti-inflammatory
drugs.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
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TABLE
1. MAPK nomenclature
Name
Alternate Names
ERK1
p44-MAPK
ERK2
p42-MAPK
SAPK-␣
SAPK-p54␣1
SAPK-p54␣2
SAPK-p46␣1
SAPK-p46␣2
SAPK-␤
SAPK-p54␤1
SAPK-p54␤2
SAPK-p46␤1
SAPK-p46␤2
SAPK-␥
SAPK-p54␥1
SAPK-p54␥2
SAPK-p46␥1
SAPK-p46␥2
p38␣
JNK2, SAPK1a
JNK2␤2
JNK2␣2
JNK2␤1
JNK2␣1
JNK3, SAPK1b
JNK3␤2
JNK3␣2
JNK3␤1
JNK3␣1
JNK1, SAPK1c
JNK1␤2
JNK1␣2
JNK1␤1
JNK1␣1
SAPK2a, CSBP1
p38␤
SAPK2b, p38-2
p38␥
p38␦
SAPK3 ERK6
SAPK4
Substrates
MAPKAP-K1, MNKs,
MSKs, Elk1
MAPKAP-K1, MNKs,
MSKs, Elk1
c-Jun, JunD, ATF2,
Elk1
c-Jun, JunD, ATF2,
Elk1
c-Jun, Jun D, ATF2,
Elk1
MAPKAP-K2/3,
MSKs, ATF2,
Elk1, MEF2C
MAPKAP-K2/3,
MSKs, ATF2
ATF2
ATF2
MAPK, mitogen-activated protein kinase. Commonly accepted
names found in the primary literature are included. ERK pathway nomenclature is also shown but is not discussed in text (195). Not all of the
names listed are included in the text.
been identified. The significance of the COOH-terminal
isoforms is not clear, but the type 1 and type 2 kinases
differ in their substrate binding affinities (63, 111, 143, 166).
C. The p38 MAPKs, a Second Stress-Activated
MAPK Group
The p38 MAPKs are a second mammalian stressactivated MAPK family. Originally described as a 38-kDa
polypeptide that underwent Tyr phosphorylation in response to endotoxin treatment and osmotic shock (117),
p38 (the ␣-isoform) was purified by anti-phosphotyrosine
immunoaffinity chromatography; cDNA cloning revealed
that p38 was the mammalian MAPK homolog most closely
related to HOG1, the osmosensing MAPK of S. cerevisiae.
Most notably, the p38s, like Hog1p, contain the phosphoacceptor sequence Thr-Gly-Tyr (117, 120). Independently and contemporaneously, two groups identified
p38␣ as a kinase activated by stress and IL-1 that could
phosphorylate and activate MAPK-activated protein kinase-activated protein kinase-1 (MAPKAP kinase-2, see
sect. IIE1), a novel Ser/Thr kinase implicated in the phosphorylation and activation of the small 27-kDa heat shock
protein HSP27 (95, 259).
Of potential clinical importance, p38␣ was also purified and cloned as the polypeptide receptor for a class of
experimental pyridinyl-imidazole anti-inflammatory drugs,
the cytokine-suppressive anti-inflammatory drugs (CSAIDs),
the most extensively characterized of which is the compound SB203580 (174). CSAIDs were originally identified
in a screen for compounds that could inhibit the transcriptional induction of TNF and IL-1 during endotoxin shock
(174). The basis for the efficacy of these compounds as
anti-inflammatory agents was their ability to bind and
directly inhibit a subset of the p38s, thereby blocking
p38-mediated activation of AP-1, a trans-acting factor
required for TNF and IL-1 induction (174). Finally, a
shorter hnRNA splicing isoform of p38 was isolated as a
kinase that could interact with the Myc binding partner
Max (362) (see sect. IIE2). With the identification of additional p38 isoforms, four p38 genes are now known
(Table 1): the original isoform, here referred to as p38␣
[also called CSAIDs binding protein (CSBP) and, somewhat confusingly, SAPK2a], p38␤ (also called SAPK2b,
and p38 –2), p38␥ (also called SAPK3 and ERK6), and
p38␦ (also called SAPK4) (95, 106, 117, 139, 140, 173, 174,
207, 259, 288, 327, 363).
In vitro assays demonstrated that only p38␣ and p38␤
are inhibited by CSAIDs; p38␥ and p38␦ are completely
unaffected by these drugs in vitro or in transfected cells
(106). The basis for this inhibition was revealed in the
crystal structure of p38␣ complexed with the SB203580.
Thr-106 in the hinge of the p38␣ ATP binding pocket
interacts with a fluorine atom in the SB203580 structure.
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TNF family [TNF, interleukin (IL)-1, CD40 ligand, CD27
ligand, Fas ligand, receptor activator of NF-␬B, RANK
ligand, etc.] as well as by vasoactive peptides (endothelin
and ANG II) (4, 16, 19, 68, 166, 178, 275, 369). p54 has
since been renamed, and the nomenclature of this family
of kinases is somewhat confusing (Table 1). Two systems
are generally accepted: stress-activated protein kinase
(SAPK) in reference to the regulation of these kinases by
environmental stress and inflammation and c-Jun NH2terminal kinase (JNK) in reference to the phosphorylation
by these kinases of the c-Jun NH2-terminal trans-activation domain.
The SAPKs are encoded by at least three genes:
SAPK␣/JNK2, SAPK␤/JNK3, and SAPK␥/JNK1 (111, 143,
166) (Table 1). As with all MAPKs, each SAPK isoform
contains a characteristic Thr-X-Tyr phosphoacceptor loop
in subdomain VIII of the protein kinase catalytic domain.
Whereas the ERK sequence is Thr-185-Glu-Tyr-187
(ERK2) or Thr-203-Glu-Tyr-205 (ERK1), that of the SAPKs
is Thr-183-Pro-Tyr-185. The expression of each SAPK gene
is further diversified by differential hnRNA splicing. Splicing within the catalytic domain at a region spanning subdomains IX and X results in type 1 and type 2 SAPKs
(␤ and ␣ JNKs, respectively, Table 1), whereas splicing at
the extreme COOH terminus yields 54-kDa (p54) and
46-kDa (p46) polypeptides (type 2 and type 1 JNKs, respectively, Table 1); thus at least 12 polypeptides have
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D. ERK5/Big MAP Kinase-1 (BMK1), a Third Class
of Stress-Activated MAPK
The novel MEK MEK5 was cloned by degenerate PCR
as part of an effort to identify new MAPK pathways and
regulators (83, 367). ERK5, a MEK5 substrate, was cloned
as part of a two-hybrid screen that employed MEK5 as
bait (367). ERK5 is a ⬃90-kDa MAPK of which only one
mammalian homolog is known. ERK5 has the sequence
Thr-Glu-Tyr in its phosphoacceptor loop. The NH2-terminal kinase domain of ERK5 is followed by an extensive
COOH-terminal tail of unknown function that contains
several consensus proline-rich motifs indicative of binding sites for proteins with SH3 domains (367).
The stimuli that recruit ERK5 have not been comprehensively characterized; however, ERK5 can be substantially activated by environmental stresses such as oxidant
stress (peroxide) and osmotic shock (sorbitol), but not by
vasoactive peptides or inflammatory cytokines (TNF) (1).
ERK5 may also lie downstream of receptor Tyr kinases.
Although mitogen activation of ERK5 was not initially
observed (1), EGF activation of ERK5 has been subsequently documented (38).
E. SAPK, p38, and ERK5 Substrates
As with the ERKs, the SAPKs, p38s, and ERK5 phosphorylate both transcription factors and other protein
kinases. Some of the protein kinase substrates (MAPKAPK2 and -K3 and PRAK) are selectively recruited by stressactivated MAPKs. Others (MNKs and MSKs), however, are
activated by both stress- and mitogen-regulated MAPKs
and, like the AP-1 transcription factor, integrate both
stress and mitogenic signaling pathways. The substrates
of stress-activated MAPKs highlight the importance of
these MAPKs to the inflammatory response.
1. Protein kinases
A) MITOGEN-ACTIVATED PROTEIN KINASE-ACTIVATED PROTEIN
(MAPKAP-KS)-2 AND -3. MAPKAP kinase-2 (MAPKAPK2) and the structurally related MAPKAP-K3 (also called
three pathway regulated kinase or 3PK) are a small family of Ser/Thr kinases that consist of an NH2-terminal
regulatory domain and a COOH-terminal kinase domain
(132, 169, 203, 283, 289, 290). They are unrelated to the
MAPKAP-K1s/Rsks, targets of the ERKs (11, 85). Along
with p38-regulated and activated kinase (PRAK, see below), MAPKAP-K2 and MAPKAP-K3 phosphorylate the
small heat shock protein HSP27 (132, 169, 203, 283, 289,
290). Nonphosphorylated HSP27 normally exists in highmolecular-weight multimers that serve as molecular chaperones. Phosphorylation of HSP27 by MAPKAP kinase-2/3
at Ser-15, Ser-78, Ser-82, and Ser-90 coincides with the
dissociation of HSP27 into monomers and dimers, and
with the redistribution of HSP27 to the actin cytoskeleton
(132). In peroxide-treated human umbilical vein endothelial cells, this redistribution of HSP27 may contribute to
triggering the reorganization of F-actin into stress fibers,
thereby affecting cell motility (132, 169). MAPKAP-K2/3catalyzed phosphorylation of HSP27 at Ser-90 appears
necessary for this process, and mutation of Ser-90 to Ala
prevents stimulus-induced changes in HSP27 oligomerization (169).
Early studies had indicated that purified ERK1 could
phosphorylate and activate MAPKAP-K2 in vitro (289).
However, ERKs likely do not represent the physiological
MAPKAP-K2 kinases. MAPKAP-K2 is activated not by insulin or mitogens, conditions wherein ERKs are strongly
activated, but by stresses and inflammatory cytokines,
conditions wherein ERKs are not appreciably activated
(95, 259). MAPKAP-K2 is phosphorylated and activated by
p38␣ and p38␤ (but not by p38␥ or p38␦) (60, 95, 259)
(Fig. 2). Activation of MAPKAP-K2 is a multistep process
wherein phosphorylation of Thr-25, catalyzed by either
p38␣ or p38␤, gates subsequent phosphorylation of
Thr-222 and Ser-272 in the kinase activation loop, a reaction also catalyzed by either p38␣ or p38␤. Together,
these phosphorylations, accompanied by an additional
autophosphorylation at Thr-334, result in activation of
MAPKAP-K2 (15). Consistent with regulation by p38␣ and
p38␤, MAPKAP-K2 activation and HSP27 phosphorylation
are inhibited by CSAIDs (15, 132, 169, 203).
MAPKAP-K3 can also phosphorylate HSP27 (169).
Although it has been shown that MAPKAP-K3 can be
activated in vivo, in overexpression experiments, by
the ERK, SAPK, and p38 pathways (189), endogenous
MAPKAP-K3 is only significantly activated by stresses
and inflammatory cytokines, and in a manner that can
KINASES
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This orients the drug to interact with His-107 and Leu-108
of the ATP binding pocket (86, 110). Substitution of Thr106, alone or in combination with His-107 or Leu-108, with
the corresponding, more bulky residues from p38␥ or
p38␦ (Met, and Pro or Phe, respectively, in both cases)
abolishes SB203580 binding. Conversely, if the amino acid
of p38␥, p38␦, or even SAPK␥ which corresponds to p38␣
Thr106 is replaced with Thr, the resulting mutants display
at least partial sensitivity to SB203580 (86, 110).
Like the SAPKs, the p38s are strongly activated in
vivo by environmental stresses and inflammatory cytokines and are inconsistently activated by insulin and
growth factors. In almost all instances, the same stimuli
that recruit the SAPKs also recruit the p38s (165). One
exception is ischemia-reperfusion. SAPKs are not activated during ischemia, but rather during reperfusion,
whereas the p38s are activated during ischemia and remain active during reperfusion (22, 165, 237). The basis
for this difference is unknown.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
be completely inhibited with CSAIDs, suggesting that
MAPKAP-K3 is, in fact, stress and not mitogen activated,
and that p38␣ and/or p38␤ are the major MAPKAP-K3
kinases in vivo (169).
B) P38-REGULATED/ACTIVATED KINASE. p38-regulated/activated kinase (PRAK) is a ⬃50-kDa Ser/Thr kinase with a
similar overall structure to MAPKAP-K2, -K3, and the
MAPK-interacting kinases (MNKs, see below). Accordingly, PRAK consists of an NH2-terminal regulatory domain and a COOH-terminal kinase domain. Like MAPKAP
-K2 and -K3, PRAK is activated selectively in response to
stress and inflammatory cytokines and is not detectably
activated by mitogens. PRAK can be activated in vivo and
in vitro by p38␣ and p38␤, and consistent with this, PRAK
activation can be blocked with CSAIDs (224). Phosphorylation of PRAK catalyzed by p38s is at Thr-182 in the
kinase domain activation loop (224). Once activated,
PRAK can phosphorylate HSP27 at the physiologically
relevant sites, and in-gel kinase assays indicate that
PRAK is an important stress-activated HSP27 kinase (224)
(Fig. 2).
C) MNKS. Cellular mRNAs contain a 5⬘-cap structure,
the N7-methylguanosine cap. The N7-methylguanosinebinding protein eIF-4E recruits mRNAs onto a scaffold
protein eIF-4G, which also binds the RNA helicase eIF-4A.
The latter, in collaboration with the RNA binding protein
eIF-4B, unwind secondary structure in the mRNA 5⬘-untranslated segment, thereby facilitating the scanning of
the mRNA by the 40S ribosomal complex to the ATG
translational initiation site. The complex of eIF-4A, B, G,
and E is known as eIF-4F (285).
The eIF-4E-eIF-4G interaction is negatively regulated
by the translational repressor protein 4E-binding pro-
tein-1 (4E-BP1 also called phosphorylated heat- and acidstable protein regulated by insulin, PHAS-I). 4E-BP1 is
multiply phosphorylated in response to insulin and mitogens resulting in the dissociation of 4E-BP1 from eIF-4E,
and the release of eIF-4E for incorporation into the eIF-4F
complex. In vivo, the phosphorylation of eIF-4E is
strongly inhibited by rapamycin and wortmannin, consistent with the fact that dissociation of 4E-BP1 is regulated
by the mammalian target of rapamycin (mTOR) (11, 285)
as well as by protein kinases downstream of PI 3-kinase
(285).
In addition, eIF-4E itself also undergoes a regulatory
phosphorylation at Ser-209 in response to both insulin/
mitogen and environmental stress. This phosphorylation
increases the affinity of eIF-4E for the 5⬘-cap by about
threefold; crystallographic data also indicate that phosphorylation of eIF-4E favors the binding of 4E to the
5⬘-cap (285).
MNK-1 and -2 are two closely related kinases that are
probably the physiologically relevant eIF-4E Ser-209 kinases. As the name implies, MNKs associate in vivo with
MAPKs and are in vitro and in vivo MAPK substrates.
MNKs are phosphorylated and activated both by ERKs 1/2
(in response to insulin and mitogens) and by the p38s (in
response to stress) (98, 331, 332). The regulation of the
MNKs by both the ERKs and p38s indicates that, as with
AP-1 (see below), MNKs are a site of integration of stress
and mitogenic signaling pathways (Fig. 2).
D) MSK1/2. Mitogen- and stress-activated protein kinases (MSKs)-1 and -2 are a recently identified family of
Ser/Thr protein kinases with an overall structure similar
to that of the MAPKAP-K1s/Rsks (85), notably, the MSKs
possess two tandem protein kinase domains. MSKs1/2
were identified in a search of databases for DNA sequences homologous to p70 S6 kinase. In vitro MSK1
will phosphorylate the synthetic peptide Crosstide (GlyArg-Pro-Arg-Thr-Ser-Ser-Phe-Ala-Glu-Gly); however, MSK1
purified from unstimulated cells possesses low Crosstide
kinase activity. In contrast, the Crosstide kinase activity
of MSK1 is activated ⬎100-fold upon incubation in vitro
with ERK2 (Fig. 2), and MSK1 is activated in vivo by
mitogens in a manner inhibitable by the MAPK pathway
inhibitor PD98059 (66). MSK1 is also a substrate for the
p38 MAPKs. Consistent with this, MSK1 is also activated
in vivo by environmental stresses [arsenite, ultraviolet
(UV) radiation, peroxide], in a manner inhibited by the
CSAID SB203580 (66). MSK1 is also activated by TNF;
however, the pharmacological inhibition of TNF activation of MSK1 is more complex than the inhibition of
mitogen or stress activation of MSK1. In HeLa cells, TNF
activates the p38s (and the SAPKs) as well as the ERKs.
However, p38 activation is more rapid, reaching a maximum at 5 min, while ERK activation is comparatively
slower, reaching an apparent maximum at 15 min. Accordingly, SB203580 completely blocks early (5 min) TNF
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FIG. 2. Downstream protein kinases and their targets regulated by
p38s. Note that MSKs and MNKs are also regulated by the extracellular
signal-regulated kinases (ERKs).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
stimulated phosphorylation of histone H3 occurs at Ser10, while HMG-14 is phosphorylated at Ser-6. Recent genetic evidence indicates that mitogenic histone H3
phosphorylation is mediated at least in part by MAPKAPK1b/Rsk2 inasmuch as cells derived from patients with
Coffin-Lowry syndrome, a loss of function mutation in the
mapkap-k1b/rsk2 gene, display a substantial deficit in
mitogen-stimulated histone H3 phosphorylation (267).
However, it is unclear if MAPKAP-K1b/Rsk2 is the
only histone H3 kinase in vivo. Thus both mitogens and
stresses can stimulate H3 and HMG-14 phosphorylation in
vivo (308), and MAPKAP-K1s/Rsks are not strongly activated by stresses (10, 164). Moreover, MAPKAP-K1s/Rsks
are poor HMG-14 kinases in vitro and are not inhibited by
the inhibitor H89 which blocks HMG-14 kinase activity in
vivo and in vitro. In contrast, MSK1 is strongly inhibited
by H89 in parallel with in vivo inhibition of histone H3 and
HMG-14 phosphorylation. In addition, in vitro, MSK1 can
phosphorylate histone H3 at Ser-10, at a rate comparable
to or better than that phosphorylated by MAPKAP-K1s/
Rsks in vitro (308).
2. Transcription factors
A) CREB HOMOLOGOUS PROTEIN/GROWTH ARREST AND DNA DAMAGE-155.
CREB homologous protein (CHOP)/growth arrest
and DNA damage-155 (GADD153) is a bZIP transcription
factor of the CREB family (113). CHOP/GADD153 is transcriptionally induced in response to genotoxic and inflammatory stresses. These treatments can also activate the
transcriptional regulatory functions of CHOP/GADD153
through stimulus-induced phosphorylation of Ser-78 and
Ser-81. CHOP/GADD153 is a transcriptional repressor of
certain cAMP-regulated genes and a transcriptional activator of some stress-induced genes. Activation by genotoxins of CHOP/GADD153 mediates in part cell cycle
arrest at G1/S, an important consequence of DNA damage
inasmuch as it allows for DNA repair before DNA replication, key to preserving genomic integrity. p38␣ is a
likely stress-activated regulator of CHOP/GADD153 function, given that p38␣, and not SAPK or ERK, can phosphorylate CHOP/GADD153 at Ser-78 and Ser-81 in vivo
and in vitro (325) (Fig. 3).
B) NUCLEAR FACTOR OF ACTIVATED T CELLS. Transcription
factors of the nuclear factor of activated T cells (NFAT)
family are distantly related to Rel/NF-␬B (250). In resting
cells, NFATs are retained in the cytosol as a consequence
of phosphorylation [catalyzed by casein kinase-I␣ and,
possibly, glycogen synthase kinase (GSK)-3] at five or six
sites (within NFAT4, this comprises a region spanning
amino acids 204 –215). NFAT phosphorylation affects conformation so as to mask the nuclear localization signal.
Agonist-induced Ca2⫹ entry recruits the Ca2⫹-dependent
phosphatase calcineurin (phosphatase 2B), which dephosphorylates NFATs, thereby exposing NFAT’s nuclear
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activation of MSK1 in HeLa cells. After 15 min of TNF
stimulation, however, SB203580 inhibition is only partial,
and MSK1 can now also be partially inhibited with the
ERK pathway inhibitor PD98059 (66) (Fig. 2).
cAMP response element binding protein (CREB) is a
bZIP transcription factor that binds and trans-activates
genes containing the cAMP response element (CRE– consensus sequence: TGACGTCA). CREB trans-activating
activity can be activated by a number of different stimuli
including mitogens, stresses, and, of course, agonists that
elevate cAMP. Activation of CREB trans-activating activity correlates with phosphorylation at Ser-133. Protein
kinase A (PKA) likely represents the major CREB kinase
recruited by cAMP (113). In addition, several protein kinases including MAPKAP-K1s/Rsks (343) and MAPKAP
-K2 (300) have been shown to possess mitogen- or stressactivated CREB Ser-133 kinase activity. MSK1 is also a
potent CREB kinase in vitro, and a substantial amount of
evidence indicates that, in fact, MSK1, and not MAPKAPK1/Rsk or MAPKAP-K2/3, is the physiologically relevant
stress- and mitogen-activated CREB kinase (66).
First, the Kcat for MSK1-catalyzed phosphorylation of
CREB is nearly 100-fold greater than that for MAPKAPK1/Rsk-catalyzed CREB phosphorylation. Moreover, the
MSK1 polypeptide has a bipartite nuclear localization signal and is localized exclusively in the nucleus, where
CREB resides. MAPKAP-K1s/Rsks are largely cytosolic,
although they can translocate to the nucleus in response
to extracellular stimuli (40). Finally, the pattern of
pharmacological inhibition of stress- and mitogen-activated CREB phosphorylation in vivo tightly corresponds
to the pattern of pharmacological inhibition of MSK1
activation. Thus activation of CREB by stress and mitogens is blocked by the broad specificity kinase inhibitor Ro318220. MSK1 is strongly inhibited in vitro by
Ro318220, whereas MAPKAP-K2 and MAPKAP-K3 are not.
Finally, CREB activation in vivo by TNF can be blocked
with SB203580 and the ERK inhibitor PD98059 with kinetics that parallel those described above for inhibition of
MSK1 (66).
A recent study suggests that MSKs may be involved in
the phosphorylation of components involved in chromatin remodeling. Both mitogens and stresses, as they stimulate gene expression, must prompt the loosening of chromatin structure from around target genes. This enables
the transcriptional machinery to gain access to genes that
are to be expressed (35, 267). This alteration in chromatin
structure involves both the acetylation and phosphorylation of histones. Many transcription factors that are phosphorylated in a stimulus-dependent manner can recruit
transcriptional coactivators that are histone acetyl transferases. In addition, protein kinase cascades phosphorylate histones themselves (35, 267). The nucleosomal proteins histone H3 and high mobility group-14 (HMG-14) are
prime targets of phosphorylation (35, 267, 308). Agonist-
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
815
FIG. 3. Regulation of transcription
factors by MAPKs. Note that AP-1 regulation involves both the direct phosphorylation of AP-1 components as well as
the transcriptional induction of AP-1
components mediated by distinct transcription factor targets of MAPKs.
lation and activation. MEKK1 inhibition of NFAT translocation occurs whether or not the SAPK phosphorylation
sites have been mutated to alanine, and the effect of
MEKK1 is not reversed by dominant inhibitors of the
SAPK pathway. Apparently, MEKK1 fosters inhibition of
NFAT nuclear translocation by stabilizing the association
between NFAT4 and the inhibitory NFAT kinase casein
kinase-I␣ (368).
Davis and colleagues (48) have also shown that
SAPKs can phosphorylate NFATc1 (also called NFAT2).
In this instance, phosphorylation is stimulated by phorbol
12-myristate 13-acetate (PMA) and ionomycin and occurs
at Ser-117 and -172. Phosphorylation inhibits or delays the
accumulation of NFATc1 in the nucleus by blocking the
binding of calcineurin, an essential step in NFATc1 activation (48). NFATc1 is crucial to the differentiation of TH
cells to the TH2 effector phenotype (see sect. VF1). Interestingly, disruption of SAPK␥ or both SAPK␥ and -␣ leads
to the preferential accumulation of TH2 cells, consistent
with the notion that SAPK-mediated inhibition of TH2
differentiation is mediated through NFATc1 (48, 76, 77)
(see sect. VF1).
C) MAX. Max is a 12-kDa helix-loop-helix (HLH)
polypeptide that interacts with the transcription factor
c-Myc enabling c-Myc to trans-activate at least a subset of
its target genes. c-Myc is a key regulator of cell proliferation, differentiation, and apoptosis. The biological functions of c-Myc are thought to depend in part on the
polypeptide binding partners with which c-Myc interacts
and the regulation of these binding partners (20). A
COOH-terminally truncated hnRNA splicing isoform of
p38␣, referred to as Max-interacting protein-2 (Mxi2), was
isolated in a yeast two-hybrid screen for Max interactors.
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localization signal and triggering NFAT nuclear translocation. Dephosphorylation of NFATs also enhances DNA
binding affinity (250). NFATs bind and trans-activate
genes with an NFAT cis-acting element (consensus sequence: T/AGGAAAAT). NFAT sites are often located
close to AP-1 sites in many promoters, allowing for the
cooperative binding and synergistic trans-activation of
numerous genes (IL-2, IL-4, IL-5, and CD40L are examples) (250). Calcineurin is a major target of the immunosuppressants FK506 and cyclosporin A, and accordingly,
inhibition of NFAT activity is an important consequence
of FK506 and cyclosporin A action (250, 270).
Serum factors can substantially inhibit Ca2⫹-mediated nuclear translocation of NFATs. Insofar as casein
kinases and GSK3 are not serum stimulated, the serumdependent inhibition of Ca2⫹-mediated NFAT translocation suggested that serum-responsive kinase cascades
might contribute to NFAT inhibition (47, 250). Davis and
colleagues (47) showed that the SAPKs could phosphorylate the NFAT family member NFAT4 (also called
NFATc3) at Ser-163 and Ser-165. This phosphorylation
correlates with an inhibition of stimulus-induced NFAT4
nuclear translocation, and on the basis of this finding, it
has been proposed therefore that the SAPK pathway antagonizes NFAT4 action (47) (Fig. 3). However, the significance of these results is somewhat unclear. Zhu et al.
(368) showed that NFAT4 mutants in which the putative
SAPK phosphoacceptor sites (Ser-163 and Ser-165) have
been changed to Ala still show serum-stimulated inhibition of nuclear translocation in many instances. These
investigators also observed that the SAPK-specific
MAP3K MEK-kinase-1 (see sect. IIG2) could indirectly,
and independently of SAPK, block NFAT4 dephosphory-
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
are phosphorylated in vivo under conditions wherein the
SAPKs are activated. Immunodepletion of SAPK from cell
extracts removes all stress- and TNF-activated c-Jun kinase (166, 237). Thus SAPKs are the dominant kinases
responsible for stress- and TNF-activated c-Jun phosphorylation (Fig. 2). JunD is also phosphorylated at Ser-90 and
Ser-100 by SAPKs, albeit less effectively than is c-Jun.
Ser-90 and Ser-100 of JunD lie within a region of the JunD
trans-activation domain similar to the phosphoacceptor
domain of c-Jun (144).
The SAPKs and p38s can both phosphorylate ATF2 at
Thr-69 and Ser-71 in the trans-activation domain. Again,
these residues are phosphorylated in vivo under conditions in which the SAPKs and p38s are activated. Phosphorylation of ATF2 at Thr-69 and Ser-71 correlates with
activation of ATF2 trans-activating activity (112) (Fig. 3).
Whether the SAPKs or p38s represent the dominant ATF2
kinases depends on the cell type and stimulus used. During reperfusion of ischemic kidney, for example, the
SAPKs are the only detectable ATF2 kinases (219),
whereas in KB keratinocytes treated with IL-1, the p38s
are the major ATF2 kinases (60).
The SAPKs, p38s, and ERK5 (primarily in response to
stress) and the ERKs (primarily in response to mitogens)
also contribute to AP-1 activation by stimulating the transcription of genes encoding AP-1 components (147, 150)
(Fig. 3). Induction of c-fos expression is one of the earliest
transcriptional events known to occur. The fos promoter
includes a cis-acting element, the serum response element (SRE), that mediates the recruitment of a heterodimeric transcription factor containing two polypeptides, the serum response factor (SRF) and a member of
the ternary complex factor (TCF) family (318) (Fig. 3).
The TCFs include Elk-1 and Sap-1a (318). The SAPKs,
and ERKs, but not p38, can phosphorylate two critical
residues in the Elk1 COOH terminus (Ser-383, Ser-389),
while the p38s can efficiently phosphorylate the corresponding residues (Ser-381 and Ser-387) on Sap1a (104,
138, 194, 337, 351). This phosphorylation enhances the
binding of TCFs to the SRF and thereby triggers transactivation at the SRE. By these processes, MAPKs activated by both stress and mitogens can convergently contribute to c-fos induction (Fig. 3).
The p38s and ERK5 can also phosphorylate the
transcription factors myocyte enhancer factor-2 MEF2
subgroup of the MCM1-agamous and deficiens-SRF
(MADS) box transcription factor family (116, 150, 365).
MEF2s (MEF2A-D) were originally identified as transcription factors that bound to AT-rich sequences (consensus: CTAAAAATAA) and trans-activated key genes
involved in myoblast differentiation; however, some
MEF2s, notably MEF2C, are widely expressed and may
regulate numerous other transcriptional regulatory
events (94). Only MEF2A and -C are MAPK substrates.
MEF2B and -D are not MAPK substrates; however,
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Max and either Mxi2 or p38␣ can form a tight complex in
vivo and in vitro, an interaction thought to be mediated by
the HLH domain of Max and a similar loop on Mxi2 and
p38␣. Max is a good substrate for both Mxi2 and p38␣
(363). Interestingly, there are no consensus proline-directed sites on Max, suggesting that the absolute proline
requirement for MAPKs may be less pronounced for p38.
The functional significance of p38-catalyzed Max phosphorylation is unclear (363) (Fig. 3).
D) ACTIVATOR PROTEIN-1. The SAPKs and p38s are the
dominant stress-activated Ser/Thr kinases responsible for
the recruitment of the activator protein-1 (AP-1) transcription factor (147, 165). ERK5 also plays a role in
stress-activated AP-1 regulation (150). AP-1 is a heterodimer comprised of bZIP transcription factors, typically c-Jun and JunD, along with members of the fos
(usually c-Fos) and ATF (usually ATF2) families. All bZIP
transcription factors contain leucine zippers that enable
homo- and heterodimerization, and AP-1 components are
organized into Jun-Jun, Jun-Fos, or Jun-ATF dimers (147)
(Fig. 4).
The presence of Jun family members enables AP-1 to
bind to cis-acting elements containing the 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TR; consensus sequence: TGAC/GTCA). ATFs, including ATF2, are
members of the CREB subfamily of bZIP transcription
factors. AP-1 heterodimers containing ATF transcription
factors can bind both to the TRE and to the CRE (113,
147). AP-1 is an important trans-activator of a number of
stress responsive genes including the genes for IL-1 and
-2, CD40, CD30, TNF, and c-Jun itself. In addition, AP-1
participates in the transcriptional induction of proteases
and cell adhesion proteins (e.g., E-selectin) important to
inflammation (147, 252).
Activation of AP-1 involves both the direct phosphorylation/dephosphorylation of AP-1 components as well as
the phosphorylation and activation of transcription factors that induce elevated expression of c-jun or c-fos.
Both events can be activated independently by several
signaling pathways (Fig. 4). Thus c-Jun is phosphorylated
in resting cells at a region immediately upstream of the
COOH-terminal, the DNA binding domain (Thr-231, Thr239, Ser-243, Ser-249). This phosphorylation inhibits DNA
binding and can be catalyzed in vivo by either glycogen
synthase kinase-3 (GSK3) or casein kinase II (CKII). Upon
stimulation of cells with AP-1 activators, the c-Jun COOHterminal phosphates are removed under conditions
wherein GSK3 is inactivated (24, 147). GSK3 is inhibited
upon mitogen stimulation by a mechanism dependent on
PI 3-kinase (58, 79).
Phosphorylation of c-Jun or ATF2 within their NH2terminal trans-activation domains correlates well with
enhanced trans-activating activity (68, 112, 166, 244). The
SAPKs can phosphorylate the c-Jun trans-activating domain at Ser-63 and Ser-73 (68, 166, 244). These residues
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
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MEF2B and -D may act in conjunction with phosphorylated MEF2A/C. Thus phosphorylation enhances the
trans-activating activity of MEF2A and -B or A and D
heterodimers (365). MEF2A is phosphorylated at Thr312 and Thr-319 by p38␣. Phosphorylation by other
MAPKs has not been observed (365). MEF2C phosphorylation by MAPKs is apparently more complex. The
p38s and ERK5 phosphorylate different sets of sites on
the MEF2C polypeptide. Thus p38s phosphorylate Thr-
293 and Thr-300, whereas ERK5 phosphorylates Ser-387
(116, 150). All three sites are phosphorylated in response to serum or stress; however, Thr-293/Thr-300
phosphorylation is sufficient for p38 activation of
MEF2C while Ser-387 phosphorylation is sufficient for
ERK5 activation of MEF2C (116, 150). A cis-element for
MEF2C resides in the c-jun promoter; thus p38 and
ERK5 activation can contribute to the induction of
c-jun expression (116) which, in turn, potentiates to
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FIG. 4. Docking sites and stress-activated protein kinase (SAPK)/p38 substrate specificity. A: SAPKs bind to the c-Jun
␦ domain, enabling phosphorylation of transcription factors with which c-Jun is dimerized, even if these dimerization
partners do not possess ␦ domain regions capable of binding SAPKs. B, top: proline-directed phosphoacceptor motifs of
c-Jun family members. Note JunB has no proline-directed phosphoacceptor sites (underlined in c-Jun and JunD), and is
therefore not a SAPK substrate. Bottom: hydrophobic SAPK docking sites in Jun family members. Note that despite the
sequence similarities, only c-Jun ␦ and JunB can associate with SAPKs. C: illustration of the selectivity of MAPKs for
substrates mediated by the CD motif of MAPKs and the polybasic docking sites (DS in the figure) for substrates. The
binding and phosphorylation of p38␣ by MKK6 and MNK1 by p38␣ are shown. D, top: polybasic docking sites of MKK6
and MNK1. Bottom: representative CD motifs of MAPKs (for details see Ref. 301).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
AP-1 activation. Indeed, MEF2A or -C, once activated
by p38s, can trans-activate the c-Jun promoter (116,
150, 365) (Fig. 3).
3. The substrate specificity of the SAPKs and p38s
is conferred by specific MAPK docking sites
on substrate proteins: the MAPK docking sites
interact with specific substrate binding motifs
on MAPK polypeptides
vitro; however, when heterodimerized with c-Jun, JunD
can undergo efficient SAPK-catalyzed phosphorylation in
vitro, and activation in transfection experiments in vivo
(144) (Fig. 4). JunB also possesses a conserved region
homologous to the SAPK binding pocket of c-Jun, and in
contrast to JunD, JunB binds SAPK well. However, JunB
does not possess the proline-directed phosphoacceptor
sites that are required for SAPK phosphorylation (144,
147) (Fig. 4A). Thus JunB is not a SAPK substrate in vivo
or in vitro (144) (Fig. 4B). However, in transfection experiments, JunB can heterodimerize with c-Jun mutants
missing the ␦-domain and, because JunB can bind SAPK,
foster SAPK phosphorylation of these mutants (144)
(Fig. 4B).
ATF2 also contains a hydrophobic pocket (amino
acids 20 – 60) similar to the c-Jun ␦-domain that binds
SAPKs and p38s. As with the c-Jun ␦-domain, the ATF2
MAPK binding site lies NH2 terminal to the phosphoacceptor sites (Thr-69 and Thr-71) (112). Likewise, Elk-1
has a MAPK docking site, the D-domain (amino acids
312–334) that lies NH2 terminal to the phosphoacceptor
sites (Ser-383, Ser-389) (337, 351). Interestingly, the
Elk-1 D-domain is essential for ERK and SAPK binding
and phosphorylation; however, p38 binding and phosphorylation, the physiological significance of which has
not been unambiguously established (138, 351), appears not to require this domain (351). The role of the
SAPK ␤-strand in determining the differential binding
of different SAPK isoforms to ATF2 or Elk1 is unknown.
A second class of MAPK binding domain generally
consists of a small stretch of basic residues (Fig. 4C). This
domain is prevalent among protein kinase substrates of
MAPKs, including MAPKAP-K1s/Rsks (ERK substrates),
MNKs, MAPKAP-K2/3, and PRAK and binds to the recently identified common docking (CD domain) domain,
an extracatalytic region rich in acidic residues, found in
all MAPKs (301). Interestingly, the basic MAPK binding
motif is found not only in many MAPK substrates, but in
a diverse array of MAPK regulators including MEKs (notably the SAPK-specific MEKs SAPK/ERK kinase-1, SEK1
and some MAPK-kinase-7, MKK7, isoforms, as well as the
p38 MEKs MKK3 and MKK6) and MAPK phosphatases
(301) (Fig. 4C). It is likely that the basic residues in these
novel MAPK binding sites interact electrostatically with
the cognate MAPK CD motifs. Moreover, differences
among the basic MAPK docking sites and MAPK CD domains likely confer a high degree of specificity among
MAPKs for their substrates and activators. This is readily
apparent in the specific activation of MAPKAP-K2, under
initial rate conditions, by p38 (see sect. IIE1), and in
the highly selective scaffolding function of SEK1 (see
sect. IIIC4).
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The phosphorylation of c-Jun by the SAPKs highlights an important point about the mechanism of substrate recognition by members of the MAPK family. All
MAPKs are “proline-directed,” phosphorylating Ser/Thr
residues only if followed immediately by proline (see sect.
I and Fig. 4). However, the specificity of MAPKs for their
physiological substrates is dictated in large part by the
presence of binding sites, often substantially distal from
the phosphorylation sites, that are specific for distinct
MAPK subgroups. These MAPK binding sites allow for the
selective interaction between MAPKs and their true in
vivo substrates.
Two general classes of MAPK binding site have been
described for MAPK substrates. The first of these to be
identified was a hydrophobic cluster of residues present
in many transcription factor substrates of MAPKs. Thus
the SAPKs, but not the ERKs or p38s, bind c-Jun quite
strongly. The SAPK binding site on c-Jun lies between
residues 32 and 52, well away in the primary sequence
from Ser-63 and Ser-73, the sites of phosphorylation (63,
143, 144, 147). The SAPK docking site overlaps with the
so-called ␦-domain (amino acids 30 –57), a hydrophobic
region initially implicated in the regulation of c-Jun oncogenicity due to its deletion in oncogenic v-Jun (accordingly, v-Jun does not bind SAPK and is not a SAPK substrate) (63, 143, 144, 147) (Fig. 4).
The binding of c-Jun to SAPK isoforms has been
mapped to a ␤-strand-like region spanning subdomains IX
and X. Interestingly, a portion of this region undergoes
differential hnRNA splicing in type I and II SAPKs (63,
111, 143, 166) (Table 1 and sect. IIE4). This ␤-strand
region also differs substantially among the three SAPK
genes, perhaps accounting for differential SAPK substrate selectivity. Thus SAPK␣ interacts most strongly
with c-Jun (63, 111, 143).
The presence of the SAPK binding site, in conjunction with the ability of c-Jun to heterodimerize with other
members of the Jun family, enables the SAPKs to phosphorylate other AP-1 constituents in vivo that, as monomers or homodimers, are poor SAPK substrates (144,
147). Thus JunD possesses domains similar to the phosphoacceptor and SAPK binding domains of c-Jun. In spite
of this, JunD binds SAPK poorly (144) (Fig. 4). Accordingly, purified JunD is not ordinarily a SAPK substrate in
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
4. The three known SAPK genes and differential
hnRNA splicing generate SAPK isoforms with
different functions
5. The regulation of AP-1 by different classes
of extracellular stimuli involves the integration
of several MAPK pathways and is mediated
by divergent AP-1 component transcription
factor subunits
The different aspects of AP-1 regulation, activation of
constituent transcription factor expression and direct
phosphorylation/activation of constituent transcription
factors, can be independently regulated by several pathways in response to different types of stimuli (Fig. 9).
Thus mitogenic stimuli, which preferentially recruit the
ERKs and inhibit GSK3, will preferentially activate AP-1
through enhancement of expression of AP-1 components
(via ERK phosphorylation of Elk-1, resulting in c-fos expression, for example), and through the relief of GSK3mediated inhibition of c-Jun DNA binding (Fig. 3).
In contrast, stresses and inflammatory cytokines
such as TNF, which preferentially activate the SAPKs and
p38s, can recruit AP-1 through the direct phosphorylation
of AP-1 components (c-Jun by the SAPKs and ATF2 by
both the SAPKs and p38s). However, stress pathways can
also promote enhanced expression of AP-1 components
through recruitment of Elk-1 (mediated by SAPK phosphorylation), which results in elevated c-fos expression,
and through p38- and ERK5-catalyzed phosphorylation of
MEF2A/C (Fig. 3). MEF2A/C can bind and trans-activate
the promoter for c-jun. Stresses can also modestly recruit
PKB/Akt, which can act to inhibit GSK3, thereby blocking
its negative regulation of c-Jun DNA binding. Finally, the
c-Jun promoter also contains an AP-1 site; thus c-jun
expression can be autoregulated by any pathway that
activates AP-1 (Fig. 3).
6. Blazing a trail to the nucleus: the regulation of
MAPKs by nucleocytoplasmic shuttling: lessons
from the ERK pathway
The ERKs, SAPKs, and p38s were first purified from
cytosolic extracts and, indeed, a substantial subset of
MAPK substrates are cytosolic (95, 117, 164, 174, 251,
259). Still, a great many nuclear substrates for MAPKs
have been identified, and the critical question is how
MAPKs localized in the cytosol could regulate nuclear
proteins such as transcription factors.
Of the mammalian MAPKs, the regulation of ERK
nucleocytoplasmic shuttling has been studied the most
extensively, and it is likely that many of the characteristics of the regulation of ERK subcellular localization may
be applicable to stress-regulated MAPKs. Early immunocytochemical examination of the subcellular localization
of ERKs revealed that upon mitogen stimulation, a substantial portion of the pool of ERK immunoreactivity
translocates to the nucleus. Similar stimulus-dependent
translocation of the SAPKs and p38s was subsequently
observed (31, 40, 247). This translocation is reversible and
terminates upon cessation of stimulus (40). The potential
importance of ERK nucleocytoplasmic shuttling became
evident when it was observed that ERK-dependent de
novo gene expression correlated with prolonged ERK
activation, conditions that coincided with nuclear translocation (26, 74, 317).
Nuclear translocation is essential for ERK-dependent
activation of gene expression and regulation of the cell
cycle. SAPK and p38 translocation may be similarly important. Thus sequestration of ERK in the cytosol, by
coexpression with a catalytically inactive form of the
MAPK phosphatase (MKP) MKP3, has no effect on ERKdependent Rsk activation or phosphorylation of an engineered cytosolic mutant of Elk-1, but strongly inhibits
ERK-dependent gene transcription and S phase entry (26).
The molecular mechanism by which ERK nuclear
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As discussed in section IIB, the SAPKs are encoded
by three genes that undergo differential hnRNA splicing to
generate at least 10, and possibly up to 12, polypeptide
species (111, 166). As noted in section IIE3, there is some
evidence that the hnRNA splicing in the SAPK ␤-strand
region spanning kinase subdomains IX-X that generates
the type 1 and type 2 enzymes (Table 1) may affect the
affinity of different SAPK splicing isoforms for substrate.
Thus SAPK-p54␣2 (JNK2␣1, Table 1) binds more strongly
to c-Jun than does SAPK-p54␣1 (JNK2␤2, Table 1). Moreover, SAPK␣ type 2 (JNK2 type ␣, Table 1) isoforms bind
more strongly to c-Jun than to ATF2, whereas SAPK␣ type
1 (JNK2 type ␤) isoforms bind more strongly to ATF2 than
to c-Jun (63, 111, 143).
In addition to differences in substrate binding among
splicing isoforms of the same SAPK gene, the different
sapk gene products themselves, SAPK␣, -␤, and -␥
(JNKs-2, -3 and -1, respectively, Table 1), may also display
differential substrate selectivity. Thus, whereas all SAPK
isoforms bind more strongly to c-Jun compared with
ATF2, SAPK␣/JNK2 isoforms are overall higher affinity
c-Jun kinases than are either SAPK␤/JNK3 or SAPK␥/
JNK1, a feature likely attributable to the high affinity for
c-Jun conferred by the ␤-strand region in domains IX-X of
the SAPK ␣-isoforms (Table 1) (63, 111, 143). It should be
noted, however, that these differences as well as those
among the splicing isoforms are modest (2- to 5-fold in
vitro), and it is unclear what the in vivo biochemical
significance of these differences is. These relatively subtle
effects may have more profound ramafications at the
cellular and organismal level. As we shall see in section V,
more dramatic differences among SAPK isoforms are observed when the biological functions of these enzymes are
examined.
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
scribed in section IIIC4, SAPKs form reversible, activationdependent associations with the SAPK-specific MEK
SEK1, complexes that may serve in part to retain inactive
SAPKs in the cytosol (301, 342). The ERKs, SAPKs, and
p38s do not possess consensus nuclear localization motifs, and the mechanism by which ERKs traverse the
nuclear pore complex is unclear. It is conceivable that
MAPKs, once liberated from elements restraining them in
the cytosol, move into the nucleus by interacting with
nuclear substrate proteins.
F. MEKs Upstream of the SAPKs, p38s, and ERK5
1. Activation of the SAPKs by SEK1 and MKK7
The SAPKs are activated upon concomitant phosphorylation at Thr-183 and Tyr-185. The MEK SAPK/ERK
kinase-1 (SEK1, also called MAPK-kinase-4, MKK4; MEK4;
JNK kinase-1, JNKK1; and SAPK-kinase-1, SKK1, Table 2)
was cloned independently by two groups who employed
degenerate PCR to identify novel MAPK signaling components. Two potential initiation methionines, separated by
34 amino acids, are present in the SEK1 cDNA sequence,
raising the possibility that two SEK1 isoforms exist that
differ at their extreme NH2 termini. Alternative splicing
isoforms of SEK1 have not been identified in vivo; however, deletion of the NH2-terminal 34 amino acids of SEK1
could affect its scaffolding function and its regulation by
the MAP3K MEK kinase-1 (MEKK1) due to loss of a
MEKK1 interaction motif and a basic amino acid-rich
SAPK binding site (see sects. IIE3 and IIIC4) (69, 266,
301, 342).
The homology shared by SEK1 and MEKs-1 and -2 (as
well as yeast MEKs) indicated that this kinase lay upstream of MAPKs. It was shown subsequently that SEK1
could phosphorylate and activate all three SAPK isoforms
in vivo and in vitro (69, 266). Dérijard et al. (69) also
showed that SEK1 could phosphorylate and activate p38
in vivo, when overexpressed, and in vitro (69). However,
the significance of p38 activation by SEK1 is unclear.
Targeted disruption of sek1 in mice has no effect on p38
activity in ES cells (226). Moreover, if SEK1 concentrations in in vitro assays are adjusted to initial rate condi-
TABLE
2. MEK nomenclature
Name
Alternate Names
Substrate(s)
MEK1
MEK2
SEK1
MAPKK1, MKK1
MAPKK2, MKK2
MKK4, JNK kinase (JNKK)-1, MEK4,
SAPK-kinase (SKK)-1
JNKK2, MEK7, SKK4
MEK3, SKK2
MEK6, SKK3
ERK1, ERK2
ERK1, ERK2
SAPKs
MKK7
MKK3
MKK6
SAPKs
p38s
p38s
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localization is regulated is still incompletely understood.
Several novel findings suggest that inactive ERK is retained in the cytosol as part of a complex with its immediate upstream activator MEK1. Formation of this complex requires a polybasic amino acid ERK binding domain
located within the NH2-terminal 32 amino acids of MEK1
(97, 301). As noted above, similar binding site for SAPK
exists on at least a subset of SAPK-specific MEKs including SAPK/ERK kinase-1 (SEK1), MAPK-kinase-7 (MKK7),
as well as the p38-specific MEKs MKK3 and MKK6 (see
sects. IIE3, IIF1, IIF2, and IIIC4). These MAPK binding sites
correspond to the basic motifs present on many MAPK
protein kinase substrates (301). MEK1 itself is retained in
the cytosol by a consensus nuclear export signal (amino
acids 33– 44). In addition, MEK cytosolic localization may
be maintained by scaffold proteins, several of which have
been identified for the SAPKs (see sect. IIIC). Dissociation
of the MEK-ERK complex requires MEK-catalyzed phosphorylation of ERK at the Thr-Glu-Tyr phosphoacceptor
region. Once dissociation of the complex occurs, activated ERK translocates to the nucleus (97).
Whereas ERK nuclear translocation requires phosphorylation to enable dissociation from MEK1, ERK
catalytic activity is not required for translocation. Both
wild-type and kinase-inactive (Lys45Arg) ERK2, phosphorylated in bacteria upon coexpression with activated
MEK1, could translocate to the nucleus upon microinjection into cells. In contrast, mutation of the ERK2
phosphoacceptor sites to nonphosphorylatable residues
(Thr185Ala/Tyr187Phe) completely abrogated the ability
of these kinases to translocate upon injection into fibroblasts (158).
Once the MEK-ERK complex dissociates, there is
some evidence that a portion of the pool of ERK (primarily an active fraction that is not dimeric, see below) can
freely diffuse into the nucleus. However, a substantial
pool of ERK is translocated into the nucleus as part of a
tightly regulated mechanism (3, 97, 158). ERK2 activationdependent dimerization is also critical for translocation.
Thus the crystal structure of activated ERK2 revealed a
dimer with the interface occurring to the rear of the
catalytic cleft between the two kinase lobes (28, 158).
Dimerization requires four Leu residues (Leu-333, Leu336, Leu-341, Leu-344) plus His-176 that forms a salt
bridge with Glu-343 (28, 158). Mutation or deletion of the
critical residues of the dimer interface has no effect on
the in vitro kinase activity or MEK-catalyzed phosphorylation of the ERK2, but abolishes completely ERK2 nuclear translocation (158). Thus phosphorylation and activation of ERK2 results in dissociation from MEK1 and
dimerization, both of which are necessary for translocation. It is noteworthy that both the SAPKs and p38s have
similar dimerization motifs and show similar ligand-dependent translocation (31, 158, 247). Indeed, as is de-
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
alternative hnRNA splicing yielding enzymes with three
different NH2 termini (␣, ␤, and ␥) and two different
COOH termini (types 1 and 2). The ␤- and ␥-isoforms bind
directly to SAPKs via an NH2-terminal extension not
present in the ␣-isoforms. Upon overexpression, the
␣-isoforms exhibit a lower basal activity and higher activation by upstream stimuli (315).
MKK7 is strongly activated by TNF and IL-1, conditions which, at best, induce modest SEK1 activation. In
contrast, SEK1 and MKK7 are activated with equal potency by osmotic shock while MKK7 is more weakly
activated by anisomycin than is SEK1 (91, 188, 218, 314,
340, 353). Thus it is plausible to argue that MKK7 represents at least a substantial portion of the biochemically
detected SAPK activating activity present in 3Y1, PC-12,
or KB cells subjected to osmotic shock or anisomycin, or
in KB cells treated with IL-1.
Although the activation patterns of SEK1 and MKK7
appear not to overlap entirely, there is evidence that SEK1
and MKK7 actively cooperate in the activation of SAPK.
Thus, whereas SEK/MKK4 is not strongly activated by
TNF, TNF cannot activate SAPK in fibroblasts derived
from sek1 ⫺/⫺ early mouse embryos, in spite of the fact
that MKK7 is expressed in these cells (102, 228). Evidence
for the basis of this difference comes from biochemical
studies of the mechanisms of SEK1 and MKK7 catalysis.
As was mentioned above, SEK1 preferentially targets
SAPK Tyr-185 and only weakly phosphorylates Thr-183
(171, 266). In contrast, MKK7 preferentially phosphorylates SAPKs at Thr-183 rather than Tyr-185 (171). Accordingly, when employed individually at low concentrations,
SEK1 and MKK7 are comparatively poor SAPK activators
in vitro, each stimulating at best a 5- to 10-fold activation
of SAPK. When added together, however, SEK1 and MKK7
synergistically activate SAPK, resulting in ⬃100-fold activation accompanied by equal phosphorylation of SAPK
Thr-183 and Tyr-185 (171) in vitro. In vivo, activation of
SAPK by TNF, for example, may involve the combined
effects of modestly activated SEK1 acting primarily on
SAPK Tyr-185 and strongly activated MKK7 acting primarily on Thr-183. Because phosphorylation of both Thr-183
and Tyr-185 is required for full SAPK activation, deletion
of sek1 would, by decreasing sharply SAPK Tyr phosphorylation, substantially compromise SAPK activation by
TNF (171).
2. Activation of the p38s by MKK3 and MKK6
MKK3 (also called MEK3 and SKK2) and MKK6 (also
called MEK6 and SKK3, Table 2) were cloned by degenerate PCR using conserved MEK sequences as templates
(59, 69, 248). Both enzymes are highly selective for p38
and do not activate SAPK or ERK under all conditions
tested (59, 60, 69, 248). MKK3 and MKK6 differ more
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tions for SAPK activation, little or no p38 activation is
observed (60, 205).
While the identification of SEK1 was encouraging,
much biochemical evidence indicated that SEK1 was not
the only SAPK-activating MEK. These studies revealed
that the spectrum of SAPK activators recruited by different stimuli depended on the stimulus used and on the cell
type. Thus fractionation on hydroxylapatite columns of
extracts of osmotically shocked 3Y1 fibroblasts demonstrated a broad peak of SAPK activating activity that was
fully resolved from a separate peak of SEK1 immunoreactivity (216). Likewise, Mono-S chromatography of KB
cell extracts showed that IL-1 failed to activate SEK1, but
stimulated a broadly eluting peak of SAPK activating activity distinct from SEK1. Similar multiple peaks of SAPK
activating activity were observed in extracts of PC-12
cells treated with arsenite or osmotic shock and in KB
cells treated with osmotic shock, UV radiation, or anisomycin (205). In osmotically shocked 3Y1 fibroblasts,
SEK1 represented a comparatively minor peak of SAPK
activating activity (216). In contrast, SEK1 was more
strongly activated by osmotic shock, UV radiation, and
arsenite in PC-12 cells (205). In KB cells, SEK1 and other
SAPK activators were activated by anisomycin, osmotic
shock, and UV radiation (205).
Genetic studies lent further support the contention
that multiple SAPK-specific MEKs existed. Thus targeted disruption of sek1, while embryonically lethal,
results in only a partial ablation of SAPK activation;
sek1 ⫺/⫺ ES cells are refractile to anisomycin and heat
shock activation of SAPK, while osmotic shock and UV
activation of SAPK are unaffected (226). Finally,
hemipterous is a Drosophila MEK required for dorsal
closure during embryogenesis, and deletion/mutagenesis of hemipterous is lethal (see sect. IVC1). Although
hemipterous is significantly homologous to SEK1,
SEK1 cannot rescue Drosophila mutants wherein
hemipterous is deficient (105, 124).
MKK7 (also called MEK7, JNKK2, and SKK4, Table 2)
was isolated contemporaneously by several laboratories.
Two of the strategies used, database mining or degenerate
PCR, sought mammalian MEKs with close homology to
hemipterous (91, 188, 218, 314, 340, 353). Alternatively,
MKK7 was cloned in a two-hybrid screen as a polypeptide
that could associate in vivo with MEK1 (124). The significance of this association is unclear. Consistent with its
structural homology to hemipterous, MKK7 can effectively rescue hemipterous lethality, whereas SEK1 cannot
(124). MKK7 displays a strong preference for SAPK, even
under conditions of high expression. This contrasts with
SEK1 which can, under conditions of high overexpression, activate p38 (69, 91, 188, 218, 314, 340, 353). MKK7
can activate all SAPK isoforms tested equally well (91,
188, 218, 314, 340, 353). MKK7, like SEK1 is subject to
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
substantially in their substrate selectivity with regard to
p38 isoforms than do SEK1 and MKK7. MKK3 preferentially activates p38␣ and p38␤ while MKK6 can activate
strongly all known p38 isoforms. Similarly, MKK3 appears
to be more restricted with regard to activation by upstream stimuli. Whereas MKK6 is activated by all known
p38 activators, MKK3, like SEK1, is more strongly activated by physical and chemical stresses (59, 60).
3. Activation of ERK5 by MEK5
2. The MEKKs
The MEKKs are the most diverse of eukaryotic
MAP3Ks, in terms of enzymology, regulation, and molecular structure. The common feature of the MEKKs is a
conserved catalytic domain homologous to that of S. cerevisiae STE11, a MAP3K that regulates both the yeast
mating pheromone and osmosensing pathways. Outside
of the kinase domains, there is little or no conservation,
and these enzymes can, in some instances, catalyze the
activation of multiple different MAPK pathways and interact with a wide array of putative regulatory proteins.
Mammalian MEKKs include MEKKs-1– 4, apoptosis signalregulating kinase-1 (ASK1), TGF-␤-activated kinase-1
(TAK1), and Tpl-2 (the product of the cot protooncogene)
(21, 32, 81, 103, 120, 134, 170, 232, 264, 297, 326, 344, 346)
(Table 3). NF-␬B-inducing kinase (NIK) is an additional
MEKK that is a specific activator of the NF-␬B pathway
(191) and is not discussed here (for review, see Refs.
258, 360).
A) MEKK1, A COMPARATIVELY SELECTIVE ACTIVATOR OF THE SAPK
PATHWAY. Johnson and colleagues (170), in an effort to
identify MAP3Ks that were upstream of the ERKs, employed a cloning strategy that exploited the existing
G. Several Divergent Families of MAP3Ks Are
Upstream of the SAPKs, p38s, and ERK5
1. General considerations
As with all MEKs, the stress-activated MEKs are regulated by Ser/Thr phosphorylation within a conserved
region the P-activation loop of subdomain VIII of the
kinase domain. The SEK1 phosphorylation sites are Ser257 and Thr-261 (69, 266), those for MKK7 are Ser-206 and
Thr-210 (␣-isoforms) (81, 188, 218, 314, 315, 340, 353),
those for MKK3 are Ser-189 and Thr-193 (266), those for
MKK6 are Ser-207 and Thr-211 (59, 248), and those for
MEK5 are Ser-311 and Thr-315 (for the long form of
MEK5) (83, 367).
The number, diversity, and complex enzymology of
Ser/Thr kinases that act as MAP3Ks upstream of the
stress-activated MAPKs are daunting. This heterogeneity
is consistent with the multiple different stimuli that recruit these MAPK pathways. The MAP3Ks upstream of
SAPK and p38 fall into three broad protein kinase families: the MEK kinases (MEKKs), the mixed lineage kinases
(MLKs), and the thousand and one kinases (TAOs)
(Fig. 5).
FIG. 5. Mammalian MAPK three-tiered core signaling modules. A, B,
and C are ordered for increasing MAP3K promiscuity (C is highest), and
the diagrams are meant to highlight the variable and often quite complex
signaling specificity of MAP3Ks. Black symbols with white writing indicate inactive MEKs.
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MEK5 (Table 2) was cloned by degenerate PCR using
conserved MEK sequences as primers (83, 367). Four
MEK5 polypeptides arise from differential hnRNA splicing. Splicing near the 5⬘-end shifts the reading frame and
generates long (450 amino acid) and short (359 amino
acid) polypeptides. The long polypeptide is localized with
the particulate, cytoskeletal fraction while the shorter
polypeptide is cytosolic. A second splicing event substitutes two 10-amino acid cassettes in a region between
subdomains IX and XI and gives rise to ␣- and ␤-MEK5
isoforms analogous to type 1 and type 2 SAPKs (Table 1)
(83, 111, 166). This region is important to substrate binding, and, accordingly, ␣- and ␤-MEK5s may differ in their
substrate specificities. ERK5 was isolated in a two-hybrid
screen to identify interactors with MEK5 and is the only
known MEK5 substrate (38, 44, 367). Thus it was recently
demonstrated that upon expression with the MAP3Ks
Tpl-2 and MEKK3 (see sect. IIG1), MEK5 is activated and
can itself activate ERK5 (38, 44).
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TABLE
3. MAP3K nomenclature
Name
Alternate Names
Raf-1
A-Raf
B-Raf
MEKK1
MEKK2
MEKK3
MEKK4
ASK1
TAK1
Tpl-2 (in rats)
MLK2
MLK3
DLK
TAO1/2
MTK1
MAPK kinasekinase-5
(MAPKKK5)
Cot (in humans)
MST
SPRK, PTK1
MUK, ZPK
PSK (?a splicing
isoform of
TAO2)
Substrates/Effectors
MEK1, MEK2
MEK1, MEK2
MEK1, MEK2
SEK1, MKK7
SEK1, MEK1
SEK1, MEK1, ?MKK3,
?MKK6
SEK1, MKK3, MKK6
SEK1, MKK3, MKK6
SEK1, MKK3, MKK6
MEK1, SEK1
SEK1, MKK7
SEK1, MKK7
SEK1, MKK7
MKK3, ?SEK/MKK7
(PSK only)
caused growth inhibition and, in some instances, apoptosis effects that were unexpected for a bona fide activator
of the mitogenic ERK pathway. Stable cell lines expressing an IPTG-inducible, truncated MEKK1 construct (catalytic domain only) were generated that bypassed the
toxic effects of constitutive MEKK1 expression (347).
IPTG induction of MEKK1 selectively activated the endogenous SAPK pathway; ERK activation was not observed
until massive overexpression of MEKK1 was achieved
(347). Similar results were obtained from transient transfection experiments if MEKK1 expression was titrated
carefully beginning at low levels of plasmid (210).
Accordingly, in vivo, MEKK1 is a highly selective
activator of the SAPKs (Fig. 5). This phenomenon is consistent with biochemical studies that indicate that MEKK1
can activate SEK1 in vitro and in vivo (347). MEKK1 can
also activate MKK7␣1, -␣2, -␤1, and -␤2 as well as -␥1 in
vivo, and in transfection experiments, of the MAP3Ks
tested, MEKK1 is the most potent overall in vivo MKK7
activator yielding MKK7 activation that is comparable to
SEK1 activation (315). However, careful in vitro biochemical and kinetic analysis of MEKK1 activation of MKK7␥1
versus SEK1 indicate that SEK1 is a preferred MEKK1
substrate (other MKK7 isoforms were not tested).
Whereas overexpressed MEKK1 can activate the ERKs
and even p38, kinetic studies of MEK1/2 and MKK3/6
activation by MEKK1 suggest that these MEKs are activated with a Kcat at least three orders of magnitude lower
that that for activation of SEK1 and two orders of magnitude lower than that for activation of MKK7␥1 (342).
The basis for this difference in in vivo and in vitro activity
is unclear and may reflect artifacts of transient expression. Alternatively, in vivo yet to be identified scaffold
proteins (JSAP1/JIP3 is a possible candidate, see sect.
IIIC2), not employed in in vitro assays using purified proteins, may enable significant MKK7 activation by MEKK1
in vivo. The in vitro selectivity of MEKK1 for SEK1 may be
due to the intrinsic scaffold properties of SEK1 (342) (see
sect. IIIC4). Few extracellular stimuli that activate MEKK1
are known. MEKK1 is significantly activated by TNF (13),
microtubule poisons (359), oxidant stress (209), and some
receptor Tyr kinase agonists (89).
B) MEKK-2 AND -3, MORE PROMISCUOUS MAP3KS. MEKK-2 and
-3 can activate the SAPKs, p38s, and the ERKs. MEKK3
can also activate ERK5. Using the same approach as that
employed in the cloning of MEKK1, Johnson et al. (142)
cloned murine MEKK-2 and -3 (Table 3). Independently,
human MEKK3 was cloned by differential display screening (21, 81). Although these enzymes are clearly STE11
homologs, as is MEKK1, MEKK-2 and -3 are more closely
related within their kinase domains to each other (⬎90%
identity) than they are to MEKK1 (⬃65% identity). MEKK2
and MEKK3 are smaller polypeptides than MEKK1 (⬃70
and 71 kDa, respectively). Both kinases have a COOHterminal catalytic domain (amino acids 362– 619 for
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knowledge of yeast signaling pathways (the mating factor
pathway of S. cerevisiae in particular) to isolate ⬃80-kDa
fragment of MEKK1 (Table 3). Thus degenerate PCR primers based on conserved elements of the STE11 sequence
and the related S. pombe MAP3K byr2 were used to
amplify mammalian homologs of the yeast kinases (120,
170). Full-length MEKK1 is a ⬃160-kDa polypeptide that
consists of a COOH-terminal kinase domain (amino acids
1221–1493) and an extensive NH2-terminal domain (amino
acids 1–1221) that includes two proline-rich segments
(amino acids 74 –149 and 233–291) containing putative
binding sites for proteins with SH3 domains, a consensus
binding site for 14 –3-3 proteins (amino acids 239 –243),
two PH domains (amino acids 439 – 455 and 643–750), and
an acid-rich motif (amino acids 817–1221) that contains
two sites for cleavage by cysteine proteases of the proapoptotic caspase family (Asp-871, Asp-874) (30, 90, 170,
344). Within the kinase domain is a binding site for Ras
the exact location of which has not been clearly defined
(260). MEKK1 can also bind Rho family GTPases (89) (see
sect. IVB1).
The 80-kDa MEKK1 fragment first isolated (amino
acids 817–1493) was initially shown in overexpression
experiments to activate the ERKs. Of note, relative to
Raf-1, much larger amounts of MEKK1 were required for
MEK1/2 activation. On the basis of these results, it was
proposed that MEKK1 was part of a distinct ERK activation mechanism (170). Inasmuch as Ste11p is recruited in
vivo as a consequence of activation by mating factor of
the heterotrimeric G protein complex Gpa1p (␣)-Ste4p
(␤)-Ste18p (␥) (120), it was suggested that MEKK1 was an
effector coupling trimeric G protein-coupled receptors to
the ERKs (170).
When MEKK1 was stably introduced into fibroblasts,
however, it was observed that expression of the kinase
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
both TCR and CD28 costimulation (or phorbol ester/calcium ionophore) (265, 293), presumably the CH12.
LX/D10 T-cell interaction involves costimulation. It is conceivable that, at the lower levels of MEKK2 incurred upon
expression from recombinant retroviruses, MEKK2 is a
preferential activator of ERK and p38, compared with
SAPK. Still, the apparent lack of activation of SAPK by
MEKK2 in this system is surprising inasmuch as recent
gene knockout studies have shown that the SAPKs are
required for TCR/CD28-stimulated release of IL-4 and IL-5
(see sect. VF ).
Expression of kinase-dead MEKK2 also weakens the
stability of APC-T cell conjugates, and wt-MEKK2
strengthens conjugate formation. This effect has been
attributed to “inside-out” signaling in which MEKK2 upregulates integrin expression on the T cells. However,
neutralizing antibodies to LFA-1, the principal integrin
involved in formation of stable T-cell APC conjugates,
abrogates conjugate formation in wt-MEKK2 overexpressing cells to the same extent as in cells expressing vector
alone (268). No potential direct upstream activators for
MEKK2 have been identified; however, APC activation of
ERK and p38 can be reversed by wortmannin, suggesting
that MEKK2 activation may be PI 3-kinase dependent
(268). The effect of wortmannin on MEKK2 translocation
was not determined.
Chao et al. (38) recently demonstrated that MEKK3
could activate MEK5 in vivo and, directly, in vitro; and
with MEK5 activation, the ERK5 pathway. Interestingly, in
these experiments, full-length MEKK3, upon transient expression, was inactive in vivo or in vitro toward MEK5,
and deletion of the NH2-terminal 11 amino acids generated a constitutively active construct. For reasons that are
unclear, this phenomenon was not observed in earlier
studies of MEKK3 activation of the SAPKs, ERKs, or p38s
(21, 38, 64, 81, 82). It is possible that the earlier studies
employed higher levels of expression resulting in partial
in vivo activation. Thus the NH2-terminal 11 amino acids
of MEKK3 may serve a negative regulatory function. Chao
et al. (38) also observed that EGF activated MEKK3’s in
vitro kinase activity and its ability to recruit ERK5 in vivo
(38). Interestingly, a dominant inhibitory form of MEKK3
(Lys391Trp) blocked EGF activation of ERK5 but not
ERK1 (38). This is likely due to the fact that Raf-1 is the
dominant EGF-activated MAP3K for the ERK pathway
(195). Thus MEKK3 is a putative effector for mitogen/Tyr
kinase regulation of all known mammalian MAPK pathways (Fig. 5).
C) MEKK4 CAN ACTIVATE BOTH THE SAPKS AND THE P38s.
MEKK4 (also called MAP three kinase-1, MTK1, Table 3)
was isolated by Johnson et al. (103) employing again a
PCR strategy based on degenerate primers derived from
STE11 and byr2. Independently, Saito and co-workers
(120, 297) cloned MEKK4 as a human cDNA that when
expressed could rescue osmosensitive yeast missing the
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MEKK2, amino acids 368 – 626 for MEKK3) preceded by
NH2-terminal noncatalytic domains. The NH2-terminal
noncatalytic portions of MEKK-2 and -3 are also significantly similar; however, these regions contain no motifs
suggestive of function or regulation (21, 81).
In contrast to MEKK1, which is strongly selective for
the SAPKs, MEKK-2 and -3 are considerably more promiscuous. In transfection experiments, each can activate
both the SAPK and ERK pathways with nearly equal potency (Fig. 5). Consistent with concomitant ERK and
SAPK activation, MEKK-2 and -3 can each activate MEK1
and SEK1 in vivo and in vitro (21, 81). MEKK3 can also
activate MKK7␣1 and, to a much lesser extent, MKK7␣2,
-␤1, and -␤2 in vivo (64, 315). The reason for the selectivity of MEKK3 for different MKK7 isoforms is unclear.
Early studies indicated that p38 was not activated by
MEKK2; however, subsequent studies have shown that
MEKK2 and MEKK3 can activate the p38s in vivo (64, 82,
268, see below), and MEKK3 can activate MKK3 and
MKK6 in vivo and in vitro (64, 82) (Fig. 5). Gene disruption studies have revealed an important role for MEKK2 in
T-cell signaling and for MEKK3 in cardiovascular development (see sect. VF ).
Recent studies indicate that MEKK2 is activated in T
cells upon stimulation with antigen-presenting cells
(APCs; see sect. VG). Thus D10 cells, a T lymphocyte line
reactive to conabumin and I-AK, were infected with a
recombinant retrovirus harboring a green fluorescent protein (GFP)-MEKK2 construct. The infected cells were
treated with CH12. LX cells, a I-AK-positive APC line, that
had been preloaded with conalbumin. Under these conditions, the D10 cell GFP-MEKK2 was activated and was
shown in live cell fluorescence imaging to translocate to
the region of the plasma membrane at the interface with
the APCs. In contrast, no such APC-dependent translocation of MEKK1 or MEKK3 was observed (269). MEKK2
activation and translocation were dependent on the APCs
being loaded with conalbumin, indicating that MEKK2
activation depended on T-cell receptor (TCR) engagement (268). Interestingly, APC treatment also resulted in
activation of D10 cell p38 and ERK, and both ERK and p38
activation by APCs was blocked with kinase-dead
(Phe502Leu) GFP-MEKK2. Direct activation of SAPK was
not measured in these studies; however, kinase-inactive
MEKK2 failed to inhibit activation of SAPK by phorbol
ester/calcium ionophore, a treatment that mimics the
TCR/CD28 costimulation characteristic of the T cell-APC
interaction and activates SAPK (see sect. VF ) (268). Thus,
although MEKK2 has been shown in overexpression assays to activate coexpressed SAPK (21), MEKK2 expressed in D10 T cells, although activated by TCR engagement, apparently does not activate SAPK (268). The
reason for this discrepancy is unclear. It is known that, in
contrast to T-cell p38s and ERKs, which are activated by
TCR engagement alone, T-cell SAPK activation requires
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
p38s. In vitro and in vivo, TAK1 can phosphorylate and
activate SEK1, MKK3, and MKK6 (217, 225, 281, 346).
E) ASK1 CAN ACTIVATE BOTH THE SAPKS AND THE P38S. Apoptosis signal-regulating kinase-1 (ASK1, also called MAPK
kinase-kinase-5, MAPKKK5, Table 3) is a sixth MEKK that
was cloned by Wang et al. (326) and, independently, by
Ichijo et al. (134). Both groups used a PCR strategy that
employed degenerate primers based on conserved elements of Ser/Thr kinase subdomains VI and VIII (134,
326). ASK1 is a ⬃150-kDa polypeptide with a centrally
located kinase domain (amino acids 677–936), an NH2terminal extension (amino acids 1– 676) that includes segments that bind polypeptides of the TNFR-associated factor (TRAF) family as well as the Fas-associated adapter
protein Daxx, and the redox sensing enzyme thioredoxin
(see sect. IVD) (36, 134, 182, 228, 263, 326). In addition,
ASK1 possesses a COOH-terminal extension (amino acids
937–1375) that has also been implicated in TRAF binding
(182, 228). ASK1 can activate the SAPKs (via SEK1) and
the p38s (via MKK-3 and -6) in vivo and in vitro (134, 326)
(Fig. 5). Endogenous ASK1 is activated by oxidant stress,
TNF, and Fas, and there is evidence that activation of
ASK1 by TNF is dependent on TNF-induced generation of
reactive oxygen species (108, 134, 182, 228, 263). ASK1 is
likely to be an important effector coupling these agonists
to the SAPKs and p38s.
Under low serum conditions, expression of ASK1
from a Zn-inducible promoter induces apoptosis in several cell lines (134). The targets recruited by ASK1 (especially if the SAPKs or p38s are involved) that promote
apoptosis are unknown. Dominant inhibitory, kinase-dead
ASK1 mutants can block apoptosis stimulated by TNF or
oxidant stress (134, 263). Thus either ASK1 is a direct
apoptogenic signaling component recruited by these ligands, or the dominant inhibitory ASK1 is sequestering a
common upstream element that regulates both ASK1 and
TNF/oxidant-induced apoptosis.
F) TPL-2 CAN ACTIVATE THE SAPK, P38S, ERKS1/2, AND ERK5.
Tumor progression locus-2 (Tpl-2) is a rat oncogene that
was identified by Tsichlis and colleagues (232) as a homolog of the human protooncogene cot (Table 3). The
oncogenic potential of Tpl-2 is activated in rat thymomas
as a result of the additive, spontaneous proviral insertion
of the Moloney leukemia virus into the infected cell genome at the Tpl-2 locus, the consequence of which is
positive selection of tumor cells and enhanced tumor
progression in vivo (232). Tpl-2 is a ⬃50-kDa protein
Ser/Thr kinase with an NH2-terminal domain, of unknown
function, which is truncated in some mRNA splicing isoforms, a central catalytic domain (amino acids 139 –394)
that is significantly homologous to the S. cerevisiae
MAP3K STE11, and a COOH-terminal regulatory domain
(amino acids 395– 467) (32, 232). Moloney leukemia virus
proviral insertions at Tpl-2 always target the last intron of
the gene and elicit the enhanced expression of a COOH-
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genes for all three MAP3Ks of the HOG1 osmoadaptation
pathway (SSK2, SSK22, and STE11). The ⬃150-kDa
MEKK4 polypeptide consists of a COOH-terminal kinase
domain (amino acids 1337–1597) and an extensive NH2terminal regulatory region that includes a putative
polyproline SH3 binding motif (amino acids 27–38) (103,
297), a binding site for GADD-45 family proteins (amino
acids 147–250, see sect. IVF2) (298), a putative PH domain
(amino acids 225–398), and a Cdc42/Rac interaction and
binding (CRIB) domain (amino acids 1311–1324, see sect.
IVF1) (103, 297). The kinase domain of MEKK4 shares
⬃55% amino acid homology with that of MEKK-1, -2, and
-3. Johnson and colleagues (103) have reported that
MEKK4 is selective for the SAPKs and can activate only
SEK1 in vivo and in vitro; however, Saito and co-workers
(297) used titered expression of increasing levels of
MEKK4 and demonstrated that MEKK4 could activate the
SAPKs (via SEK1) and the p38s (via MKK3 and MKK6)
with equal potency in vivo and in vitro. In contrast, Davis
and colleagues (315) reported that MEKK4 selectively
activated the p38 pathway in vivo (Fig. 5). The reason
for these discrepancies is unclear. MEKK4 may be an
effector for stress pathways activated by genotoxins (see
sect. IVF2).
D) TAK1 CAN ACTIVATE BOTH THE SAPKS AND THE P38S.
Yamaguchi et al. (346) cloned TAK1 (Table 3) in a novel
screen for mammalian MAP3Ks. This screen employed a
mutant strain of S. cerevisiae wherein STE11 was deleted, and a mutant form of the mating factor pathway
MEK STE7, STE7 P368, was expressed. STE7 P368 is a gainof-function mutant that still requires Ste11p for full activity; however, the selectivity of Ste7P368p for upstream
activators is more relaxed than that of wt Ste7p and
constitutively active Raf-1, MEKK1, and, by extension,
presumably other MAP3Ks, can substitute for Ste11p in
⌬STE11/STE7 P368 mutant cells (120, 346). Thus the
⌬STE11/STE7 P368 mutant is a facile reagent for the identification of novel MAP3Ks; and TAK1 was cloned as an
additional MAP3K that could substitute for Ste11p in
⌬STE11/STE7 P368 cells. TAK1 is a ⬃60-kDa polypeptide
with an NH2-terminal kinase domain (amino acids 30 –
294) preceded by a short regulatory motif (amino acids
1–22) and followed by a COOH-terminal extension (amino
acids 295–557) of unknown function (346). The NH2-terminal regulatory motif appears to serve an inhibitory role
inasmuch as full-length TAK1 cannot substitute for Ste11p
in ⌬STE11/STE7 P368 cells, whereas TAK1⌬1–22 can (346).
Two hybrid screening with TAK1 amino acids 1–22 as bait
identified TAK1 binding proteins (TABs)-1 and -2, two
regulatory proteins that bind the TAK1 NH2-terminal regulatory motif and are essential for coupling TAK1 to upstream signals (see sect. IVE ) (225, 279). Endogenous
TAK1 is activated by TGF-␤, IL-1, and TNF. In overexpression experiments, TAK1 can activate both the SAPKs and
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
specifically (44). Consistent with this, ectopic expression
of Tpl-2 expression activated ERK1, SAPK (␥), p38␥, and
ERK5 but not p38␣ or -␦. Correspondingly, coexpression
with Cot activated MEK1, MKK6, MEK5, and SEK1. The
selective activation of p38␥ was curious, insofar as MKK6
can activate all known p38 isoforms in vitro and, in transfection experiments in vivo (44). Mechanisms of sequestration, such as scaffold proteins, may isolate Tpl-2 and
MKK6 from all p38 isoforms other than ␥. In correspondence with the broad effector specificity of Tpl-2, Tpl-2dependent transformation requires p38, SAPK, and ERK5.
Thus Tpl-2-induced NIH3T3 cell focus formation is completely blocked by dominant inhibitory MEK5, MKK6, or
JIP1 (44). Extracellular stimuli that recruit Tpl-2 in vivo
are unknown.
3. The mixed lineage kinases: MLK3, MLK2, and DLK
selectively activate the SAPKs
The mixed lineage kinases (MLKs) are a small family
of protein Ser/Thr kinases that share a general structural
configuration wherein an NH2-terminal kinase domain is
followed by one to two leucine zippers, a Cdc42/Rac
interaction and binding (CRIB) domain (see sect. IIIA2),
and a COOH-terminal proline-rich domain with several
consensus SH3 binding motifs. Four MLKs have been
identified: MLK1; MLK2 (also called MKN28 cell-derived
Ser/Thr kinase, MST, Table 3); MLK3 (also called SH3
domain-containing proline-rich kinase, SPRK, or protein
Tyr kinase-1, PTK1); and dual leucine zipper kinase (DLK,
also called MAPK upstream kinase, MUK, or zipper-containing protein kinase, ZPK, Table 3). MLK2 and MLK3, in
addition to the common features described above, contain SH3 domains NH2 terminal to the kinase domains (78,
101, 127).
Although the MLKs are clearly Ser/Thr specific in
vitro and in vivo, as their names suggest, the kinase
domains of the MLKs bear structural similarities to both
Ser/Thr and Tyr kinases. Thus, for example, MLK1 contains a Lys residue (Lys-129 in the sequence His-ArgAsp-Leu-Lys) in subdomain VIb that is characteristic of
Ser/Thr kinases; however, two Trp residues in MLK1 subdomain IX (Trp-192 and Trp-199) are highly conserved
among Tyr kinases, as is a motif in subdomain XI (Met241Glu-Asp-Cys-Trp-Asn-Pro-Asp-Pro-His-Pro-Ser-Arg-ProSer-Phe255) which conforms to a conserved region in Tyr
kinases (Met-X-X-Cys-Trp-X-X-Asp/Glu-Pro-X-X-Arg-ProSer/Thr-Phe, where X is any amino acid) (78).
Three of the four known MLKs have been assayed for
activation of MAPK pathways: MLK3, MLK2, and DLK. All
three are potent activators of the SAPKs in vivo in transfection experiments (88, 121, 122, 249, 315). In contrast to
the generally more promiscuous MEKKs, the MLKs are
entirely SAPK pathway specific. ERK, p38, and NF-␬B are
not activated by MLK-2, -3, or DLK, except under condi-
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terminally truncated protein, both through enhanced transcription and mRNA stabilization. It is likely, therefore,
that the COOH-terminal regulatory domain of Tpl-2 exerts
a negative effect on Tpl-2 activity. In support of this idea,
the free Tpl-2 kinase and COOH-terminal domains, when
expressed together in Sf9 cells, interact in vivo, as demonstrated in coimmunoprecipitation experiments. Moreover, transgenic mice expressing the COOH-terminally
truncated protein develop T-cell lymphomas, whereas expression of the wild-type protein is without effect (32).
Tsichlis and colleagues demonstrated that transient
expression of Tpl-2 activated the ERK pathway in parallel
to Raf-1, and possibly downstream of Ras (233). Subsequently, Ley and co-workers (264) as well as Tsichlis and
co-workers (32) showed that expression of Tpl-2 activated the SAPKs and ERKs with equal potency. Ley and
co-workers (264) then demonstrated that, consistent with
its homology to STE11, Tpl-2 was a MAP3K that could
directly activate MEK1 and SEK1 in vivo and in vitro
(Fig. 5). While overexpression of full-length Tpl-2 activates both the SAPKs and ERKs, expression of the oncogenic COOH-terminally truncated Tpl-2 results in substantially greater SAPK and ERK activation, further
supporting the contention that the COOH-terminal domain negatively regulates Tpl-2 activity (32).
A recent study by Chiariello et al. (44) revealed a far
broader substrate specificity for Tpl-2. Thus transient expression of Tpl-2 induces c-jun transcription and transformation of NIH3T3 cells. c-Jun expression is required
for Tpl-2 transformation inasmuch as TAM67, a dominant
inhibitory mutant c-Jun construct wherein the ␦-domain
and phosphoacceptor sites have been deleted, strongly
blocks Tpl-2, but not v-Ras transformation (44). Insight
into the mechanism by which Tpl-2 recruits the c-jun
promoter came from studies employing the SAPK binding
portion of the scaffold protein JNK-interacting protein-1
(JIP1, see sect. IIIC1), a potent and specific inhibitor of
SAPK-dependent signaling. Tpl-2 induction of c-Jun is
dependent on activation of the ERK5/MEF2C pathway as
well as on the SAPKs. Thus expression of the JIP1 SAPK
binding pocket blocked MEKK1 induction of a c-jun-Luc
reporter completely, while only partially blocking Tpl-2
induction of c-jun-Luc. Promoter bashing experiments
indicated that MEKK1, Tpl-2, and Ser311Asp/Thr315Asp
(constitutively active, DD-MEK5) MEK5 could induce
c-jun-Luc, and deletion of the AP-1 site crippled MEKK1
and, to a slightly lesser extent, Tpl-2 trans-activation of
c-jun-Luc. In contrast, deletion of the MEF2C site on the
c-jun reporter crippled completely Tpl-2 and DD-MEK5
induction of c-jun-Luc without affecting MEKK1 induction of c-jun-Luc. SAPK-dependent and -independent
trans-activation of c-jun-Luc was further demonstrated
with DN constructs; thus Tpl-2 transactivation is blocked
with DN MEK5, MKK6, and ERK as well as expression of
JIP1. All of these DN constructs are shown to be acting
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
4. TAO kinases are novel MAP3Ks that regulate the
p38s and are structurally homologous to both Ste20
and MAP3Ks
Cobb and colleagues (133) recently identified the
TAO kinases (TAO1 and TAO2), a novel family of 1,001
amino acid Ser/Thr kinases, as part of a scheme to identify additional mammalian kinases homologous to S. cerevisiae STE20, a proximal kinase thought to be important
in the regulation of the Ste11p ⬎Ste7p ⬎Fus3p/Kss1p
yeast mating pheromone MAPK core signaling module
(42, 120, 133). A possible splicing isoform of TAO2, referred to as prostate-derived STE20-like kinase (PSK, Table 3), that includes an extended COOH-terminal tail, but
is otherwise identical to TAO2, was cloned independently
by Moore et al. (214) as a kinase expressed at elevated
levels in prostate tumors. TAOs consist of NH2-terminal
kinase domains and very large (700 amino acids) COOHterminal extensions of poorly characterized function. The
kinase domains of TAOs are significantly homologous to
Ste20p (40% identity) and the GCKs (43% identity with
GCK) both established mammalian Ste20-like kinases (42,
120, 133, 163, 214). However, there is appreciable identity
with the MLK2 kinase domain (33% overall), especially
within the substrate binding motifs (133).
TAO-1 and -2 appear to be specific activators of p38.
The purified kinase domain of TAO1 will directly phosphorylate and activate SEK1, MKK3, and MKK6 in vitro;
however, in vivo only MKK3 is activated. Moreover, when
expressed in Sf9 cells from a recombinant baculovirus,
TAO1 will interact in vivo with and coimmunoprecipitate
with MKK3. Thus, although TAO1 bears significant homology to Ste20s and GCKs, unlike kinases of the Ste20 and
GCK groups, which are thought to regulate MAP3Ks (see
sect. IV, B3 and G1), TAO1 appears to be a direct MAP3K
selective for MKK3 (133) (Fig. 5). The ability of TAO1 to
catalyze directly the activation of MEKs may be due to the
kinase domain homology with MLK2 in the substrate binding region. The high in vivo selectivity of the TAOs for
MKK3 may be due to the presence of a specific MKK3
binding site. Cobb and colleagues (42) identified a region
on the TAO2 polypeptide (amino acids 314 – 451) that
selectively binds MKK3 in vitro and in vivo. Interestingly,
this region is rich in acidic amino acid residues, similar to
the CD loop of MAPKs, a region that binds to basic MAPK
interaction motifs on MAPK regulators and substrates
(Fig. 5D, sect. IIE3) (301). Thus an analogous acidic amino
acid-rich/basic amino acid-rich interaction may govern
the TAO-MKK3 interaction. It will be important to determine if the NH2-terminal basic MAPK interaction motif on
MKK3 (301) is important for binding TAOs. A similar
acidic amino acid-rich site is present in TAO1 (133), although its binding to MKK3 was not tested.
PSK, the TAO2 splicing isoform, appears to activate
selectively the SAPK pathway; ERK and p38 are not activated in transfection experiments (214). This difference in
pathway selectivity is curious given that the MKK3 interaction loop found in TAO2 (42) is present in PSK (214).
The COOH-terminal tail unique to PSK may account for
the altered specificity of PSK; however, this has not been
demonstrated.
III. PUTATIVE ELEMENTS UPSTREAM OF
STRESS-ACTIVATED MITOGEN-ACTIVATED
PROTEIN KINASE CORE PATHWAYS
A. Regulation of the SAPKs and p38s by Rho
Family GTPases
1. General considerations
Sections IIB to IIG have described much of what we
know of mammalian stress-activated MAP3K 3 MEK
3 MAPK core signaling modules. The following five sections (sects. IIIA to IIIE) will describe some of the known
proximal signaling components thought to couple to
these core modules.
Monomeric GTPases of the Ras superfamily are potent upstream activators of signal transduction pathways,
and Ras is pivotal to the activation of the ERK pathway.
The activation of Ras by guanine nucleotide exchange
factors (GEFs) and the inactivation of Ras by GTPase
activating proteins (GAPs) has been extensively reviewed
(23, 201, 202). Ras regulates ERK predominantly through
triggering the activation of the MAP3K Raf-1 (195). The
regulation of Raf-1 by Ras is a paradigm for MAP3K
regulation, and studies of Raf-1 regulation point to three
crucial events that govern MAP3K activation: binding to
an upstream activating protein accompanied by translocation to the membrane, phosphorylation, and homoli-
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tions of extreme overexpression (Fig. 5). MLK3, MLK2,
and DLK are all established MAP3Ks. Thus, in vitro and in
vivo, MLK3, MLK2, and DLK can activate SEK1 to a degree
commensurate with that catalyzed by MEKK1 (121, 122,
249, 309, 315). In addition, MLK3 and DLK can activate
MKK7 in vivo in transfection experiments, and in vitro,
albeit with differential isoform selectivity in vivo. Thus, in
both instances, MKK7␤1 and -␤2 are strongly activated in
vivo while ␣-isoforms are modestly activated (315). Although this selectivity is likely due to the structural differences in the NH2 termini of the ␣- and ␤-isoforms of
MKK7 (discussed in sect. IIF1), which, in turn, may affect
interactions with MLK3 and/or scaffold proteins such as
JNK interacting proteins (JIPs) or JNK/SAPK interacting
protein-1 (JSAP1/JIP3, section IIIC), the significance and
molecular basis of this selectivity are unknown. MLK2 can
also activate MKK7␣1. Indeed, MLK2 is a stronger
MKK7␣1 kinase than it is a SEK1 kinase (122, 315).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
filopodia, with regard to the control of cellular proliferation and differentiation, are incompletely understood;
however, the ability of Rho family GTPases to trigger
these cytoskeletal changes correlates at least in part with
Rho family GTPase transforming capability. Thus Rac,
Rho, and Cdc42Hs all appear necessary for the entry of G0
cells into the cell cycle, and expression of constitutively
active, GTPase-deficient mutants of Rac, Rho, or Cdc42Hs
will drive quiescent cells into the cell cycle in the absence
of serum. Conversely, ras transformation is blocked by
Asn-17, exchange-deficient mutants of Rac or Cdc42, or
by effector loop mutations that also abrogate cytoskeletal
functions (114) (Fig. 6A).
Constitutively active, GTPase-deficient (Val-12) mutants of Rac1, Cdc42Hs, or Chp are potent activators of
the SAPKs (8, 52, 211, 229). In addition, Val12-Rac1 and
Cdc42Hs can activate p38 (12, 364). RhoA generally does
not activate SAPK or p38 but can in certain cell types
(notably 293 cells) (306). Consistent with these findings,
expression of the Dbl protooncogene product or FGD1,
Rho GEFs that recruit Rac and Cdc42, also results in
strong SAPK activation (52, 366). Transforming alleles of
FIG. 6. G protein regulation of stress-activated MAP3Ks. A: Ras
superfamily regulation of MAP3Ks. B: trimeric G protein regulation of
SAPKs.
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gomerization. Thus Raf activation requires binding to
GTP-Ras, an event that results in membrane translocation
(195). At the membrane, Raf is phosphorylated by several
protein kinases including Tyr kinases such as Src and
Ser/Thr kinases such as p21-activated kinase (PAK)-3, an
effector for Rac1, a Rho family GTPase that is itself a Ras
effector (87, 118, 160, 198). Phosphorylation of Raf-1 results in structural alterations that change the binding to
proteins of the 14 –3-3 family. These structural changes
are necessary for Raf-1 activation (320). Ras-dependent
oligomerization is also necessary for Raf-1 activation
(190). In the same vein, activator protein binding/membrane translocation, phosphorylation, and oligomerization may be important in the regulation of stress-activated
MAP3Ks.
Whereas Ras itself can directly influence the ERK
pathway by recruiting Raf-1, at least some stress-activated
MAPK core pathways may be regulated by members of
the Rho subfamily of the Ras superfamily. The Rho subfamily is comprised of mammals of the Rho (RhoA-E),
Rac (Rac1 and -2), and Cdc42 (Cdc42Hs, G25K, Tc10, and
Chp) groups (8, 23, 114). As with other members of the
Ras superfamily, Rho subfamily GTPases are active in the
GTP-bound state (promoted by GEFs) and inactive in the
GDP-bound state (promoted by GAPs) (23, 201, 202). Rac,
Cdc42Hs, and other Rho subfamily GTPases share a large
number of GEFs, belonging to the Dbl family, including
Ost, a GEF for RhoA and Cdc42Hs; Tiam-1, a GEF for
Rac1 and Cdc42Hs; Lbc, a GEF for RhoA; and faciogential
dysplasia-1 (FGD1), a GEF selective for Cdc42Hs (33).
All Rho family GEFs consist of a conserved Dbl
homology (DH) domain that is necessary for promoting
GDP dissociation and a plekstrin homology (PH) domain.
Inositol phospholipids, including inositol 3,4,5-trisphosphate, the product of PI 3-kinase activity, are putative
targeting signals for PH domain-containing polypeptides
(79). Activation of PI 3-kinase requires Ras (255) and,
accordingly, Rho family GEFs themselves may be subject
to regulation by Ras 3 PI 3-kinase (33, 118). Indeed,
mitogen activation of Rac1 requires activation of PI 3-kinase (118).
Among the first recognized physiological functions
for the Rho family was regulation of the actin cytoskeleton (33, 114). Thus fibroblasts in culture will form actin
stress fibers that associate with the focal adhesions
which, in turn, bind the cells to the substratum. Formation of these stress fibers requires RhoA (114). Within
minutes of mitogen treatment of cells, transient changes
in the actin cytoskeleton occur. Notably, any dense perinuclear actin filaments present in the resting cells are
disassembled, and the cells form peripheral filopodia (actin microspikes) and lamellopodia (membrane ruffles)
from the free actin (114). Filopodium formation requires
Cdc42Hs. Lamellopodium formation requires Rac1 (114).
The physiological functions of lamellopodia and
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
2. Coupling Rho GTPase targets to SAPK and p38 core
signaling pathways
As mentioned above, proteins of the Ras superfamily
activate their targets in part through a direct binding
interaction between the target protein and the effector
loop of the GTP-charged G protein (201, 202). This binding results in membrane translocation and, in the case of
Ras-Raf, is a necessary prerequisite for subsequent activation events such as homoligomerization and phosphorylation by upstream kinases (160, 190, 198). Most, but not
all, direct targets for Rac1 and Cdc42Hs contain a socalled Cdc42/Rac interaction and binding (CRIB) domain
that interacts directly with the effector loops of Rac1 and
Cdc42Hs (27). Several polypeptide species including protein kinases and adapter proteins are candidate Rac1 and
Cdc42Hs effectors that couple to the SAPKs and p38s.
A) PAKS. STE20 encodes a S. cerevisiae Ser/Thr protein kinase with an NH2-terminal regulatory domain containing a CRIB motif that binds Cdc42Sc, the yeast
Cdc42Hs ortholog (120). Ste20p is thought to regulate the
mating pheromone MAPK core signaling module Ste11p
3 Ste7p 3 Fus3p/Kss1p, and genetic studies suggest that
Ste20p is upstream of Ste11p, although the mechanism of
Ste20p action in this process has yet to be defined completely (120). Genetic epistasis studies provisionally placing Ste20p upstream of the MAP3K Ste11p led to the
notion that Ste20-like kinases in mammals might couple
Rho GTPases MAPK core signaling modules (120).
The PAKs (PAK1, also called ␣PAK; PAK2, also called
␥PAK or hPAK65; PAK3, also called ␤PAK; and PAK4,
Table 4) are a family of mammalian kinases that are
structural and functional orthologs of Ste20p (2, 120, 192,
192, 197, 305). PAK1 and PAK2 were originally purified as
protein kinases that selectively bound GTP-Rac1 and
GTP-Cdc42Hs (193, 197, 305), and in vitro and in vivo, the
kinase activity of PAKs is activated upon binding GTPRac1 or GTP-Cdc42Hs through a process that involves an
obligatory autophosphorylation event (197). PAK2 can
also undergo activation during apoptosis through a mechanism that involves caspase 9-mediated cleavage at Asp212, a process that removes the NH2-terminal regulatory
domain (including the CRIB motif) (175). The significance
of this cleavage-mediated activation is unclear, although
PAK2 may mediate some morphological functions associated with apoptosis such as cell shrinkage (175).
A major function of the PAKs (PAK1 in particular) is
to serve as effectors for Cdc42 and Rac in the regulation
of the actin cytoskeleton (271, 272). It is conceivable that,
as is the case with Raf-1 activation by PAK3, stressactivated MAP3Ks might, once translocated to the membrane upon binding GTP-loaded Rho family GTPases, undergo activating phosphorylation catalyzed by PAKs
contemporaneously located at the membrane as a consequence of binding GTP-Rac1 or Cdc42. Indeed, several
reports have indicated that constitutively activated forms
of PAKs can activate coexpressed SAPKs and p38s (12, 25,
236, 364). Thus, for example, mutation of Leu-107 to Phe,
within the CRIB motif of human PAK1, results in a constitutively active construct that can activate coexpressed
SAPK (25). Moreover, addition to cell-free extracts of
Xenopus oocytes, of a PAK1 mutant wherein the NH2terminal regulatory domain has been deleted also results
in substantial SAPK activation (236). However, SAPK activation by coexpressed PAKs has not been universally
observed. Moreover, the activation of SAPK by wild-type
PAKs is generally small, and expression of PAKs does not
TABLE
4. GCK/PAK nomenclature
Name
GCK
GCKR
GLK1
HPK1
NIK
Misshapen
PAK1
PAK2
PAK3
Alternate Names
Kinase homologous to
Ste20 (KHS)
NIK (not to be confused
with NF-␬B-inducing
kinase), HPK1/GCKrelated kinase (HGK)
Msn
␣-PAK
␥-PAK, hPAK65
␤-PAK
Substrates/Effectors
MEKK1, ?MLK3
MEKK1
?
MEKK1, MLK3
MEKK1
?
?
?
Raf-1
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ras and EGF (through Ras) may signal to the SAPKs via
Rac1 inasmuch as Asn-17 Rac1 can block the activation of
SAPK by these stimuli. In addition, Rac1 is also involved
in SAPK activation in response to CD3-CD28 T-cell costimulation (137, 229). Cdc42Hs, in contrast, is activated
by lysophosphatidic acid, bombesin, and, in spreading
melanoma cells, engagement of chondroitin sulfate proteoglycan (80, 114).
While Rac1 and Cdc42Hs can each regulate both
MAPK pathways and the cortical actin cytoskeleton, it
appears that these functions are mediated by distinct
pathways. Support for this idea comes from the use of
effector loop mutants (334). The effector loop of all Ras
superfamily GTPases lies adjacent to the guanine nucleotide binding site (23, 201, 202). Conformational changes
that occur upon GDP-GTP exchange expose the effector
loop enabling the binding of target proteins. Point mutations in the effector loops of Ras superfamily proteins can
disrupt the binding of selective effector proteins resulting
in the selective loss of functions attributable to those
proteins that can no longer bind (23, 114, 201, 202, 334).
Thus Phe37Ala Rac1 is unable to stimulate membrane
ruffling but still activates SAPK. Conversely, neither
Tyr40Cys Rac1 nor Cdc42Hs can activate SAPK, but each
is still able to induce, respectively, membrane ruffling and
filopodium formation (168).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
B. Regulation of the SAPKs and p38s by
Heterotrimeric G Proteins: Implications for
Cardiovascular Diseases
A number of ligands that bind to seven pass membrane receptors can strongly recruit the SAPKs. These
include potent vasoactive compounds, suggesting a major
role for the SAPKs in cardiovascular regulation. Thus
ANG II and endothelin-1 are strong SAPK agonists in
several cell types including cardiomyocytes (ANG II and
endothelin-1), hepatocellular carcinomas (ANG II), and
pulmonary epithelia (endothelin-1) (46, 92, 275, 369). In
addition, the SAPKs can be activated upon engagement of
the m1 muscarinic acetylcholine receptor and the thrombin receptor (51, 92).
The ␣- and ␤␥-subunits of heterotrimeric G proteins
couple seven pass receptors to intracellular effectors.
Trimeric G protein ␣-subunits are divided into four subfamilies based on primary sequence conservation and
shared effectors: Gi␣, Gq␣, Gs␣, and G12␣ (23, 115, 154).
There are 5 different mammalian ␤-subunits (␤1–5) and 10
different ␥-subunits (␥1–10) known (115).
Receptor engagement fosters GTP binding to the
␣-subunit that results in dissociation of the ␣-subunit
from the ␤␥-complex. Free ␣ and ␤␥ can each bind and
regulate target proteins. Although the subunits of trimeric
G proteins do not assemble completely indiscriminately,
the sheer number of different ␣-, ␤-, and ␥-subunits suggests that an extremely diverse array of heterotrimeric
G protein complexes can be assembled within a cell
(115, 154).
Of the ligands that engage seven pass receptors and
activate the SAPKs, not much is known regarding the
subset of trimeric G proteins to which these receptors
couple. The ANG II and thrombin receptors recruit primarily the Gi␣ family ␣-subunit G0␣ and possibly the G12␣
family ␣-subunits G12␣ and G13␣ (115).
The ability of some G␣ subunits to transform cells
and activate the ERKs (100), coupled with the observation
that ANG II, endothelin I, and other ligands that bind to
trimeric G protein-coupled receptors, could recruit the
SAPKs (92, 275, 369), led to the examination of trimeric G
protein subunits as SAPK activators. From these studies,
it is evident that G protein ␣- and ␤␥-subunits can both
recruit the SAPKs and that the degree of activation as well
as the mechanism (␣- or ␤␥-driven) are cell and stimulus
dependent.
Thus transient overexpression of GTPase-deficient
mutants of the G12␣ family ␣-subunits G12␣ and G13␣, or
the Gq␣ family ␣-subunits G16␣ or Gq␣ results in modest
(PC12 cells) to strong (293 cells) SAPK activation. Strong
activation of SAPK is also observed in NIH3T3 cells transformed upon stable overexpression of GTPase-deficient
G12␣ and G13␣ (50, 119, 243). p38 was not tested in these
studies (Fig. 6B).
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synergize with Rac or Cdc42 to activate further SAPK
(89). Thus it is not clear if PAKs are true effectors coupling Rho family GTPases to the SAPKs and p38s.
B)
MAP3KS.
Several established stress-sensitive
MAP3Ks, including MEKK1 (89) (discussed in sect. IVB1),
MEKK4 (89, 103) (discussed in sect. IVF1), MLK2, and
MLK3 (27, 222) (discussed in sect. IVG2), are putative
effectors for Rac1 and Cdc42Hs (Fig. 6A). However,
whereas the PAKs are clearly activated by Rac1 and
Cdc42Hs, the functional role of the interactions between
these MAP3Ks and Rho family GTPases is, in most cases,
unclear.
C) PLENTY OF SH3 DOMAINS. Plenty of SH3 domains
(POSH) is a novel ⬃90-kDa polypeptide that consists of
four SH3 domains (amino acids 139 –190, 198 –254, 457–
511, and 838 – 892), a domain rich in putative polyproline
SH3 binding sites (amino acids 368 – 405), but no CRIB
motif. In spite of this, however, POSH interacts directly
with Rac1 (but not Cdc42Hs, Ras, or Rho) in yeast twohybrid assays and a GTP-dependent manner in vitro. The
Rac binding domain has been localized to amino acids
292–362, a domain that appears thus far to be unique to
POSH (302). Transient expression of POSH in COS1 cells
results in potent SAPK activation. In addition, POSH expression triggers nuclear translocation of NF-␬B and apoptosis (302). POSH recruitment by Rac1 effector mutants correlates tightly with the ability of these Rac1
mutants to activate the SAPKs. Thus, in contrast to MLK3
and MLK2, POSH can interact with Phe37Ala-Rac1, a Rac1
effector mutant that cannot elicit lamellopodium formation but does activate coexpressed SAPK (168, 302). However, POSH cannot interact with Tyr40Cys Rac1, a second
Rac1 effector mutant that does not activate the SAPKs in
vivo (168, 302). These results suggest that POSH, but not
the MLKs, is a likely effector for Rac1 activation of the
SAPKs. However, as is discussed in section IVG2, there is
good evidence implicating MLK3 as an effector for
Cdc42Hs. The mechanism by which POSH recruits the
SAPKs is unclear. It is plausible to speculate that the SH3
domains of POSH may serve a scaffolding function, binding MAP3Ks such as MEKK1 or MLKs, that contain SH3
binding sites.
D) SUMMARY. To summarize, PAKs as well as MEKK-1
(see sect. IVB1) and -4 (see sect. IVF1) can interact with
both Rac1 and Cdc42Hs. Constitutively active PAK mutants can, in some but not all instances, modestly activate
the SAPKs in vitro; however, kinase-dead MEKKs cannot
block this activation, calling into question the original
hypothesis, based on studies of yeast signaling (120), that,
in response to activated Rho family GTPases, PAKs would
recruit MEKKs to activate the SAPKs and p38s. In contrast, MLK3 and MLK2 are likely effectors for Cdc42Hs
(but not Rac1) (see sect. IVG1) while POSH is a candidate
adapter protein that couples Rac1 to the SAPKs.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
the relay of signals from trimeric G proteins to ERKs, and
transient expression of Pyk2 and SAPK also results in
significant SAPK activation (75, 312). Thus agonists such
as ANG II could recruit the SAPKs by direct Gq␣-mediated
PLC-␤/Ca2⫹-dependent activation of Pyk2 (Fig. 6B). It is
attractive to speculate that Btk and Pyk2 recruit Ras and,
possibly, Rac1, thereby triggering activation of SAPK;
however, data in support of this hypothesis are not yet
available. PLC-␤ activation also results in the generation
of diacylglycerol and activation of classical PKCs; however, evidence implicating classical PKCs in coupling the
SAPKs or p38s to seven pass receptors is unavailable
(165, 166).
C. Regulation of the SAPKs by Scaffold Proteins
Given the large number of MAP3Ks and additional
upstream regulators of the SAPKs and p38s, the organization of individual MAP3K 3 MEK 3 MAPK core signaling components into groups subject to regulation by select activators, and able to recruit select effectors, is vital
to the maintenance of signaling specificity, efficiency, and
integrity. In lower eukaryotes, this sequestration is mediated by scaffolding proteins that collect specific MAP3K 3
MEK 3 MAPK core modules into organized clusters (120,
242). These scaffolding proteins may be distinct polypeptides such as the S. cerevisiae scaffold protein Ste5p
(Fig. 7A), or, alternatively, as with the yeast MEK Pbs2p,
the signaling components themselves may, in addition to
signaling activity, possess intrinsic scaffold properties
(120, 242) (Fig. 7B).
Scaffold proteins for mammalian stress-activated
MAPK pathways are now being identified. As with yeast,
these include proteins whose sole apparent function is
that of a scaffold, others which are multipurpose binding
proteins, and still others which are also components of
MAP3K 3 MEK 3 MAPK core modules.
1. JIPs: distinct scaffold proteins that may regulate
MLK 3 MKK7 3 SAPK core signaling modules
JNK interacting protein-1 (JIP1) is the founding member of a novel family of mammalian scaffold proteins that
was identified from a two-hybrid screen that sought
polypeptides that could interact with SAPK␥/JNK1. A
⬃60-kDa polypeptide, JIP1, consists of an NH2-terminal
domain that binds SAPKs, but not ERKs or p38s (amino
acids 143–163), and a COOH-terminal SH3 domain (amino
acids 491– 600). In between is a proline-rich segment (amino acids 281– 448) with several consensus putative SH3
binding sites (72). JIP1 can bind elements at all three
levels of a SAPK core signaling module: the MAP3Ks
MLK3 and DLK (but not MEKK1), a MEK, MKK7 (but not
SEK1), and SAPK. However, it is unclear if this binding is
at all dynamic or regulated in any way. MLK3 binds JIP1
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Activation of SAPK by these constitutively active G
protein ␣-subunits has not been universally observed, and
it is evident that G protein ␤- and ␥-subunits can also
activate coexpressed SAPK and may, in fact, represent a
major mechanism by which trimeric G proteins signal to
the SAPKs (53). For example, in one study of COS-7 cells,
SAPK is weakly activated upon transient coexpression
with GTPase-deficient G13␣, G12␣, or Gq␣, and trimeric G
protein signaling to the SAPKs may be mediated by ␤␥subunits. Thus coexpression in COS-7 cells of G␤1 with
G␥1 results in much stronger SAPK activation (53). Furthermore, recruitment of SAPK by the m1 or m2 muscarinic acetylcholine receptor can be reversed with a construct expressing the G protein ␤-subunit binding domain
of the ␤-adrenergic receptor kinase (␤-ARK), a scavenger
for free G␤␥ subunits (53). Weaker ␣-subunit activation of
the SAPKs may not be a general feature of all COS cells
(see below), and, as noted above, other studies using
different cell types have observed substantial SAPK activation upon coexpression with G12␣ or G13␣ (50, 243).
Several candidate mechanisms have been proposed
for the coupling of heterotrimeric G proteins to the
SAPKs; however, there is little really definitive evidence
in favor of any of these models (Fig. 6B). Inasmuch as
Asn-17 Ras or Rac blocks G12␣ activation of SAPK, a role
for Ras and Rac has been proposed for G12␣ activation of
SAPK. The role of Ras as an effector for G12␣ may be cell
dependent, however. Thus Asn17-Ras can block signaling
from GTPase-deficient (Gln229Leu) G12␣ to SAPK in
NIH3T3 cells and in stably transfected COS cells but not
in HEK293 cells (50, 243). In contrast, Asn17-Rac can
inhibit Gln229Leu-G12␣ activation of SAPK in both
NIH3T3 and HEK293 cells (50, 243). Moreover, Asn17-Rac
can also inhibit activation of SAPK by free G␤1␥1 (53).
G13␣ signaling, at least in COS cells, may also require
Ras given that Asn17-Ras blocks SAPK activation by
Gln226Leu GTPase-deficient G13␣ (50).
Nonreceptor Tyr kinases may also couple trimeric G
proteins to the SAPKs. Thus the Bruton’s Tyr kinase (Btk)
directly binds and is activated by G12␣ (141), a putative
effector of the receptor for ANG II, a good activator of the
SAPKs (115, 369). Ectopic overexpression of Btk also
activates coexpressed SAPK (153). The mechanism by
which G12␣ binding activates BTK is unclear. BTK has a
PH domain and, once recruited to the membrane by G12␣
may be an effector for PI 3-kinases, downstream of Ras,
trimeric G proteins that recruit Ras or trimeric G protein
␤␥-subunits themselves (Fig. 6B). Gq␣ and free G␤␥ subunits can also activate phospholipase C-␤ (PLC-␤). Inositol trisphosphate generated as a consequence of PLC-␤
activity releases intracellular Ca2⫹ stores into the cytosol
(115). Pyk2 is a nonreceptor Tyr kinase that is activated in
vivo by elevations in intracellular free Ca2⫹ and in some
cells (hepatocellular carcinoma cells) by ANG II (75, 369).
Pyk2, acting in concert with Src, has been implicated in
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
Volume 81
via the JIP1 SH3 domain, whereas MKK7 binding to JIP
requires a central region (amino acids 283– 471) of the
JIP1 polypeptide that overlaps with the proline-rich region (72, 336). JIP1 can also bind the mammalian GCK
homolog hematopoietic progenitor kinase-1 (HPK1) (but
not GCK-related, another GCK homolog, see sect. IIID)
(72, 336). The binding of HPK1 is likely to be indirect,
mediated by the binding of HPK1 to MLK3 (see sect.
IVG1); however, the ability of JIP1 to potentially link HPK1
to a complete core signaling module fits nicely with the
idea of JIP1 as a true scaffold protein.
The exact biological function of JIP1 is unclear, and
the binding of endogenous JIP1 to endogenous MAPK
signaling components has not been demonstrated; however, coexpression of JIP1 with MLK3 or MKK7 enhances
the ability of these proteins to foster SAPK activation in
vivo. In contrast, overexpression of JIP1 inhibits activation of SAPK by extracellular stimuli including TNF and
UV radiation, and there are no known stimuli whose
activation of SAPK is enhanced by JIP expression (336). It
is likely that JIP1 serves to organize SAPK core signaling
modules so as to permit regulation by selective sets of
upstream stimuli. Accordingly, overexpression of JIP1
could nonspecifically sequester endogenous MLK3, DLK,
MKK7, or SAPK, enhancing the ability of recombinant,
ectopically expressed MLK3, DLK, or MKK7 to activate
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FIG. 7. Regulation of the SAPKs and p38s by scaffold proteins. A:
distinct scaffold proteins. Yeast Ste5p is the model for lower eukaryotes.
B: core elements with scaffold properties. In yeast, this is exemplified by
Pbs2p; in mammals by SEK1. In A, we have also illustrated the interactions mediated by SEK1 scaffold function that may occur contemporaneously with regulation by discrete scaffold proteins.
April 2001
MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
2. JNK/SAPK associated protein-1/JIP3 is a second
SAPK pathway scaffold protein
Murine JNK/SAPK-associated protein-1 (JSAP1) was
isolated by Ito et al. (136) as a polypeptide that interacts
with SAPK␤/JNK3 in a yeast two-hybrid screen, and, independently, by Kelkar et al. (155) as an interactor with
SAPK␥/JNK1 (Kelkar et al. refer to the protein as JIP3).
JSAP1/JIP3, like SAPK␤, is localized selectively in brain
and heart (a small amount of JSAP1/JIP3 message is
detectable in lung) (136, 155). Two alternative splicing
forms of JSAP1/JIP3 may exist: a larger clone (JIP3b)
expressed in brain and a smaller clone (JIP3a) found in
heart. JSAP1/JIP3 is a large polypeptide (⬃144 kDa) containing a leucine zipper motif (amino acids 392– 427, JIP3a
numbering, amino acids 424 – 459 JIP3b numbering) as
well as several coiled-coiled motifs (amino acids 25–155,
265–315, 385– 424, 459 –515, and 585– 615, JIP3b numbering). No other recognizable structural domain features are
present (136, 155).
Biochemical studies of JSAP1/JIP3 have yielded con-
flicting and contradictory results. Both Ito et al. and
Kelkar et al. demonstrate that JSAP1/JIP3 can bind all
three SAPK isoforms, but binding to SAPK␤ is strongest.
Binding to other MAPKs (p38 and ERK) is not observed
(136, 155). The SAPK␤ binding region of JSAP1/JIP3 was
mapped to amino acids 115–233 by Ito et al. (136) and
refined further to amino acids 202–213 by Kelkar et al.
(155). Amino acids 202–213 are strikingly conserved with
a segment of the SAPK binding region identified for JIP1
(amino acids 153–164) and suggest a consensus sequence
(Arg-X-X-Arg-Pro-Thr-Ser/Thr-Leu-Asn-Val/Leu-Phe-Pro,
where X is any amino acid) for SAPK binding to JIP1 and
JSAP1/JIP3 (136, 155). The basic amino acids in this SAPK
binding domain are reminiscent of the basic MAPK docking sites present in many MAPK substrates and regulators,
that bind to MAPK CD motifs (see sect. IIE3; Fig. 4, C and
D) (301). It will be important to determine if the CD motif
of SAPK is necessary for binding JIP1 and JSAP1/JIP3.
Outside of the SAPK binding segment, the sequences of
JIP1 and JSAP1/JIP3 are divergent. Three potential SAPK
proline-directed phosphorylation sites (amino acids 234,
244 and 255) lie just downstream of the SAPK binding site
on JSAP1/JIP3. A truncated JSAP construct containing
these sites is efficiently phosphorylated, likely at all three
sites, in vitro by SAPK; however, this has no apparent
effect on SAPK binding (136, 155).
In contrast, Ito et al. (136) report that prior activation
of SAPK in vivo (by expression with truncated, active
MEKK1) significantly reduces the binding of SAPK to
either unphosphorylated or in vitro phosphorylated
JSAP1/JIP3; however, Kelkar et al. (155) report the apparent opposite: prior activation of SAPK by UV radiation
increases binding to JSAP1/JIP3 (155). This difference
may be due to different modes of SAPK activation
(MEKK1 vs. UV radiation), which may enhance or disrupt
the binding of the expressed SAPK to scaffold proteins
other than JSAP1/JIP3. In any case, activation of the SAPK
pathway may regulate differentially the association of
SAPK with JIP3.
Consistent with its role in organizing SAPK signaling
modules, JSAP1/JIP3 can, like JIP1 and JIP2, associate
with upstream elements of SAPK core signaling modules.
However, interactions between endogenous JSAP1/JIP3
and endogenous SAPK pathway components have not
been assayed, and there is substantial disagreement as to
which recombinant, ectopically expressed SAPK pathway
components interact with JSAP1/JIP3. Ito et al. (136)
observe that in cotransfection/coimmunoprecipitation
and in vitro pull down experiments, there is a stable
association between JSAP1/JIP3 and SEK1 (but no binding to MKK7 or MKK6). Curiously, Ito et al. (136) also
observe that JSAP1/JIP3 can bind MEK1. The significance
of MEK1 binding is unclear. Increasing the level of SEK1
expression has no effect on MEK1 binding; likewise, increasing MEK1 expression has no effect on SEK1 binding
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the SAPKs, but inhibiting the ability of stimuli that normally recruit these proteins independently of JIP1 to activate the SAPKs. Alternatively, overexpression of JIP1
could disrupt the normal stoichiometry of JIP1 and its
effectors. By definition, for a scaffold protein to function
appropriately, its level in a cell must be tightly regulated
such that it can bind its effectors at an appropriate stoichiometry. Thus, if the concentration of an ectopically
expressed scaffold protein such as JIP1 exceeds the concentration of the endogenous signaling components it
regulates, a situation arises, the prozone effect, where no
single scaffold protein molecule is associated with all of
the signaling effectors it must coordinately regulate to
perform its function. At this point, the overexpressed
scaffold protein becomes an inhibitor of signaling.
Recently, the cloning of JIP2 was reported. JIP2 is
structurally and functionally quite similar to JIP1 and is
almost exclusively localized in brain. As with JIP1, JIP2
interacts in vivo with MLK-2 and -3 (but not MEKK family
MAP3Ks), with MKK7 (but not MEK1, MKK3, SEK1, or
MKK6), and with SAPK-␣, -␤, and -␥ (but not p38 or ERK)
(355). There are significant differences in the JNK binding
properties of JIP1 and JIP2, however. JIP1 binds all SAPK
isoforms more strongly than does JIP2, and both JIPs
preferentially interact with the SAPK-␥ isoform (355).
JIP1 and -2 can potentiate (2.5- to 3-fold) activation of
SAPKs-␣, -␤, or -␥ by coexpressed MLK3, and the JIP
proteins (endogenous and ectopically expressed) can homo- and heterodimerize. Immunofluorescence studies indicate that JIP proteins are localized in the cytoplasm and
peripheral membrane, with JIP1 more localized in membrane projections of PC12 pheochromocytoma cells. The
significance of this localization is unclear (355).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
SAPK once it is activated (136). In a similar vein, Kelkar et
al. (155) observed that MLK3 activation of all three SAPK
isoforms is strongly enhanced upon coexpression with
JSAP1/JIP3.
In cotransfection/coimmunoprecipitation experiments,
JSAP1/JIP3 can also homodimerize and heterodimerize
with JIP2, but not JIP1 (155). The significance of these
interactions is unclear.
The signaling pathways that recruit JSAP1/JIP3-containing signaling modules are not known. However,
Kelkar et al. (155) observed that treatment of PC12 cells
with NGF significantly induces expression of JIP3 protein
coincident with the differentiation of these cells to a
neuronlike phenotype. Immunolocalization of JSAP1/JIP3
demonstrates that the protein is localized in axonal
growth cones. In contrast, NGF withdrawal or suspension
from the substratum, conditions which trigger PC12 cell
apoptosis, correlate with caspase-3-mediated cleavage
and degradative loss of endogenous JSAP1/JIP3, and
JSAP3/JIP1 is a substrate of caspase-3 in vitro. Taken
together, these results suggest that conditions supporting
PC12 survival and differentiation coincide with JSAP1/
JIP3 expression (155). Inasmuch as the SAPKs have been
implicated in brain development (see sect. VC), the role
JSAP1/JIP3 plays in neuronal SAPK regulation remains an
important question.
3. The 280-kDa actin binding protein-280 may be a
scaffold protein for TNF and lysophosphatidic acid
activation of the SAPKs in melanoma cells
The 280-kDa actin binding protein-280 (ABP-280)
(also called filamin) is a 280-kDa actin binding protein.
Actin binding is mediated by the ABP-280 NH2 terminus.
This region is followed by two flexible hinge regions, one
two-thirds along its length and the other immediately
adjacent to a COOH-terminal homodimerization domain.
Structurally, ABP-280 is rod shaped, and aside from the
actin binding domain, the various protein interaction domains are distributed among 24 repeats of an ⬃98-amino
acid structure. Notably, repeat 24 itself mediates homodimerization. ABP-280 cross-links actin filaments into
orthogonal arrays and contributes to the structure of the
cortical actin meshwork, functions attributable to ABP280’s flexible rod-shaped structure (291). Recent studies
indicate that ABP-280 may also serve as a signaling scaffold protein.
In a yeast two-hybrid screen employed to identify
novel SEK1 interactors, Marti et al. (196) identified ABP280 as a SEK1 binding protein. SEK1 binds ABP-280 in
vivo and in vitro, and the binding requires a COOH-terminal region of ABP-280 consisting of repeats 21–23C (amino acids 2282–2454) (196). Binding is quite specific, and
ERK1, p38␣, and MEK1 bind ABP-280 at best very weakly
compared with SEK1 binding. However, MEKK1 and
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to JSAP1/JIP3, suggesting, but not proving, that the SEK1
binding site is distinct from the MEK1 binding site. The
SEK1 binding site has been mapped by Ito et al. to the
COOH-terminal region of JSAP1/JIP3 (amino acids 1054 –
1305, JIP3a numbering) (136). In contrast, Kelkar et al.
(155) observed no interaction between JSAP1/JIP3 and
SEK1 (or MEK1 for that matter), but observed a strong
interaction between JSAP1/JIP3 and MKK7. In both instances JIP3a was used, making it unlikely that these
differences are due to the JSAP1/JIP3 splicing isoform
used (Fig. 7B).
Ito et al. (136) also observed that JSAP1/JIP3 also
binds full-length in vitro translated MEKK1 in in vitro
binding assays. In vivo coimmunoprecipitation assays using various MEKK1 truncation mutants indicate that residues 1– 640 of MEKK1 are essential for this interaction;
however, it is noteworthy that for technical reasons, Ito et
al. (136) were unable to detect binding between MEKK1
and JSAP1/JIP3 in vivo. In in vitro assays using 35S-labeled
full-length MEKK1, Ito et al. (136) mapped MEKK1 binding to two contact points on JSAP1/JIP3: amino acids
1–305, a weak interaction domain, and amino acids 434 –
1053, a stronger interaction domain. Ito et al. also observed that JSAP1/JIP3 can weakly interact with a construct consisting of the catalytic COOH-terminal domain
of Raf-1 (Raf-C), a MAP3K of the ERK cascade. MEKK1
expression has no competitive effect on Raf-C binding to
JSAP1/JIP3, indicating that the binding site for Raf-1 is
distal from the MEKK1 binding site. The significance of
the Raf-1-JSAP1/JIP3 interaction is unknown (136). It is
conceivable that this interaction is indirect, mediated by
MEK1. Moreover, binding of full-length Raf-1 to JSAP1/
JIP3 has not been observed. In contrast, Kelkar et al. (155)
observe no binding between JSAP1/JIP3 and MEKK1,
likely due to the use of a truncated MEKK1 construct
missing the NH2-terminal 640 amino acids, shown by Ito
et al. to be necessary for MEKK1 binding (136, 155). Raf-1
binding to JSAP1/JIP3 was also not observed by Kelkar et
al. (155), raising the possibility that the finding of Ito et al.
(136), that Raf-1 binds JSAP1/JIP3, is an expression artifact. Instead, Kelkar et al. observe that JSAP1/JIP3 binds
MLK3 (but not MLK2 or DLK) (155) (Fig. 7B).
Consistent with their binding results, Ito et al. (136)
demonstrate that JSAP1/JIP3 expression significantly enhances the ability of MEKK1 and SEK1 to activate SAPK␤
and, to a lesser extent, SAPK␣ and -␥. Activation by
MEKK1 of MEK1 signaling to SAPK is not affected by
coexpression with JSAP1/JIP3, in spite of the fact that
MEK1 is a weak MEKK1 substrate. The effect of JSAP1/
JIP3 on ERK activation by MEKK1/MEK1 was not tested.
The turnover of activated SEK1 by JSAP1/JIP3 was also
not tested; however, the observation that MEKK1 activation of SAPK reduces SAPK’s affinity for JSAP1/JIP3 suggests that JSAP1/JIP3 organizes signaling complexes containing MEKK1, SEK1, and inactive SAPK, releasing the
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4. SEK1 has intrinsic scaffold properties
In addition to its properties as a MEK, SEK1 possesses intrinsic scaffold properties, and as is the case for
the yeast MEK Pbs2p and its upstream activators and
effectors (242) (Fig. 7B), the intrinsic scaffold properties
of SEK1 enable it to form specific, sequential dynamic
complexes with both an upstream activator and a substrate. Thus the NH2 terminus (amino acids 1–77) of
inactive, dephosphorylated SEK1 can specifically bind
one of its upstream activators, MEKK1 (Fig. 7B). A potential splicing isoform of SEK1 wherein the first 34 amino
acids have been deleted has not been tested for MEKK1
binding. MEKK1 does not readily interact in in vivo or in
vitro with MEK-1 or -2, MKK-3 or -6 or with the ␥1-isoform
of MKK7 under conditions in which each MEK is expressed separately with MEKK1. As a consequence, none
of these MEKs is as good an in vitro substrate of MEKK1
as is SEK1 (342). However, while MKK7␥1 does not interact with MEKK1 and is a modest MEKK1 substrate in
vitro, several MKK7 isoforms (␣1, ␣2, ␤1, and ␤2) are
strongly activated by MEKK1 in vivo (see sect. IIF1) (188,
315). These MKK7 isoforms are either NH2 terminally
deleted or truncated (315), and their binding to MEKK1
has not been ascertained.
The SEK1-MEKK1 complex can be generated in vitro.
Under these conditions, the addition of ATP to the SEK1MEKK1 complex results in SEK1 activation which, in
turn, triggers dissociation of the SEK1-MEKK1 complex
(342) (Fig. 7B). Phosphorylated and activated SEK1 then
forms a second specific complex, with SAPK, again mediated by the SEK1 NH2-terminal MEKK1 binding domain.
This MEKK interaction motif also contains the polybasic
MAPK docking site that interacts selectively with the
SAPK CD domain (see sect. IIE3) (301). Upon phosphorylation and activation of SAPK, this second complex dissociates (342) (Fig. 7B).
D. Regulation of the SAPKs by the GCKs
1. General considerations
GCK is the founding member of a novel family of
stress-regulated Ser/Thr protein kinases some of which
are selective activators of the SAPKs (163). Accumulating
evidence suggests that those GCKs that recruit the SAPKs
do so by binding MAP3Ks and elements upstream of
MAP3Ks (163, 359). Thirteen mammalian GCKs have been
cloned. In addition, there are Drosophila, C. elegans, and
Dictyostelium homologs, as well as two S. cerevisiae
genes with known phenotypes (163). All GCKs possess
NH2-terminal kinase domains that are distantly related to
those of the PAKs and Ste20p. The kinase domains are
followed by extensive COOH-terminal regulatory domains
(CTDs) (163). The distant sequence homology between
the kinase domains of the GCKs, Ste20p, and the PAKs led
to the initial placement of the GCKs in the Ste20 family.
However, the domain arrangement of the GCKs coupled
with the fact that these kinases do not possess CRIB
motifs and are not directly activated by Rho family GTPases indicate that the GCKs should be considered a distinct protein kinase family (163).
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SAPK can coprecipitate with ABP-280 if, and only if, SEK1
is present (196). As discussed below, SEK1 itself has
scaffold properties (342) and may permit the formation of
MEKK1-SEK1-SAPK-ABP-280 complexes that can be specifically recruited by upstream stimuli (Fig. 7B). Activation of SEK1 with MEKK1 has no effect on the binding of
SEK1 to ABP-280; however, ABP-280 appears to be important for the activation of SEK1 and the SAPKs in vivo
in melanoma cells, in response to certain specific extracellular stimuli, notably TNF (196).
M2 cells are a human melanoma cell line that has
spontaneously lost ABP-280 expression. After plating,
these cells show exaggerated and prolonged membrane
blebbing, poor spreading, and defective directed cell motility. M2A7 cells are M2 cells wherein ABP-280 has been
reconstituted to physiological levels, correcting the defects in motility observed for M2 cells (61, 62). SAPK
activation by TNF and lysophosphatidic acid (LPA) is
strikingly abrogated in M2 cells, while activation by arsenite, anisomycin, or hyperosmolarity are unaffected. In
contrast, activation of SAPK by TNF and LPA is restored
in M2A7 cells (196). Thus ABP-280 may serve as a scaffold
protein to coordinate the proper activation of the SAPKs
by a select subset of activators (TNF and LPA) to the
exclusion of others. It is important to note that the loss of
TNF activation of SAPK was not observed in other cells
wherein ABP-280 expression was ablated. Thus this phenomenon may be cell specific (196).
Recently, it was reported that, in addition to binding
SEK1, ABP-280 can bind TNF receptor-associated factor-2
(TRAF2), an adapter protein implicated in TNF and stress
activation of the SAPKs (see sect. IIIE) (179). TRAF2
consists of an NH2-terminal RING effector domain, a central Zn finger domain, and two tandem COOH-terminal
TRAF domains (see sect. IIIE1). ABP-280 binding requires
the RING and Zn finger domains of TRAF2. Amino acids
1644 –2118 of ABP-280, just upstream of the SEK1 binding
site (amino acids 2282–2454), contain the TRAF2 binding
domain. Overexpression of ABP-280 in transfected 293
cells abrogates TRAF2 activation of SAPK (and NF-␬B);
however, TRAF2 activation of SAPK is also lost in M2
cells (179). Given that SAPK activation by TNF is lost
specifically in M2 cells, and not in other cells (196), the
293 cell result may be attributable to sequestration of
endogenous 293 cell SAPK activating machinery from the
expressed TRAF2, although this inhibition could be attributable to a prozone effect (see sect. IIIC1) (Fig. 7B).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
nine (PEST) motifs and at least two polyproline putative
SH3 domain binding sites. Of particular importance, all of
the group I kinases possess a highly conserved ⬃350amino acid region, the GCK homology domain, at the
COOH-terminal end of the CTD. The GCK homology domain can be subdivided into a somewhat hydrophobic,
leucine-rich domain and a 142- to 152-amino acid stretch,
the COOH-terminal (CT) motif. The leucine residues in
the Leu-rich domains are not organized into leucine zippers, nor are these domains sufficiently hydrophobic for
membrane insertion. The COOH-terminal-most putative
SH3 binding sites of GCK, GCKR, GLK, and HPK1 lie at
the NH2-terminal end of the cognate Leu-rich domains.
Such a binding site is not evident in the Leu-rich segments
of NIK, TNIK or Nrk which are closely related to each
other, but more divergent from the other mammalian
group I GCKs (73, 96, 131, 145, 151, 159, 163, 277, 294,
319, 354).
GCK, GCKR, GLK, and NIK are all activated in vivo by
TNF, and TNIK may also be a TNF effector (73, 96, 239,
277, 354), and the CT extensions of the CTDs of GCK,
GCKR and GLK along with that of HPK1 are quite homologous (Fig. 8A) (73, 131, 151, 159, 277, 319). The CTs of
NIK, TNIK, and Nrk, while similar to those of the other
mammalian group I GCKs, are closer in sequence to each
other (96, 145, 294, 354); however, the activation and
function of Nrk have not been analyzed (145). Thus structurally, NIK, TNIK, and Nrk form one arm of the mammalian group I GCKs while GCK, GCKR, GLK, and HPK1
form a second arm of the mammalian group I GCKs.
Studies of GCK and GCKR indicate that the CT motif
is required for binding proteins of the TNF receptorassociated factor (TRAF) family, and, possibly, for gating
the binding of MAP3Ks (278, 359) (see sects. IVB3 and
IVC). The notion that GCKs can both potently activate the
SAPKs as well as stably associate with SAPK core signaling module elements and their upstream activators, in a
manner dependent at least in part on the kinase activity of
the GCKs (see sects. IVB3 and IVC), suggests that the
group I GCKs may coordinately regulate MAPK core signaling modules in conjunction with additional upstream
elements.
2. Group I GCKs: selective regulators of the SAPKs
FIG. 8. Germinal center kinase (GCK) regulation of MAPK pathways.
A: structure of GCK, a typical group I GCK family enzyme. B: regulation
of MAP3Ks by GCKs and regulation of GCKs by upstream elements
including Tyr kinases, cytokines, and stress.
A) GCK. GCK (Table 4) was identified in a subtractive
screen for genes preferentially expressed in B-cell germinal centers. Thus, although GCK is present in all tissues
examined, its distribution in B lymphocyte follicular tissue is restricted largely to the germinal centers and not
the surrounding mantle zones (151). Germinal centers are
sites of B-cell maturation (notably Ig class switching) and
selection, processes that are driven by ligands of the TNF
family (CD40L, CD30L, TNF, in particular) (34, 200). For
example, mice in which the gene for the type 1 TNF
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GCKs can be subdivided into two groups based on
overall sequence conservation and function: group I GCKs
are structurally similar within both the kinase and CTD
regions to GCK itself and are specific upstream activators
of the SAPKs (163) (Fig. 8A). Group II GCKs are more
closely related to the S. cerevisiae GCK Sps1p and are
activated in vivo by extreme stresses. Few downstream
effectors of group II GCKs have been identified (163). A
putative third GCK subgroup consists thus far only of
JNK/SAPK inhibitory kinase (JIK) (303).
Seven mammalian group I GCKs have been identified
to date: GCK itself, GCK-related (GCKR, also called kinase homologous to Ste20, KHS, Table 4), GCK-like kinase (GLK), hematopoietic progenitor kinase-1 (HPK1),
and Nck-interacting kinase [NIK, also called HPK1/GCKlike kinase, HGK; the GCK NIK should not be confused
with NF-␬B inducing kinase (191), which is also abbreviated NIK], TRAF and Nck interacting kinase (TNIK), and
NIK-related kinase (Nrk) (Table 4) (73, 96, 131, 145, 151,
159, 277, 294, 319, 354).
There is remarkable structural similarity among the
CTDs of group I GCKs. The CTDs of all group I GCKs
include two or more proline/glutamic acid/serine/threo-
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293 cells, can completely inhibit TNF activation of the
SAPKs (277) (see sect. IVC).
C) GLK. GLK (Table 4) is a third type I GCK homolog
that is, like GCK and GCKR, a selective activator of the
SAPKs. The CTD of GLK is strikingly conserved with
those of GCK and GCKR, especially within the GCK homology domain. Consistent with this close similarity, endogenous GLK is activated in vivo by TNF (73).
D) HPK1. HPK1 (Table 4) was identified in a subtractive
screen for genes preferentially expressed in cells of the
hematopoietic lineage. HPK1 shares many of the structural features of group I GCKs, although it is more distantly related to GCK than is GCKR and GLK. HPK1 has
four consensus SH3 domain binding sites (131, 159, 163).
The COOH-terminal-most SH3 binding site interacts with
the SH3 domain of the mixed lineage kinase (see sect.
IIG3) MLK3 (159) (discussed in sect. IVG1). Transient
overexpression of HPK1 results in vigorous activation of
the SAPKs. p38 is activated only very weakly, and no ERK
activation is observed upon expression of HPK1 (131,
159, 163).
Two recent studies indicate that HPK1 may be
regulated by SH2/SH3 adapter proteins that couple to
receptor and/or nonreceptor Tyr kinases. Thus the NH2terminal two SH3 binding sites of HPK1 interact with
the COOH-terminal SH3 domain of SH2/SH3 adapter
protein Grb2 (5), which is also a key regulator of Ras
(195). The Grb2-HPK1 interaction is not affected by
extracellular stimuli; instead, treatment of cells coexpressing Grb2 and HPK1 results in a translocation of
the Grb2-HPK1 complex to the membrane. The significance of the HPK1-Grb2 interaction is still somewhat
unclear, however, given that coexpression with Grb2
has no effect on HPK1 activity (5).
In contrast, Crk and CrkL, two closely related SH2/
SH3 adapter proteins, can bind to the second and fourth
polyproline SH3 binding sites of HPK1. As with the Grb2HPK1 interaction, the Crk(CrkL)-HPK1 interaction enables the EGF-dependent recruitment of HPK1 to the EGF
receptor (181). Support for the idea that the Crk(CrkL)HPK1 interaction is physiologically relevant comes from
the observation that coexpression of Crk or CrkL with
HPK1 results in HPK1 activation and synergistic SAPK
activation (181). Although the SAPKs are usually only
modestly activated by mitogens such as EGF that signal
through receptor Tyr kinases, the Grb2-HPK1 and Crk/
CrkL-HPK1 interaction could explain in part the regulation of the SAPKs by any of several nonreceptor Tyr
kinases such as Btk and Pyk2 that strongly activate the
SAPKs in vivo (see sect. IIIB) (Figs. 6B and 8B).
In this regard, it is noteworthy that Btk and Pyk2 are
putative effectors for trimeric G protein-coupled receptors (75, 141). Several MAP3Ks upstream of the SAPKs
[the SAPK-specific MEKK1 (see sect. IVB1) and MLK3 (see
sect. IVG2) for example] bind Rac1 in a GTP-dependent
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receptor has been disrupted display little or no germinal
center formation (200). Accordingly, it was logical to
propose that GCK, which is selectively expressed in the
germinal center, might be an effector for TNF family
ligands and might signal to the SAPKs.
Indeed, endogenous GCK, upon immunoprecipitation
from several B cell lines, is significantly activated in vivo
by TNF (239). Moreover, transient expression of GCK
results in potent SAPK activation in the absence of external stimulus. In contrast, expression of GCK does not
result in activation of the ERKs, p38s, or NF-␬B (239, 277).
Thus, like MEKK1 and the MLKs (see sects. IIG2 and IIG3),
GCK is a specific SAPK activator.
Although capable of undergoing activation in vivo by
upstream stimuli, both endogenous and recombinant
GCK, like many stress-activated MAP3Ks, exhibits substantial basal activity in in vitro assays, or upon expression in vivo, unless expressed at extremely low levels
(151, 239, 359). This finding suggests that GCK is activated
in vivo either by limiting concentrations of an inhibitor
lost during immunoprecipitation or stoichiometrically titered out upon GCK overexpression. Alternatively, GCK
might be regulated by oligomerization, a process that
could be mimicked by immunoprecipitation and/or overexpression. In either case, the CTD is the region of the
GCK polypeptide likeliest to mediate GCK regulation and
substrate recognition (163). In support of this idea, transient expression of the free GCK-CTD, or a GCK construct
(Lys44Met) that is completely devoid of kinase activity
results in SAPK activation (239, 359). As discussed in
sections IVB3 and IVC, the GCK-CTD binds both GCK
regulators and effectors.
B) GCKR. Using a database mining strategy, Shi and
Kehrl (277) identified GCKR (also called kinase homologous to STE20, KHS, Table 4) as a protein kinase highly
similar to GCK. Independently, Tung and Blenis (319)
cloned GCKR as part of a screen to identify novel mammalian homologs of STE20. GCKR is ⬃60% identical to
GCK throughout its primary sequence. Most notably, the
GCK homology domain (Leu-rich and CT motifs) of the
CTD is highly conserved (151, 163, 277, 319). Like GCK,
GCKR is a selective activator of the SAPKs. The ERKs,
p38s and NF-␬B are not activated upon expression of
GCKR (277, 319). For reasons that are unclear, both endogenous and recombinant GCKR possess considerably
lower basal activity than GCK both in vivo and in vitro,
and in contrast to GCK itself, transient expression of the
GCKR-CTD does not result in significant SAPK activation
(277, 319).
Endogenous GCKR is activated in vivo by TNF,
CD40, and UV radiation (45, 277, 278). UV activation of
GCKR is likely mediated by UV-induced clustering of TNF
receptors (256). It is likely that GCKR is an important
effector for TNF, at least in some cell types. In support of
this, antisense constructs of GCKR, when expressed in
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
F) TNIK. TRAF2 and Nck-interacting kinase (TNIK) is
quite similar in structure overall to NIK (⬃90% identity at
the amino acid level within the kinase domain, amino
acids 1–306, ⬃90% within the Leu-rich and CT motifs,
amino acids 1019 –1360, and ⬃53% within the remainder
of the CTD, amino acids 307–1018). As with NIK, TNIK
expression results in potent and specific SAPK activation.
In contrast to NIK and GCK, however, the TNIK CTD is
equally potent at SAPK activation as is full-length TNIK
(96). TNIK’s nonconserved intermediate region, amino
acids 307–1018, contains both the binding site for TRAFs
and Nck (below). In addition to full-length TNIK (TNIK1),
at least seven hnRNA splicing isoforms of TNIK may exist.
These splicing variations are all deletions within the intermediate region of the TNIK CTD, upstream of the
Leu-rich/CT motif (TNIK2, ⌬447– 475; TNIK3, ⌬537–591;
TNIK4, ⌬795– 802; TNIK5, ⌬447– 475/537–591; TNIK6, ⌬447–
475/795– 802; TNIK7, ⌬537–591/795– 802; and TNIK8, ⌬447–
475/537–591/795– 802) (96). TNIK can interact with Nck in
vivo, although the role of this interaction is unclear. Several of the splicing deletions disrupt one potential SH3
binding site at amino acids 590 –593. However, it has not
been determined which potential SH3 binding site (amino
acids 590 –593, which corresponds to the SH3 binding site
on NIK at amino acids 574 –579, or a second potential site
at amino acids 669 – 673, which corresponds to the SH3
binding site on NIK at amino acids 611– 616) is necessary
for Nck binding. TNIK can also interact with TRAF2,
although there is no evidence that TNIK is regulated by
TNF family agonists. The TRAF binding region has been
localized to the intermediate region upstream of the Leurich motif of the CTD. The impact of TNIK mRNA splicing
on TRAF binding is unknown (96). It is tempting to speculate that the multiplicity of TNIK isoforms permits multiple forms of interactions with upstream activators, or
the generation of endogenous, inhibitory TNIK species,
missing specific binding sites, which serve to sequester
TNIK from its effectors.
3. Group II GCKs: activation by extreme stresses
Mammalian group II GCKs include Ste20-like oxidant
stress-activated kinase-1 (SOK1), kinase responsive to
stress (Krs)-1, mammalian sterile twenty-like-1 (MST1)/
Krs2, MST2, MST3, and lymphocyte-oriented kinase
(LOK) (55, 57, 162, 238, 269, 304). These enzymes are
structurally more similar to S. cerevisiae Sps1p than to
group I kinases. Group II GCKs are less well understood,
and, with the exception of MST1/Krs2 (109), apparently
do not activate any of the known MAPK pathways. Although group II GCKs share substantial catalytic domain
homology with group I GCKs, their CTDs differ significantly from those of the group I enzymes.
Like group I GCKs, mammalian group II kinases are
essentially ubiquitously expressed (with one exception,
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manner. Rac1 has also been implicated in trimeric G
protein signaling to SAPK; and C3G, a GEF for Rho family
GTPases, is, like HPK1, recruited by Crk/CrkL (33). Accordingly, it is possible that one function of HPK1 translocation is to colocalize it and, possibly, other MAP3K
activators, with effector MAP3Ks simultaneously translocated to the plasma membrane perhaps via binding to Ras
superfamily GTPases (163).
E) NIK. Murine NIK (also called HPK1/GCK-like kinase, HGK, Table 4) was cloned as part of a two-hybrid
screen to identify novel polypeptides that interact with
the SH2/SH3 domain-containing adapter protein Nck. A
human homolog was cloned by degenerate PCR using
primers based on sequences conserved in the GCK family
(294, 354). Of the mammalian group I GCKs, NIK, and its
close relatives TNIK (below) and Nrk, are the most distantly related. Still, functionally speaking, NIK shares
many of the same features of the group I GCKs. Notably,
NIK transient overexpression strongly and specifically
activates the SAPKs (294, 354). Moreover, as with GCK,
expression of the free NIK CTD significantly activates the
SAPKs, albeit to a lesser degree than does wild-type
NIK (294).
NIK contains two consensus SH3 binding motifs
(amino acids 574 –579 and amino acids 611– 616), both of
which can interact with the SH3 domains of Nck (294).
Nck is thought to be involved in the regulation of the actin
cytoskeleton, and one function of NIK may be to mediate
changes in cell motility and shape. Indeed, a Drosophila
ortholog of NIK, Misshapen, is required for embryonic
dorsal closure (see sect. IVC1) (295). NIK can be activated
by TNF (354), and recent studies of Misshapen signaling
indicate that it (and, by extension, NIK), like GCK and
GCKR, is an effector for TNF receptor-associated factors
(TRAFs) (163, 183, 278, 359) (see sect. IVC).
As with the Crk/CrkL-mediated activation of HPK1 by
EGF (181), the interaction between NIK and Nck couples
NIK to Eph family Tyr kinase receptors. The EphRs are
critical to embryonic patterning in the nervous and vascular systems. NIK is substantially activated by several
Eph family Tyr kinases, notably EphB1R and EphB2R
(14). Stimulation of EphB1R leads to the formation of a
complex consisting of NIK, Nck, and p62dok (14). p62dok is
a PH-domain-containing adapter that is Tyr phosphorylated by activated EphRs and other Tyr kinases. Upon Tyr
phosphorylation, p62dok recruits SH2 domain-containing
effectors (355) and RasGAP (201, 202). Within the NIKNck-p62dok complex, NIK is associated with the SH3 domains of Nck, and the SH2 domain of Nck itself is associated with Tyr phosphorylated p62dok. Studies with
dominant inhibitory NIK constructs indicate that NIK is
critical to several downstream consequences of EphB1R
including SAPK activation and activation of integrins (14)
(Fig. 8B).
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4. JIK, an inhibitor of mitogen-activation of SAPK:
a third class of GCK
JIK was recently identified as a novel member of the
GCK family. The sequence of JIK differs significantly from
group I and II GCKs, and, given its apparently novel
function, JIK may represent a third GCK subgroup. JIK
was cloned in a two-hybrid screen for interactors with
Eps8, an SH3 domain-containing protein identified as a
substrate for the EGF receptor (303). Nevertheless, stimulation of cells with EGF does not result in the translocation of JIK to the EGFR complex. Remarkably, however, JIK Ser/Thr kinase activity is almost completely
inhibited upon treatment of cells with EGF. Coincident
with this, transient overexpression of JIK blocks the modest (2-fold) activation of SAPK incurred by EGF, but not
the more robust SAPK activation stimulated by UV light or
anisomycin (303). On the basis of these results, it is
conceivable to speculate that in vivo, a component of
EGF regulation of SAPK involves relieving an inhibition
mediated by JIK. JIK has no effect, positive or negative,
on ERK, p38, or ERK5 activity (303).
E. Regulation of the SAPKs and p38s by Adapter
Proteins That Couple to TNF Family Receptors
Upon binding ligand, receptors of the TNF family
(the TNFR family) can elicit a wide variety of inflammatory responses and are critical to immune cell development, innate and acquired immunity, as well as the pathogenesis of a number of diseases such as arthritis, septic
shock, and, possibly, type 2 diabetes mellitus. Accordingly, this family of receptors is among the most important activators of the SAPKs, p38s. The TNFR superfamily
includes TNFR1, TNFR2, the lymphotoxin-␤ (LT-␤) receptors, CD40, CD27, Fas, receptor activator of NF-␬B
(RANK, also called osteoprotegerin, OPG, or TNF-related
activation-induced cytokine, TRANCE, receptor), the
death receptors (DRs)-3–5, the TNF-related apoptosis inducing ligand receptors (TRAILRs 1– 4, TRAILR2 is the
same as DR5), CD30, Ox40, 4 –1BB, and the p75 neurotrophin receptor (p75NTR). The receptors for IL-1 and the
Toll-like receptors (TLRs, which, among other things, include receptors for endotoxin) are structurally completely divergent from the TNFR family; however, their
mode of signaling is quite similar to TNFRs and, accordingly, these receptors are often grouped with the TNFR
superfamily. Several viral genes also encode TNFR family
polypeptides; the latent membrane protein-1 (LMP1) of
Epstein-Barr virus is an example. With the exception of
the IL-1R and the TLRs, most TNFR superfamily receptors
share a modest structural similarity within their extracellular domains, in keeping with the structural conservation
of many TNF superfamily ligands. In contrast, despite the
similarities in TNFR superfamily signaling programs and
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LOK, which is selectively expressed in lymphocytes) and
possess significant basal activity when immunoprecipitated from endogenous sources, or when overexpressed
(55, 57, 162, 238, 269, 304). However, Krs1, MST1/Krs2,
and SOK1 can be activated substantially in vivo by different environmental stresses. Krs1 and MST1/Krs2 are activated by extreme heat shock, high concentrations of arsenite, the general protein kinase inhibitor staurosporine,
and the phosphatase 2A inhibitor okadaic acid (56, 304).
MST1/Krs2 can also be activated in vitro by phosphatase
2A (56). Thus these two group II GCKs may be activated
by contemporaneous phosphorylation and dephosphorylation. MST1/Krs2 is also activated in vivo upon engagement of the proapoptotic receptor Fas (109). SOK1, as its
name implies, is strongly activated by oxidative stress
(238). SOK1 is also activated by ischemic injury and depletion of the cellular ATP pool (240). In all cases, SOK1
activation appears to require the generation of reactive
oxygen intermediates as well as elevated levels of cytosolic free Ca2⫹ (240). Stimuli that recruit LOK and MST3
are unknown (162, 269).
Certain of the group II GCKs display enzymologic
properties that may yield clues as to the mechanisms of
regulation of kinases of the entire GCK family. Thus SOK1
and MST3 autoactivate upon autophosphorylation in vitro
(238, 269), and SOK1 and MST1/Krs2 spontaneously homodimerize in vivo (56, 238), suggesting that oligomerization and autophosphorylation may play a role in GCK
family kinase activation.
MST1/Krs2 is the only group II GCK for which
potential downstream targets have been identified.
MST1/Krs2 is activated by caspase-3-mediated cleavage
at a consensus Asp323-Glu-Val-Asp-Ser caspase cleavage site that neatly bisects the enzyme into a free,
active catalytic domain moiety and a free regulatory
domain (109). MST1/Krs2 cleavage occurs in response
to two of its known upstream activators: staurosporine
and engagement of the proapoptotic TNF receptor family receptor Fas. Expression of high levels of wild-type
MST1/Krs2 or the active MST1/Krs2 kinase domain activates coexpressed SAPK, and p38 MAPKs indicating
that, in spite of the fact that MST1/Krs2 is not a group
I GCK, the SAPKs and p38s may be MST1/Krs2 effectors
(109). However, wild-type MST1/Krs2 activates SAPK
and p38 to the same degree as does the free MST1/Krs2
kinase domain, despite the fact that caspase cleavage
of MST1/Krs2, which releases its kinase domain, apparently results in MST1/Krs2 activation (109). Moreover,
it is not clear if Fas or oxidant activation of SAPK and
p38 coincides with or is preceded by cleavage of endogenous MST1/Krs2, a situation that would be expected if MST1/Krs2 were a bona fide upstream activator of SAPK and p38.
839
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
modes of receptor activation, the intracellular extensions
of TNFR family receptors are quite divergent (7, 17, 18,
284, 316, 323).
1. The protein recruitment model for TNFR
family signaling
and the TRAF domains of TRAF2 (below) (7, 129, 322,
323) (Fig. 9A).
B) TRAFS. The TRAFs are an emerging family of proinflammatory signal-transducing adapter proteins that are
important for the activation of a number of pathways in
response to TNF family ligands (7, 323). Six mammalian
TRAF polypeptides (TRAFs 1– 6) have been identified.
The TRAFs consist of two tandem COOH-terminal TRAF
domains (TRAF-N followed by TRAF-C), a central zinc
finger motif and, with the exception of TRAF1, an NH2terminal RING finger motif (7, 323). The TRAF domains
are responsible for binding upstream activators such as
TRADD, as well as some TRAF effectors. The function of
the Zn finger domains is unclear; however, the RING
fingers are important for the activation of downstream
effectors (7, 13, 96, 185, 182, 223, 253, 278, 299, 323, 359)
(Fig. 9).
Details of the structural features required for TRAF
binding to upstream proteins are emerging. TRAF-1 and -2
were purified and cloned based on their interactions with
the intracellular extension of TNFR2, with which both
proteins interact directly (7, 257, 323). Since then, the
repertoire of polypeptides that bind TRAFs has expanded
considerably, and a greater understanding has developed
of the structural basis of TRAF proteins binding to their
activators/effectors. Crystallographic studies have shown
that the TRAF domains of TRAF2 trimerize upon interaction with trimerized upstream activating receptors (204,
231). In these arrangements, TRAF domains bind to one of
two consensus sequences: Pro/Ser/Ala/Thr-X-Gln/Glu-Glu,
the predominant site, or a secondary sequence, Pro-XGln-X-X-Asp, where X is any amino acid. These sites are
present in TNFR family receptors and some TRAF2 binding proteins (356).
A notable exception to this rule is TRADD, which couples TNFR1 to TRAF2 (130, 129). TRADD contains neither
consensus TRAF2 TRAF domain binding site (130, 356).
Thus other TRAF domain binding motifs likely exist, and
these other sites may link TRADD to TRAF2. This leaves
open the possibility that, once TRAF2 is bound to TRADD,
the regions of the TRAF2 TRAF domains that interact with
the consensus TRAF2 binding motifs are free to interact
with other proteins. Conversely, TRAF2 associated with a
“consensus” TRAF2 binding site on one of its binding partners may have additional sites on its TRAF domains free to
associate with other proteins (Fig. 9B). Thus TRAF domains
could conceivably associate contemporaneously with upstream and downstream polypeptides each with distinct
TRAF domain binding motifs.
C) RECEPTOR INTERACTING PROTEIN. Receptor interacting
protein (RIP) was cloned as a polypeptide that could
interact in a two-hybrid screen with the death domaincontaining receptor Fas. Indeed, ectopic expression of
RIP results in rapid apoptosis and, in parallel, activation
of NF-␬B (287, 310). Subsequent studies using cell lines in
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Receptors of the TNFR family possess no intrinsic
enzymatic activity. Instead, upon binding ligand, these
receptors homotrimerize or heteroligomerize with receptor accessory proteins. Receptor oligomerization is
thought to trigger conformational changes that initiate
signal transduction. The protein recruitment hypothesis
poses that receptor oligomerization enables the binding
of signal transducing polypeptides that recruit downstream targets (322, 323).
A) DEATH DOMAINS AND SIGNALING. Several TNFR family
receptors (TNFR1, Fas, TRAILR and the DRs, in particular) contain an 82- to 102-amino acid extension, the death
domain (301–304). Death domains mediate homotypic
and heterotypic protein-protein interactions and are critical for nucleating receptor-effector complexes and implementing several signaling programs including apoptosis (322, 323). The type-1 TNFR (TNFR1) can recruit all of
the known signaling pathways activated by TNF. Signal
transduction by TNFR1 is now understood in considerable detail. Upon ligand-induced TNFR1 trimerization, the
TNFR1 death domain binds the death domain of the platform adapter protein TNFR-associated death domain protein (TRADD) (130, 322, 323). In contrast, TNFR2 does not
possess a death domain, and, upon trimerization, instead
binds directly to TNFR-associated factor (TRAF) proteins
through a TRAF domain binding site (below) (257).
TRADD consists of a COOH-terminal death domain
and an NH2-terminal domain, the TRAF interaction domain, that binds TRAF proteins (see below). TRADD is a
critical proximal component in the recruitment by TNFR1
of downstream targets. Consistent with this, overexpression of TRADD can activate many TNF signaling pathways including apoptosis and NF-␬B (130). In contrast,
although TRADD can recruit TRAF2 and receptor interacting protein (RIP) (129, 128), two key upstream activators of the SAPKs and p38s (185, 223, 253, 359) (see sect.
IIIE2), TRADD overexpression does not activate the
SAPKs (185, 223, 253). The reason for this is unknown.
The interaction between the TRADD death domain and
that of TNFR1 is thought to trigger the binding of additional death domain proteins to the TRADD death domain. An example is Fas-associated death domain protein
(FADD), an adapter protein that couples TNFR1 to the
apoptotic machinery. FADD associates with TRADD
through a homotypic interaction between the two proteins’ death domains (129). TRAF proteins are also recruited to the TRADD-TNFR1 complex through an interaction between the TRAF interaction domain of TRADD
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
841
which expression of the endogenous RIP gene is ablated
have shown that RIP is not involved in Fas proapoptotic
signaling but is instead critical TNF activation of NF-␬B, a
contention supported by subsequent gene disruption
studies (156, 310). RIP consists of a COOH-terminal death
domain, an NH2-terminal Ser/Thr kinase domain, and an
intermediate domain that likely forms a coiled-coil structure (287).
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FIG. 9. Coupling of tumor necrosis factor receptor
(TNFR)-associated factors (TRAFs) to upstream activators and the SAPKs and p38s. A: TRAF2 couples from
TNFR1, TNFR2, and the stress receptor Ire1. TRAF6 is
essential to interleukin-1 activation of the SAPKs. B: hypothetical scheme for TRAF coupling to effectors and
activators. On the left, TRAF2 associates with TRADD via
a TRAF interaction domain on TRADD that does not possess a conventional TRAF domain binding motif. Accordingly, effector proteins with conventional TRAF domain
binding motifs (cTBD in the figure) could associate and
undergo regulation mediated by the TRAF2 RING domain.
Alternatively, effectors could associate directly with the
TRAF2 RING domain. On the right, the cTBD of TNFR2
binds the TRAF2 TRAF domain, leaving free sites on the
TRAF domain that interact with novel TBDs.
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
RIP does not interact directly with TNFR1. Instead,
the RIP death domain binds to that of TRADD in a TNFdependent manner (128). The RIP kinase and intermediate domains can also bind to the TRAF domains of TRAF2
(299), and TNF treatment is thought to result in the formation of a TRADD-RIP-TRAF2 complex at the membrane (Fig. 9A).
2. TRAF-2, -5, -6, and RIP can activate the SAPKs
and p38s
independently of RIP (see sect. IV, B3, C, and D). A
similar situation may exist for p38.
3. TRAF2 also couples ER stresses to the SAPKs
A recent study has implicated TRAFs in the coupling
of endoplasmic stress (ER stress) signaling to the SAPKs
(321). Stress in the ER is induced by environmental perturbations such as oxidants, heat, ionizing radiation, or
chemical denaturants such as tunicamycin (152). These
stresses lead to the accumulation of misfolded proteins in
the ER lumen. Misfolded proteins are sensed by a novel
family of enzymes, the Ire1s, mammalian homologs of S.
cerevisiae IRE1, the product of the inositol auxotrophy
gene IRE1 (also called ERN1) (152, 215, 274, 311, 328).
Ire1p possesses both endoribonuclease and protein Ser/
Thr kinase activity, each of which is required for biological function (152, 311).
In mammalian cells, the ER stress response can be
triggered by tunicamycin (152), and tunicamycin leads to
the robust activation of the SAPKs (166). Tunicamycin
and other ER stresses lead to the oligomerization and
activation of mammalian Ire1p (311, 328), which in turn
activates SAPK. Moreover, overexpression of mammalian
Ire1␣ is sufficient to activate SAPK, and ER stress activation of SAPK is ablated in ire1␣-/- cells and is blocked by
kinase-dead Ire1p (321).
Urano et al. (321) performed a yeast two-hybrid
screen using the intracellular extension of Ire1p as bait
and, surprisingly, isolated TRAF2. Consistent with the
possibility that TRAF2 is a genuine effector for Ire1, ER
stress leads to the association of endogenous TRAF2 and
endogenous Ire1p, and dominant inhibitory TRAF2 can
block activation of SAPK by overexpressed Ire1p (321).
Inasmuch as Ire1p activation involves homoligomerization, it is likely that this triggers the binding and oligomerization-dependent activation of TRAF2 (152, 274, 321).
Thus TRAFs may serve in a broad range of signaling
pathways, transducing not only cytokine signals but ER
stress signals as well (Figs. 8 and 9A).
4. IL-1 and TLR signaling pathways also follow the
protein recruitment model and employ TRAF6
IL-1 likely requires TRAF6 to signal to the SAPKs, and
targeted disruption of traf6 severely blunts IL-1 activation
of the SAPKs and NF-␬B (186). IL-1 signaling, like that of
TNFR1, follows the protein recruitment paradigm (Fig.
9A). IL-1 binding to its receptor (IL-1R) triggers heterodimerization of the IL-1R with the IL-1R accessory
protein (IL-1RAcP), a transmembrane protein necessary
for IL-1 signal transduction. The IL-1R-IL-1RAcP heterodimer then recruits the death domain adapter protein
MyD88 (333).
In a manner similar to RIP recruitment by TRADD,
the death domain of receptor-bound MyD88 binds Ser/Thr
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Inasmuch as TRAFs and RIP appear to be constitutively active upon overexpression, nonspecific artifacts of
overexpression could lead to incorrect inferences concerning function. Still, in spite of this, overexpression
studies have provided valuable insight into the possible
mechanisms by which the TRAFs and RIP act.
Transient overexpression of TRAF2 results in potent SAPK and p38 activation (185, 223, 253, 359). The
RING domain appears necessary for relaying signals
from TRAFs to downstream effectors, and deletion of
the TRAF2 RING finger domain abrogates completely
the ability of TRAF2 to recruit the SAPKs and p38s.
Moreover, ⌬RING-TRAF2 can act as a dominant inhibitor of TNF activation of the SAPKs and p38s (185, 223,
253, 359). Targeted disruption of traf2, or transgenic
expression of ⌬RING-TRAF2 abolishes TNF activation
of SAPK; however, IL-1 activation of SAPK is unaffected (p38 was not tested) (176, 357). Thus TRAF2 is a
key rate-limiting signal transducer for coupling TNFRs
to the SAPKs and, possibly, the p38s. TRAF-5 and -6,
when expressed ectopically, can also strongly activate
the SAPKs; however, overexpression of TRAF-1, -3, and
-4 does not result in SAPK activation (286). Inasmuch
as TRAF2 appears to be the dominant TRAF coupling
the TNFRs to the SAPKs, activation of the SAPKs by
TRAF-5 and -6 may couple the SAPKs to other signaling
pathways.
RIP, like TRAF2, is, when overexpressed, a potent
activator of both SAPK and p38. The RIP intermediate
domain is both necessary and sufficient for this function (185, 359). Expression of a RIP construct wherein
the intermediate domain is deleted (RIP-⌬ID) effectively inhibits TNF activation of both SAPK and p38
(359). Moreover, RIP and TRAF2 can interact directly in
vivo (299), raising the possibility that TRAF2 and RIP
act collaboratively to recruit the SAPKs. However, gene
disruption studies indicate that deletion of rip does not
affect TNF activation of SAPK but, instead, prevents
activation of NF-␬B (156, 310), suggesting that RIP is
not rate limiting for SAPK activation by TNF, an idea
supported by the observation that several elements
upstream of the SAPKs, including GCK, GCKR, ASK1,
and MEKK1 also bind and are activated by TRAFs
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
5. Daxx may couple FasL to the SAPKs:
conflicting findings
Fas is a widely expressed cell death receptor that,
among other things, is crucial to immune cell regulation
where it governs in part the apoptotic elimination of
autoreactive T lymphocytes. Engagement of Fas by FasL
triggers apoptosis through a process that involves the
adapter protein Fas-associated death domain protein
(FADD). The death domain of FADD interacts with that of
activated Fas to signal apoptosis through activation of the
caspase family of proapoptotic cysteine proteases (284,
323, 358).
Daxx is a novel protein that was isolated in a twohybrid screen for Fas interactors. On the basis of this
interaction, it was proposed that Daxx served as an
adapter, coupling Fas to the SAPKs (352). Daxx overexpression can also trigger apoptosis, suggesting a FADDindependent apoptotic pathway emanating from Fas. The
SAPKs appeared to be required for Daxx-induced cell
death but only in a subset of the cell lines tested. Thus
Daxx-induced apoptosis was blocked by kinase-inactive
SEK1 in 293 and L929 cells, but not in HeLa cells (352).
Daxx does not possess a death domain. Instead, a COOHterminal death domain interaction motif (amino acids
625–739) of Daxx may bind directly to the Fas death (see
below) domain, but not to the death domain of FADD.
Expression of a construct consisting solely of the Fas
binding region of Daxx was shown to block both Fasinduced SAPK activation and apoptosis (352). As discussed in section II, Daxx may recruit the SAPKs by
directly binding and activating ASK1 (36).
Whereas these original studies implicated Daxx in
Fas-induced cell death and SAPK activation, more recent
studies call these findings into question. Thus Fas mutants
wherein the death domain fails to interact with FADD are
completely unable to trigger apoptosis in response to
FasL, despite the fact that Daxx binding and Fas-induced
SAPK activation by these mutant receptors are unimpaired (37). More importantly, targeted disruption of
daxx is embryonic lethal, with lethality occurring as a
result of massive cellular apoptosis (208). Taken together,
these results argue in favor of an antiapoptotic role for
Daxx, rather than a proapoptotic role. Finally, the exact
role of Daxx as an effector for Fas is also unclear. Several
laboratories have cloned Daxx independently of its association with Fas; and in some instances, Daxx has been
isolated in yeast two-hybrid screens as an interactor with
nuclear proteins such as DNA methyltransferase. Paradoxically, specific antisera were used in one study to
demonstrate a predominantly nuclear localization for
Daxx, and in another Fas-dependent association of endogenous Daxx with endogenous, cytosolically localized
ASK1 (37, 208).
Studies by Reed and co-workers (313) further sup-
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kinases of the IL-1R-associated kinase (IRAK) family.
IRAKs (IRAK-1 and -2) consist of a short NH2-terminal
extension required for binding to the MyD88 death domain, a central kinase domain, and a COOH-terminal extension necessary for recruiting downstream target proteins (29, 221).
As with TRAF6, ectopic expression of IRAKs activates NF-␬B and the SAPKs, and disruption of irak1
substantially diminishes IL-1-stimulated activation of
NF-␬B (13, 146, 307). Upon recruitment to the IL-1R complex, IRAK1 binds TRAF6 in a reaction involving the
TRAF6 TRAF domains and the COOH-terminal effector
region of IRAK1, forming a MyD88-IRAK1-TRAF6 complex analogous to the TNFR-associated complex of
TRADD, RIP, and TRAF2 (Fig. 9) (7, 333). The functional
interrelationship between TRAF6 and IRAK1 in SAPK and
NF-␬B activation is unclear.
Structurally, IRAKs are orthologs of Drosophila pelle,
which encodes a Ser/Thr kinase recruited by Toll, a Drosophila IL-1-like signaling pathway involved in innate immunity and in the establishment of dorsoventral polarity
(135). Toll itself shares significant structural similarity
within its intracellular extension with IL-1R, in spite of the
fact that the extracellular extensions of these receptors
are quite divergent (6, 135).
Mammalian toll-like receptors (TLRs) have been
isolated that share an even greater overall similarity
with toll (6). These receptors are central to the acute
phase response and to the pathogenesis of septic
shock, a major cause of mortality among hospitalized
patients. Both the TLRs and toll possess extracellular
leucine-rich repeats, as well as intracellular extensions
homologous to that of IL-1R (6). Consistent with this
structural similarity, TLRs and toll likely serve to recognize a subset of structurally conserved molecules
present on the surfaces of bacterial pathogens; binding
of these bacterial proteins initiates the acute phase
response (6, 17, 17). Thus genetic complementation
studies recently revealed that TLR4 is the main component of a receptor complex for the lipid A component of
endotoxin, a major causative agent for septic shock (17,
18, 177, 235). Initiation of TLR signaling requires the
formation of a multiprotein complex analogous to the
IL-1R-IL-1RAcP complex. Thus lipid A is thought to
interact with and trigger the formation of a functional
complex that consists of TLR4, the cell surface protein
CD14 and a third polypeptide, monocyte-derived-2
(MD-2) (17, 18, 177, 235, 280). Activated TLR receptor
complexes then recruit intracellular effector proteins.
Thus TLR2 is thought to signal through TRAF6 and
IRAK1, thereby recruiting NF-␬B and AP-1, and prompting the release of TNF and IL-1 to initiate the acute
phase response (350).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
IV. MOLECULAR MECHANISMS COUPLING
STRESS-ACTIVATED MAP3Ks TO
UPSTREAM SIGNALS
A. General Considerations
Section II outlined the basic elements of known mammalian stress-activated MAP3K 3 MEK 3 MAPK core
modules, while section III described upstream components thought to couple to these core pathways. Still,
despite considerable progress in the identification of
SAPK, and p38-activating MAP3Ks, and the identification
of proximal signaling components, such as Ras superfamily and trimeric GTPases, GCKs and TRAFs, that are
thought to couple to SAPK and p38 core pathways, the
molecular mechanisms by which stress-activated
MAP3Ks are regulated by events at the cell membrane
remain obscure. What little is known suggests that stressactivated MAP3Ks either 1) bind directly to upstream
activating regulatory proteins or 2) couple to other protein kinases that, in turn, bind upstream regulatory molecules, and 3) activation of these MAP3Ks by upstream
components involves in part regulated oligomerization.
B. MEKK1 May Lie Downstream of Ras and Rho
Family GTPases, GCK Family Kinases, and
TRAFs and May Be Targeted to an Apoptotic
Pathway Upon Caspase Cleavage
Three mechanisms for regulation of MEKK1 are suggested by existing data: 1) recruitment by Ras and Rho
family GTPases, 2) site-specific cleavage to generate an
active fragment that triggers apoptosis, and 3) binding to
TRAFs and GCK family kinases that, in turn, couple to
complexes associated with TNFR family receptors.
1. Ras superfamily GTPases and elements coupled to
receptor and nonreceptor Tyr kinases
MEKK1 can interact with Ras, Rac1, and Cdc42Hs in
a GTP-dependent manner (89, 260). Moreover, kinasedead forms of the MEKK1 catalytic domain (⌬1–1221/
K1253M) can block activation of SAPK by Rac1 and
Cdc42Hs (89). MEKK1 does not contain a consensus CRIB
motif, and deletion of the entire regulatory domain does
not affect Rac1 or Cdc42Hs binding; the interaction of
MEKK1 and Ras or Rho family GTPases likely requires the
MEKK1 kinase domain (89, 260).
In apparent contrast to the activation of Raf-1 upon
coexpression with active Ras, coexpression of Ras superfamily GTPases with MEKK1 has no detectable effect on
MEKK1 activity, largely due to MEKK1’s high degree of basal
activity, and the functional significance of the GTPaseMEKK1 interaction is unclear. Indeed, MEKK1 and SEK1
form a tight complex in vivo (342) (see sect. IIIC4), and,
accordingly, inhibition of G protein signaling by K1253M
MEKK1 could merely reflect sequestration of SEK1. It is
possible that one function of GTP-dependent Ras, Rac, or
Cdc42 binding to MEKK1 is to translocate MEKK1 to sites of
activation by other polypeptides or second messengers, or
to sites where substrates are located.
The GTP-dependent coupling of MEKK1 to Ras superfamily GTPases indicates that MEKK1 may be an effector for those agonists that recruit the SAPKs through
Ras, Rac1, or Cdc42Hs-dependent mechanisms. These include agonists coupled to Tyr kinases such as mitogens
and agents that signal through heterotrimeric G proteins
(195). In this regard, it is noteworthy that endogenous or
recombinant MEKK1 can interact with the SH2/SH3
adapter protein Grb2 (241). This interaction requires the
COOH-terminal SH3 domain of Grb2 and amino acids
233–531 of MEKK1 (amino acids 233–291) which includes
the COOH terminal of two SH3 binding sites (see sect.
IIG2) (241, 344). EGF recruits the Grb2-MEKK1 complex
to the Tyr-phosphorylated form of the SH2 adapter protein Shc, already bound to the Tyr phosphorylated EGFR
(241). EGF also recruits to the membrane complexes of
either Grb2 or Crk/CrkL and the GCK family kinase HPK1
(a putative regulator of MEKK1, see sect. IIID2 and Refs.
131, 159) as well as the Ras-activating Grb2-mSOS complex to the EGFR (5, 181, 195); and Eph Tyr kinases
recruit and activate NIK/Nck, another potential MEKK1
activator (see sect. IIID2) (14). On the basis of these
results, it is conceivable that activation of MEKK1 (and,
perhaps by extension other MAP3Ks) by Tyr kinases may
involve the coordinated recruitment of MEKK1 and its
activators to the membrane, followed by their binding to
MEKK1 and contribution to its activation (Fig. 8B).
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port the idea that Daxx is a nuclear protein. These studies
indicate that Daxx associates with PML oncogenic domains (PODs), regions of chromatin that bind PML proteins, a class of oncogenic transcriptional regulators.
Daxx association with PODs correlates with transcriptional repression and enhancement of Fas apoptosis that
is apparently completely independent of any regulation of
the SAPKs (313). In contrast to the original characterization of Daxx as a Fas binding SAPK activator, Reed and
co-workers (313) observed that Daxx could not associate
with Fas and did not, upon transient overexpression,
activate the SAPKs. The reasons for these discrepancies
are unclear and might merely reflect trivial issues such as
transfection levels, in situ masking of epitopes to which
anti Daxx antisera are directed, or the cell types in which
the experiments were performed. Clearly, however, more
work is needed before the role of Daxx in signaling to the
SAPKs is established.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
2. Caspases and regulation of MEKK1 during anoikis
more sensitive to proapoptotic environmental stresses
(360). Moreover, cardiomyocytes from embryoid bodies
derived from MEKK1 knockout ES cells are more prone to
undergo apoptosis in response to oxidant stress (209).
These findings suggest a protective rather than proapoptotic role for MEKK1.
3. Regulation of MEKK1 by GCKs
Several group I GCKs (GCK itself, HPK1 and NIK) can
interact with MEKK1 in vivo and in vitro, and there is evidence that MEKK1 is an effector for these GCKs (131, 294,
359). In all cases, the binding of MEKK1 requires the CTD of
the GCK family kinase polypeptide. Although the binding of
MEKK1 to the HPK1 CTD has not been mapped in detail, the
binding of MEKK1 to GCK and NIK has been well characterized.
NIK binds only full-length MEKK1. Deletion of the
first 719 amino acids of MEKK1 abolishes NIK binding,
and a truncation construct of MEKK1 consisting solely of
amino acids 1–719 will interact in vivo with amino acids
948 –1233 of MEKK1. MEKK1 binding has been mapped to
the COOH-terminal GCK homology domain (amino acids
948 –1233) of NIK, and accordingly, deletion of this domain prevents the binding of MEKK1 (294).
The GCK-MEKK1 interaction is somewhat more complex. GCK can interact in vivo and in vitro with both
endogenous or recombinant, full-length MEKK1. One
GCK binding site on MEKK1 has been mapped to residues
817–1221 of the MEKK1 polypeptide. Deletion of the kinase domain of GCK or the region of GCK that includes
the NH2-terminal PEST domain has no effect on the binding of a MEKK1 construct consisting of amino acids 817–
1493. However, deletion of the CT motif of GCK’s GCK
homology domain abolishes MEKK1[817–1221] binding
while subsequent deletion of the Leu-rich region of the
GCK homology domain restores binding. Deletion of the
COOH-terminal PEST motif of GCK (amino acids 404 –
447) again abolishes binding to MEKK1[817–1221] but has
no effect on the binding of GCK to full-length MEKK1 (D.
Chadee and J. M. Kyriakis, unpublished data). These results suggest that amino acids 1– 817 of MEKK1 can interact with GCK within a region that includes the middle
PEST domain (Fig. 8A) while amino acids 817–1221 require at least the domain spanning the COOH-terminal
PEST domain for binding (Fig. 8A). Both full-length and
truncated MEKK1 binding to GCK also requires the CT
extension of GCK’s GCK homology domain which, as
described below, may relieve an inhibition to MEKK1
binding conferred by the Leu-rich region (359).
The GCK homology domains of group I GCKs are
strikingly conserved. Why, then, does MEKK1 binding to
NIK and GCK appear to be so different? First, it is conceivable that the binding of MEKK1 to either GCK or NIK
involves multiple contact points both within and outside
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Although endogenous, full-length MEKK1 when assayed in vitro displays high basal activity; cleavage of the
NH2-terminal regulatory domain results in a modest but
significant increase in activity, suggesting that the MEKK1
NH2 terminus exerts an inhibitory effect on activity in the
resting state that is relieved by upstream stimuli (344).
Many cells undergo apoptosis upon dissociation from the
substratum, a process referred to as anoikis. The apoptosis characteristic of anoikis was recently shown to be
regulated in part by MEKK1. There is evidence that during
anoikis MEKK1 is regulated by controlled proteolysis of
the MEKK1 polypeptide at Asp-871 and Asp-874 (30). This
cleavage is catalyzed by caspases. Caspases are a family
of at least 11 related cysteine proteases that are vital to
the regulation of apoptosis and inflammation. Caspases
cleave at Asp residues within the consensus sequence
Asp-X-X-Asp (358). Caspase-3 and -7 are activated preferentially in response to apoptotic stimuli, rather than inflammatory episodes, and can be inhibited with the fluromethyl ketone-derivatized synthetic peptide DEVD-fmk.
Caspase-7 is also inhibited by the cowpox viral protein
CrmA (358). A role for caspase-7 and, possibly, caspase-3
in MEKK1 cleavage during anoikis was inferred from
studies showing inhibition of MEKK1 proapoptotic activity by DEVD and CrmA. Moreover, caspase-3 and -7 can
cleave MEKK1 at Asp-871/Asp-874 in vitro. Caspase cleavage irreversibly disinhibits MEKK1 and apparently unmasks a latent apoptogenic activity. Thus mutant MEKK1
polypeptides wherein the consensus caspase cleavage
sites were mutated (Asp-871/Asp-874 3 Glu-871/Glu-874)
were resistant to activation during anoikis and conferred
resistance to apoptosis (30).
Although caspase cleavage renders activation of
MEKK1 irreversible, actual activation of MEKK1 likely
does not require prior MEKK1 cleavage. Moreover,
MEKK1 induction of apoptosis is SAPK independent.
Thus expression of caspase-resistant MEKK1 still results
in robust SAPK activation. Moreover, activation of the
MAP3K activity of MEKK1 occurs before caspase cleavage (338). In addition, kinase-dead SEK1 does not reverse
apoptosis incurred upon expression of the free MEKK1
catalytic domain (142). Caspase cleavage of MEKK1 results in a MEKK1 fragment that is free to migrate within
the cell. Apparently, this diffusability, and not SAPK activation, is key to MEKK1’s proapoptotic activity (338).
The proapoptotic function of MEKK1 may be unique
to anoikis. Thus, although MEKK1 is cleaved by caspases
during anoikis, and when ectopically overexpressed, recombinant MEKK1 corresponding to those generated in
vivo as a consequence of caspase activity can induce
apoptosis, recent genetic studies suggest that the proapoptotic function of MEKK1 is not widespread. mekk1-/ES cells have been generated. Surprisingly, these cells are
845
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
4. Binding and activation of MEKK1 by TRAFs
MEKK1 can also interact in vivo with TRAF-2 and -6, in
the absence of coexpressed GCKs (although the possibility
that endogenous GCKs participate in stabilizing the MEKK1TRAF2 complex cannot be ruled out, see sect. IVC), and the
interaction with TRAF2 is stimulated by TNF and requires
the TRAF2 RING effector domain (see sect. IIIE1) (13).
Yeast two-hybrid screening and coimmunoprecipitation suggested initially that TRAF proteins could homoligomerize (299). Crystallographic studies indicate that the
TRAF domains of TRAF2 exist as a trimer when complexed with the trimerized intracellular TRAF binding
region of TNFR2 or CD40 (204, 231). This result suggests
that TRAFs oligomerize, when complexed with their up-
stream activators, and it has been suggested that TRAFs
are themselves activated to bind their effectors upon
oligomerization.
Oligomerization of TRAFs is sufficient to trigger
MEKK1 binding. FK506 is a macrolide immunosuppressant that binds to a receptor protein FK506 binding protein-12 (FKBP12). This complex, in turn, can bind to and
inhibit the calcium-sensitive phosphatase calcineurin. By
this process, activation of NF-ATs during T-cell activation
is inhibited, thus accounting for the immunosuppressive
activity of FK506 (270). Fusion proteins were prepared
consisting of the RING and Zn finger loops of TRAF-2 and
-6 linked to the FK506 binding domain of FKBP12. These
constructs, when expressed in transfected cells, were
forced to oligomerize upon addition of FK1012, a nonimmunosuppressive dimeric analog of FK506 (13). In a manner analogous to the activation of ERK by forced dimerization of FKBP12-Raf-1 (190), these FKBP-TRAF
constructs, when expressed in transfected cells, could
stimulate SAPK activity and AP-1-driven gene expression
upon incubation with FK1012 (13).
MEKK1 associates selectively with oligomerized
TRAF2, in vivo in a manner that requires the TRAF2 RING
domain and MEKK1 amino acids 1– 817. Thus addition of
FK1012 to cell expressing FKBP12-TRAF2 results in the
formation of insoluble FKBP-TRAF2 aggregates that can be
recovered by centrifugation. When MEKK1 is coexpressed
with FKBP12-TRAF2, the MEKK1 coprecipitates with the
FKBP-TRAF2 in a FK1012-dependent manner. Expression of
a truncated FKBP12-TRAF2 construct wherein the NH2-terminal RING domain is deleted abolishes this coprecipitation.
Transiently expressed full-length MEKK1 generally undergoes progressive proteolysis (see sect. IVB2), and it was
observed that only full-length MEKK1, and neither recombinant MEKK1[817–1493] nor MEKK1 fragments generated
spontaneously in vivo from expressed full-length MEKK1
coprecipitated with FKBP-TRAF2 in an FK1012-dependent
manner (13). Given the observation that TRAF2 oligomerize
upon interacting with their upstream activators, it is plausible to suggest that, in vivo ligand-driven TRAF oligomerization may prompt MEKK1 binding. The FK1012-dependent
FKBP12-TRAF2-MEKK1 interaction is consistent with this
idea.
Further support for the idea that MEKK1 is a TRAF2
effector comes from the observation that transiently expressed MEKK1 and TRAF2 can associate in vivo in a
TNF-dependent manner. Again, this association requires
the TRAF2 RING domain. Moreover, coexpression of
TRAF2 and MEKK1 also results in significant MEKK1
activation (13). These results imply a comparatively simple, linear mechanism whereby TRAF2, through its RING
effector domain, directly binds and activates MEKK1.
However, as is discussed in the next section, the situation
is likely to be more complex.
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the Leu-rich segments. Indeed, the complex binding of
full-length MEKK1 versus MEKK1[817–1221] supports this
idea. Second, NIK and the related TNIK are the most
divergent of the group I GCKs (163). The Leu-rich region
of the NIK and TNIK GCK homology domains are considerably shorter than those of GCK, GCKR, GLK, and HPK1
due to the presence of an additional, highly conserved
stretch of ⬃60 amino acids at the NH2-terminal end of the
GCK, GCKR, GLK, and HPK1 Leu-rich motifs that is absent in NIK and TNIK (73, 96, 131, 145, 151, 159, 163, 277,
294, 319, 354). This extension may perform an inhibitory
role that is relieved upon the binding of GCK upstream
activators to the CT region. Accordingly, deletion of the
CT region, by abolishing the possibility of disinhibition of
GCK, would prevent MEKK1 binding.
In this regard, it is noteworthy that full-length, wildtype GCK binds MEKK1 less stably than does the free
GCK CTD (359), and kinase-inactive GCK binds MEKK1
even more poorly than does wild-type GCK. Conversely,
wild-type GCK is the best in vivo activator of the SAPK
pathway (359). Thus activation of GCK could both enable
MEKK1 binding and prompt more efficient MEKK1 activation and turnover (163, 359).
It is possible that MEKK1 is an effector for GCK, NIK,
and HPK1. In support of this, dominant negative constructs of MEKK1 can block GCK, NIK, and HPK1 activation of SAPK; of note, the GCK-binding fragment of
MEKK1 (amino acids 817–1221) (359) but not the SEK1binding fragment of MEKK1 (amino acids 1221–1493)
(342) will inhibit GCK signaling without affecting the
kinase activity of GCK (359), suggesting that interfering
with the binding of endogenous MEKK1 to GCK inhibits
GCK signaling. Exactly how GCK family kinases might
activate MEKK1 is still unclear. GCK can phosphorylate
MEKK1 in vitro; moreover, the binding of MEKK1 to GCK
may allow for the regulated targeting of MEKK1 to site(s)
of activation. Alternatively, GCK’s interaction with
MEKK1 may foster activation of MEKK1 by an oligomerization-dependent mechanism (see sect. IVC).
Volume 81
April 2001
MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
C. GCKs May Collaborate With TRAFs to Activate
MAP3Ks Including MEKK1
signaling from TRAF2 to GCKs and MEKK1 and may
enable the indirect association of TRAF2 and GCK/GCKR.
TRAF-associated NF-␬B activator (TANK) was isolated in
a screen for TRAF3 interactors and was shown to interact
with all TRAF isoforms in vivo. A 21-amino acid fragment
in the middle of the TANK polypeptide (amino acids
169 –190) is required for TRAF binding. At low levels of
expression, recombinant TANK synergistically enhances
TRAF2 activation of NF-␬B, while at higher levels of
expression, TANK inhibits NF-␬B activation (43), a phenomenon suggestive of a prozone effect (see sect. IIIC1),
suggesting that TANK might perform a scaffold function.
TANK also synergistically increases TRAF2 activation of
SAPK, and expression of amino acids 190 – 413 of TANK,
which lie downstream of the TRAF binding domain, can
potently block both NF-␬B and SAPK activation incurred
by TRAF2 (45). Although TANK itself does not interact
with GCKR, the interaction between GCKR and TRAF2 is
strongly enhanced upon coexpression of TANK, and dominant inhibitory GCKR blocks SAPK activation by coexpressed TRAF2 or TRAF2 plus TANK, suggesting that
TANK’s stimulation of TRAF activation of SAPK involves
stabilization of the TRAF2-GCKR complex (45).
Although biochemical and genetic data (see sect. IVC1)
suggest that GCKs either relay signals from TRAFs to
MAP3Ks or collaborate with TRAFs to activate MAP3Ks
(183, 278, 359), biochemical evidence also indicates that
TRAF2 and MEKK1 interact apparently independently of
coexpressed GCKs (13). How might these conflicting results
be reconciled? It is possible, of course, that both mechanisms operate separately and in parallel. Alternatively, the
TRAF2-MEKK1 interaction may be comparatively weak and
might be stabilized or potentiated by GCKs once the GCKs
themselves are activated by TRAFs. Both the TRAF-MEKK
and GCK-MEKK binding results are consistent with the notion that a complex consisting of a group I GCK and MEKK1
could interact in such a way as to permit a regulatory
binding of MEKK1 to the TRAF2 RING domain. Another
possibility is that both TRAFs and GCKs might contribute
contemporaneous, yet independent inputs to MEKK1 activation, requiring the obligate formation of a TRAF-group I GCK
family-MEKK1 complex.
Just what constitutes group I GCK and MEKK1 activation is still nebulous. The results with FKBP-MEKK1 (13)
implicate oligomerization in MEKK1 activation, but this has
yet to be demonstrated. The fact that GCK’s kinase activity
enables maximal recruitment of the SAPKs (239, 359) suggests that phosphorylation may also contribute to MEKK1
activation; however, whether or not this might be MEKK1
activating autophosphorylation or phosphorylation catalyzed by GCKs (or, indeed if phosphorylation is at all important to MEKK1 regulation) is somewhat enigmatic. Support
for a role for autophosphorylation in the regulation of
MEKK1 comes from the finding that the activity of a truncated MEKK1 construct (either amino acids 817–1493 or
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The group I GCKs, GCK itself, GCKR, GLK and NIK
are activated by TNF in vivo (73, 239, 277, 354), and there
is evidence that GCK, GCKR and, possibly, NIK and TNIK
(see sect. IVC1) are TRAF effectors (96, 183, 278, 295, 359).
In particular, GCKR is activated by TNF and upon coexpression with TRAF2, binds TRAF2 in vivo (277, 278).
Furthermore, despite the observation that TRAF2 and
MEKK1 can associate apparently independently of coexpressed GCK family kinases (13), there is evidence that
GCKs, TRAFs, and, perhaps additional components TNFR
family complexes, collaboratively activate MAP3Ks upstream of the SAPKs and p38s. Thus 1) antisense GCKR
constructs will block TNF activation of SAPK in 293 cells,
and some GCKs associate in vivo with TRAFs (96, 183,
278, 359); 2) dominant inhibitory MEKK1 can block GCK,
NIK, HPK1, and GCKR activation of SAPK (131, 277, 295,
359); and 3) genetic studies of Drosophila implicate GCK
family kinases as TRAF effectors (see sect. IVC1) (183).
As with MEKK1, GCK, GCKR, and TNIK can bind
TRAF2 in vivo. The binding of GCK, GCKR, or TNIK to
TRAF2 requires the TRAF domains of TRAF2. The GCK
homology domain CT motifs of GCK and GCKR are necessary for TRAF binding (96, 163, 278, 359). In addition,
deletion of the NH2-terminal PEST motif of GCK (Fig. 8A)
hampers TRAF2 binding (359). Neither GCK nor GCKR
possesses a strong consensus TRAF2 TRAF domain binding motif (151, 277, 356) in the regions mapped as necessary for TRAF binding, suggesting that, either the interactions with TRAF2 are indirect or, as with TRADD (356),
are direct but involve other motifs.
Coexpresson with TRAF2 activates GCKR, and deletion of the TRAF2 RING domain cripples TRAF2’s ability
to activate coexpressed GCKR (277). From these results,
it is plausible to speculate that the TRAF2 TRAF domains
bind GCK and GCKR while the RING domains mediate
GCK/GCKR activation. Thus a stable interaction with the
TRAF2 RING domain is not an absolute requirement for
all TRAF2 effectors, although the RING domain is essential for regulation of TRAF effectors.
In contrast, the association of TNIK and TRAF2 requires an intermediate segment (amino acids 306 –1017)
that lies between the kinase domain and the GCK homology domain, while the GCK homology domain appears to
be dispensable (96). This result is similar to the binding of
the Drosophila group I GCK Misshapen (which is closer in
sequence to TNIK and NIK than are even the other group
I GCKs) to Drosophila TRAF in the dorsal closure SAPK
pathway (see sect. IVC1) (295). TNIK possesses four consensus TRAF2 TRAF domain binding sites (TNIK amino
acids 326 –329, 832– 835, 838 – 841, and 839 – 842), which
are conserved in NIK (96, 295, 356).
A fourth molecular component may also regulate
847
848
JOHN M. KYRIAKIS AND JOSEPH AVRUCH
1. Drosophila SAPK and the regulation of dorsal
closure: parallels with mammalian signaling
from TRAFs to GCKs
Analysis of the genetic basis of Drosophila morphogenesis has identified a signaling pathway, the dorsal
closure pathway, that includes components homologous
to the TRAFs and GCKs which recruit a SAPK ortholog
(135, 183, 295). These findings support the contention that
TRAFs recruit GCKs to signal to the SAPKs. During Drosophila embryogenesis, organogenesis and germ band retraction are followed by dorsal closure, the establishment
of a contiguous dorsal epidermis. The epidermal primordia of early Drosophila embryos is ventral, and the dorsal
surface is covered by a primitive tissue, the aminoserosa.
Dorsal closure involves a concerted ventrodorsal cell
sheet movement that positions and eventually fuses the
lateral epidermal primordia over the aminoserosa. This is
followed by degeneration of the aminoserosa. Several key
components of a Drosophila SAPK pathway are necessary for dorsal closure including the products of basket
(bsk, also called DJNK, a Drosophila SAPK), hemipterous
(hep, a Drosophila ortholog of MKK7), Djun (Drosophila
c-Jun), and Dfos (Drosophila c-Fos). Biochemical and
genetic evidence indicate that Hep, Bsk, and DJun are
directly linked in a signaling pathway. Additional components include DRac and DCdc42, which, as in mammals,
regulate cytoskeletal function and may in part regulate
Bsk. The ultimate consequence of DJun activation is expression of decapentaplegic (dpp), a member of the
TGF-␤ family that is similar to BMP4. dpp is expressed in
leading-edge epidermal cells during dorsal closure (135).
Misshapen (msn) is a Drosophila ortholog of NIK
and TNIK (96, 294, 295). Recent studies from Skolnik
and colleagues (295) implicate msn in dorsal closure
signaling and suggest that Msn acts in parallel with
dRac to recruit Bsk. Deletion or mutational inactivation
of msn results in dorsal closure defects that are strikingly similar to the bsk phenotype (135, 295), and a
significant percentage of either doubly heterozygous
msn ⫺/⫹, bskt ⫺/⫹ or msn ⫺/⫹, and hep ⫺/⫹ flies
exhibit a dorsal-open phenotype, with the severity of
the phenotype correlating well with the strength of the
bsk or hep allele, findings consistent with the idea that
Msn signals to Bsk,. Moreover, constitutively active
Djun rescues not only the bsk phenotype, but the msn
phenotype as well. Expression of msn in mammalian
cells activates coexpressed SAPK (295).
Of particular interest, a Drosophila TRAF (Dtraf)
was recently identified in a yeast two-hybrid screen for
Msn interactors. DTRAF also activates coexpressed SAPK
in mammalian cells, and kinase-dead Msn can block
DTRAF activation of SAPK in mammalian cells (183).
DTRAF binds to the Msn CTD at a region of the Msn
polypeptide (amino acids 293–587) that corresponds to
the region of TNIK that binds TRAF2, and to the domain
that includes the PEST1 sequence of GCK (amino acids
293–313), a region which, in addition to the GCK-CT extension, is required for TRAF2 binding (163, 183, 359) (see
sect. IVC). Although inactivating dtraf mutants exhibit a
dorsal-open phenotype, genetic evidence placing DTRAF
upstream of Msn is not yet available (183). However,
these results suggest that a signaling pathway strikingly
similar to the TRAF2 3 GCK/GCKR 3(MEKK1) 3 SAPK
pathway (see sects. IVB3 and IVC) exists in Drosophila to
regulate in part dorsal closure (96, 183, 277, 295, 359).
Moreover, given the close homology between NIK, TNIK,
and Msn, plus the observation that NIK is activated by
TNF (354), it is plausible to speculate that NIK and TNIK
are effectors for mammalian TRAFs.
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1221–1493) requires phosphorylation at Thr-1381 and Thr1393 for activity. As with many kinase regulatory phosphoacceptor sites, both of these residues lie within the activation loop just upstream of kinase subdomain VIII. ⌬1–
1221 MEKK1 autophosphorylates at Thr-1381, in vitro, and
mutagenesis of either Thr-1381 or Thr-1393 to Ala abolishes
activity (65, 282). Moreover, 14 –3-3 proteins interact with
the NH2-terminal noncatalytic region of MEKK1 (see sect.
IIG2). Although the significance of this interaction is unclear,
binding of 14 –3-3 proteins to their targets requires prior
phosphorylation of the target proteins, suggesting that extracatalytic phosphorylation sites on MEKK1 exist (90). What
is still unclear is the mechanism by which this MEKK1
phosphorylation is triggered, specifically, is the autophosphorylation cotranslational, with additional steps initiating
activation of the full-length protein, or is autophosphorylation triggered by aggregation alone, or are additional phosphorylations of the full-length kinase necessary to disinhibit
the autophosphorylation mechanism?
As for GCKs, all group I GCKs, as well as JIK contain
a conserved Thr residue (Thr-173 for GCK, Thr-178 for
GCKR, Thr-175 for HPK1, Thr-164 for GLK, Thr-190 for
NIK, Thr-191 for TNIK, and Thr-181 for JIK) which lies just
upstream of the Ala-Pro-Glu conserved residues in the
subdomain VIII activation loop (73, 96, 151, 159, 277, 294,
303, 319, 354). Mutagenesis of Thr-178 of GCKR to Ala
renders GCKR completely inactive, and this construct can
dominantly inhibit TNF- and TRAF2 activation of coexpressed SAPK (277). This finding suggests that Thr-178 of
GCKR (and, by extension, the analogous residues in the
remainder of the GCK family) might be sites that must be
phosphorylated for activation. Inasmuch as TRAFs are
oligomerized upon interaction with receptors (204, 231),
the clustering of GCKs at oligomerized TRAFs may elicit
GCK phosphorylation (autophosphorylation?) and activation, triggering MEKK1 binding, and oligomerization-dependent (13) activation (Fig. 10A).
Volume 81
April 2001
MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
849
D. ASK1 Is an Effector for TRAFs, Specifically,
a TNF Effector Recruited by TRAF2:
Role of Cellular Redox in ASK1 Regulation
The coupling of group I GCKs to MAP3Ks and TRAFs
provides an attractive model for the regulation of the
SAPKs by TNFR family receptors. However, the GCKs, as
well as MEKK1 and MLK3 (see sects. IIG and IIID), are
highly selective for the SAPKs, and, therefore, these models do not account for TNFR family regulation of the p38
pathway. Recruitment of ASK1 provides a mechanism for
TRAF recruitment of p38.
ASK1 is a MAP3K that can activate both the SAPKs
(through activation of SEK1) and the p38s (through MKK3
and MKK6). Moreover, the MAP3K activity of ASK1 itself
is activated by TNF (134) (see sect. IIG2). ASK1 is also
activated upon Fas engagement and may be an effector
for Daxx (36).
1. Recruitment of ASK1 by TRAF2 is redox dependent
and blocked by reduced thioredoxin
Expression of TRAF-2, -5, or -6 results in vigorous
SAPK and p38 activation, and TRAF2 is required for SAPK
activation by TNF, while TRAF6 is required for IL-1 activation of SAPK (7, 176, 185, 186, 223, 253, 286, 323, 357,
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FIG. 10. Models for cytokine regulation of MAP3Ks. A:
TNFR1 signaling to GCK and MEKK1 involves TRAF2
binding to GCK and MEKK1. MEKK1 may then be regulated by membrane recruitment, oligomerization, and
phosphorylation. B: TNFR1 regulation of ASK1 is redox
dependent. TRAF2 activation triggers a ROS pulse that
causes dissociation of Trx from ASK1. ASK1 then is activated by binding TRAF2, and consequent TRAF2-dependent oligomerization. C: TRAF6-dependent activation of
TAK1 by interleukin-1. Note that activation requires TAB1.
850
JOHN M. KYRIAKIS AND JOSEPH AVRUCH
mutant TRAF2 construct missing the RING effector domain, triggers the production of ROS (182). TNF fosters
the dissociation of ASK1 from Trx by a process that can
be blocked with free radical scavengers (263).
Recent results suggest that Trx sequesters ASK1 from
TRAF2 until TRAF2-dependent ROS production stimulates the dissociation of the ASK1-Trx complex (182) (Fig.
10B). Trx is abundant in cells and serves as a catalytic
antioxidant (125, 126). In contrast, TRAF2 and ASK1 are
low-abundance polypeptides (134, 257). Expression of
Trx at levels in excess of coexpressed TRAF2 and ASK1
completely reverses the ASK1-TRAF2 interaction. The interaction between ASK1 and TRAF6 is also reversed upon
coexpression of excess Trx. The inhibition of the ASK1TRAF2 interaction can be reversed upon the subsequent
administration of oxidant (H2O2) (182). Taken together,
these results suggest that TRAF2 engagement by the
TNFR complex triggers the production of ROS, which
oxidize Trx causing its dissociation from ASK1, thereby
enabling the binding of TRAF2 to the free ASK1 polypeptide. How is it possible, then, to observe an interaction
between overexpressed ASK1 and TRAF2, in the absence
of excess Trx, without addition of oxidant? It is likely that
overexpression of ASK1 and TRAF2 enables the ASK1TRAF2 interaction against a background of endogenous
Trx 1) because the overexpressed ASK1 titers out endogenous Trx, creating a pool of free ASK1 to which TRAF2
can bind, and/or 2) overexpression of TRAF2 generates a
ROS pulse in vivo resulting in oxidation of endogenous
Trx, thereby enabling the ASK1-TRAF2 interaction (182).
A likely consequence of Trx dissociation from ASK1 is
dimerization-dependent ASK1 activation. Upon overexpression, ASK1 spontaneously dimerizes in vivo, and TNF promotes the dimerization of endogenous ASK1 by a mechanism that requires ROS and can be reversed with free radical
scavengers (108). Moreover, expressed fusion proteins of
ASK1 and DNA gyrase can be forced to dimerize in vivo
upon administration of the binary DNA gyrase-binding drug
coumermycin. This coumermycin-induced dimerization results in substantial activation of coexpressed SAPK (108).
TRAF2 is known to homodimerize in vivo (204, 231, 299),
and coexpression of ASK1 and TRAF2 enhances the level of
recoverable ASK1 homomers in a reaction that requires the
TRAF2 RING domain (i.e., is not observed upon expression
of TRAF2⌬RING) and is blocked by free radical scavengers
(182) (Fig. 10B).
2. ASK1 recruitment by Daxx: conflicting results
As was noted in section IIIF5, Daxx is a novel adapter
protein that couples Fas to SAPK activation. In certain
cell types, Daxx-induced apoptosis requires activation of
the SAPKs (352). Chang et al. (36) observed that Fas
engagement could activate endogenous ASK1. These investigators also observed that coexpression of Daxx and
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359) (see sect. III, E1, E2, and E4). Recombinant ASK1 can
interact in vivo with recombinant TRAF-2, -5, and -6.
Moreover, endogenous TRAF2 and ASK1 interact in vivo
in a TNF-dependent manner (123, 182, 228). There is some
disagreement as to the domains of ASK1 and TRAF2
involved in the ASK1-TRAF2 interaction. Nishitoh et al.
(228), Hoeflich et al. (123), and Liu et al. (182) observed
that deletion of the TRAF2 TRAF domains (especially
TRAF-N) seriously compromises ASK1 binding. However,
whereas Nishitoh et al. (228) and Liu et al. (182) observed
that the free TRAF domains of TRAF2 can bind ASK1,
Hoeflich et al. (123) observed no interaction between
ASK1 and the free TRAF2 TRAF domains. Moreover, Hoeflich et al. (123) observed that the TRAF2 RING effector
domain may be important to ASK1 binding and found that
deletion of this domain also substantially reduces ASK1
binding, a phenomenon not observed by Nishitoh et al.
(228) or Liu et al. (182). The basis for these differences is
unclear and cannot be attributed to cell type, transfection
conditions, or choice of plasmid.
Both Nishitoh et al. (228) and Liu et al. (182) observed that the COOH-terminal noncatalytic domain of
ASK1 (amino acids 937–1375) interacts with TRAF2
(through the TRAF domains). However, Liu et al. (182)
also observed that the NH2-terminal 460 amino acids of
ASK1 can interact with the TRAF domains of TRAF2.
Moreover, coexpression of this fragment of ASK1 blocks
TRAF2 and TNF activation of SAPK, suggesting that this
domain is relevant to ASK1 activation by TRAF2.
Coexpression of ASK1 and TRAF2 also results in
activation of ASK1 (182, 228). Given that ASK1 is a potent
activator of p38 (134), the TRAF2-ASK1 interaction might
also mediate the activation of p38 by TNF.
The regulation of ASK1 by TRAF2 is redox dependent
(Fig. 10B). Thus addition of free radical scavengers prevents the TNF-dependent association of endogenous
ASK1 and TRAF2 (182). Yeast two-hybrid screening has
revealed that the redox sensing oxidoreductase thioredoxin (Trx) is an endogenous inhibitor of ASK1. This
inhibition requires that Trx be in a reduced state. Thus
treatment of cells with oxidant stresses (H2O2) triggers
the dissociation of Trx from ASK1 and the activation of
ASK1 in vivo (263). TNF can trigger the release of reactive
oxygen intermediates (ROIs); however, the biochemical
mechanism by which this process is regulated is still
nebulous, especially in nonphagocytic cells. TNF treatment is known to generate a mitochondrial pulse of reactive oxygen intermediates (ROIs) with kinetics (maximal
1 h) even slower than the comparatively slow activation
of ASK1 by TNF (detectable within 10 min and maximal at
20 min) (107, 108). An additional more rapid onset ROI
pulse of unknown origin may be generated upon recruitment of TRAF2 inasmuch as activation of the SAPKs by
coexpressed TRAF2 is partially reversed with free radical
scavengers (223), and expression of TRAF2, but not a
Volume 81
April 2001
MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
E. TAK1 Is a Target for TGF-␤ and IL-1 Through
Its Association With TAB1
TAK1 was cloned as part of a screen to isolate novel
mammalian MAP3Ks that could rescue S. cerevisiae deficient in STE11, a MAP3K of the mating factor pathway
(346) (see sect. IIG2). TAK1 can recruit both the SAPKs
(via activation of SEK1) and the p38s (via activation of
MKK3 and MKK6). Substantial evidence suggests that
TAK1 couples the SAPKs to the TGF-␤ superfamily receptors (217, 346). The TGF-␤ superfamily [TGF-␤, the activins, bone morphogenic proteins (BMPs) and others]
regulates numerous morphogenic and developmental processes. Signaling by TGF-␤ superfamily receptors has
been reviewed elsewhere (9, 199).
TGF-␤ and BMP-2 and -4 can activate the SAPKs in
some cell lines; the MAP3K activity of TAK1 itself is
activated by TGF-␤ and BMP-4 in murine osteoblastic
cells (MC3T3-E1). Moreover, transient ectopic expression
of TAK1 can stimulate trans-activation of reporter genes
driven by the TGF-␤-sensitive plasminogen activator inhibitor-1 (PAI1) promoter (217, 346, 345). The small NH2terminal extension of TAK1 plays an inhibitory role. Thus,
in resting cells, recombinant, ectopically expressed TAK1
is inactive; however, deletion of the NH2-terminal 22
amino acids from the TAK1 polypeptide results in constitutive activation (346).
TAK1 binding protein-1 (TAB1) is an essential cofactor
required for activation of TAK1 by upstream stimuli. A 55kDa polypeptide with no obvious structural features indicative of biochemical properties, TAB1 was cloned in a twohybrid screen for interactors with the NH2-terminal 22amino acid regulatory domain of TAK1 (279). Coexpression
of full-length TAB1 with TAK1 results in TAK1 activation,
assayed using the S. cerevisiae STE11 rescue assay (see
sect. IIG2), or in transfected cells using either the PAI1
reporter system or p38 activation as a readout (279).
Recently, a yeast two-hybrid screen to identify novel
TAB1 interactors identified the human X chromosomelinked inhibitor of apoptosis (XIAP) as a TAB1 binding
protein (345). XIAP is a member of an emerging family of
polypeptides, the inhibitors of apoptosis (IAPs). IAPs
were originally identified as baculovirally encoded proteins and were subsequently shown to exist in mammalian
cells where they function as inhibitors of proapoptotic
signaling from TNFR family receptors. All IAPs contain
three baculoviral IAP repeat (BIR) motifs followed by a
COOH-terminal RING domain (71). The NH2-terminalmost of the three XIAP BIR motifs is required for binding
TAB1, while the RING domain is required for interacting
with type I BMP receptors (345). Given that different
domains of XIAP interact with TAB1 and BMPR type I, it
is not surprising that trimeric complexes of BMPR-I,
XIAP, and TAB1 can be isolated (345). XIAP also interacts
directly with the type I receptors for BMP-2 and -4. The
physiological significance of the XIAP-TAB1-TAK1 complex was demonstrated when it was observed that XIAP
expression in Xenopus embryos could mimic the ventralizing effects of BMP, while expression of the TAB1 binding region of XIAP (see below) could block the expression
of ventral mesoderm markers elicited by a constitutively
active mutant type I BMP receptor (345).
Other members of the IAP family can be recruited to
TNFR complexes. Thus cIAP1 and cIAP2 interact with
TRADD and TNFR1 (71). The interaction between XIAP and
TAB1 raises the possibility that IAPs may serve as adapters
that recruit MAP3Ks to receptors of the TNFR family. In this
regard, a recent study has also implicated TAK1 in IL-1
signaling to the SAPKs. Thus both IL-1 and coexpressed
TRAF6 can activate TAK1. In the latter instance, TAB1 is
required for activation (Fig. 10C). Moreover, coexpressed
TRAF6 and TAK1 can coimmunoprecipitate in a reaction
that requires TAB1 (225). The mechanism by which TRAF6
activates TAK1/TAB1 is unknown.
F. MEKK4 Is a Putative Effector for Rho Family
GTPases and May Be Activated by DNA
Damage Through a Direct Interaction
With GADD45 Homologs
1. MEKK4 is a possible effector for Rho GTPases
MEKK4, a MAP3K that can activate both the SAPK
pathway (through activation of SEK1) and the p38 pathway
(through MKK3 and MKK6) (103, 297) (see sect. IIG2), possesses a putative CRIB motif in its NH2-terminal regulatory
domain (amino acids 1311–1324). Consistent with this, there
is some evidence that MEKK4 may be a target for Rho family
GTPases (89, 103). MEKK4 can bind Rac1 and Cdc42Hs in
vitro and in vivo; however, these interactions appear to be
GTP independent (89, 103). Moreover, while kinase-inactive
MEKK4 can inhibit activation of SAPK by constitutively
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ASK1 results in substantial activation of ASK1. Moreover,
both endogenous and recombinant Daxx can associate
directly in vivo and coimmunoprecipitate with ASK1 in a
reaction that is Fas dependent and requires the NH2terminal 648 amino acids of ASK1. Kinase-inactive ASK1
effectively blocks both Daxx-induced apoptosis and
SAPK activation (36). Thus ASK1 is a putative downstream target of Daxx that couples Fas to the SAPKs.
A critical caveat of these findings comes from recent
examinations of the subcellular localization of Daxx. As
was mentioned in section IIIF5, Daxx has been isolated in
yeast two-hybrid screens as an interactor with nuclear
proteins such as DNA methyltransferase (208). Moreover,
in apparent contrast to the ASK1 coimmunoprecipitation
results described above (36), anti-Daxx antisera were
used in cell fractionation studies to demonstrate a predominantly nuclear localization for Daxx (208, 313).
851
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
active Rac1 and Cdc42Hs, as with MEKK1 (89), there are no
data indicating whether or not the activity of MEKK4 is
actually modified as a consequence of binding Rac1 or
Cdc42Hs. In the absence of these data, results using kinaseinactive MEKK4 need to be interpreted cautiously as these
dominant inhibitors may merely sequester downstream effectors shared by MEKK4 and other MAP3Ks.
2. Recruitment of MEKK4 by ionizing radiation and
other genotoxic stresses
ins and the role played by the Abl Tyr kinase in genotoxin
signaling. Thus Kharbanda et al. see strong activation of
the SAPKs by ␥-radiation and chemical genotoxins
(CDDP, mitomycin-C) that apparently requires c-Abl and
does not occur in abl ⫺/⫺ mouse fibroblasts. These investigators were able to restore genotoxin activation of
the SAPKs into abl ⫺/⫺ cells upon reintroduction of c-Abl
cDNA (157, 230). In contrast, Liu et al. (184) observed at
best modest SAPK activation by ␥-radiation and CDDP
and substantial activation by mitomycin-C; however, activation of the SAPKs by these stimuli was unaffected in
fibroblasts wherein c-abl was disrupted (184). The reason
for this discrepancy is unclear, as is the relationship, if
any, between ATM and Abl in signaling from DNA damage
to the SAPKs. Moreover, these studies do not indicate
which MAP3Ks couple the SAPKs to genotoxin signals.
A series of recent studies provides some possible clues as
to the identity of the MAP3K 3MEK 3MAPK core pathways
recruited by genotoxins. As with the abl ⫺/⫺ studies, however,
there have been conflicting findings. Results from Saito and
co-workers (298) indicate that MEKK4 is recruited by genotoxins that transcriptionally induce proteins of the GADD45 family. GADD genes are a set of transcripts that are rapidly induced
by DNA damage and are thought to play a role in coordinating
the cellular response to genotoxins (93, 149). GADD153/CHOP,
described in section IIE2, is one example. Gadd45 was one of
the first GADD genes to be identified, although its function has
remained obscure. Expression of GADD45 is the culmination
of a signaling pathway that requires prior expression of the
tumor suppressor protein p53 which trans-activates the
gadd45 (gadd45␣) gene. p53 expression is, in turn, driven by
the activity of the ATM gene product (149). This is particularly
noteworthy given the observation, cited above, that cell lines
derived from AT patients with null or inactivating ATM mutations are resistant to SAPK activation by genotoxins but not by
TNF (273).
Yeast two-hybrid screening using MEKK4 as bait identified three gadd45 homologs (GADD45␣, which is the original GADD45, plus GADD45␤ and -␥) as direct MEKK4 interactors. These results were confirmed in experiments
wherein MEKK4 was overexpressed with recombinant
GADD45s, and GADD45␥ is the strongest MEKK4 interactor
of the three GADD45 isoforms. A broad segment in the
middle of the GADD45 polypeptides (amino acids 24 –147 of
GADD45␥) binds to a small domain (amino acids 147–250)
at the NH2 terminus of the MEKK4 polypeptide (298).
Evidence favoring a role for GADD45s in genotoxin
regulation of MEKK4 signaling to the SAPKs and p38s
comes from the observation that all three gadd45 genes
are transcribed in response to the same genotoxic stimuli
shown to upregulate gadd45␣ (93, 298). Moreover,
Takekawa and Saito (298) observed that ectopic overexpression of all three GADD45 polypeptides results in substantial activation of both the p38 and SAPK, a reaction
that can be reversed upon coexpression with kinase-dead
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Most chemotherapeutic and radiation treatment protocols employed to combat cancer kill cells by eliciting
irreversible DNA damage and consequent apoptosis of
tumor cells. An unwanted side effect of these treatments
is the collateral killing of normal cells. Accordingly, there
is a considerable need to understand how genotoxins
trigger cell death so as to enable the improvement of
conventional anticancer treatments.
In general, genotoxic stress triggers rapid cell cycle
arrest, and, to maintain genomic integrity, either the initiation of DNA and general cellular repair, or, if the damage is too great, apoptosis. Genotoxins such as UV and
␥-radiation, methylmethane sulfonate (MMS), cytosine arabinoside (AraC), N-acetoxy-2-acetylaminofluorene, cisplatinum (CDDP), and mitomycin-C have in some instances been shown to activate the SAPKs, p38s, and
other stress-activated signaling pathways (41, 70, 157, 184,
230, 273). However, attempts to identify the spectrum of
genotoxic stresses capable of activating the SAPKs, and
to identify the upstream components that couple these
stresses to the SAPKs, have yielded conflicting results.
Activation of the SAPKs by UV radiation (most studies have employed UV-C, a component of the UV spectrum that does not penetrate the Earth’s upper atmosphere) is rapid, and it is generally agreed that UV
signaling to protein kinase cascades does not require DNA
damage per se. Instead, UV triggers the clustering and
activation of cell surface mitogen and cytokine receptors,
a process likely driven by the formation of disulfide
bridges between adjacent receptor proteins (256).
In contrast, activation of the SAPKs by ␥-radiation
and most chemical genotoxins proceeds slowly, and most
investigators believe that recruitment of the SAPKs by
these stimuli requires DNA damage as a triggering event
(41, 157, 180, 230). Thus, for example, the ataxia telangiectasia mutated (ATM) gene product, a polypeptide
implicated in the cellular response to DNA damage, is
necessary for SAPK activation in response to a number of
radiant and chemical genotoxins (93, 149, 273).
In addition to ATM, the Tyr kinase protoncoprotein
c-Abl has been implicated in signaling from DNA damage
to the SAPKs and p38s. In this instance, however, there is
considerable disagreement among investigators as to the
degree of SAPK activation incurred by different genotox-
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
(p53 positive), treated with ionizing radiation sufficient to
induce GADD45 expression, there is little or no SAPK
activation (276). It must be borne in mind, however, that
while a role for GADD45s in the regulation by genotoxins
of the SAPKs (via MEKK4 or otherwise) is controversial,
there may be physiological circumstances, such as certain
developmental stages, during which GADD45s are expressed under conditions wherein they can recruit
MEKK4, and through MEKK4, the SAPKs, and p38s.
G. Regulation of MLK3 by Group I GCKs and
Cdc42Hs: Role of Dimerization Through
the Leu Zippers
1. Regulation of MLK3 by the group I GCK HPK1
There is evidence that the mixed lineage kinase
MLK3 (see sect. IIG3) is an effector for HPK1 (159) (see
sect. IIID2). Thus not only can HPK1 associate in vivo with
MEKK1, but MLK3 was identified in a yeast two-hybrid
screen for HPK1 interactors (159, 309). HPK1 binds MLK3
in an interaction that requires the NH2-terminal SH3 domain of MLK3 (amino acids 1–93) and the COOH-terminal
two of the four SH3 binding domains of HPK1 (amino
acids 433– 442 and 468 – 474) (159). Expression of kinasedead MLK3 abrogates activation of SAPK by coexpressed
HPK1, suggesting that MLK3 is an HPK1 effector (159). In
contrast, the NH2-terminal SH3 binding domains of HPK1
interact with Grb2 and Crk/CrkL. Crk/CrkL activates
HPK1 in vivo (181) (see sect. IIID2).
2. Regulation of MLK3 by Cdc42Hs
Although the role of Rho family GTPases in the regulation
of MEKK1 and MEKK4 is still nebulous (see sect. IV, B1 and
G1), MLK3 and MLK2, two mixed lineage kinase activators of
the SAPKs, contain CRIB motifs and can interact directly with
Cdc42Hs and Rac1 in a GTP-dependent manner.
Although MLK-2 and -3 may be Cdc42Hs effectors
(below), it is unlikely that they are Rac targets (222, 302).
Thus Phe37Ala-Rac1 is an effector loop mutant of Rac1
that retains its ability to recruit the SAPK pathway in vivo
(168). However, neither MLK3 nor MLK2 can interact with
Phe37Ala-Rac1 in vivo either in COS1 cells or in a yeast
two-hybrid assay (168, 302). As discussed in section IIIA2,
Phe37Ala-Rac1 can bind POSH, and POSH is a likely
candidate effector coupling Rac1 to the SAPKs (302).
In contrast, there is evidence that MLK3 is a Cdc42Hs
effector. As with all MLKs (see sect. IIG3), MLK3 possesses a leucine zipper (amino acids 400 – 487) (101).
Leucine zippers frequently mediate protein-protein binding; for example, the bZIP transcription factors in AP-1
dimerize via leucine zipper interactions. MLK3, when
overexpressed, will spontaneously dimerize in vivo. The
leucine zipper domain of MLK3 is required for this ho-
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MEKK4. Kinase-dead MEKK4 can also inhibit activation of
p38 by genotoxic stress that transcriptionally induce
gadd45 gene expression. In addition, expression of all
three GADD45 polypeptides activates MEKK4 in vivo
(298). Most importantly, however, addition of purified,
recombinant GADD45 proteins to immunoprecipitated
MEKK4 activates MEKK4 in vitro, suggesting that the
GADD45s are direct MEKK4 activators (298).
Ectopic overexpression of GADD45 proteins is apoptogenic in HeLa cells, a reaction that may require in part
MEKK4 insofar as either kinase-dead MEKK4 or the
GADD45 binding domain of MEKK4 (amino acids 147–
250) significantly blocks GADD45-induced apoptosis,
while an in-frame deletion of the GADD45 binding domain
of MEKK4 renders kinase-dead MEKK4 incapable of
blocking apoptosis (298).
Taken together, these results implicate GADD45s as
activators of the SAPKs, p38s, and apoptosis via an
MEKK4-dependent mechanism. The requirement for
GADD45 transcription also explains the slow kinetics of
SAPK and p38 activation by many genotoxins (41, 157,
184, 230). The fact that SAPK activation by genotoxins is
ablated in ATM null or mutant cells (273) can also be
explained by the observation that GADD45 expression
(and, hence, subsequent activation of the SAPKs) is also
blunted in these cells (149).
However, although expression of GADD45s is upregulated by stimuli that can activate the SAPKs, missing
from the studies implicating MEKK4 in coupling genotoxic stress to the SAPKs are experiments showing that
GADD45 proteins are present at times when MEKK4 is
activated, and contemporaneously with SAPK activation.
In this regard, recent findings apparently conflict with the
observation that DNA damage activates SAPK through
GADD45␣ and, perhaps, other GADD45 homologs. Thus
studies from Shaulian and Karin (276) as well as Holbrook
and co-workers (324) indicate that none of the endogenous GADD45 homologs is present at detectable levels
when SAPK is fully activated by DNA damaging chemicals
(MMS). Moreover, gadd45␣ knockout cells show adequate SAPK activation by MMS and other genotoxic
stresses, although this experiment does not rule out the
possibility that this SAPK activation is mediated by
GADD45␤ or -␥, both of which are also induced by MMS.
Still, incubation with cycloheximide (employed at concentrations too low to activate SAPK) to block induction
of all GADD45 isoforms also fails to prevent MMS activation of SAPK (276). In contrast to the results of Saito,
Holbrook and co-workers (324) do not observe activation
of SAPK by coexpressed GADD45␣, -␤, or -␥. The results
of Holbrook and colleagues may reflect the effects of
modest GADD45 expression, whereas greater levels of
GADD45 may promote SAPK activation (298). However, it
is unclear if, under physiological circumstances, such
high levels of GADD45 are attained. Even so, in fibroblasts
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
V. BIOLOGICAL FUNCTIONS OF THE STRESSACTIVATED PROTEIN KINASE AND P38
PATHWAYS IN THE STRESS RESPONSE,
CELL CYCLE CONTROL EMBRYOGENESIS,
AND IMMUNE SYSTEM MATURATION
A. The SAPKs Can Participate in Regulating
Apoptosis in Response to Environmental
Stress Through AP-1-Regulated
Transcriptional Induction of FasL
Withdrawal of NGF or nutrients from differentiated
PC-12 cells or primary cultures of neurons results in
apoptosis, and there is some evidence that this apoptotic
program requires the SAPKs and p38s (178a, 341). Thus
NGF withdrawal-induced apoptosis in PC-12 cells correlates with the activation of the SAPKs and p38s and a
decrease in ERK activity. Conversely, overexpression of
constitutively active MEKK1 in PC-12 cells both activates
the SAPKs and promotes apoptosis even in the presence
of NGF (341). Similarly, constitutively active forms of
MKK3, when overexpressed with p38, can promote apoptosis in the presence of NGF. Reciprocally, and in apparent contrast to fibroblasts where SEK1-K129R fails to
block MEKK1-induced apoptosis (142) (see sect. IVB2),
nonphosphorylatable, dominant inhibitory mutants of cJun can prevent MEKK1-induced PC-12 cell apoptosis,
and the expression of dominant interfering mutants of
MKK3 can prevent apoptosis incurred by NGF withdrawal
(178a, 341). Insofar as a constitutively active, oncogenic
form of MEK1 can prevent apoptosis induced by NGF
withdrawal, it has been suggested that the decision to
initiate apoptosis in PC-12 cells depends at least in part on
the balance of SAPK and p38 versus ERK activity (341).
Insight into the mechanism by which the SAPKs
might promote apoptosis in response to nutritional/trophic factor withdrawal or genotoxic stress came from the
results of Green and colleagues (148). Jurkat cells treated
chemotherapeutic genotoxins (etoposide, tenoposide) or
UV irradiation rapidly undergo apoptosis coincident with
the transcriptional induction and cell surface expression
of FasL (148). The binding of newly expressed FasL to
existing constitutively expressed Fas triggers an autocrine apoptotic program in response to these genotoxins.
The promoter for FasL contains AP-1 and NF-␬B cisacting elements, and inhibition of AP-1 activation (with
dominant inhibitory SAPK to block AP-1 activation or
with a dominant negative c-Jun construct, TAM67, in
which the trans-activating ␦-domain is deleted) prevents
FasL induction (148). Similarly, overexpression of a nondegradable I␬B mutant prevents FasL induction. Taken
together, these results indicate that genotoxic stresses
promote apoptosis by recruiting the SAPKs and NF-␬B
activation machinery which, in turn, activate AP-1 and
NF-␬B-mediated induction of FasL (148). Induction of
FasL is not restricted to genotoxic stresses. Nutrient withdrawal in cultured cerebellar granule neurons, or NGF
withdrawal PC-12 cells, also results in induction of FasL
by a SAPK-dependent mechanism (178a).
Studies of heat shock-dependent apoptosis have revealed both a role for the SAPKs in this process and a
novel mode of SAPK regulation. TR-4 cells are a thermotolerant subline of the murine fibroblast line RIF-1. Comparative studies of the apoptogenic effects of heat shock
and genotoxic (cisplatinum) stress on these cells has
implicated the SAPKs in the regulation of stress-induced
TR-4 or RIF cell death (362). Whereas TR-4 and RIF-1 cells
express identical amounts of SAPK polypeptide, TR-4 cell
SAPKs are not activated by heat shock, despite the ability
of UV radiation to activate of the SAPKs in both cell lines.
Furthermore, whereas heat shock and cisplatinum readily
kill RIF-1 cells, these treatments are not apoptogenic in
TR-4 cells; RIF-1 and TR-4 cells, however, are equally
sensitive to UV-C radiation (362). This finding is consistent with the observation that deletion of sek1 abolishes
heat shock activation of SAPK (226) (see sect. IIF1). Moreover, if the thermosensitive RIF-1 cells are stably transfected with a mutant of SEK1, wherein the phosphoacceptor sites are changes to nonphosphorylatable residues
(SEK-Ser257Ala/Thr261Leu, SEK-AL), the activation of
SAPK, but not p38, to all stimuli is inhibited. RIF-1 cells
expressing SEK-AL acquire resistance to the cytocidal
effects of UV-C as well as to heat shock and cisplatinum,
indicating that activation of the SAPK pathway is required
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modimerization, insofar as deletion of this domain abrogates in vivo MLK3 homodimerization (180).
The MLK3 leucine zipper is also critical for signal
transduction. Deletion of this domain, even in the presence of a fully competent kinase domain, completely
inhibits the ability of MLK3 to recruit the SAPK pathway
in vivo and converts the truncated construct into a dominant inhibitor of wild-type MLK3 signaling. Of particular
importance, coexpression of MLK3 with Val12-Cdc42Hs
substantially enhances the ability of MLK3 to homodimerize (180). From these results, it is plausible to speculate
that GTP-Cdc42Hs binds and promotes MLK3 (or MLK2)
homodimerization and activation.
Grb2, Crk, and CrkL can mediate the mitogen-stimulated recruitment of HPK1 to autophosphorylated Tyr kinase
receptors at the plasma membrane. Different regions of the
HPK1 polypeptide mediate the interactions with SH2/SH3
adapters and MLK3 (5, 181), and one function of the recruitment of HPK1 to the membrane might be to position associated MLK3 in the vicinity of GTP-Cdc42, fostering MLK3
dimerization and activation. Alternatively, HPK1 (and GCK)
may bind MLK3 and foster its dimerization-dependent activation independently of Cdc42Hs.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
B. The p38s Can Trigger Cell Cycle Arrest at G1/S
as Part of a Signaling Pathway in Late G1
Mediated by Cdc42Hs and/or MEKK3
The Rho family GTPases RhoA, Rac1, and Cdc42Hs
are required for the reentry of quiescent G0 cells into the
G1 stage of the cell cycle transition. In addition, Rho
family GTPases are important effectors required for the
oncogenicity of transforming alleles of ras. Thus microinjection, in G0, of constitutively active mutants of Rac1
or Cdc42Hs can prompt quiescent Swiss 3T3 fibroblasts to
transit from G0 into G1, and continuous, ectopic expression of Cdc42Hs can transform NIH3T3 and Rat-1 fibroblasts. Moreover, inhibition of Cdc42Hs or Rac1 significantly reduces Ras-mediated transformation of Rat-1
fibroblasts (229, 245, 246).
However, in cells already committed to the cell cycle,
Cdc42Hs, signaling through p38, in contrast to its role in
quiescent cells, may have a role in cell cycle inhibition
(213). Thus microinjection of p38␣, MKK3, or MKK6, but
not SAPK, into NIH3T3 cells synchronized in early G1
(with expression first detectable in mid G1) arrests cells at
the G1/S boundary. p38␣-induced cell cycle arrest is prevented by kinase-dead MKK3, implicating active p38 in
this process (213). Expression of Cdc42Hs, but not Rac1
or RhoA, in early G1 also potently arrests cells at G1/S in
several cell lines (NIH3T3 fibroblasts, Mv1-Lu mink lung
epithelial cells, and MG63 osteosarcoma cells). The
Cdc42Hs-induced growth arrest of G1-committed cells can
be effectively blocked with dominant inhibitory, kinasedead MKK3, again implicating the p38s in Cdc42Hs-mediated cell cycle arrest (213). Thus Cdc42Hs serves as a
transforming/growth-promoting protein in quiescent cells,
or as a growth suppressor in cells already committed to
the cell cycle. Inasmuch as p38 is a common target of
Rac1 and Cdc42Hs, different Cdc42Hs targets may collaborate with p38 in G1/S growth arrest versus growth promotion.
The mechanisms by which Cdc42Hs couples to p38 to
inhibit the cell cycle are unknown. A noteworthy candidate is MEKK3. Thus Siebenlist and colleagues (82) observed that expression of an estrogen-inducible estrogen
receptor MEKK3 fusion construct blocks cell proliferation incurred by activated Ras (82).
C. The SAPKs and Brain Development
Gene disruption studies have revealed specific roles
for the different SAPK isoforms in the brain. These studies reveal functions related to stress responses and brain
development.
Kainic acid is a glutamate receptor agonist that rapidly induces seizures similar to those of epilepsy. In addition, kainate also causes widespread hippocampal neuronal apoptosis similar to that which occurs in stroke.
Whereas SAPK␣/JNK2 and SAPK␥/JNK1 are ubiquitously
expressed, SAPK␤/JNK3 is selectively expressed in brain,
heart, and testis. Flavell and colleagues (348) disrupted
the gene for SAPK␤ in mice and observed that the knockout animals were markedly resistant to kainate seizures.
More importantly, however, deletion of sapk␤ protected
these animals from kainate-induced neuronal apoptosis
(348). Hippocampal cell death is a major clinical indicator
in stroke and Alzheimer’s disease. It will be important to
determine if ischemia or Alzheimer’s-induced cell death
correlates with SAPK␤ activation. It will also be important to determine if hippocampal cell death induced by
kainate involves upregulation of FasL (see sect. VA).
The establishment of a role for SAPK␤ in the brain’s
response to stress prompted the investigation of the role
of the SAPKs in normal brain development and function.
Kuan et al. (161) and Sabapathy et al. (262) generated
mice with sapk␥/sapk␤, sapk␣/sapk␤, and sapk␥/sapk␣
double knockouts. Studies of these mice revealed a pivotal role for the combined effects of SAPK␣ and -␥ in
regional-specific apoptosis during early brain development (161, 262). In contrast, inasmuch as the sapk␥/sapk␤
and sapk␣/sapk␤ animals were viable, deletion of sapk␣
or sapk␥ alone is insufficient to affect brain development,
and SAPK␤ likely is not a rate-limiting component in brain
development, but, as described above, may serve in the
response to stress (348).
The sapk␥/sapk␣ double knockout was embryonic
lethal and resulted in an abnormally prominent hindbrain
exencephaly. Normally, the closure of the hindbrain neural tube is an essential step in the process of cephalic
neurulation. Cell death is important to hindbrain neural
tube closure. However, compared with wild-type or single
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for efficient cell death induced by these stress agonists
(362).
Heat shock activation of the SAPKs appears to bypass the “traditional” three-tiered core module. Although
SEK1 is required for heat shock activation of SAPK (226)
(see sect. IIF1), SEK1 is not actually activated by heat
shock per se. Instead, heat shock suppresses a Tyr phosphatase that ordinarily inactivates SAPK, thereby allowing basal SEK1 Tyr kinase activity (SEK1 and SAPK activity is never entirely absent, even in the resting state) to
slowly activate SAPK (206). A consequence of heat stress
is the transcriptional induction of the chaperone protein
HSP70. HSP70 expression reactivates this SAPK-targeted
Tyr phosphatase resulting in SAPK inactivation (206). In
this regard, it is noteworthy that TR-4 cells constitutively
express high levels of HSP70, and HSP levels are not
transcriptionally regulated by heat stress, accounting perhaps for the inability of heat shock to activate SAPK in
these cells (362).
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
D. The SAPKs and p38s Are Required for the
Progression of Cardiomyocyte Hypertrophy
in Response to Pressure Overload
and Vasoactive Peptide
Pressure overload cardiac hypertrophy (a consequence of hypertension) and cardiomyocyte apoptosis
and necrosis (arising from ischemic injury) are the leading causes of cardiovascular morbidity and mortality in
the developed world. At the cellular level, hypertension
subjects cardiomyocytes to mechanical shear stress. In
addition, hypertension elicits the release of the vasoactive
peptides endothelin-1 and ANG II. Hypertensive mechanical strain, acting directly, and through vasoactive peptides, in turn, can elicit cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy is marked by cell enlargement,
increased protein synthesis (as a function of DNA synthesis), as well as the formation of rigid cytoskeletal actomyosin fibrillar networks and the expression of embryonic genes such as atrial natriuretic factor (ANF).
Cardiomyocyte contractility is compromised as a result of
these changes, leading ultimately to heart failure (92).
Recent results from Olson and co-workers (212) have
implicated the Ca2⫹/calmodulin-dependent phosphatase
calcineurin (CaN/PP2B), signaling through NF-AT3, in
cardiac hypertrophy. However, the situation is likely to be
much more complicated.
Chien and colleagues (329, 330) have observed that
markers of cardiomyocyte hypertrophy (cell enlargement,
actomyosin fibrils, and ANF expression) could be induced
in cultured neonatal rat cardiomyocytes upon ectopic
expression from recombinant adenoviruses of constitutively active forms of MKK6 and MKK7. Force and colleagues (46) extended these results to known physiological activators of cardiomyocyte hypertrophy. Thus
expression of kinase-dead Lys129Arg-SEK1 from recombinant adenoviruses can block completely cardiomyocyte
hypertrophy (cell enlargement, actomyosin fibrils, and
ANF expression) incurred in neonatal rat cardiomyocytes
by endothelin-1 (46).
E. The SAPKs, p38s, and
Ischemic/Ischemic-Reperfusion Injury
A potential role for the SAPKs and p38s in the pathogenesis of ischemic injury came with the initial observation that the SAPKs were activated selectively during the
reperfusion of ischemic kidney (237). Subsequently, it
was demonstrated that the same held true for reperfused
heart (22). In contrast, the p38s are activated during ischemic injury and remain activated during reperfusion (22).
In the heart, ischemic infarcts are marked by the
death (both apoptotic and necrotic) of cardiomyocytes, as
well as by fibrosis surrounding the site of vessel occlusion. In contrast, to expression of active MKK6 and MKK7,
which foster hypertrophy, expression of active MKK3 mutants causes cardiomyocyte apoptosis (329, 330) in a cultured cardiomyocyte cell model. MKK6 activates all forms
of p38, whereas MKK3 preferentially recruits p38␣ and
p38␤. These results suggest that the SAPKs, p38␥, and
p38␦ are critical for promoting cardiomyocyte hypertrophy, whereas p38␣ and p38␤ are more important for
eliciting cardiomyocyte apoptosis (330).
The mechanisms by which reperfusion, as occurs in
surgical bypass or balloon angioplasty, incurs cell injury
remain largely enigmatic. It is generally accepted that
reoxygenation of ischemic/atherosclerotic tissues results
in the production of ROS, triggering oxidant stress-induced upregulation of inflammatory cell adhesion molecules such as the selectins and vascular cell adhesion
molecule-1 (VCAM-1). The expression of adhesion molecules attracts neutrophils and macrophages, contributing
to a local pathogenic inflammatory response which includes the release of inflammatory cytokines of the TNF
family. Oxidant stress itself also upregulates TNF expression. Together, these effects are thought to contribute to
cardiomyocyte apoptosis as well as to vascular restenosis,
common unwanted side effects of bypass or angioplasty.
Several recent studies have shown that E-selectin and
VCAM-1 expression are AP-1 dependent (252), suggesting
that the SAPKs, which are activated during reperfusion episodes, may initiate reperfusion injury. Inasmuch as ASK1 is
activated both by oxidant stress, and by TNF, through an
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knockouts, sapk␥/sapk␣ double knockouts exhibited reduced pynknotic nuclei at the lateral edges of the converging hindbrain, an indication of reduced apoptotic cell
degeneration (161, 262). Thus SAPK␣ and -␥ collaborate
to foster appropriate developmental hindbrain apoptosis.
In contrast, the sapk␥/sapk␣ double knockout, while
decreasing hindbrain developmental apoptosis, increased
forebrain apoptosis [assayed as pynknotic nuclei or terminal deoxynucleotidyl transferase-mediated dUTP nick
end labeling (TUNEL) positive cells, a measure of apoptotic internucleosomal cleavage], suggesting that the SAPKs
suppress apoptosis in the forebrain (161, 262). This increased apoptosis was attributed to excess activation of
caspase-3, as determined by an observed increase in staining with the CM1 antibody which recognizes the cleaved,
17-kDa active form of caspase-3, but not its inactive 35kDa precursor (161, 262). Thus SAPK␣ and -␥ together
exert opposing effects with regard to fore- and hindbrain
developmental apoptosis. The basis for this difference is
nebulous. It is conceivable that the cell context, SAPK␣
and -␥ substrates present, or the spectrum of pathways
activated in conjunction with SAPK␣ and -␥ during development determines the ultimate effects of SAPK␣ and -␥
on apoptosis.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
F. MEKK3 and Cardiovascular Development
Recent genetic studies suggest that stress-activated
MAPK pathways are also critical not only to the response
of cardiovascular cells to extracellular stress but to developmental cues that regulate cardiovascular development. Thus mice in which mekk3 is disrupted die in utero
at embryonic day 11. In these animals there is a severe
disruption of blood vessel development and angiogenesis.
Interestingly, this occurs without a loss of expression of
vascular endothelial cell growth factor or of its receptor
(349). The development of embryonic but not maternal
blood vessels in the placenta is particularly impaired in
mekk3 ⫺/⫺ embryos, indicating an intrinsic defect in the
endothelial cells (349). Inasmuch as MEKK3 was shown
to activate MEF2C, a transcription factor critical to cardiovascular development, via the p38 pathway (349), it is
plausible to speculate that MEKK3 regulation of p38 is
especially important to embryonic angiogenesis.
G. The SAPKs Are Key Regulators of T-Cell
Maturation, Activation, and Protection
From FasL Apoptosis
1. T-cell receptor signaling and T-cell maturation
Three key gene disruption studies indicate a complex
role for the SAPKs in T-cell maturation and function.
Antigen presenting cells (APCs), such as macrophages
and dendritic cells, present antigen to naive T cells
through the T-cell receptor (TCR), the CD3 subunit of
which, in conjunction with CD28, recruits the TCR/CD28
costimulatory pathway. Activation of the costimulatory
pathway culminates in the clonal proliferation and maturation of CD4⫹/CD8⫹ double positive cells to CD4⫹ TH
cells and in the production of certain cytokines such as
IL-2. While stimulation of either the TCR (CD3) or CD28
alone is insufficient for SAPK activation, TCR/CD28 costimulation results in strong SAPK and, hence, AP-1 activation. AP-1, in turn, is necessary for T-cell maturation
and the development of acquired immunity (discussed
below). IL-2 transcription is robustly activated by TCR/
CD28 costimulation and is a particularly prominent target
for the SAPKs (147, 293) inasmuch as the IL-2 promoter
contains AP-1 and CRE elements (targets for the SAPKs
and p38s) along with NF-␬B and NF-AT sites.
Upon initial differentiation, CD4⫹ TH cells may then
become either TH1 or TH2 effector cells. TH1 cells produce
interferon-␥ and promote the Ig class switching of B cells
to those isotypes that engender phagocytosis by macrophages. Thus TH1 cells stimulate phagocytosis-dependent
host responses and are important for the elimination of
cells infected with viruses and other intracellular microbes (cell-mediated immunity). TH2 cells, on the other
hand, produce IL-4 and IL-5. IL-4 induces B-cell class
switching to IgE or, in humans, IgG4, neither of which can
activate phagocytosis. IL-5 instead activates eosinophils.
Thus TH2 cells contribute to phagocytosis-independent,
IgE/eosinophil-mediated defenses (humoral immunity),
such as those against helminth parasites.
Differentiation to TH1 or TH2 cells and, hence the
type of immune response, are governed in part by the
cytokine milieu to which the immature TH cells are exposed during antigen presentation. Thus, in addition to
presenting antigen, some APCs (macrophages and dendritic cells in particular) produce IL-12. IL-12 promotes
differentiation to TH1 cells (the type I cytokine immune
response), while mature T cells (TH2 cells in particular,
which can act as a second type of APC) produce IL-4,
leading to further differentiation of TH to TH2 cells.
SAPK␥ suppresses the differentiation of TH lymphocytes into TH2 cytokine-producing cells (76). Thus immature TH cells from mice in which sapk␥ was disrupted
were refractory to costimulatory SAPK activation, despite
the continued presence of SAPK␣. Thus in the TH stage,
before differentiation into TH1/TH2 cells, SAPK␥ is the
only Jun kinase recruited by costimulation. Costimulation
of the sapk␥ knockout cells elicited indistinguishable levels of IL-2 production, suggesting that SAPK is not a
rate-limiting pathway in TCR/CD28-stimulated IL-2 production (76). On the other hand, costimulation triggered
enhanced proliferation in the knockout cells compared
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oxidant stress-dependent mechanism, it will be important to
ascertain if ASK1 can elicit cardiovascular injury.
In contrast, a recent study has demonstrated that
MEKK1 suppresses oxidant stress-induced apoptosis in ES
cell-derived cardiomyocytes (209). Thus mekk1 ⫺/⫺ ES
cells were stimulated to differentiate into embryoid bodies
from which cardiomyocytes were isolated and cultured. Disruption of mekk1 rendered these cardiomyocytes considerably more sensitive to the cytocidal effects of oxidant stress
(H2O2), increasing oxidant stress-induced apoptosis. Coincident with this, oxidant stress- and hypoxia-induced activation of SAPK was abrogated in the mekk ⫺/⫺ cells. p38 and
ERK activity were unaffected (209). Thus MEKK1, through
SAPK, apparently acts to protect cardiomyocytes from oxidant stress-induced cytotoxicity. Oxidant stress causes cardiomyocytes to release TNF, triggering a TNF-induced
caspase apoptotic cascade. Oxidant stress-induced TNF release was blunted in mekk1 ⫺/⫺ cells, indicating that
MEKK1 negatively regulates the expression of TNF (209).
This result is in contrast to results indicating that SAPKs are
necessary for TNF release from macrophages stimulated
with lipopolysaccharide (acting presumably through TLR4,
see sect. VG). The basis for this difference is unclear, but, as
is discussed above for developmental apoptosis in the brain
(see sect. VC), this difference highlights the ability of the
SAPKs to regulate apparently opposing biological processes
depending on the cell type.
857
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JOHN M. KYRIAKIS AND JOSEPH AVRUCH
2. SEK1 and protection from FasL apoptosis
Early in T- and B-lymphocyte development, ablation
of autoreactive cells is mediated in part by FasL-stimulated apoptosis. Penninger and colleagues (226) disrupted
sek1 in mice and observed that this deletion abrogated
SAPK activation by anisomycin and heat shock in cultured ES cells (see sect. IIF1). However, deletion of sek1
was early embryonic lethal. The lethality incurred upon
deletion of sek1 is likely due to impaired hepatogenesis.
Thus sek1 ⫺/⫺ embryos die early in embryogenesis, subsequent to the establishment of the primitive vasculature,
coincident with early hepatogenesis. Lethality is accompanied by excessive apoptotic death in the liver, again
indicating an antiapoptotic role for SEK1 (102, 227). To
circumvent this problem, and examine the role of SEK1
(and, hence, the SAPKs) in immune cell function, rag2deficient blastocysts were microinjected with sek1 ⫺/⫺
ES cells to produce chimeric mice in which a SEK1deficient immune system developed against a wild-type
background. T lymphocytes isolated from the chimeras
were strikingly hypersensitive to FasL-induced apoptosis,
suggesting that, consistent with the impaired hepatogenesis in sek1 ⫺/⫺ mice, SEK1 and its substrates actually
protect T cells from FasL-induced apoptosis (102, 227).
H. The SAPKs and p38s Promote the Stabilization
and Enhanced Translation of mRNAs Encoding
Proinflammatory Proteins
Much of the preceding discussion of the mechanisms
of SAPK and p38 signaling has focused on the role of
these MAPK pathways in the phosphorylation of transcription factors and the regulation of gene transcription.
However, as mentioned in section IIE1, p38 can phosphorylate the MNKs, potentially activating their translational
enhancement function. Several recent studies have highlighted roles for the SAPKs and p38s in the posttranscriptional and translational control of gene expression.
The signal-induced stabilization of mRNAs encoding
proinflammatory proteins contributes to the robust and
efficient induction of genes in the inflammatory response.
Thus proinflammatory stimuli such as bacterial lipopolysaccharide, IL-1, and TNF can trigger the stabilization of
mRNAs encoding secondary cytokines (IL-6, IL-8) and
prostaglandin biosynthetic enzymes [cyclooxygenase-2
(COX2)], and the disappearance of these mRNAs in the
presence of the transcriptional inhibitor actinomycin D is
substantially reduced in the presence of inflammatory
cytokines or LPS.
COX2 is an inducible isoform of prostaglandin H
synthase and is the rate-limiting enzyme in extracellular
signal-regulated inflammatory prostaglandin biosynthesis.
Treatment of human monocytes with LPA, IL-1, or TNF
stabilizes the COX2 mRNA in a reaction that is substan-
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with controls. TCR engagement alone did stimulate IFN-␥
production, a hallmark of TH1 differentiation; however,
CD28/TCR costimulation, which recruits SAPK (293),
failed to enhance IFN-␥ production (76). In contrast, TCR
engagement alone stimulated the production of exaggerated levels of the TH2 markers IL-4, IL-5, and IL-10. Thus
deletion of sapk␥ renders TH cells hyperresponsive to
TCR engagement in the absence of costimulation and
favors production of TH2 marker cytokines (76). As noted
in section IIE2, SAPK␥ may suppress TH2 differentiation
by inhibiting the activation of NFATc1 (48, 77).
While SAPK␥ contributes to TH1 cell differentiation
by suppressing the differentiation of TH lymphocytes into
TH2 cytokine-producing cells (76), MKK3 and its p38 targets contribute to TH1 development by fostering production of IL-12 (187). Macrophages and dendritic cells produce IL-12 as part of the type I cytokine immune
response. As noted above, this response contributes to
the maturation and differentiation of naive T cells into
TH1 cells. Flavell and colleagues (187) deleted the mkk3
gene. These mice were viable and fertile but showed
severely impaired macrophage and dendritic cell IL-12
production. In addition, these mice were defective in
interferon-␥ production, and antigen-driven differentiation of naive T cells was significantly reduced. Accordingly, deletion of mkk3 cripples the type I cytokine immune response (187). In cultured macrophage cells,
pharmacological inhibition of the p38 pathway blocks
IL-12 gene transcription, indicating that a significant component of p38’s effect on IL-12 expression is transcriptional.
SAPK␣/JNK2 is apparently required for peripheral
T-cell activation. Mice were generated wherein sapk␣ was
deleted (261). The requirement for SAPK␣ differs from
that for SAPK␥. Whereas deletion of sapk␥/jnk1 enhanced
costimulatory proliferation and TCR-stimulated IL-4 production, while having no effect on TCR-stimulated IL-2
production, deletion of SAPK␣ reduced TCR-mediated
proliferation and production of IL-2, IL-4, and interferon-␥, markers of peripheral T-cell activation (76, 261).
B-cell activation in the sapk␣ knockout was unimpaired.
In addition, CD3-induced apoptosis of immature (CD4⫹/
CD8⫹) T cells was reduced in sapk␣ ⫺/⫺ animals. Thus in
apparent contrast to SAPK␥, which suppresses T-cell differentiation, SAPK␣ is required for efficient peripheral
T-cell activation (76, 261). MEKK2 has been implicated in
APC-directed TCR signaling to MAPKs (p38s, and ERKs,
but not SAPKs, see sect. IIG2). It is unclear if MEKK2 is
actually recruited by TCR alone or by costimulation (although the lack of SAPK activation would argue in favor
of the former) (268). It will be important to determine if
MEKK2 couples the TCR/CD28 costimulatory pathway to
SAPK␣.
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS
higher than those necessary to completely inactivate p38,
suggesting that the drug was affecting SAPK activity. In
contrast, there was a close correspondence between the
concentration of drug needed to reverse IL-2 message
stabilization and to prevent in vivo c-Jun phosphorylation
which is exclusively catalyzed by SAPKs (39). Consistent
with a role for the SAPKs in mRNA stabilization, expression of constitutively active MKK7 or MEKK1, but not
constitutively active MKK6 or Raf-1, triggered IL-2 message stabilization. Dominant inhibitory MKK7 blocked
MEKK1- or CD3/CD28-stimulated IL-2 message stabilization (39).
The ability of lipopolysaccharide to trigger the acute
phase response depends in large part on the transcriptional induction of TNF-␣ (signaled through the mammalian toll-like receptors, see sect. IIIE4), and enhanced
TNF-␣ mRNA translation. Swantek et al. (296) used a
TNF-␣ translational reporting system to demonstrate that
a dominant inhibitory SAPK-␤ construct could block lipopolysaccharide-enhanced TNF translation (296). Interestingly, dexamethasone, a potent anti-inflammatory synthetic corticosteroid which has long been known to
directly block AP-1 activity (147), could also strongly
inhibit lipopolysaccharide activation of SAPK, but not
activation of ERK or p38. Thus SAPKs are necessary not
only for enhancement of TNF-␣ gene expression, but for
increased translation of TNF-␣ (147).
VI. CONCLUDING REMARKS
During the past six years, our understanding of
stress-activated signaling pathways has expanded dramatically. It has become apparent in the intervening time that
these pathways can no longer be considered simple and
linear. Instead, these mechanisms form complex signaling
networks. At this time, the two most compelling issues in
the study of stress-activated MAPK pathways are MAP3K
3 MEK 3 MAPK core pathway regulation and biological
function.
A. Oligomerization, Adapter/G Protein Binding,
Membrane Translocation, and Phosphorylation
as Themes in MAP3K Regulation
Although a large number of potential MAP3K 3 MEK
3 MAPK core pathways, and their possible upstream activators and downstream targets have been identified,
there is still a troubling lack of clarity concerning MAP3K
regulation. In the case of stress-regulated MAP3Ks, this is
due in large part to the high basal activity of these enzymes, making analysis of their activation difficult. It is
becoming evident, however, that most MAP3Ks are regulated at least in part by four key processes: oligomerization, adapter protein binding, consequent membrane
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tially inhibited by SB203580. In these cells, at the doses of
SB203580 used, p38 (presumably the ␣- and ␤-isoforms)
and MAPKAP-K2 activity were significantly inhibited, and
SAPK activity was unaffected. Thus it can be concluded
that proinflammatory stimulation of COX2 mRNA stabilization is p38 dependent (67).
The mechanism by which p38s target mRNAs for
stabilization has only begun to be elucidated. Rapidly
turning over mRNAs such as those encoding COX2, interferon-␥, IL-1, IL-2, IL-3, IL-6, and IL-8 frequently contain
AU-rich elements (AREs) in the 3⬘-untranslated region
which are characterized by multiple copies of the sequence AUUUA. These elements, if transferred to stable
mRNAs, such as that for ␤-globin, confer destabilization
in resting cells and signal-induced stabilization. Winzen et
al. (339) demonstrated that transient overexpression of
active MEKK1 enhanced the stability of a chimeric mRNA
containing ␤-globin and a 161 nt ARE from the IL-8 message. This reaction was not blocked by dominant interfering constructs of the SAPK, ERK, or NF-␬B pathways. In
contrast, expression of MKK6 enhanced mRNA stability,
and MEKK1-induced stabilization could be reversed by a
kinase-inactive, dominant inhibitory mutant of p38␣
(339). It can be inferred from these findings that the
overexpressed MEKK1 was acting nonspecifically to recruit p38 which, in turn, triggered the stabilization of
AU-rich mRNAs. Winzen et al. (339) also observed that a
constitutively active form of MAPKAP-K2 could trigger
mRNA stabilization through AREs, suggesting that p38activated mRNA stabilization is mediated by MAPKAP-K2,
a p38 substrate (95, 259, 339). The mechanism by which
MAPKAP-K2 couples to ARE-mediated mRNA stabilization remains to be elucidated.
Despite the fact that AREs can regulate mRNA stability, this regulation is often differentially manifested.
Thus T-cell activation stabilizes several ARE-containing
cytokine mRNAs, including that for IL-2, while the mRNA
for c-fos, which also contains an ARE, remains labile. This
raises the possibility that additional elements can confer
signal-induced mRNA stabilization. As mentioned above,
induction of IL-2 expression is a hallmark of T-cell activation and requires costimulation mediated by the TCR
(CD3) and CD28 (293). A significant component of IL-2
induction is at the transcriptional level; however, the
signal-induced stabilization of IL-2 mRNA increases further the expression of IL-2 by activated T cells. The
SAPKs were recently shown by Chen et al. (39) to regulate
the stabilization of the IL-2 message through a cis-element
spanning the 5⬘-UTR and the beginning of the coding
region (39). Pretreatment with SB202190, a CSAID similar
to SB203580 that blocks p38 and, at high concentrations,
SAPK, or cyclosporin, which blocks CD3/CD28 activation
of SAPK (293) (and p38), prevented stabilization. However, the concentrations of SB202190 required to achieve
inhibition of IL-2 mRNA stabilization were considerably
859
860
JOHN M. KYRIAKIS AND JOSEPH AVRUCH
B. MAPK Pathway Biology
Genetic models were indispensable in the dissection
of the Ras-ERK pathway. Although until recently there
has been a comparative paucity of genetic models from
which to draw conclusions as to stress-activated MAPK
pathway regulation, new emerging genetic models such as
the dorsal closure pathway in Drosophila, coupled with
the completion of the C. elegans and other genome sequencing projects, should make it possible to understand
the epistatic relationships between MAP3Ks and their
upstream activators.
Placing these pathways in the context of human disease pathology will be more difficult. In this instance,
pharmacological inhibitors such as PD98059 or SB203580,
plus mouse knockout and transgenic approaches, will
enable a greater understanding of how mammalian stressactivated MAPK pathways relate to disease and will identify important new drug targets.
The original draft of this article was completed in March
2000.
Address for reprint requests and other correspondence:
J. M. Kyriakis, Diabetes Research Laboratory, Massachusetts
General Hospital East, 149 13th St., Charlestown, MA 02129
(E-mail: [email protected]).
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MAMMALIAN MAPK SIGNAL TRANSDUCTION PATHWAYS