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The MKK7 gene encodes a group of c-Jun
NH2-terminal kinase kinases
Cathy Tournier
University of Massachusetts Medical School
Alan J. Whitmarsh
Julie Cavanagh
University of Massachusetts Medical School
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Tournier, Cathy; Whitmarsh, Alan J.; Cavanagh, Julie; Barrett, Tamera; and Davis, Roger J., "The MKK7 gene encodes a group of c-Jun
NH2-terminal kinase kinases" (1999). Open Access Articles. 1445.
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The MKK7 gene encodes a group of c-Jun NH2-terminal kinase kinases
Authors
Cathy Tournier, Alan J. Whitmarsh, Julie Cavanagh, Tamera Barrett, and Roger J. Davis
Rights and Permissions
Citation: Mol Cell Biol. 1999 Feb;19(2):1569-81.
This article is available at eScholarship@UMMS: http://escholarship.umassmed.edu/oapubs/1445
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1999, p. 1569–1581
0270-7306/99/$04.0010
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 19, No. 2
The MKK7 Gene Encodes a Group of c-Jun NH2-Terminal
Kinase Kinases
CATHY TOURNIER, ALAN J. WHITMARSH, JULIE CAVANAGH, TAMERA BARRETT,
AND ROGER J. DAVIS*
Howard Hughes Medical Institute and Program in Molecular Medicine and Department of Biochemistry and
Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received 27 April 1998/Returned for modification 3 June 1998/Accepted 4 November 1998
(13). The MAPKKK and MAPKK that activate the ERK MAP
kinases include c-Raf-1 and MEK1, respectively (63). The cRaf-1 protein kinase activity is regulated by the small GTPase
Ras, which induces translocation of c-Raf-1 to the plasma
membrane, where it is thought to be activated (63). In contrast,
JNK and p38 MAPK appear to be activated by small GTPases
of the Rho family (3, 10, 49, 59, 91). The mechanism by which
Rho GTPases activate the JNK and p38 MAPK signaling pathways is unclear. Although Rho GTPases interact with the PAK
group of STE20-related protein kinases, it appears that JNK
and p38 MAP kinase activation may be mediated, in part, by
the mixed-lineage group of protein kinases (MLK) (62, 74) or
by the scaffold protein POSH (72). STE20-like protein kinases
represent possible targets for other upstream signals that lead
to JNK activation. Among the STE20-like protein kinases, the
hematopoietic progenitor kinase 1 (HPK1) (2, 37, 41, 79) and
the kinase homologous to STE20/SPS1 (KHS) (78) appear to
specifically activate JNK. There is evidence for significant complexity in the mechanism of initiation of the JNK and p38
MAPK signaling pathways because of the large number of
MAPKKK protein kinases that contribute to stress-activated
MAPK signaling (19, 38). Whether there is a general or a
specific role for Rho family GTPases in the activation of the
JNK and p38 MAP kinase signaling pathways has not been
established.
The protein kinases that have been reported to act as MAPKKKs for the JNK signaling pathway include the MEK/ERK
kinase (MEKK) group, the MLK group, TPL-2, ASK1, and
TAK1 (19, 38). The MEKK group of MAPKKKs includes
MEKK1 (43, 50, 86), MEKK2 (6, 11), MEKK3 (6, 11, 15),
MEKK4/MTK1 (25, 71), and MAPKKK5/MEKK5 (80). The
MLK group of MAPKKKs includes MLK1 (14), MLK2/MST
(14, 33), MLK3/SPRK (62, 74, 75), dual-leucine zipper-bearing
kinase (DLK)/MUK (18, 32), and leucine zipper-bearing ki-
Mitogen-activated protein kinases (MAPKs) are components of pathways that relay signals to particular cell compartments in response to a diverse array of extracellular stimuli (38,
42, 63, 83). Activated MAPK can translocate to the nucleus
and phosphorylate substrates, including transcription factors,
thereby eliciting a biological response. At least three groups of
MAPKs have been identified in mammals: ERK (extracellular
signal-regulated kinase), JNK (c-Jun N-terminal kinase; also
known as stress-activated protein kinase), and p38 MAPK
(also known as cytokine-suppressive anti-inflammatory drugbinding protein). ERK contributes to the response of cells to
signals initiated by many growth factors and hormones through
a Ras-dependent pathway (63). In contrast, JNK and p38
MAPK are activated by environmental stresses, such as UV
radiation, osmotic shock, heat shock, protein synthesis inhibitors, and lipopolysaccharide (38, 83). The JNK and p38 MAP
kinases are also activated by treatment of cells with proinflammatory cytokines, including interleukin-1 (IL-1) and tumor
necrosis factor alpha (TNF-a) (38, 83). MAPKs are involved in
the control of a wide spectrum of cellular processes including
growth, differentiation, survival, and death (38, 63).
MAPKs are activated by conserved protein kinase signaling
modules which include a MAPK kinase kinase (MAPKKK)
and a dual-specificity MAPK kinase (MAPKK). The MAPKKK
phosphorylates and activates the MAPKK which, in turn, activates the MAPK by dual phosphorylation on threonine and
tyrosine residues within a Thr-Xaa-Tyr motif located in protein
kinase subdomain VIII (38, 63). Separate protein kinase signaling modules are used to activate different groups of MAPKs
* Corresponding author. Mailing address: Howard Hughes Medical
Institute, Program in Molecular Medicine, University of Massachusetts
Medical School, 373 Plantation St., Worcester, MA 01605. Phone:
(508) 856-6054. Fax: (508) 856-3210. E-mail: [email protected].
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The c-Jun NH2-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK)
group and is an essential component of a signaling cascade that is activated by exposure of cells to environmental stress. JNK activation is regulated by phosphorylation on both Thr and Tyr residues by a dualspecificity MAPK kinase (MAPKK). Two MAPKKs, MKK4 and MKK7, have been identified as JNK activators. Genetic studies demonstrate that MKK4 and MKK7 serve nonredundant functions as activators of JNK
in vivo. We report here the molecular cloning of the gene that encodes MKK7 and demonstrate that six
isoforms are created by alternative splicing to generate a group of protein kinases with three different NH2
termini (a, b, and g isoforms) and two different COOH termini (1 and 2 isoforms). The MKK7a isoforms lack
an NH2-terminal extension that is present in the other MKK7 isoforms. This NH2-terminal extension binds
directly to the MKK7 substrate JNK. Comparison of the activities of the MKK7 isoforms demonstrates that
the MKK7a isoforms exhibit lower activity, but a higher level of inducible fold activation, than the corresponding MKK7b and MKK7g isoforms. Immunofluorescence analysis demonstrates that these MKK7 isoforms are detected in both cytoplasmic and nuclear compartments of cultured cells. The presence of MKK7 in
the nucleus was not, however, required for JNK activation in vivo. These data establish that the MKK4 and
MKK7 genes encode a group of protein kinases with different biochemical properties that mediate activation of
JNK in response to extracellular stimuli.
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TOURNIER ET AL.
MATERIALS AND METHODS
Materials. Human TNF-a and IL-1a were from Genzyme Corp. [g-32P]ATP
was obtained from Amersham Corp. Recombinant glutathione S-transferase
(GST)–c-Jun and GST-JNK1 were purified from bacteria as described elsewhere
(12). Mammalian expression vectors for MKK4 isoforms (13) and MKK7a1 (76)
have been described elsewhere. MKK4b includes an additional 34 amino acids
fused to the NH2 terminus of MKK4 (13). The expression vectors pCMV-HAMEK1 and pCMV-HA-DN3-MEK1-EE were provided by N. Ahn (47). The
expression vectors pEBG-KHS (78), pSRa-HPK1 (37), pMT2T-MEKK3 (15),
pCDNAI-MTK1/MEKK4 (71), pCDNA3-MLK3 (74), and pSRa-DLK (33) were
provided by J. Blenis, T. Tan, U. Siebenlist, H. Saito, S. Gutkind, and J. Avruch,
respectively.
Molecular cloning of MKK7 isoforms. Genomic DNA clones corresponding to
the MKK7 locus were isolated by screening a l FixII murine genomic library
(Stratagene, Inc.), using the MKK7a1 cDNA as a probe. The genomic sequence
of MKK7 was obtained with an Applied Biosystems model 373A machine. To
identify additional MKK7 isoforms, a mouse testis cDNA library cloned in the
phage lZAPII (Stratagene, Inc.) was screened by using the MKK7a1 cDNA as
a probe. Positive clones were plaque purified and sequenced. The MKK7 isoforms were subcloned into the mammalian expression vectors pCDNA3 (Invitrogen Inc.) and pEBG (86). The Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-AspLys; Immunex Corp.) was fused to the NH2 terminus of the MKK7 isoforms by
insertional overlapping PCR (35). A nuclear export signal (NES) sequence (23)
corresponding to residues 32 to 51 of MEK1 (Ala-Leu-Gln-Lys-Lys-Leu-GluGlu-Leu-Glu-Leu-Asp-Glu-Gln-Gln-Arg-Lys-Arg-Leu-Glu) was inserted following the Flag epitope, by using a PCR-based procedure to create NES-MKK7
isoforms. Bacterial expression of MKK7 was performed with the vector pGEX4T-1 (Pharmacia-LKB Biotechnology, Inc.).
FISH. Lymphocytes isolated from male mouse spleen were used for chromosome fluorescence in situ hybridization analysis (FISH) (30, 31). Metaphase
chromosomes spread on a glass slide were air dried, baked at 55°C (1 h), and
digested with RNase A. The DNA was denatured for 2 min at 70°C in 70%
formamide in 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and
dehydrated with ethanol. DNA prepared from a mouse MKK7 genomic clone
was biotinylated and used as the hybridization probe (30). The FISH and 49,6diamidino-2-phenylindole (DAPI) staining were recorded on film. The assignment of the FISH mapping data with chromosomal bands was achieved by
superimposing the FISH signals with images of the DAPI-banded chromosomes.
Tissue culture and transfection assays. COS-7 and 293 cells were cultured at
37°C in Dulbecco’s modified Eagle’s medium supplemented with 10% heatinactivated fetal bovine serum (Life Technologies, Inc.), 2 mM glutamine, 100 U
of penicillin per ml, and 100 U of streptomycin per ml in a humidified environment with 5% CO2. Transient transfections were performed with the LipofectAMINE reagent (Life Technologies) according to the manufacturer’s recommendations. After 36 h, the cells were serum starved for 1 h and treated with
UV-C (80 J/m2), anisomycin (5 mg/ml), TNF-a (20 ng/ml), or IL-1a (2 to 10
ng/ml) for 30 min prior to lysis. Cell extracts were prepared in lysis buffer (20 mM
Tris [pH 7.4], 10% glycerol, 1% Triton X-100, 0.137 M NaCl, 25 mM b-glycerophosphate, 2 mM EDTA, 1 mM sodium orthovanadate, 10 mg of leupeptin per
ml, 10 mg of aprotinin per ml, 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 15,000 3 g (15 min at 4°C). The concentration of total soluble protein
in the supernatant was quantitated by the Bradford method (Bio-Rad).
Binding assays. GST-tagged MKK7 proteins were isolated by incubation with
glutathione (GSH)-Sepharose (Pharmacia-LKB Biotechnology) in lysis buffer (4
h at 4°C). The beads were washed five times with lysis buffer, and the presence
of bound JNK was examined by immunoblot analysis.
Protein kinase assays. Epitope-tagged MAPKK was immunoprecipitated from
cell extracts by incubation for 3 h at 4°C with the Flag-specific monoclonal
antibody M2 (IBI-Kodak) bound to protein G-Sepharose beads (PharmaciaLKB Biotechnology). GST-tagged MAPKK was isolated by incubation for 3 h at
4°C with GSH-Sepharose beads (Pharmacia-LKB Biotechnology). The complexes were washed twice with lysis buffer and three times with kinase buffer (25
mM HEPES [pH 7.4], 25 mM b-glycerophosphate, 25 mM MgCl2, 0.5 mM
dithiothreitol, 0.1 mM sodium orthovanadate). MAPKK activity was measured at
30°C for 20 min in 30 ml of kinase buffer containing 0.5 mg of GST-JNK1, 2 mg
of GST–c-Jun, and 50 mM [g-32P]ATP (10 Ci/mmol; 1 Ci 5 37.6 GBq). The
reactions were terminated by addition of Laemmli sample buffer. Proteins were
resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE 10% polyacrylamide gel) and identified by autoradiography. The incorporation of [32P]phosphate into GST–c-Jun was quantitated by PhosphorImager
analysis.
JNK protein kinase assays were performed with extracts of cells transfected
with HA-JNK1. The JNK was immunoprecipitated with an antibody to the HA
epitope tag (12CA5; Boehringer Mannheim). Protein kinase activity was measured in the immune complex with c-Jun as the substrate (12).
Western blot analysis. Proteins were resolved by SDS-PAGE (10% gel), electrophoretically transferred to an Immobilon-P membrane (Millipore Inc.), and
probed with the Flag-specific monoclonal antibody M2 (1:4000; IBI-Kodak) or a
monoclonal antibody to MKK4 (1:1000; Pharmingen). Immune complexes were
detected by enhanced chemiluminescence (Lumiglo; Kirkegaard & Perry Laboratories).
Immunofluorescence microscopy. COS-7 cells were seeded onto glass coverslips coated with poly-L-lysine (Sigma Chemical Co.); 36 h after transfection, the
cells were fixed for 10 min with 4% paraformaldehyde in phosphate-buffered
saline (PBS) and then permeabilized for 5 min with 0.2% Triton X-100 in PBS.
After incubation for 15 min with 3% (wt/vol) bovine serum albumin (BSA) in
PBS, the coverslips were incubated for 1 h with primary antibodies in PBS with
3% BSA. The primary antibodies were rabbit anti-MKK4 (K-18; 1:100; Santa
Cruz Biotechnology), goat anti-MKK7 (C-18; 1:100; Santa Cruz), rabbit antiphosphorylated (on Thr-183 and Tyr-185) JNK (phospho-JNK) (9251; 1:100;
New England Biolabs Inc.), and monoclonal antibodies (1:500) to Flag (M2) and
HA (12CA5). Immune complexes were detected with Texas red-conjugated
anti-mouse immunoglobulin (Ig), Texas red-conjugated anti-goat Ig, rhodamineconjugated anti-rabbit Ig, and fluorescein-conjugated anti-rabbit Ig antibodies
(1:100; Jackson ImmunoResearch, Inc.) in 3% BSA in PBS. After nuclei were
stained for 1 min with DAPI (1:10,000; Molecular Probes, Inc.), the coverslips
were mounted in Vectashield (Vector Laboratories, Inc.). All procedures were
performed at room temperature. Fluorescence microscopy was performed with a
Zeiss Axioplan microscope.
Nucleotide sequence accession numbers. The sequences of the murine MKK7
cDNA clones have been deposited in GenBank with accession no. U93030,
AF060943, AF060944, AF060945, AF060946, and AF060947.
RESULTS
Molecular cloning of MKK7 isoforms. MKK7 has recently
been identified as a specific activator of the JNK group of
MAPKs. Northern blot analysis demonstrated the presence of
at least two MKK7 mRNAs (2.2 and 4.2 kb) in adult murine
tissues (76), an observation which suggested that MKK7 may
be expressed as a group of alternatively spliced isoforms. To
characterize members of the MKK7 group, we exhaustively
screened a mouse testis cDNA library by using our original
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nase (LZK) (64). Binding sites for the Rho family GTPases
Cdc42 and Rac1 have been described for MLK1, MLK2, and
MLK3 (but not DLK or LZK) (7). It has also been reported
that MEKK1 and MEKK4 (but not MEKK2 or MEKK3) bind
directly to the Rho family GTPases Cdc42 and Rac1 (20).
These interactions between Rho GTPases and MAPKKKs may
contribute to the effects of Rho GTPases on JNK activation.
MAPKKK protein kinases that do not interact with Rho GTPases may mediate the effects of Rho-independent signals that
lead to JNK activation.
Several MAPKKs have been identified. ERK is activated by
MEK1 and MEK2 (1); p38 MAPK is activated by MKK3 (13),
MKK4 (13, 45), and MKK6 (28, 53, 61, 68); JNK is activated by
MKK4 (13, 45, 65). The existence of JNK activators distinct
from MKK4 was suggested by chromatographic fractionation
of cell extracts (48, 52) and by the results of targeted disruption
of the MKK4 gene (58, 87). We (76) and others (21, 34, 36, 44,
46, 55, 77, 84, 89) have recently characterized the novel JNK
activator MKK7. In contrast to MKK4, which activates both
JNK and p38 MAPK, the MKK7 protein kinase selectively
activates only JNK. Interestingly, the two JNK activators DMKK4 (Drosophila MKK4) (29) and Hep/D-MKK7 (26, 67)
are conserved in Drosophila. Genetic analysis demonstrates
that D-MKK4 and Hep/D-MKK7 serve nonredundant functions in the fly (38). Similarly, gene disruption experiments
demonstrate that MKK4 is an essential gene in the mouse,
indicating that MKK7 is unable to fully substitute for the function of MKK4 (24, 57, 58, 70, 87). These data demonstrate that
MKK4 and MKK7 serve nonredundant functions as activators
of the JNK MAP kinases in vivo.
We report here the molecular cloning of the MKK7 gene and
the characterization of MKK7 isoforms that are created by
alternative splicing. The MKK7 isoforms are differentially activated by upstream signals, and their regulation differs from
that of the MKK4 isoforms. These data establish that JNK
activation is mediated by a family of protein kinases that are
formed by the alternative splicing of two genes.
MOL. CELL. BIOL.
VOL. 19, 1999
MKK7 ENCODES A GROUP OF JNK KINASES
1571
MKK7 clone (MKK7a1) as a probe. This analysis led to the
identification of additional isoforms: MKK7a2, MKK7b1,
MKK7b2, MKK7g1, and MKK7g2 (Fig. 1). The predicted
amino acid sequences are identical except at the NH2 terminus
(a, b, and g isoforms) and at the COOH terminus (1 and 2
isoforms).
We isolated a murine genomic clone that encodes the
MKK7 protein kinase isoforms. FISH analysis led to the mapping of the gene to mouse chromosome 11 region A2 (Fig. 2).
Sequence analysis of the gene demonstrated the presence of 14
exons (Fig. 3). Sequence comparison of the gene with cDNA
encoding the MKK7 isoforms demonstrated that these isoforms are created by the usage of alternative exons. This analysis indicated that MKK7 is a complex gene that expresses a
group of MKK7 protein kinases.
Initiation codons in frame with upstream termination
codons are located in separate exons in the 59 region of the
MKK7 gene. The a isoforms are created by using an initiation
codon located in exon 4 in frame with an upstream termination
codon located in exon 3 (Fig. 3). In contrast, the initiation
codon (together with an upstream in-frame termination
codon) used to create the b and g isoforms is located in exon
1. Alternative splicing which includes or excludes exon 2 distinguishes the b and g isoforms (Fig. 3).
Alternative splicing in the 39 region of the gene changes the
usage of termination codons to create a truncated COOH
terminus (1 isoforms) and an extended COOH terminus that
includes 33 additional amino acids (2 isoforms). The truncated
COOH terminus results from the presence of an in-frame
termination codon and a polyadenylation signal in exon 13
(Fig. 3). Alternative splicing within exon 13 immediately prior
to this termination codon fuses the open reading frame to
sequences located in exon 14. This fusion creates the longer
COOH terminus that is characteristic of the 2 isoforms. A
termination codon and a polyadenylation signal are located in
exon 14.
Comparison of protein kinase activities of MKK7 isoforms.
We used a coupled protein kinase assay in vitro to examine the
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FIG. 1. Primary structures of MKK7 protein kinase isoforms. The primary sequence of the mouse MKK7 protein kinase isoforms deduced from the sequences of
cDNA clones are presented in single-letter code. Residues that are identical to those in MKK7g2 (.), deletions (2), and termination codons (#) are indicated.
1572
TOURNIER ET AL.
protein kinase activities of MKK7 isoforms that were isolated
from transfected mammalian cells. This assay employs bacterially expressed JNK1 as a substrate for the MKK7 protein
kinases. The activation of JNK1 by MKK7 was examined by
using bacterially expressed c-Jun as a substrate for JNK. Control experiments indicated that MKK7 does not phosphorylate
c-Jun (data not shown). These assays demonstrated that the
activities of the MKK7b and MKK7g isoforms were similar
and that the alternative splicing at the COOH terminus of
MKK7 (1 and 2 isoforms) did not markedly alter protein kinase activity (Fig. 4). In contrast, activities of the MKK7a
isoforms were lower than those of the MKK7b and MKK7g
isoforms. Quantitation of the MKK7 activity by PhosphorImager analysis indicated that MKK7b1 was 37-fold more active
than MKK7a1.
The difference in protein kinase activity of the MKK7 isoforms was detected under basal conditions (Fig. 4). To determine whether similar differences could be detected following
activation, we examined the effect of a strong activator of the
JNK signaling pathway (MEKK1). We found that MEKK1
caused marked activation of all the MKK7 isoforms. The largest increase was observed for MKK7a1 and MKK7a2, which
were activated by 34- and 20-fold, respectively (Fig. 4). The
MKK7b and MKK7g protein kinases were activated more
modestly (three- fivefold). These data demonstrate that the
MKK7b and MKK7g isoforms are more active than MKK7a
isoforms (under basal conditions and following activation) but
that the MKK7a isoforms are more inducible following stimulation. The low basal activity of the MKK7a isoforms accounts, in part, for their greater activation.
Together, these data demonstrate that the activity of MKK7
isoforms is affected by alternative splicing of the NH2-terminal
region but not by alternative splicing of the COOH-terminal
region. However, the inclusion or exclusion of exon 2 within
the NH2-terminal region of MKK7 (b and g isoforms) did not
alter MKK7 protein kinase activity in these assays.
The NH2-terminal region of MKK7b interacts with JNK.
The observation that activities of the MKK7a isoforms were
lower than those of the MKK7b and MKK7g isoforms suggests
that these MKK7 isoforms may differentially interact with the
substrate JNK. To test this hypothesis, we examined the interaction of MKK7a and MKK7b isoforms with JNK1 following
FIG. 3. Schematic representation of the structure of the MKK7 gene. The MKK7 gene is formed by 14 exons. The alternative splicing that creates the a, b, g, 1,
and 2 isoforms of MKK7 is illustrated. The coding (black) and noncoding (grey) regions and excluded exons (striped) are shown. Initiation codons (ATG), termination
codons (p), and polyadenylation signals (●) are indicated.
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FIG. 2. The MKK7 gene is located on mouse chromosome 11. The MKK7
gene was identified by FISH analysis of murine metaphase chromosomes by
using an MKK7 genomic clone as a probe. The FISH signal is illustrated on the
left (arrow). The corresponding DAPI signal indicating chromosome 11 is shown
on the right. Detailed comparison of DAPI-banded chromosome 11 with the
FISH signal indicated that the MKK7 gene is located in region A2.
MOL. CELL. BIOL.
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MKK7 ENCODES A GROUP OF JNK KINASES
1573
FIG. 5. Interaction of JNK with MKK7 isoforms. (A) Selective binding of
MKK7b isoforms to JNK. Epitope-tagged JNK1 and either GST (Control) or
GST-tagged MKK7a1, MKK7a2, MKK7b1, or MKK7b2 were expressed in
COS-1 cells. Protein expression levels, monitored by immunoblot analysis of the
cell lysates, were similar in all transfections. The MKK7 protein kinases were
isolated from cell extracts by incubation with GSH-agarose. The binding of JNK1
was examined by immunoblot analysis with an antibody to the HA epitope tag.
(B) JNK binds to the NH2 terminus of MKK7b. Bacterially expressed GST
(Control) or a GST fusion protein (residues 1 to 73 of MKK7b) was immobilized
on GSH-agarose and incubated with extracts prepared from COS-7 cells expressing epitope-tagged JNK1. The agarose beads were washed, and the amount of
bound JNK1 was examined by immunoblot analysis.
expression in COS-7 cells. We precipitated the MKK7 protein
kinases from cell lysates, and examined the presence of JNK1
in the precipitates by immunoblot analysis (Fig. 5A). These
assays demonstrated that MKK7b isoforms, but not MKK7a
isoforms, coprecipitated with JNK. This binding interaction
may contribute to the higher activities of MKK7b isoforms
than of MKK7a isoforms (Fig. 4).
The MKK7b and MKK7a isoforms differ structurally by
virtue of an NH2-terminal extension that is present in MKK7b
but not MKK7a. This NH2-terminal region of MKK7b therefore accounts for the binding interaction observed between
JNK and MKK7b. The mechanism by which this NH2-terminal
region alters the binding to JNK is unclear. The NH2-terminal
region may act indirectly by altering the conformation of another region of the MKK7 protein kinase that binds JNK.
Alternatively, JNK may bind directly to the NH2 terminus of
MKK7b. To test the latter hypothesis, we expressed the NH2terminal region of MKK7b (residues 1 to 73) as a GST fusion
protein in bacteria. This recombinant protein was found to
bind JNK (Fig. 5B). These data demonstrate that the MKK7b
isoforms bind JNK and indicate that this is mediated, in part,
by a direct interaction between JNK and the NH2-terminal
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FIG. 4. Effects of MKK7 homologs on JNK activation. (A) COS-7 cells were
transfected with expression vectors encoding Flag epitope-tagged MKK7a1,
MKK7a2, MKK7b1, MKK7b2, MKK7g1, or MKK7g2. The expression of each
of these MKK7 isoforms was examined by immunoblot (IB) analysis using the
Flag-specific monoclonal antibody M2. The MKK7 isoforms were immunoprecipitated, and their activities were measured in the immune complex by a coupled
protein kinase assay (KA) using recombinant GST-JNK1 and GST–c-Jun as
substrates. The phosphorylated c-Jun was detected after SDS-PAGE by autoradiography and was quantitated by PhosphorImager analysis. The effect of cotransfection with an empty expression vector (2) or with an MEKK1 expression
vector (1) is shown. Similar results were observed in three separate experiments.
(B) MKK7 activity, presented as relative protein kinase activity. (C) MKK7
activation caused by MEKK1 (fold activity over MKK7 activity in the absence of
MEKK1).
region of MKK7b that is absent in MKK7a. The presence of a
region in the NH2 terminus of MKK7b that binds JNK is
consistent with previous studies that have implicated the NH2terminal region of MAPKK in the determination of signaling
specificity to distinct MAPK isoforms (4).
Regulation of MKK7 isoforms by MAPKKKs. The protein
kinases MEKK1, MLK3, and DLK preferentially activate JNK
(32, 33, 50, 62, 74, 75, 86), MEKK3 activates both JNK and
ERK (6, 11, 15), while MEKK4 has been reported to activate
both JNK and p38 MAPK (25, 71). Since MEKK1 causes
differential activation of MKK7 isoforms (Fig. 4), we examined
whether other MAPKKKs, including members of the MEKK
and MLK groups (19), could also cause differential activation
of MKK7 isoforms.
To test the effects of various MAPKKK isoforms on activation of MKK7 isoforms, we examined MEKK and MLK protein kinases in cotransfection assays. Control experiments
demonstrated that MEKK1, MEKK3, MLK3, and DLK caused
similar increases in JNK activity (Fig. 6). However, MEKK4
caused no JNK activation (Fig. 6), in contrast to a previous
report (25). Instead, MEKK4 selectively activated p38 MAPK
(data not shown). These data identify MEKK4 as an activator
of the p38 MAPK pathway (71).
To compare the levels of activation of MKK4 and MKK7
isoforms by different MAPKKKs, we performed coupled protein kinase assays. MKK4 and MKK7 activity was detected with
bacterially expressed JNK1 as the substrate, and JNK activation was assessed by measurement of the phosphorylation of
c-Jun. Protein immunoblot analysis demonstrated that similar
amounts of MKK7 were examined in all assays (Fig. 7). While
MEKK1, MEKK3, MLK3, and DLK efficiently activated JNK
(Fig. 6), these MAPKKKs displayed differences in the ability to
activate MKK4 and MKK7 isoforms. For example, MEKK1
was the most potent activator of MKK7 isoforms, whereas
MEKK3 was the most potent activator of MKK4 isoforms (Fig.
7). MEKK1, MLK3 and DLK caused similar increases in
MKK4 and MKK4b activity. The extent of activation of MKK7
isoforms by MEKK3 was largest for MKK7a1, while the
MKK7b2 isoform was only weakly activated by MEKK3. However, the MKK7b isoforms were activated by the mixed-lineage
kinases DLK and MLK3 more strongly than by MEKK3. The
decreased electrophoretic mobility of MKK7a2 (and MKK7b
isoforms to a lesser extent) caused by MEKK1 and MEKK3
was not caused by MLK3 or DLK (Fig. 7). Together, these data
demonstrate that MEKK1, MEKK3, MLK3, and DLK are
capable of selectively activating MKK4 and MKK7 protein
kinase isoforms.
Consistent with the observation that MEKK4 did not acti-
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MOL. CELL. BIOL.
vate JNK (Fig. 6), we found that MEKK4 did not activate any
of the MKK4 or MKK7 isoforms (Fig. 7).
Regulation of MKK4 and MKK7 isoforms by STE20-like
protein kinases. Several STE20-like protein kinases have been
reported to be capable of activating the JNK pathway (19).
Among them, KHS (78) and HPK1 (37, 41, 79) appear to be
specific for the JNK signaling pathway. We therefore examined
the abilities of KHS and HPK1 to activate MKK4 and MKK7
isoforms. Cells were transiently transfected with expression
vectors encoding epitope-tagged MKK4 or MKK7 isoforms,
together with expression vectors for either KHS or HPK1.
Control experiments demonstrated that the KHS and HPK1
protein kinases caused similar levels of JNK activation (Fig. 6).
The amount of MKK4 and MKK7 protein expression was
examined by Western blot analysis (Fig. 8). The MKK4 and
MKK7 protein kinases were immunoprecipitated, and their
activities were measured by a coupled protein kinase assay.
KHS and HPK1 caused similar increases in the activities of
MKK7b1, MKK7b2, and MKK4. However, differences in the
activation of MKK4b and MKK7a isoforms were detected.
Indeed, HPK1 was the most potent activator of MKK7a1 and
MKK7a2, while KHS was the most potent activator of
MKK4b.
Regulation of MKK4 and MKK7 protein kinases by extracellular stimuli. To identify the nature of the MKKs involved
in the regulation of the JNK cascade in response to specific
extracellular stimuli, we examined the effects of environmental
stresses and proinflammatory cytokines on activation of MKK4
and MKK7 protein kinase activities. We found that MKK4 and
MKK7 isoforms were selectively regulated by upstream kinases
(Fig. 7 and 8). Therefore, we examined the effects of UV-C
radiation, anisomycin, TNF-a, and IL-1a on the regulation of
MKK7 and MKK4 isoforms. The MKK4 and MKK7 isoforms
FIG. 7. Activation of MKK4 and MKK7 protein kinases by MAPKKKs. To
examine the abilities of MAPKKKs to activate MKK isoforms, COS-7 cells were
cotransfected with mammalian expression vectors encoding tagged MKK7a1,
MKK7a2, MKK7b1, MKK7b2, MKK4, or MKK4b together with either an
empty expression vector (Control) or an expression vector encoding MEKK1,
MEKK3, MEKK4, MLK3, or DLK. MKK protein expression was monitored by
immunoblot (IB) analysis. The MKK4 and MKK7 isoforms were immunoprecipitated, and their activities were measured by a coupled protein kinase assay
(KA) using recombinant JNK1 and c-Jun as substrates. The phosphorylated
c-Jun was detected after SDS-PAGE by autoradiography and was quantitated by
PhosphorImager analysis. MAPKK activity is presented as relative protein kinase
activity. The levels of MAPKK activation caused by MEKK1, MEKK3, MEKK4,
MLK3, and DLK were 27-, 19-, 1.8-, 5.0-, and 7.0-fold (MKK7a1); 32-, 10-, 1.4-,
13-, and 6.5-fold (MKK7a2); 5.6-, 2.0-, 0.3-, 4.2-, and 4.5-fold (MKK7b1); 13-,
2.6-, 1.3-, 7.5-, and 5.1-fold (MKK7b2); 42-, 88-, 2.0-, 38-, and 39-fold (MKK4);
and 53-, 77-, 2.2-, 48-, and 36-fold (MKK4b), respectively. Similar results were
obtained in three separate experiments.
were isolated, and their activity was measured by a coupled
kinase assay. TNFa and IL-1a selectively activated the MKK7
isoforms and only weakly activated MKK4 (Fig. 9). In contrast,
UV-C radiation and anisomycin caused selective activation of
MKK4 compared to MKK7 (Fig. 9). These data indicate that
the MKK4 and MKK7 isoforms are selectively regulated by
specific extracellular stimuli. This selective regulation is consistent with the genetic evidence for nonredundant roles of
MKK4 and MKK7 in Drosophila and mammals (38).
Subcellular localization of MKK4 and MKK7 protein kinases. JNK has been reported to accumulate in the nucleus
upon treatment of cells with stimuli known to activate the JNK
signaling pathway (8, 40, 51). We therefore examined the subcellular localization of MKK4 and MKK7 by immunofluorescence analysis. These studies demonstrated that the endoge-
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FIG. 6. Effect of MEKK and STE20 protein kinases on JNK activation. To
examine the ability of MAPKKKs and STE20 homologs to activate JNK1, COS-7
cells were cotransfected with a mammalian expression vector encoding HAtagged JNK1 together with an empty expression vector (Control) or an expression vector encoding either MEKK1, MEKK3, MEKK4, MLK3, DLK, KHS, or
HPK1. The expression of JNK1 was examined by immunoblot (IB) analysis using
a monoclonal antibody to the HA epitope tag. JNK1 was immunoprecipitated
and its activity was measured in the immune complex by a protein kinase assay
(KA) using recombinant GST–c-Jun as the substrate. The phosphorylated c-Jun
was detected after SDS-PAGE by autoradiography and was quantitated by PhosphorImager analysis. JNK activity is presented as relative protein kinase activity.
The levels of JNK activation caused by MEKK1, MEKK3, MEKK4, MLK3,
DLK, KHS, and HPK1 were 28-, 29-, 2.0-, 24-, 30-, 5.3-, and 4.3-fold, respectively.
Similar results were obtained in three separate experiments.
VOL. 19, 1999
nous MKK4 and MKK7 protein kinases were distributed in
both the cytoplasm and the nucleus (Fig. 10A). Exposure of the
cells to UV-C radiation or treatment with IL-1a did not cause
marked changes in the distribution of the endogenous MKK4
or MKK7 protein kinases.
To examine the subcellular localization of individual MKK4
and MKK7 isoforms, we transfected cells with epitope-tagged
MKK4 and MKK7 and also performed control experiments
with epitope-tagged wild-type MEK1 and constitutively activated MEK1 (DN3-MEK1-EE). The activated MEK1 was constructed by replacing the two activating phosphorylation sites
with glutamic acid residues and by deletion of the NH2-terminal NES (22, 23, 39, 47). Immunofluorescence analysis demonstrated that, as expected, wild-type MEK1 was restricted to
the cytoplasm, while activated MEK1 was present in the nucleus (Fig. 10B). Under the same experimental conditions, the
MKK4 and MKK7 isoforms were detected at low levels in the
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FIG. 9. Regulation of MKK4 and MKK7 protein kinases by extracellular
stimuli. 293 cells were transiently transfected with expression vectors (pEBG)
encoding either MKK7a1, MKK7a2, MKK7b1, MKK7b2, MKK4, or MKK4b;
36 h after transfection, the cells were untreated (Control) or treated with UV-C
(UV; 80 J/m2), anisomycin (ANISO; 5 mg/ml), TNF-a (20 ng/ml), or IL-1a (2
ng/ml). The cells were harvested after incubation at 37°C (30 min). MKK4 and
MKK7 were isolated by using GSH-Sepharose beads, and the MKK activity was
measured by a coupled protein kinase assay using recombinant JNK1 and c-Jun
as substrates. The radioactivity incorporated into c-Jun was quantitated after
SDS-PAGE by PhosphorImager analysis. MAPKK activity is presented as relative protein kinase activity. The levels of MAPKK activation caused by UV,
anisomycin, TNF-a, and IL-1a were 1.4-, 1.5-, 2.2-, and 1.9-fold (MKK7a1); 2.8-,
1.8-, 3.9-, and 2.5-fold (MKK7a2); 2.4-, 2.1-, 2.5-, and 4.1-fold (MKK7b1); 2.0-,
1.7-, 2.8-, and 2.9-fold (MKK7b2); 4.7-, 3.2-, 0.9-, and 0.9-fold (MKK4); and 2.6-,
2.3-, 0.9-, and 0.9-fold (MKK4b), respectively. Similar results were obtained in
three separate experiments.
cytoplasm and appeared to preferentially accumulate in the
nucleus (Fig. 10B). The higher level of nuclear accumulation of
the transfected MAPKK than of the endogenous MAPKK may
reflect the overexpression of the recombinant proteins. Exposure of the cells to UV-C radiation or treatment with IL-1a did
not induce changes in the localization of MKK4 or MKK7
(data not shown).
Effect of nuclear exclusion on the activity of MKK7. The
distribution of the MKK7 protein in the cytoplasm and the
nucleus differs from that described for the ERK MAPK activator MEK1. While the endogenous MKK7 protein appears to
preferentially accumulate in the nucleus (Fig. 10A), MEK1 is
excluded from the nucleus. The mechanism of nuclear exclusion is mediated by the presence of an NES in the NH2terminal region of MEK1 (23). Disruption of the NES causes
nuclear accumulation of MEK1 and marked potentiation of
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FIG. 8. Activation of MKK4 and MKK7 protein kinases by STE20 homologs.
To examine the abilities of KHS and HPK1 to activate MKK7 isoforms, COS-7
cells were cotransfected with Flag-tagged MKK7a1, MKK7a2, MKK7b1,
MKK7b2, MKK4, or MKK4b together with an empty expression vector (Control) or an expression vector for either KHS or HPK1. Protein expression was
monitored by immunoblot (IB) analysis. Flag-tagged MKKs were immunoprecipitated, and MKK activity was measured in the immune complex by a coupled
protein kinase assay (KA) using recombinant JNK1 and c-Jun as substrates. The
phosphorylated c-Jun was detected after SDS-PAGE by autoradiography and
quantitated by PhosphorImager analysis. MAPKK activity is presented as relative protein kinase activity. The levels of MAPKK activation caused by KHS and
HPK were 15- and 31-fold (MKK7a1); 3.9- and 15-fold (MKK7a2); 4.0- and
3.5-fold (MKK7b1); 5.4- and 4.4-fold (MKK7b2); 3.2- and 2.9-fold (MKK4); and
4.4- and 2.4-fold (MKK4b), respectively. Similar results were obtained in three
separate experiments.
MKK7 ENCODES A GROUP OF JNK KINASES
MOL. CELL. BIOL.
TOURNIER ET AL.
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MKK7 ENCODES A GROUP OF JNK KINASES
VOL. 19, 1999
DISCUSSION
The genes that encode the JNK activators MKK4 and
MKK7 are located on mouse chromosome 11. The MKK7 gene
was mapped to mouse chromosome 11 (Fig. 2). It is interesting
that the MKK4 gene is also located on this chromosome (81).
The localization of both of the genes that encode JNK activators on the same mouse chromosome suggests that these genes
might be linked. This is an interesting possibility because
MKK4 is a candidate tumor suppressor gene that is mutated in
certain tumors (73), suggesting that the MKK7 gene may also
be a target of genetic alterations in disease processes.
Although the mouse MKK4 and MKK7 genes reside on the
same chromosome, it does not appear that this relationship is
present in all species. Thus, in Drosophila, the D-MKK4 (29)
and D-MKK7 (26) genes are located on different chromosomes. Indeed, it appears that the D-MKK3 gene (and not the
D-MKK4 gene) is tightly linked to the D-MKK7 gene in the fly
(29). The colocalization of the genes that encode the JNK
activators MKK4 and MKK7 on one mouse chromosome is
therefore not evolutionarily conserved.
The MKK7 gene encodes a group of protein kinases. The
MKK7 gene includes 14 exons. Alternative splicing leads to the
inclusion or exclusion of exons located in the 59 and 39 regions
of the gene, resulting in the expression of a group of MKK7
isoforms that differ in their NH2 and COOH termini (Fig. 3).
These MKK7 protein kinase isoforms act as activators of JNK
MAPK. Comparative studies demonstrate that the MKK7 isoforms differ in the extent of activation in response to different
upstream components of the JNK signaling pathway (Fig. 7 to
9).
Sequences in the NH2 termini of MAPKK protein kinases
have been reported to mediate specific interactions with other
components of MAPK pathways (4). Similarly, specificity determinants have been identified in the NH2- and COOH-terminal regions of MAPK pathway components, including PBS2
and MEKK1 (60, 69, 85). It is therefore interesting that multiple cDNA clones with different 59 regions have been reported
for MKK3, MKK4, MKK5, and MKK6 (13, 17, 28, 54, 61, 65,
90). Whether these different cDNA clones correspond to fully
processed mRNA is unclear because the corresponding genes
have not been characterized. In contrast, it is established that
the usage of alternative exons within the MKK7 gene leads to
the formation of multiple protein kinase isoforms (Fig. 3). It is
therefore likely that the expression of a group of alternatively
spliced isoforms is a common property of MAPKK genes. The
possible role of the divergent NH2-terminal sequences of
MAPKK isoforms as specificity determinants for MAPKK
function (4) suggests that different MAPKK isoforms may have
different physiological functions. In this study, we demonstrate
that the NH2-terminal extension that is present in the MKK7b
isoforms, but not MKK7a isoforms, binds to the substrate JNK
and may account, in part, for the differences in activity between
these MKK7 isoforms (Fig. 4 and 5). This binding to JNK is
consistent with the presence of consensus primary sequence
motifs for JNK interaction (Leu-Xaa-Leu) in the NH2-terminal region of MKK7b (88).
The MKK7 group of protein kinases are present in the
cytoplasm and the nucleus. Studies of the ERK MAPK signaling pathway demonstrate that the ERK MAPKs are present in
the cytoplasm of quiescent cells and that the ERKs accumulate
in the nucleus following activation (9, 27, 66). Similarly, it has
been reported by several groups that JNK accumulates in the
nucleus following activation (8, 40, 51). In contrast to the
activation-induced nuclear accumulation of the ERK MAPKs,
the ERK activators MEK1 and MEK2 are cytoplasmic enzymes (1). A nuclear export sequence in the NH2-terminal
region of MEK1 appears to mediate rapid export out of the
nucleus and, therefore, the accumulation of MEK1 in the cytoplasm (22, 23, 39). In contrast, we found that the JNK activator MKK7 was present in both the cytoplasm and the nucleus
(Fig. 10). This nuclear location was observed for endogenous
MKK7 (Fig. 10A) and for multiple MKK7 isoforms (Fig. 10B).
Nuclear localization was also observed for the JNK activator
MKK4. The mechanism that accounts for the nuclear location
of MKK4 and MKK7 is unclear because the sequences of these
FIG. 10. Subcellular distribution of MAPKK isoforms. (A) Endogenous MKK4 and MKK7 (red) were detected by immunofluorescence analysis using primary
antibodies specific for these MKK isoforms. The cells were untreated (Control) or treated (30 min) with UV (80 J/m2) or IL-1a (10 ng/ml). DNA was visualized by
staining with DAPI (blue). The scale bar (white) represents 20 mm. (B) Epitope-tagged MKK7a1, MKK7a2, MKK7b1, MKK7b2, MKK4, MKK4b, MEK1, and
DN3-MEK1-EE were detected by using a primary monoclonal antibody to the epitope tag and a Texas red-labeled secondary antibody. The scale bar (white) represents
10 mm.
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signaling (22). These data suggest that the NES may be important for the normal control of MEK1 activity.
To test whether the presence of an NES would affect the
properties of MKK7, we fused the NES of MEK1 to the NH2terminal region of MKK7b (Fig. 11A). Immunofluorescence
analysis demonstrated that while MKK7b2 preferentially accumulated in the nucleus, the NES-MKK7b2 was excluded
from the nucleus (Fig. 11B). Similar results were obtained in
experiments using MKK7b1 and NES-MKK7b1 (data not
shown).
The presence of an NES may alter the activation of MKK7
by cytokines or by environmental stresses. We therefore examined the effects of UV-C radiation and IL-1a on the activation
of MKK7b2 and NES-MKK7b2 (Fig. 11C), using a coupled
protein kinase assay to measure the MKK7 protein kinase
activity. Western blot analysis demonstrated the expression of
similar amounts of MKK7b2 and NES-MKK7b2. Protein kinase assays demonstrated that both MKK7b2 and NESMKK7b2 were activated by UV-C radiation and IL-1a. These
data demonstrated that extracellular stimuli can activate
MKK7 proteins that are preferentially located either in the
nucleus (MKK7b2) or in the cytoplasm (NES-MKK7b2).
The presence of an NES might also alter the effect of MKK7
on JNK. To examine JNK activation, we performed immunofluorescence analysis using an antibody that binds to JNK
phosphorylated on Thr-183 and Tyr-185. Control experiments
demonstrated that the exposure of cells to UV-C radiation
caused increased staining by the phospho-JNK antibody (data
not shown). Expression of either MKK7b2 or NES-MKK7b2
also caused increased staining by the phospho-JNK antibody.
Immunofluorescence analysis demonstrated that the activated
JNK accumulated in the nucleus (Fig. 11B). Quantitative assays of protein kinase activity using biochemical methods demonstrated that MKK7b1, MKK7b2, NES-MKK7b1, and NESMKK7b2 caused a similar level of JNK activation (Fig. 11D).
These data indicated that recombinant MKK7 molecules that
are preferentially located in the nucleus (MKK7b2) or in the
cytoplasm (NES-MKK7b2) are equally capable of activating
the JNK MAPK. This observation contrasts with the previous
finding that constitutively nuclear MEK1 mutants cause enhanced signaling by the ERK MAPK (22).
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MOL. CELL. BIOL.
protein kinases do not include an obvious nuclear localization
sequence. It is possible that the absence of an NES may contribute to the localization of MKK4 and MKK7 in the nucleus.
Thus, the subcellular location of the JNK activators MKK4 and
MKK7 differs from that of the ERK activators MEK1 and
MEK2.
Recent studies indicate that the subcellular organization of
the p38 MAPK pathway differs from that of both the ERK and
JNK signaling pathways. The major p38 MAPK activators
(MKK3 and MKK6), like the activators of ERK (MEK1 and
MEK2), are both preferentially located in the cytoplasm (5).
However, unlike ERK, which exhibits activation-induced redistribution from the cytoplasm to the nucleus, the p38 MAPK
is prelocalized in the nucleus and is rapidly exported to the
cytoplasm upon activation (5). The mechanism of nuclear export appears to be mediated, at least in part, by the nuclear
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FIG. 11. Effect of an NES on the properties of MKK7. (A) Schematic representation of MKK7b and NES-MKK7b. Both proteins were constructed with an
NH2-terminal Flag epitope (grey box). The nuclear export signal (NES) of MEK1 (residues 32 to 51) was inserted following the Flag epitope to create NES-MKK7b.
(B) The MKK7 proteins and HA-JNK1 were expressed in COS-7 cells. Transfected cells were identified by immunofluorescence analysis using antibody M2, which binds
the Flag epitope on the MKK7 proteins. Activated JNK in the transfected cells was examined by using an antibody to phospho-JNK [JNK(P)]. The MKK7 (red) and
phospho-JNK (green) were detected with secondary antibodies conjugated to Texas red and fluorescein, respectively. DNA was visualized by staining with DAPI (blue).
The scale bar (white) represents 10 mm. (C) Regulation of MKK7b2 and NES-MKK7b2 by extracellular stimuli. COS-7 cells expressing MKK7b2 or NES-MKK7b2
were untreated (Control) or treated (30 min) with UV-C (80 J/m2) or IL-1a (10 ng/ml). The expression of MKK7 was examined by immunoblot (IB) analysis using
the Flag-specific monoclonal antibody M2. MKK7 activity was measured in a coupled protein kinase assay (KA). The radioactivity incorporated into c-Jun was
quantitated after SDS-PAGE by PhosphorImager analysis. MAPKK activity is presented as relative protein kinase activity. The levels of MAPKK activation caused by
UV and IL-1a were 3.0- and 3.7-fold (MKK7b2) and 1.5- and 1.8-fold (NES-MKK7b2), respectively. (D) Activation of JNK1 by MKK7b and NES-MKK7b. COS-7
cells were cotransfected with plasmids expressing HA-tagged JNK1 together with an empty vector (Control) or an expression vector encoding Flag-tagged MKK7b1,
NES-MKK7b1, MKK7b2, or NES-MKK7b2. The expression of MKK7 and JNK1 was monitored by immunoblot (IB) analysis using antibodies to Flag and HA,
respectively. JNK1 activity was measured by immune complex kinase assay (KA) using the substrate c-Jun. The radioactivity incorporated into c-Jun was quantitated
after SDS-PAGE by PhosphorImager analysis. JNK activity is presented as relative protein kinase activity. The levels of JNK activation observed for MKK7b1,
NES-MKK7b1, MKK7b2, and NES-MKK7b2 were 21-, 27-, 25-, and 31-fold, respectively.
MKK7 ENCODES A GROUP OF JNK KINASES
VOL. 19, 1999
ACKNOWLEDGMENTS
We thank N. Ahn, J. Avruch, J. Blenis, S. Gutkind, H. Saito, U.
Siebenlist, and T. Tan for providing reagents, and we thank K. Gemme
for administrative assistance.
This work was supported by grant CA53896 from the National Cancer Institute. R.J.D. is an Investigator of the Howard Hughes Medical
Institute.
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substrate MAPKAP kinase 2, which is also exported from the
nucleus following activation (5, 16). Together, these data indicate that there are marked differences between the subcellular
organization of the ERK, JNK, and p38 MAPK pathways.
The presence of MKK4 and MKK7 in the nucleus suggests
that these MAPKK may activate JNK in this compartment of
the cell. Indeed, Mizukami et al. (51) have recently reported
that JNK may be activated in the nucleus during ischemiareperfusion injury to the heart. However, it is possible that
MKK4 and MKK7 may also activate JNK in the cytoplasm.
Evidence in favor of this hypothesis was obtained from studies
of mutated MKK7 protein kinases that are excluded from the
nucleus (Fig. 11). We found that cytoplasmic MKK7 protein
kinases efficiently activated JNK in vivo (Fig. 11D) and that the
activated JNK in these cells accumulated in the nucleus (Fig.
11B). These data indicate that MKK7 (and possibly MKK4)
can activate JNK in the cytoplasm and that the activated JNK
redistributes from the cytoplasm to the nucleus.
The presence of MKK4 and MKK7 in the nuclei of quiescent and stimulated cells indicates that these MKK isoforms
may also be activated in the nucleus. Fanger et al. have reported that MEKK1 is located in the nucleus (20). However,
other MAPKKK that activate the JNK signaling pathway appear
to be preferentially located in the cytoplasm. For example, MLK2
is detected in punctate structures along microtubules (56),
MEKK2 and MEKK3 are located on Golgi-associated vesicles
(20), while MLK3 and DLK were identified in the cytoplasm by
immunofluorescence microscopy (data not shown). The cytoplasmic location of these MAPKKKs raises questions concerning the mechanism of action of nuclear MKK4 and MKK7.
Clearly MAPKKKs and MAPKKs must interact and therefore
have to be present in the same cellular compartment. The
simplest explanation is that although MKK4 and MKK7 are
present in the nucleus, a cytoplasmic population of molecules
may arise from rapid shuttling across the nuclear membrane.
This model implies that some MAPKKKs (e.g., MEKK1) may
directly activate MKK4 and MKK7 in the nucleus, whereas
others (e.g., MLK3) may activate a cytoplasmic population of
MKK4 and MKK7 that rapidly shuttles to the nucleus.
Further studies are required to identify the mechanisms
used to determine the subcellular distribution of components
of the JNK signaling pathway. Such mechanisms may include
scaffold proteins that interact with JNK and MKK7 (82). The
expression of scaffold proteins may contribute to the selective
control of JNK signaling in specialized differentiated cells.
1579
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MKK7 ENCODES A GROUP OF JNK KINASES