Download Aspects of growth factor signal transduction in the cell cytoplasm

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Journal of Cell Science 107, 747-752 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
COMMENTARY
Aspects of growth factor signal transduction in the cell cytoplasm
B. A. Panaretto
CSIRO, Division of Animal Production, Box 239, Blacktown, NSW 2148, Australia
INTRODUCTION
Significant advances have been made during the last decade in
the structure of growth factors, the cloning of their membrane
receptors, recognition of autophosphorylation of their intracellular tyrosines when receptor ligand complexes form and the
translation of signals into phosphorylation of serines and
threonine residues in proteins throughout the cell. The first
steps by which a number of extracellular signals control
cellular proliferation, migration, differentiation and transformation clearly operate through cell-surface receptors and cytoplasmic tyrosine kinases.
Progress in defining some components of growth factorinduced signal transduction in cytoplasm is discussed here and
includes the roles of molecular binding domains and other
cytoplasmic serine/threonine kinases in the signalling
pathways. The central problem of specific growth factor signalling remains but understanding of events in the cytoplasm
that help in resolving this matter is included.
Recent research on phospholipase C γ (PLCγ), phospholipid
metabolism and calcium/protein kinase C-propagated signals
when growth factor-ligand complexes (particularly EGF) form
has been reported (Clark and Dunlop, 1991; Allbritton et al.,
1992; Davis, 1992; Peppelenbosch et al., 1992; Spaargaren et
al., 1992; Lin et al., 1993; Berridge, 1993) and is not included.
SH2 AND SH3 DOMAINS OF RECEPTOR-BINDING
PROTEINS
Activated mammalian growth factor receptors associate with
several cytoplasmic proteins that bind to specific phosphorylated tyrosines within non-catalytic regions of the receptors.
These receptor-binding proteins, including PLCγ1, phosphatidylinositol (PI) 3-kinase, GAP and the Src family tyrosine
kinases, are receptor targets (Koch et al., 1991; Pawson and
Gish, 1992) and share two modular domains of about 95 and
45 amino acids, respectively, called the SH2 and SH3 domains
(Src homology domains). The amino acid sequences of several
SH2 domains, both enzymatic and non-catalytic ‘adaptor’
molecules, e.g. sem-5, in several signalling proteins are
published (Koch et al., 1991; Overduin et al., 1992a,b; Pawson
and Gish, 1992; Waksman et al., 1992).
Binding of SH2 domains to cytoplasmic tyrosine
phosphorylated proteins
The three-dimensional solution structure of the SH2 domain of
c-Abl (Overduin et al., 1992a,b), the SH2 domain of the p85a
subunit of phosphatidylinositol-3-OH kinase (Booker et al.,
1992) and the crystal structure of the phosphotyrosine recognition domain SH2 of v-src complexed with tyrosine phosphorylated peptides (Waksman et al., 1992) have been determined.
Koch et al. (1991) suggested that the binding of SH2
domains to activated growth factor receptors exemplifies the
general reaction between these domains and tyrosine-phosphorylated proteins. The evidence presented by these authors
is based upon experiments showing the binding in vitro and in
vivo of the transforming oncoprotein that lacks a tyrosine
kinase domain, with tyrosine-phosphorylated cellular polypeptides in transformed cells and the necessity for an SH2 domain
in the oncoprotein for binding. The mechanism by which transforming oncoproteins enhance tyrosine phosphorylation is
unknown but may be a balance between the enhancement of
an endogenous tyrosine kinase and/or the blocking of a
tyrosine phosphatase. To date phosphotyrosine phosphatase
(PTP) expression in mammals is largely confined to
haematopoietic tissue. However a murine PTP (Syp; Feng et
al., 1993) binds through its two SH2 domains to autophosphorylated epidermal growth factor receptor (EGFR) and plateletderived growth factor receptor (PDGFR) in stimulated cells
and is phosphorylated on tyrosine; the gene is widely expressed
in embryonic and adult mice. Feng et al. (1993) discount the
suggestion that Syp may act as a negative regulator by dephosphorylating receptor autophosphorylation sites. An alternative
hypothesis examined by these authors is that Syp is a positive
regulator of signal transduction by dephosphorylating
inhibitory phosphotyrosine sites; they cite the findings of
Zheng et al. (1992) who showed that phosphorylation of Tyr527 in the COOH-terminal tail of c-Src blocks c-Src tyrosine
kinase activity. Vogel et al. (1993) reported that PTP 1D,
which also contains two SH2 domains, did not dephosphorylate receptor tyrosine kinases, indeed PTP 1D was phosphorylated on tyrosine in cells stimulated through the β subunit
of the PDGFR and that this reaction was associated with its
enhanced catalytic activity. They suggest that the interaction
between the SH2 domains of PTP 1D and phosphotyrosine
residues in the cytoplasmic domain of the β subunit of the
PDGFR prevents dephosphorylation and protects the receptor
from inactivation. PTP 1D was phosphorylated on tyrosine and
could activate a positive signalling pathway by dephos-
Key words: growth factor, signalling, cytoplasm, specificity
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B. A. Panaretto
phorylating other phosphotyrosines that were suppressing signalling potentials of other peptides; Src tyrosine kinases were
cited as an example. Superti-Furga et al. (1993) propose, on
the basis of experimental induction of c-Src in yeast, that its
SH3 domain is required for stabilising the interaction between
the SH2 domains and the phosphorylated (Tyr-527) tail of cSrc kinase.
SH3 domains and functions
The solution structure of the SH3 domains of Src kinase (Yu
et al., 1992), human phospholipase C γ (Kohda et al., 1993),
the crystal structure of the SH3 domain of human Fyn, a
member of the Src family (Noble et al., 1993), and the SH3
domain of a subunit of phosphatidylinositol 3-kinase (Booker
et al. 1993) have been determined and probable ligand-binding
sites identified; the predicted secondary structure of SH3
domains (Benner et al., 1993) closely followed the publication
of the crystal structure (Musacchio et al., 1992). The last
authors suggest that where proteins contain multiple SH3
domains, variations in the N-terminal Ala-Leu-Tyr-Asp-Tyr
motif might allow recognition of different related proteins.
SH3 domains occur in a variety of proteins that comprise,
or are associated with, the cytoskeleton or membranes (BarSagi et al., 1993) possibly by association with the Rho/Rac
family of G proteins (Bourne et al., 1991) that control
membrane ruffling, the formation of actin stress fibres and
focal contacts (Ridley and Hall, 1992; Ridley et al., 1992;
Pawson and Gish, 1992). The cDNA product 3BP-1, cloned
from a mouse cell line, binds to the Abl and Src SH3 regions,
and supports this hypothesis (Cicchetti et al., 1992). 3BP-1 has
71% homology with GAP-rho protein (partial sequence taken
from Diekmann et al., 1991) over a 17 amino acid region as
well as homologies with Bcr and human n-chimaerin over a
128 amino acid region. Ren et al. (1993) localised the Abl-SH3
binding region of 3BP-1 to a nine to ten amino acid stretch rich
in proline residues. The emerging principle is that activity of
small G proteins may be regulated by the interaction of SH3
domains with guanine nucleotide exchange factors and
GTPase-activating proteins (Pawson and Gish, 1992). This is
consistent with the functions of sem-5/GRB2 (Lowenstein et
al., 1992; Rozakis-Adcock et al., 1993 and Chardin et al.,
1993) and the expressed protein of the Drosophila gene drk
(Olivier et al., 1993; Simon et al., 1993) discussed below.
Duchesne et al. (1993) have confirmed that GAP is an effector
of Ras in Xenopus oocytes and that the amino acid domain 275351 is essential for signal transduction.
Specificity in growth factor signalling
Understanding of some early elements involved in specificity
in growth factor signalling is developing. One principle is that
the amino acid sequence of the autophosphorylation site in a
receptor determines the binding of specific SH2-containing
proteins (Pawson and Gish, 1992).
High affinity binding of an SH2 domain requires that the
phosphorylated tyrosine be embedded within a specific
sequence of amino acids. For example the SH2 domains of
PLCγ, PI 3-kinase and GAP each bind to different autophosphorylated sites of the β subunit of PDGFR. Fantl et al. (1992)
assigned binding sites for SH2-containing proteins to welldefined regions of the PDGFR. PI 3-kinase and GTPase-activating protein (GAP) bound to different phosphotyrosine-con-
taining sequences separated by 20 amino acids within the
kinase insert of the receptor. Kashishian et al. (1992) reported
that of the three phosphorylation sites in the kinase insert of
the β subunit of the PDGFR, tyrosines 740 and 751 are
involved in the receptor-stimulated binding of PI 3-kinase and
tyrosine 771 is required for the efficient binding of GAP. Fantl
et al. (1992) and Kashishian et al. (1992) ascribed the methionine or valine residues at positions +1 and the methionine
residues +3 or +4 relative to the C-terminal phosphotyrosines
731 and 751 of the PDGFR as possibly specifying binding to
PI 3-kinase while the methionine +1 with respect to phosphotyrosine 771 specified binding to GAP.
Three-dimensional structures of the v-src oncogene product
complexed with two different phosphotyrosol pentapeptides
have been determined (Waksman et al., 1992) and give
important insights into the interactions. The phosphotyrosine
protrudes into a pocket to interact with lysine 203 and arginines
175 and 155 (numbered according to v-src; Takeya and
Hanafusa, 1983) in the SH2 domain. The invariant arginine
175 is buried deep and reacts with the terminal guanidium
nitrogen and two phosphate oxygens. Substitution of this
critical residue with lysine in Abl and N-terminal GAP SH2
domains results in a complete loss of binding activity (Mayer
et al., 1992; Marengere and Pawson, 1992). Arginine 155,
which is on the surface, makes two contacts, one to a phosphate
oxygen and one polar interaction with the tyrosine ring. Lysine
203 also binds to the tyrosine ring by polar interaction
(Waksman et al., 1992); the phosphotyrosine is sandwiched
between Arg-155 and Lys-203, while the invariant Arg-175
grasps the phosphate (Pawson and Gish, 1992). The ability of
SH2 domains to distinguish phosphotyrosine-containing
proteins from those with phosphoserine and phosphothreonine
lies in the fact that, in the last two, the phosphate group is
attached to shorter side chains that cannot reach into the
binding pocket to react with the buried arginine.
It is evident that there are elegant ways of achieving specificity in signal transduction but, as Pawson and Gish (1992)
have remarked, much remains to be learned. For example, in
1991, Mohammadi and his colleagues identified tyrosine 766
in a phosphorylated peptide in a conserved region of the FGFR
that was shown to be a binding site for PLCγ (Mohammadi et
al., 1991). Mohammadi et al. (1992) and Peters et al. (1992)
announced that a point mutant in which Tyr-766 was replaced
with a phenylalanine was unable to associate with and tyrosine
phosphorylate PLCγ or stimulate hydrolysis of phosphatidylinositol and calcium mobilisation; despite this, the mutant
receptor was autophosphorylated and phosphorylated several
cellular proteins and stimulated DNA synthesis.
ADDITIONAL GENE PRODUCTS IN THE
TRANSDUCTION OF SIGNALS TO THE NUCLEUS
Several gene products are involved in signal transduction to
the nucleus and include the guanosine triphosphate-binding
(GTP) proteins such as the products of Ras genes, the serine
kinase Raf and MAP2 serine/threonine kinases. Three forms of
MAP2 kinase have been purified from fibroblasts, including
insulin- and nerve growth factor (NGF)-activated protein
kinases ERK 1 and ERK 2 (extracellular signal-regulated
kinases), which are closely related (Boulton et al., 1991); and
Growth factor signal transduction
myelin basic protein (MBP) kinase, which phosphorylates
myelin basic protein in vitro on a threonine residue.
The ordinal arrangement of these enzymes in cytoplasmic
signalling is being clarified for several systems but the contribution of this research to clarifying the specificity of growth
factor signalling in the cytoplasm is equivocal. Chao (1992)
exemplified the point by remarking that many known
metabolic activities in PC12 cells in response to EGF or NGF
stimulation fail to explain their different biological effects.
Ras and GTPase-activating protein (GAP)
The three mammalian Ras genes, H, K and N encode 21K
proteins, p21ras, critical for cellular proliferation and differentiation. The GTP-bound form of Ras is thought to be responsible for the effect. Pai et al. (1989) have determined the crystal
structure of the p21Ha ras GTP complex and Milburn et al.
(1990) and Schlichting et al. (1990) compared three-dimensional structures of ras proteins in both the GTP- and GDPbound forms.
Two types of GAP have been cloned from human placenta
(Trahey et al., 1988) and from bovine brain and the interaction
of the latter with normal and oncogenic p21ras reported (Vogel
et al., 1988). Provision for specificity in signalling is now
emerging in the report of specific GAPs published recently by
Strom et al. (1993). These authors cloned a gene GYP (GAP
for Ypt6 protein) encoding a protein of 458 amino acids, which
is highly specific for Ypt6 protein and shows little or no crossreactivity with other members of the Ypt/Rab GTPase family
or with H-ras p21.
Control of the activity states of Ras in several
systems
The necessity to control the activity state of Ras is essential for
normal cellular biology in order to avoid neoplasia. Satoh et
al. (1992) have differentiated six categories of signal transductions involving ras proteins.
The p21K products of Ras are a well-characterised subgroup
of small GTPases; more than 40 small GTPases related to Ras,
Rho and Rac have been identified (Bourne et al., 1991;
Valencia et al., 1991). Downregulation of c-Ras by GAP was
exemplified by Zhang et al. (1990). Posttranslationallymodified ras proteins are believed to be compromised by the
loss of cell membrane anchoring (Willumsen et al., 1986).
Tsai et al. (1989) showed in vitro that certain phospholipids
and their breakdown products blocked the ability of GAP to
stimulate GTPase activity; however the contrasting roles of
arachidonic acid metabolism in EGF- and PDGF-dependent
mitogenesis (Handler et al., 1990) need to be clarified.
A number of other models for regulating the activation state
of Ras have been proposed. One of increasing importance is
by stimulating GNRP (guanine nucleotide releasing factor or
GRF); a GRF for p21ras (p140Ras-GRF) has been cloned from
rat brain (Shou et al., 1992) and two widely-expressed genes
have been cloned from mice that are the equivalent of the
Drosophila exchanger gene, son of sevenless, Sos (Bowtell et
al., 1992). The method of activating ‘Ras exchangers’ is
unknown.
Relationships between activated ras proteins and growth
factor receptors are now being clarified. Clark et al. (1992)
isolated a gene, sem-5, from C. elegans that encodes a protein
composed almost exclusively of SH2 and SH3 domains; sem-
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5 is required in C. elegans for sex myoblast migration, differentiation of cells within the vulval lineage and for larval
survival. These authors suggested that two genes are required
for signal transduction in vulval cells; let-23 encodes a transmembrane tyrosine kinase, similar to EGFR, thought to be the
target for the gonadal cell-signalling molecule and encoding a
ras protein (Han and Sternberg, 1990), possibly let-60/Ras,
which is activated and acts downstream of let-23. While sem5 has no enzymatic activity, it acts as an ‘adaptor’ molecule
between let-23 and let-60/Ras. The model proposed by these
authors is that let-23 tyrosine kinase and sem-5 interact through
the SH2 and SH3 domains and then downstream through a postulated protein that directly modifies let-60/Ras. The carboxyterminal tail of let-23 contains several potential autophosphorylation sites, one of which has a sequence similar to Tyr-920
in the kinase domain of the EGFR whose phosphorylation
induces high-affinity binding of PLCγ SH2 domains. An allele
of sem-5 in which an SH2 serine residue required for phosphotyrosine binding was substituted by asparagine gave a
partial vulvaless phenotype. Both SH2 domains of sem-5 are
required for normal function.
The growth factor receptor-binding protein GRB2 containing one SH2 and two SH3 domains has been isolated and
exhibits striking structural homology with sem-5 (Lowenstein
et al., 1992). They showed by immunoblotting experiments
that GRB2 associates with tyrosine-phosphorylated EGFR and
PDGFR via its SH2 domain and suggest that GRB2 transmits
the EGF and PDGF signals in a similar way to the proposed
pathway for C. elegans vulval induction discussed above.
Evidence supporting this hypothesis has now come from
several sources including Rozakis-Adcock et al. (1993), Li et
al. (1993), Gale et al. (1993), Olivier et al. (1993), Simon et
al. (1993), Buday and Downard (1993) and Chardin et al.
(1993). Rozakis-Adcock et al. (1993) and Li et al. (1993)
reported that the SH3 domains of GRB2 bind, respectively, to
the proline-rich carboxy-terminal tail of mSos in rat fibroblasts
and hSos1, the human homologue of the Drosophila GNRP for
Ras. Significantly, the GRB2-mSos1 complex binds to the
autophosphorylated EGF receptor, and mSos is phosphorylated. Thus GRB2 appears to link tyrosine kinases to a RasGNRP in mammalian cells (Rozakis-Adcock et al., 1993).
Additional support linking receptor tyrosine kinases to Ras by
a complex consisting of GRB2-hSos 1 comes from Chardin et
al. (1993). Gale et al. (1993) showed that overexpression of
GRB2 potentiates EGF-induced activation of Ras by
enhancing the rate of GNRP on Ras.
The Drosophila drk gene encodes a protein with a single
SH2 and two flanking SH3 domains homologous to sem5/GRB2 (Olivier et al., 1993; Simon et al., 1993). The
expressed protein of drk correlated with binding of its SH2
domain to autophosphorylated receptor tyrosine kinase and
localisation of drk to the plasma membrane. In vitro drk bound
directly to the C-terminal tail of Sos, which is required for
sevenless signalling, and has a domain related to CDC25 and
is likely to act as a Ras GNRP thus coupling receptor tyrosine
kinases to Sos and activating Ras.
Rozakis-Adcock and her colleagues (1993) state that the
SH3 domains of sem-5/Grb2 in C. elegans, Drosophila and
mammals, through their interaction with hSos1, provide a
major cellular signalling pathway.
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B. A. Panaretto
Raf and MAP kinases
The immediate downstream targets for Ras in mammalian cells
are unknown. The activation states of a set of kinases including
c-Raf, MEK (a protein kinase that lies upstream of MAP
kinase; Crews et al., 1992), MAP kinase and RSK (ribosomal
S6 kinase(s)) are rapidly and perhaps directly regulated by Ras
proteins. These four cytoplasmic protein (Ser/Thr) kinases
appear to be arranged in a linear cascade with c-Raf-1 closest
to the signal generated by Ras (Zhang et al., 1993). Morrison
et al. (1988) showed that phosphorylation occurred rapidly in
the serine/threonine-specific kinase activity of Raf in mouse
3T3 cells treated with PDGF, acidic FGF, EGF and phorbol
12-myristate 3-acetate. Kolch et al. (1991) showed that PKCmediated Raf-1 protein kinase was required for the seruminduced growth of NIH/3T3 cells; investigating this
mechanism further Kolch et al. (1993) concluded that phosphorylation was induced at several sites including a mandatory
serine at position 499 as a prerequisite for the autophosphorylation of Ser-259 that is also required for activation by PKCα.
Raf-1 activates MAPK-kinase in NIH/3T3 cells (Dent et al.,
1992) and appeared to be the immediate upstream activator of
MAPK-kinase in vivo (Kyriakis et al., 1992). Howe et al.
(1992) also concluded that raf protein kinase is upstream of
MAP kinases and is either a MAP kinase kinase kinase or a
MAP kinase kinase kinase kinase. Experiments by Zhang et al.
(1993), using purified polypeptides in vitro and in a yeast
expression system, have determined that the amino-terminal
regulatory region of Raf-1 protein binds directly to GTP-Ras
and inhibits Ras-GAP; a finding confirmed by Warne et al.
(1993).
MAP kinase kinases have been isolated from several sources
including Xenopus oocytes (Kosako et al., 1992), epidermal
growth factor-stimulated A431 cells (Seger et al., 1992), and
rabbit muscle, the last phosphorylating in Thr-183 and Tyr-185
on MAP kinase and autophosphorylates on Tyr, Thr and Ser
residues (Nakielny et al., 1992). The primary structure of yet
another MAP kinase isolated from rat kidney has been determined and found to have high similarity scores to four yeast
protein kinases (Wu et al., 1993). A cytosolic ras p21dependent ERK-kinase stimulator (REKS) has been postulated
to intervene between GTP-bound ras p21 and MAP kinase
(Itoh et al., 1993). The MAP-2-related protein kinase ERT that
phosphorylates the EGFR at Thr-669 contains a sequence, ProLeu-Ser/Thr-Pro, that is recognised by the ERT protein kinase
(Alvarez et al., 1991). In addition to the specificities discussed
above, Wu et al. (1993) have suggested that specific activator
proteins will exist for different MAP kinases.
Wood et al. (1992) have discussed the relationship between
activated Ras, MAP kinases and Raf in NGF-stimulated PC12
cells and Moodie et al. (1993) have reported the formation of
complexes, possibly through an as yet unidentified protein,
between activated Ras and MAPKK and Raf-1 protein in rat
brain cytosol.
CONCLUSION
The main task is to identify the ways specific growth factor
signalling is achieved and the understanding of cytoplasmic
determinants of this may increase as the functions of additional
molecules and domains in the signalling pathways in developmental and cell biology are discovered. Examples include the
Rap proteins (Bokoch, 1993) (the formation of a non-productive complex between Rap-1b and Raf has been suggested by
Zhang et al. (1993) as the basis of the antagonism observed
between Ras and Raf1 a/b), the pleckstrin homology (PH)
domains discussed by Mayer et al. (1993) and the activation of
the insulin receptor that, unlike EGFR which complexes
directly to GRB2-Sos, requires the interaction of GRB2-Sos
with insulin receptor substrate-1 and Shc, an SH2 domain-containing protein, in order to link the receptor to Ras signalling
pathways (Skolnik et al., 1993). Knowledge of the structures
of molecules in the signalling pathways provides opportunities
to define reactive surfaces as exemplified by the differentiation
of regions of p21ras that are most likely involved in determining affinity for GAP from those essential for ‘exchanger’ activation (Polakis and McCormick, 1993). Cantley et al. (1991)
and Waksman et al. (1992) have proposed manipulating known
structures of SH2 complexes to design general SH2 inhibitors
in order to reveal effects on cellular responses to growth factors
as well as providing some clues for understanding the specificities of particular SH2 domains.
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