SH2 and SH3 domains

REVIEW

SH2 and SH3 domains T. Pawson* and J. Schlessingert TDepartment

*Division of Molecular and Developmental Mount Sinai Hospital, 600 University of Pharmacology, New York University

Biology, Samuel Lunenfeld Research Institute, Avenue, Toronto, Ontario MS6 1X5, Canada Medical Center, 550 First Avenue, New York, NY 10016,

USA

The Src-homology domains SH2 and SH3 are protein modules that determine the specificity of protein-protein interactions in the transduction of signals from the cell surface to the nucleus. Introduction

The stimulation of cells with growth factors that bind to, and activate, receptor protein-tyrosine kinases (RTKs) can elicit a remarkably diverse array of cellular responses, with the precise response depending on the identity of the RTK and the nature of the stimulated cell [ 11. Activation of tyrosine kinases can induce cells to proliferate and migrate, as in the case of fibroblasts treated with platelet-derived growth factor (PDGF), to differentiate, as noted for neuronal precursors stimulated with nerve growth factor (NGF), or to turn on metabolic pathways, as occurs in cells exposed to insulin. Furthermore, RTK over-expression, or gainof-function mutations in RTK genes, can contribute to cancer; and loss-of-function RTK mutations can result in inherited disorders such as insulin-resistant diabetes or piebaldism. The mechanisms by which RTKs activate intracellular signalling pathways is therefore of the greatest interest, both for understanding the control of normal cell growth, metabolism and development, and for defining the alterations in signal transduction that occur in cells with aberrant tyrosine kinase activity. There is a considerable body of evidence indicating that many of the protein substrates of RTKs have in common one or two copies of a motif of approximately 100 amino acids that was originally found in cytoplasmic tyrosine kinases related to c-Src - and hence called the Src homology 2 (SH2) domain - although these SH2containing polypeptides may otherwise be structurally and functionally quite distinct from one an other [ 21. The binding of a ligand to the extracellular domain of an RTK induces dimerization of the receptor, leading to activation of the intrinsic tyrosine kinase and intermolecular autophosphorylation [ 11. One function of receptor autophosphorylation is to act as a molecular switch to induce a physical association between SH2containing cytoplasmic signalling proteins and the activated receptor 121. SH2 domains directly recognize phosphorylated tyrosine residues; they also have independent binding sites for the residues surrounding the phosphotyrosine within a polypeptide chain. Receptor autophosphorylation therefore creates SH2-binding sites on the receptor; the receptor 434

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sequences flanking the phosphotyrosine dictate which particular SH2 domains will bind with high alfinity to which tyrosine-phosphorylated receptor. This general scheme can be illustrated more precisely for the PPDGF receptor and the epidermal growth factor (EGF) receptor, as shown in Figure 1. In both cases, the SH2-binding autophosphotylation residues are clustered within non-catalytic regions of the receptor’s cytoplasmic domain [ 1,3-71. The cytoplasmic portion of the PPDGF receptor contains autophosphotylation sites, and hence binding sites for SH2 domains, in the juxtamembrane region, in an insert in the kinase domain and in the carboxyterminal tail - none of which forms part of the kinase domain itself. Phosphorylated tyrosine residues Y1021 and Y1009 in the tail bind to phospholipase Cyl (PLC-~1) and to the phosphotyrosine phosphatase Syp, respectively [S-lo]. Within the kinase insert, W71 binds to the GTPase-activating protein (GAP) of the GTPase Ras, and both W40 and W51 of the kinase insert are involved in binding to phosphotidylinositol 3-kinase (PU-kinase), apparently with each site engaging one of the two SH2 domains of the PIS-kinase ~85 subunit. W51 in the kinase insert region is also essential for binding of Nck, an adaptor protein containing one SH2 and three SH3 domains (R Nishimura, W Li, A Kashishia, A Mondino, M Zhou, J Cooper and J Schlessinger, unpublished data). In the juxtamembrane region, Y579 and Y581 are implicated in binding to c-Src, a cytoplasmic tyrosine kinase [ 11 ] Experiments in which single tyrosine autophosphorylation sites are mutated to phenylalanine have shown that these interactions are indeed specific, and are required to couple the activated receptor to intracellular signalling pathways. For example, a mutant PPDGFR with phenylalanine instead of tyrosine at residue 1021 binds poorly to PLC-)11 and does not stimulate the hydrolysis of phosphatidylinositol bisphosphate [9]. However, the F1021 mutant receptor continues to interact normally with other SH2-containing signalling proteins. These results show that SH2 domains bind with high affinity to specific receptor autophosphotylation sites, thereby mediating the initial interactions of RTKs with their substrates. 1993,

Vol

3 No

7

SH2 and SH3 domains

REVIEW

‘.

Fig. 1. SH2-binding sites on the f3PDCF are given in the single letter amino-acid

receptor code.

and

PDCF

ECF

receptor

EGF receptor.

At least three mechanisms can be envisaged by which such intimate association might stimulate the function of the SH2-containing target proteins. First, binding to a receptor might drive re-localization of soluble cytosolit signalling proteins to the plasma membrane. This may be especially important if the substrates of the signalling molecule are also membrane-bound, as is the case for enzymes such as PLC-~1, PI3kinase, GAP and Grb2/Sos (see below). Second, physical binding of ki nase substrates, such as PLC-~1, to RTKs can markedly lower the Michaelis constant, K,, for phosphoryla tion, making the SH2-containing proteins preferred substrates of receptor kinase activity. In the case of PLC-)I~, tyrosine phosphotylation is required for activation of the enzyme in uiuo [ 121. Finally, physical association between the SH2 domain of a signalling protein with phosphotyrosine-containing sites may alter the conformation of the signalling protein, directly stimulating its activity. There is evidence to support this proposition in the case of PI3kinase, the activity of which can be stimulated in vitro by binding of PI3kinase to appropriate phosphopeptides [ 13,141. This mechanism of activation may be particularly pertinent for signalling proteins that have two SH2 domains; the presence of more than one SH2 domain within a single polypeptide may not only allow direct activation. but also expansion of the repertoire of phosphotylated

The

residues

carboxy-terminal

to the

receptor

SH2-binding

phosphotyrosines

sites with which the signalling protein can interact. We shall review here recent findings that have shed light on how SH2 domains achieve specificity while recognizing short phophotyrosine-containing peptides, the determination of the structure and specificity of 913 domains, and the ways in which these two types of protein modules mediate interactions between the many proteins involved in the transduction of signals from the cell surface.

Specificity

in binding

phosphotyrosine

of SH2 domains residues

to

SH2-binding sites have been delined using several complementary techniques. Initial efforts took the empiri cal approach of identifying receptor autophosphorylation sites responsible for association with specific signailing proteins. Subsequently, short phosphotyrosinecontaining peptides of identical sequence to these autophosphotylation sites were found to bind to the relevant SH2containing proteins with high al%nity. These studies led to the identification of a consensus binding site for the two SH2 domains of PI3kinase, pY(M/V)(D/E/P)M, in which the methionine residue in the third position ( + 3) relative to the tyrosine seemed particularly important [15,16]. The SH2 domains of

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~85 bind to phosphorylated peptides containing this sequencewith very high affiity (dissociation constant Q = 0.3-3 rlM) [ 17,181. Taking advantage of these findings, Cantley and coworkers devised a scheme to investigate SW2 binding specificity using a library of synthetic peptides containing phosphotyrosine, in which the residues at the + 1, + 2 and + 3 positions relative to the phosphotyrosine were equally likely to be any amino acid except tryptophan or cysteine (161. This degenerate peptide library was then chromatographed on columns made up of immobilized, bacterially-expressed SH2 domains from different signalling proteins. In this assay, the SH2 domains of the p85 subunit of PW-kinase selected peptides with residues at the + 1 to + 3 positions similar to those seen in physiological PIS-kinase-binding protein sequences, suggesting that this approach can yield physiologically relevant data. Notably, only methionine was selected at the + 3 position by the SH2 domains of ~85. In addition, the more carboxyterminal of the two SH2 domains of PLC-yl selected a sequence similar to the known PLC-yl-binding sites - pY(L/I)IP, [8,19] on the PPDGF and EGF receptors, whereas the specificity of the more amino-terminal SH2 domain of PLCyl resembled the PLC-yl-binding site on the fibroblast growth factor (FGF) receptor [20]. Application of this new technique to SH2 domains for which the binding specificity is not known generally resulted in the preferential selection of defined amino acids at one or more of the f 1, + 2 or + 3 sites. Some SHZ domains, such as those from ~85, show the most selectivity at the + 1 and + 3 positions. However, the SH2 domain from the nematode protein Sem-5 an ‘adaptor’ protein made up of only SH2 and SH3 domains, which is the homologue of the mammalian Grb2 protein - showed a very strong preference for asparagine at the + 2 position but less discrimination at the + 1 and -t- 3 positions. This is consistent with the identification of asparagine at the + 2 position of sites on the EGF receptor, insulin receptor substrate protein IRS1 and SHC proteins, all of which are known to bind to the SH2 domain of Grb2 ([21] ; J McGlade, unpublished observations). It is important to note that, in this assay, SH2 domains frequently do not bind exclusively to peptides containing a single amino acid at a particular position; hence, a particular SH2 domain can potentially bind to a range of distinct sites, presumably with different affinities. This variability in the sites bound by a particular SH2 domain is strikingly demonstrated by the SH2 domain of c-Src, which is predicted by affinity chromatography to have an optimal binding sequence of pYEE1, and indeed binds with high affinity (& = 4nM) to such a peptide [ 161. However, the physiological binding site on the PDGF receptor for the SH2 domain of c-Src appears to be pYI(p)YV, in which both Y579 and Y581 are phosphorylation sites [ 111. Furthermore, both genetic and biochemical evidence suggests that the SH2

domain of c-Src interacts with an inhibitory tyrosine phosphorylation site - Y527 in the sequence context PQYQPDE -within the carboxyterminal tail of c-Src itself. When phosphorylated, this inhibitory tyrosine contributes to the repression of c-Src tyrosine kinase activity [22], The interaction between pY527 and the SH2 domain appears to be rather weak, and may occur through an intramolecular interaction within the intact protein, rather than between neighbouring c-Src molecules. This interaction, and hence the inhibition of c-Src tyrosine kinase activity, may then be reversed by dephosphorylation of Y527 or by engagement of the SH2 domain by a phosphotyrosine residue that binds with a higher a&&y. As SH2 domains may therefore bind distinct sites with differing affinities in duo, the sites identified using the degenerate peptide library should only be taken as guides in the identification of natural SH2-binding sites. As a corollary, it seems likely that phosphorylated residues on some RTKs will have the potential to bind to more than one SH2-containing protein. The successes obtained using alhnity chromatography to identify SH2-binding peptides indicate that SH2 domains must have independent binding sites for phosphotyrosine and the three adjacent residues, as an obligatory combination of residues in the + 1 to + 3 positions would not have yielded detectable binding partners in the assay used. This hypothesis has been directly tested and confirmed by structural analysis of the SH2 domains of c-Src and the related kinase Ick bound with high allin@ to a peptide with the motif pYEE1. The phosphotyrosine binding pocket of these SH2 domains is formed by highly conserved, primarily basic residues that contact the phosphate oxygens or the electron density of the tyrosine ring [23-251. A second pocket, lined principally by hydrophobic residues, encompasses the isoleucine at the + 3 position, The binding sites for the residues at the + 1 and + 2 positions involve charged amino acids on the surface of the SH2 domain. As anticipated, the residues of the SH2 do main involved in recognition of amino acids at the + 1 to + 3 sites are rather variable between proteins, potentially accounting for the individual binding specificities of distinct SH2 domains. Knowledge of the SH2-domain residues involved in ligand-binding provides another means to predict which amino acid sequence a particular SH2 domain will bind. It remains possible that additional residues either amino-terminal or carboxy terminal to the four amino-acid binding sites described above are also involved in recognition by SH2 domains ([ 19,20,26] ; R Nishimura, W Li, A Kashishian, A Mondino, M Zhou, J Cooper and J Schlessinger, unpublished data),

Structure

and specificity

of SH3 domains

Many of the proteins involved in the transmission of signals from receptor protein-tyrosine kinases contain,

SH2

and

SH3

REVIEW

domains

in addition to SH2 domains, another conserved motif of 50-75 residues, the SH3 domain [1,3,4,27]. The two types of domain are frequently found within a single protein, although certain proteins contain only SH3 or SH2 domains. Moreover, the location of the SH3 domains within their host protein varies. Proteins with enzymatic activities, such as c-Src, PLCyl or Ras-GAP, contain only a single SH3 domain, but the adaptor proteins Crk and Grb2, which lack catalytic sequences, contain two SH3 domains, while the adaptor protein Nck contains three SH3 domains [ 1,3,4]. SH3 domains have also been found in cytoskeletdl proteins, such as myosinlB, fodrin and the yeast actin-binding protein ABP-1 [ 28,291, Although the physiological role of SH2 domains is relatively well understood, much less is known about the biological function of SH3 domains. The current view is that SH3 domains function, in part, as protein-binding modules, and that they are involved in linking signals transmitted from the cell surface by protein-tyrosine kinases to ‘downstream’ effector proteins. It has been shown that deletion of the SH3 domains of the c-Src or c-Abl tyrosine kinases leads to activation of their oncogenic capacity, presumably reflecting a loss of regulation of kinase activity and interaction with proteins [30-321. It is therefore thought that the SH3 domains of these cytoplasmic tyrosine kinases have a regulatory role, mediating interactions between the kinases and proteins with regulatory functions. This idea is consistent with the identification of a protein, termed 3BP1, that binds in z&-o to the SH3 domain of the cvoplasmic kinase c-Abl and that also has a region with sequence homology to the GAP for the Ras-related GTPase, Rho [33]. A recent study shows that the SH3 domains of PLC-yl and Grb2 are responsible for the intracellular targeting of these proteins to the microfilament network and to membrane ruffles, respectively 1341. In addition, the SF13 domain of v-Src binds to 1’6kinase, an enzyme implicated in the regulation of protein traffic 1351. It is therefore possible that, like SH2 domains, SH3 domains may be involved in targeting signalling proteins to their site of action in the plasma membrane or other subcellu~dr compartments, as well as in the regulation of protein movement within the cell. The crystal structure of the %I3 domain of spectrin, and the solution structures of the SH3 domains of cSrc, PLC-)/l and 1’13.kinase subunit ~85, have recently been described (see Fig, 2). In spite of relatively lim ited sequence similarity, the overall structures of these SH3 domains are very similar [3&40]. The amino- and carboxyterminal residues are in close proximity to each other in all four structures, indicating that SI-I3 domains are independent structural modules, which can be inserted at different locations within a protein. The SH3 structures contain between five and eight p strands, which form a barrel-like structure, The conserved aliphatic and aromatic residues in all four SH3

structures form a hydrophobic pocketi and conserved carboxylic amino acids are located in loops adjacent to the pocket. The SH3 domains of ~85 contain an additional 15 amino-acid insert containing three short helices [ 9,401. A preliminary analysis, by nuclear magnetic resonance (NMR), of the binding of a short proline-rich peptide to the SH3 domain of C-SK is consistent with the idea that the conserved hydrophobic pocket in all four SH3 domains may function as a binding site for cellular ligands (Fig. 2) [ 37,401. The variations between proteins in amino-acid sequence within the hydrophobic pocket and surrounding loops may constitute the structural basis of the binding specificity of the various SH3 domains for their putative ligands.

Fig. 2. Model for the bindq of a proline-nch peptide to an SH3 domain. The SH3-binding peptide APTMPPPLPPNS, generated by molecular modeling CC Cish, personal communication) and illustrated for clarity as a gold ribbon, is shown docked along one surface of the c-Src SH3 domain. The NMR solution structure of the c-Src SH? domain is displayed as a red ribbon, with green coloring used to hIghlight rcgons that undergo chemical shift changes upon peptlde binding and arc therefore likely to be Involved in ligand-binding (NMR data used in the modelllng was taken from 137,401). Note the modular structure of the SH3 domaln.

SH2 and SH3 domains of Crb2 link Ras-activator SOS to tyrosine kinases

The first insight into the nature of a potential bindingsite for SH3 domains came from the cloning of the 3UP 1 protein, achieved using the SH3 domain of the kinase c-Abl as a specific probe with which to screen expression libraries [33]. It was demonstrated that short, proline-rich peptides, derived from 3UPl or from

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other proteins, are able to bind to the SH3 domain of CAbl. Similar proline-rich sequences have been identified in other proteins. One such protein, SOS(Son-of-sevenless), acts as a guanine-nucleotide releasing factor for the Ras GTPase; SOSwas initially identified in a genetic screen in Drosophila, as a component of a signalling pathway that is initiated by RTKs l41-441. Detailed genetic analysis in both the nematode Caenorhabditis elegans and the fruit fly Drosophila have identified the gene products that are involved, in both organisms, in a signalling pathway leading from receptors at the cell surface to the activation of Ras and thence to downstream protein-serine/threonine kinases that control cellular differentiation [ 41,42,4&48], These genetic analyses revealed that the main components of this pathway are highly conserved between species, in each case including a receptor tyrosine kinase, a small adaptor protein composed of SH2 and SH3 domains (Sem-5 in C: elegans), a guanine-nucleotide releasing factor for Ras (SOSin Drosophila), and the Ras protein itself (Table 1). Both human [49] and Drosophila [48] adaptor proteins homologous to Sem-5 - Grb2 and Drk, respectively - have been identified: cDNAs were cloned using the phosphotylated tail of the EGF receptor as a probe to screen expression libraries. Both human Grb2 and Drosophila Drk proteins are able to rescue sem5 loss-of-function mutations in C:elegans (Stem J, Marengere LEM, Daly RJ, Lowenstein EJ, Kokel M, Batzer A, Oliver P, Pawson T, Schlessinger J, unpublished data). These studies indicate that the main components of the Ras signalling pathway are conserved in worms, flies and mammals, in terms of both structure and function (Table 1). Biochemical studies in mammalian cells provide a consistent and complementary picture to the conclusions of the genetic analyses of C elegans and Drosophila. It has been shown that human SOS possesses guaninenucleotide releasing activity for H-Ras [44] and that over-expression of Grb2 enhances this activity [50]. Table

1. Similarites

in the

Ras signalling

pathways C.

of different

eiegans

Tyrosine kinase receptor

Let-23

4 SH3-SH2-SH3 adaptor protein

I Sem-5

4 Guanine-nucleotide exchange factor 4 Ras CTPase

I ?

I

Ras

Grb2 binds, through its SH3 domains, to at least two proline-rich sequences in the carboxy-terminal tail of SOS ([48,51-541; and M Stern, LEM Marengere, RJ Daly, EJ Lowenstein, M Kokel, A Batzer, P Oliver, T Pawson and J Schlessinger, unpublished data). Moreover, amino-acid substitutions in the SH3 domains of Grb2, corresponding to those found in sem5 loss-offunction mutations, reduce or abolish binding of Grb2 to SOS.This indicates that a stable association between Grb2 and SOSrequires interactions of SOSwith both of the SH3 domains of Grb2. Furthermore, a short syn thetic peptide, with the sequence PPPVPPRRR, blocks the binding of Grb2 to SOS,suggesting that this prolinerich sequence corresponds to the ligand of at least one of the SH3 domains of Grb2. Other proline-rich peptides, with sequences modelled on that of the tail of SOSalso competed for Grb2 binding, suggesting that they may correspond to additional Grb2 binding sites [51,52]. In mammalian cells, EGF-receptor activation results in tyrosine autophosphorylation, creating a binding-site for the SH2 domain of Grb2 at ~I068 of the carboxy terminal tail (Fig. 1; [ 11 and A Batzer and J Schlessinger, unpublished observations). The Grb2-Sos complex binds to the EGF receptor, thus recruiting SOSto the plasma membrane where Ras is located (Fig. 3a). There is also evidence that stimulation by EGF, PDGF, FGF or insulin leads to phosphorylation of SOS on serine and threonine residues. However, no change in the guanine-nucleotide releasing activity of SOSis detected after EGF stimulation. It appears, therefore, that the translocation of SOSto the plasma membrane may enable activation of Ras simply by increasing its local concentration, rather than by altering SOSactivity. Additional proteins are involved in the control of Ras signalling when it is stimulated by other receptor and non-receptor tyrosine kinases. Cellular transformation with the oncogenic kinase v-Src leads to tyrosine phosphorylation of She, a protein that species. Drosphiia

Sevenless I Drk

I I

Mammal ECF

receptor I Crb2

I I

so5

mSos I, mSos2

Ra!,

Ras

In each case, an activated receptor tyrosine kinase is proposed to bind to the SH2 domain of an turn, interacts through its SH3 domain with a guanine-nucleotide releasing factor to stimulate the Active Ras then activates a series of serine/threonine-specific and dual specificity protein kinases, phosphoryiation of transcription factors. The homologue of SOS in C. elegans may be encoded by

adaptor protein which, in GTPase activity of Ras. leading ultimately to the the Let-347 gene. 0 1993 Current

Biology

SH2

and

SH3

REVIEW

domains

1 (b)

I

SH2

domain

SH3

domain

n

Tyrosine

@@

Kinase

residue &main

=

Prolinr-rich

0

Cdc25

sqeuence similarity 0 1993 Current Biology

Fig. 3. Crb2

and SOS couple tyrosine binding of the SH2 domain of Crb2. Crb2, is thereby brought close to the factor Cdc25, then activates Ras by within the sequence YVNV, leading is proposed to stimulate SOS-mediated

kinases to Ras activation. (a) ECF-induced autophosphorylation at Y1068 of the ECF receptor induces SOS, which is bound through proline-rich motifs in its carboxy-terminal tail to the SH3 domains of membrane. The amino-terminal domain of SOS, which is similar to the guanine-nucleotide releasing exchange of GDP for CTP. (b) The oncogenic kinase Arc phosphorylates the She protein at Y317, to an association beteen the GrbZ%os complex and tyrosine-phosphorylated She. This interaction guanine-nucleotide exchange on Ras, although the mechanism of SOS activation is unclear.

contains one SH2 domain [55]. Over-expression of She itself can induce transformation of fibroblasts and neuronal differentiation of PC12 phaeochromocytoma cells [ 55,561. Moreover, She-induced differentiation of PC12 cells is prevented by expression of a dominant inhibitory Ras protein, indicating that She acts in the control of Ras signalling. Indeed in v-Src~transformed rodent cells, She is phosphorylated on tyrosine and is found associated with the Grb2-Sos complex through the SH2 domain of Grb2 (Fig. 3b). A similar complex of She, Grb2 and SOS is detected in cells stimulated by insulin, FGF and other growth factors [21,57,58]. Moreover, insulin induces the formation of a complex between the tyrosine-phosphoqlated protein IRS1 and Grb2-Sos [58]. Hence, stimulation with insulin leads to association of the Grb2-Sos complex with two tyrosine phosphorylated proteins, IRS1 and She. While the importance of She in signalling by insulin is currently unknown, the significance of IRS1 as an SH2-docking protein is well established.

SH’L-docking

proteins

In some instances, tyrosine kinases may employ an intermediate SH2-docking protein, rather than binding directly to SH2containing signalling proteins. One example of such a docking protein is provided by IRSI, a protein that is phosphorylated on multiple tyrosine residues in insulin-stimulated cells [ 591. Phosphorylated IRS1 binds to the SH2 domains of PW-kinase through pYMXM motifs [13,60], to the SH2 domains of Grb2 through a pYVN1 site 1211, to Nck (E Skolnik and J Schlessinger, unpublished observations) and to the Syp tyrosine phosphatase (also called SH-PTP2 or PTPID) [6I] - and no doubt to other, as yet uniden tified, signalling proteins (Fig. 4). Indeed, a single IRS1 molecule can be found in association with both Grb2 and PW-kinase. The use of a separate docking protein, with more potential SH2-binding sites than found on any single RTK, may be a means by which the insulin

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receptor couples to many, diverse intracellular nalling pathways.

sig

will require intensive investigation. Although a considerable amount is known about the biochemistry and structure of SH2/SH3-containing signalling proteins, much less is known about their biological functions. In a few cases there are genetic clues - the invertebrate adaptor proteins Sem-5 and Drk are required for the development of specific cells and in the formation of an adult organism, apparently through their involvement in signalling pathways that also include Ras. The SH2-containing protein-tyrosine phosphatase PTPlC is mutated in Motheaten mutant mice, which suffer from a deficiency in production of B cells and T cells and from an autoimmune disorder, implicating PTPlC in the de velopment and regulation of lymphoid cells [ 641. A re lated SH2-containing tyrosine phosphatase, Corkscrew, is required for embryonic development in Dmsopbib [ 651. However, further genetic analyses in both vertebrates and invertebrates are required to unravel the cellular phenotypes controlled by signalling proteins with SH2 and SH3 domains. Acknowle&emenlx of the SH3 domain

We thank Gerald Gish for the computer and the ligand seen in Figure 2

modeling

References 1. Fig. 4. The proteins.

IRS1 docking

protein

binds

multiple

A variation on this theme is observed during the activation of lymphoid cells by engagement of their surface antigen receptors, and in the stimulation of mast cells by occupancy of the high affinity IgE (immunoglobulin) receptor [62]. Activation of these cells induces the rapid stimulation, probably by receptor dimerization, of cytoplasmic tails of accessory proteins of the relevant antigen receptor. These accessory proteins such as the [, E, y and 6 chains of the T-cell receptor each contain one or more copies of a conserved motif, characterized by two tyrosine residues separated by ten amino acids, the tyrosine-phosphotylzion of which is proposed to provide binding sites for the SH2 domains of signalling proteins, which, in turn, stimulate the biochemical pathways leading to T-cell or B-cell activation, or mast cell degranulation.

2.

tyrosine

KOCII

kinases.

CA, ANIXRSON

Neuron

factor signaling 1992, 9.383~391.

11, IMORAN MF,

by recep-

El.1.E C, PAWSON T: SH2

and SH3 domains: toplasmic signaling 3. i.

elements that control interactions of cymolecules. Science 1991. 252, 66%674. PAWSON T, GISH GD: SH2 and SH3 domains: from structure to function. Cell 1992,72:35%362. MAYER BJ, BALXIMORE D: Signaling through SH2 and SH3 domains. Trends Cell 8101 1993, 3:%13.

5.

KN.l.4lisKks

6.

activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor !3 subunit. Mol Cell Bid 12:253625/i/t. FANTI. WJ, ESCOfVDO JA, h4AKTlN GA, ‘1‘1IRCK CW, IXL KOSAIUO RI, MCCORMICR I;, WILIIAMS LT: Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell 1992, 69:413--123.

7/.

A, KWHISHL~N

A, COOPEK JA, VAJXJS M.

GTPase-

MARC;OUS B, Li N, KOCH A, MOHA,UMADI M, H[IRWITZ I>, Uluucf ZILBEKSTEIN A, PAWSON T, SCHLESSIN(;ER J, The tyrosine

I

A,

phosphorylated carboxy terminus a binding site for tiAP and PLCy, 8.

9

Conclusion

Recent work has yielded a clear picture of the structural and biochemical functions of SH2 domains. Our understanding of SH3 domains is at an earlier stage, but the determination of the structures of several SH3 domains, the identification of an SH3-binding motif, and the analysis of at least one physiological SH3-mediated interaction - betieen Grb2 and SOS- presages rapid progress in this area. The details of the network of protein-protein interactions that are regulated by SH2 and SH3 domains appear to be extremely complex, and

SCHLESSINGEK J, IJLLKICH A: Growth

tor

SH2-containing

10.

is

of two C-terminal autophosphorylation sites in the PDGF !.% receptor: involvement in the interaction with phospholipase C-y. 1AWO .I 1992 11:3911-3919. VALIUS M, BAZENI:‘~ C, KAZLWSK.V A: Tyrosines 102 1 and 1009 are phosphorylation sites in the carboxy terminus of the platelet derived growth factor receptor p subunit and are required for binding of phospholipase Cyand a Wkilodalton protein, respectively. Mel Cell Biol 1993, 13:133-143. K.~ZIAIISKAS A, FEN(;

G S, P.~WSON T, VALILIS M: The

64

Kd

that associates with the PDGF receptor subunit via 1009 is the SH2 containing phosphotyrosine phosSyp/SH-PTP2/FTPID/SH-mP3. IVoc NatI Acmd ,Sci

USA 1993, in press. MORI SM, RONNSTRAND I., YOKOTE K, ENGS~ROM A. COIIRTNKIXF SA. CIAESSON-WELSH I., HEIDIN (X-1: Identification of two jux-

tamembrane autophosphorylation tor: involvement in the interaction kinases. .EMBC) .I 1993, in press. 12.

EGF receptor 1990,9:4575”t380.

RONNX’IXAND L, MORI S, ARIUDSSON A-K, ERIKSSON A, WEKNSTED~ C, HELLMAN II, CL~ESSON-WELSII L, HELDIN, C-H: Identification

protein tyrosine phatase 11.

of the LMBOJ

sites in the PDGF with Src family

KIM HK, KIM JW, ZILEERST~IN A, MAR(;OLIS SCHLFSSINGBR J. RHEE SG: PDGF stimulation

B,

of

P-receptyrosine KIM

IG,

inosjtol

SH2 -

and

SH3

domains

REVIEW

phospholipid hydrolysis tyrosine residues 783 13.

BACKER JM,

MARS

requires PLC-~1 phosphorylation and 1254. Cell 1991, 65:435-441.

MG

JR, SHOELSON

SE, CHIN

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34.

R.I L!T AL: SH2 domains recognize specific phosphopeptide sequences. Cell 1993. 72~767-778. FFIUER S, Zirorl M, Hu P, URENA J, UWCII A, CHAUDH~~U M, WHITI: M. SHOEISON SE, SCHIESSINGER J: SH2 domains exhibit high affinity binding to tyrosine phosphorylated peptides yet also exhibit rapid dissociation and exchange. ~Wol Cell mid 1993 13:144~-1455.

2.3.

25

26

27

28

29

30.

CICCHETT~

THOMAS

SM, BRUGGE

on the variants

functional Of C-SK

P, MAVER BJ, THIEL

JS: Effects

activities MJ! cell

of Rio/

G, BALTIMOR!Z D: Identificatiotl

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