Signalling by the p60c-src family of protein—tyrosine kinases

~

Int. J. Biochem. Cell Biol. Vol. 27, No. 6, pp. 551-563, 1995

Pergamon

1357-2725(95)00024-0

Copyright ~ 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1357-2725/95 $29.00 + 0.00

MINIREVIEW

Signalling by the p60 c-src Family of Protein-Tyrosine Kinases P. K E F A L A S , I T. R. P. B R O W N ,

P. M. B R I C K E L L l*

1The Medical Molecular Biology Unit, Department o f Molecular Pathology', and :Department o f Immunology, University College London Medical School, The Windeyer Building, Cleveland Street, London W I P 6DB, U.K. The c-arc gene family has nine known members (blk, c-fgr, fyn, hck, lck, lyn, c-arc, c-yes and yrk), each encoding a cytoplasmic protein-tyrosine kinase (PTK) believed to be involved in signal transduction. The c-arc PTKs contain three domains (SH1, SH2 and SH3) that are found in many other signalfing proteins. The SH1 domain has PTK activity, whilst the SH2 and SH3 domains are involved in mediating protein-protein interactions by binding to phosphotyrosine-containing and proline-rich motifs, respectively. The expression patterns of the c-arc PTKs suggest that they function in a broad range of biological situations, in many cases regulating the behaviour of terminally-differentiated, post-mitotic cell types. Targeted disruption of members of the c-arc family in transgenic mice has confirmed important roles for p56I¢kand p59 fy~r~in T-lymphocyte maturation and activation, but has also revealed unexpected roles for p60~'s'cin bone maintenance and for p59 sy"{n~in learning and memory. There is increasingly detailed information about the biochemical nature of the signalling pathways in which the c-arc PTKs operate and about the other signalling proteins with which they interact. The c-arc PTKs can associate with activated receptor PTKs, including the receptors for platelet-derived growth factor and epidermal growth factor, by means of SH2-phosphotyrosine binding. The c-arc PTKs also associate with transmembrane proteins that lack PTK activity, frequently by means of interactions involving their unique amino-terminal sequences. Keywords: c-arc Proto-oncogene

Protein-tyrosine kinase

Signal transduction

lntracellular signalling

Int. J. Biochem. Cell Biol. (1995) 27, 551-563

members (Table 1) named blk, c-fgr, fyn, hck, lck, lyn, c-yes (for review see Brickell, 1992) The c-sre family of genes encodes cyto- and the recently discovered y r k (Sudol et al., plasmic protein tyrosine kinases (PTKs) that 1993). are believed to transduce and amplify exoThe past few years have witnessed significant genous signals received by cells. The first breakthroughs in our understanding of how the PTK to be discovered was the product of the catalytic activities of the c-sre PTKs are reguRous sarcoma virus (RSV) oncogene, p60 ~-~r" lated, of the identities of the regulatory mol(for review see Jove and Hanafusa, 1987) and ecules that control their activity and of their it was later shown that the product of the role in transducing and amplifying exogenous cellular proto-oncogene c-are also has tyrosine signals in a wide variety of biological situations. kinase activity (for review see Wilks, 1990). In this article we will first discuss our current The c-arc gene proved to be the prototype of knowledge of the structure of the e-src PTKs. a gene family that has at least eight other We will then focus on clues to the biological roles of these proteins that have come from studies of their normal expression patterns and *To whom correspondence should be addressed. Received 23 May 1994; accepted 2 March 1995. from their targeted disruption ("knock-out") INTRODUCTION

551

552

P. Kefalas et al. Table 1. The c-src gene family Gene Protein product(s) '/blk p5&tk c -fgr p58~t~ fyn p59I~"IB/,p59/~"ln hck p59h'k lck p56h* lyn p53t~, p56TM c-src p60"~r"(3 isoforms) c-yes p62"-~'~' yrk p6@'k Genes with the prefix "c-'" were originally discovered as retroviral oncogenes.

other in the SH3 domain (Pyper and Bolen, 1990), two splice variants of p59 ty", named p59 ~y"(B) and p59¢y"(n, which differ from each other in the catalytic domain (Cooke and Perlmutter, 1989) and two splice variants of the lyn gene product, named p53 ty"and p56 ~v",which differ from each other in the unique aminoterminal region (Stanley et al., 1991). The unique a m i n o - t e r m i n a l region

in transgenic mice. Finally, we will describe work that has identified components of the signalling pathways in which the c-src PTKs are involved.

S T R U C T U R E OF T H E c - s r c P R O T E I N - T Y R O S I N E K1NASES

General structural f e a t u r e s

The amino acid sequences of the proteins encoded by the s-src gene family have been predicted from the nucleotide sequences of the corresponding cDNA clones. These analyses show that the proteins share the basic architecture illustrated for p60 '-~" in Fig. 1. The proteins have unique amino-terminal sequences ranging between 60 and 90 amino acids in length, and share 60 to 75% amino acid identity over the rest of their length. The conserved region can be subdivided into three domains, homologues of which are found in a range of other proteins. These are the SH2 and SH3 domains (for Src Homology 2 and 3) and the catalytic (SH1) domain. The proteins also contain two conserved tyrosine residues that are themselves targets of phosphorylation. One of these (Tyr416 in p60 c-~") lies within the catalytic domain and can be autophosphorylated. The other (Tyr-527 in p60 os'c) lies to the carboxyl-terminal side of the catalytic domain and is involved in regulating its PTK activity. Several members of the c-src gene family encode more than one protein product, as a result of alternative splicing. There are three splice variants of p60 "~', which differ from each Gly-2 NH2,] unique region

While the amino-terminal region is in general highly diverged, all family members retain a glycine residue at position 2. In p60 ...... and a number of other family members, this residue has been shown to be the site of the posttranslational addition of the 14-carbon saturated fatty acid myristic acid (for reviews see Resh, 1990; Gordon et al., 1991). It is likely that all of the family members are myristoylated. Myristoylation is necessary, though not sufficient, for the localization of the proteins to the inner surface of the plasma membrane and other cytoplasmic membranes, and the localization of these proteins to the inner surface of cell membranes appears to be a requirement for their function. The catalytic ( S H 1 ) domain

The catalytic domain comprises approx. 260 amino acids (residues 260-516 in p60 ..... ) and catalyses the phosphorylation of proteins on tyrosine residues. Extensive analyses of the catalytic domains of a range of PTKs have shown that these are not uniformly conserved but rather consist of alternating regions of high and low conservation (for review see Hanks et al., 1988). The S H 2 and S H 3 domains

The SH3 domain of p60 "~'~ extends from residue 86 to residue 136, while the SH2 domain extends from residue 137 to residue 241. Sequences related to SH2 or SH3 have been identified in a growing number of proteins, as shown in Fig. 2 (for reviews see Brickell, 1992; Fry et al., 1993; Mayer and Baltimore, 1993; Pawson and Schlessinger, 1993). Some of these, including p150 cabl, p98 'fps, pS0 c~* and Zap-70 (Chan et al., 1992), have PTK activity. Others, Tyr-416

SH3

SH2

catalytic domain

Fig. l. Structural features of p60c-s''.

Tyr-527

553

Signalling by the p60c-*'' family of protein-tyrosine kinases p60 c-src

Zap-70 p50 csk p150 c-abl

p98 c-fps PI 3-kinase p85 PLC¥1

• ,,,

//

I

I

t

H

,

t

,,-

/,/-

ras GAP

a-spectrin

////

1 1

//~_._._

Fig. 2. Structures of some of the proteins that contain SH1 domains (m), SH2 domains ([~) or SH3 domains ( I ) .

including the p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase), phospholipase C-71 (PLC-y I) and ras GTPase-activating protein (ras GAP) lack PTK activity but have other enzyme activities. Yet others, such as Grb-2 and ~-spectrin, have no enzyme activity. FROM STRUCTURE TO FUNCTION

The c a r b o x y l - t e r m i n a l O'rosine residue

As noted above, each member of the p60 '-~'" family contains a carboxyl-terminal tyrosine residue (tyr-527 in p60 "-s~') which is a major site of phosphorylation in vh, o. A number of pieces of evidence have demonstrated that the phosphorylation state of this residue regulates the tyrosine kinase activity of the protein, thus, p60 ..... is inactive when Tyr-527 is phosphorylated and active when it is dephosphorylated (for review see Hunter, 1987). Nada et al. (1991) identified a PTK that can phosphorylate Tyr-527 of p60 ''~"~. This protein° named p50 ~k, resembles the p60 '~" PTKs in some respects (Fig. 2) but differs from them in that it has no amino-terminal membrane attachment motif, no autophosphorylated tyrosine residue and no carboxyl-terminal regulatory tyrosine residue. The p50 "sk PTK can also phosphorylate the carboxyl-terminal tyrosine residues of other p60 c-~r"family members and so down-regulate their activity (Bergman et al., 1992; Okada et al., 1991). However, it remains

possible that a family of p50'~k-like PTKs is responsible for negatively regulating p6W s'c family members in vivo. Activation of the p60 ~~" PTKs by dephosphorylation of their carboxyl-terminal tyrosine residues is thought to be catalysed by one or more phosphotyrosine phosphatases (PTPases). In support of this, the transmembrane glycoprotein CD45, which has a cytoplasmic PTPase domain, has been shown to activate p5U ~k (for reviews see Mustelin and Burn, 1993; Brickell, 1992) and p59 ~'" (Shiroo et al., 1992) in T-lymphocytes. Other, as yet unidentified, PTPases presumably activate p60 ...... family members in cell types that do not express CD45. One candidate for such a role is the transmembrane PTPase PTP~ (Zheng et al., 1992). The emerging picture is therefore that the PTK activity of a particular p60 '~'' family member in a particular cell type at a particular time is regulated by a balance between the activities of a p5@~k-like PTK and a PTPase. The dephosphorylation of Tyr-527 in p60' .... and the consequent increase in PTK activity is accompanied by phosphorylation of Tyr-416, which appears to be the result of autophosphorylation (for review see Brickell, 1992). Other members of the p60 ..... family have an equivalent autophosphorylated tyrosine residue, and mutation of this residue results in a decrease in PTK activity. It has therefore been suggested that autophosphorylation may result in

554

P. Kefalas et al.

conformational changes that allow better access of other substrates to the catalytic site (Hanks et al., 1988). Complex patterns of phosphorylation, involving serine, threonine, and tyrosine residues, have been identified in the aminoterminal regions of p60 ..... members, but the significance of these phosphorylation events in vivo is not clear (for a review see Brickell, 1992). S H 2 and S H 3 domains

Considerable advances in understanding the functions of SH2 and SH3 domains have been made in the last few years. These advances resulted from the finding that the sequences encoding SH2 SH3 domains could readily be expressed in E. coli, yielding isolated protein domains that folded correctly and retained their function. Proteins that bound to SH2 or SH3 domains have been identified by incubating western blots of cell extracts with labelled domains (Mayer et al., 1991), by passing cell extracts down affinity columns carrying bound domains (Gout et al., 1993) and by screening cDNA expression libraries with labelled domains (Cichetti et al., 1992). SH2 domains have been found to bind with high affinity to selected phosphotyrosinecontaining proteins. Whilst phosphotyrosine is an essential component of the sites to which SH2 domains bind, the surrounding amino acids are important in determining the affinity of binding. Thus, particular SH2 domains show specificity for particular phosphotyrosinecontaining peptides. For example, the consensus high-affinity binding site sequence for the SH2 domain in the p85 subunit of PI3-kinase is YxxM (where x can be a wide range of residues), whilst for the SH2 domain in Grb-2 it is YxNx (for reviews see Fry et al., 1993; Pawson and Schlessinger, 1993). As discussed above (Fig. 2), SH2 domains are found in a wide range of signalling proteins. There is growing evidence that as a result they play critical roles in PTKdependent pathways, by mediating the assembly of multimeric protein complexes through SH2phosphotyrosine interactions (for reviews see Fry et al., 1993; Mayer and Baltimore, 1993; Pawson and Schlessinger, 1993). The observation that SH2 domains bind to phosphotyrosine-containing proteins led Matsuda et al. (1990) to suggest a mechanism for the inhibition of p60 ' ' ' PTK activity by phosphorylation of Tyr-527. They proposed that intramolecular binding of the SH2 domain

a)

b)

.,

~ active

mTyr-527 Fig. 3. Model for the regulation of p60 c-'rc PTK activity. (a) Inactive p60 '-~' in which there is intramolecular binding of the SH2 domain to phosphorylated Tyr-527 (O--). (b) Activation of the catalytic domain by dephosphorylation of Tyr-527. SHI domain, II; SH2 domain, D; SH3 domain, m; myristic acid, k~.

to phosphorylated Tyr-527 results in a protein conformation that inhibits the activity of the catalytic domain, possibly by preventing the access of substrates (Fig. 3a). Dephosphorylation of Tyr-527 would result in unfolding of the p60 ...... molecule, with consequent activation of its PTK activity towards its substrates (Fig. 3b). Liu et al. (1993) subsequently showed that the SH2 domain of p60 "-~r' does indeed bind with moderate affinity to the carboxylterminal sequence that contains Tyr-527. Interestingly, there is a class of p60 c'r' SH2 domain mutants that exhibit increased PTK activity even when Tyr-527 is phosphorylated (O'Brien et al., 1990). These mutations presumably render the p60 c'r' SH2 domain incapable of binding to Tyr-527. In support of this idea, recent studies of the three-dimensional structure of SH2 domains suggest that some of these mutations would disrupt the overall structure of the SH2 domain whilst others would disrupt the phosphotyrosine-bindng site (Fry et al., 1993). SH3 domains recognize a proline-rich motif. For example, the provisional consensus sequence of the high-affinity binding site for the SH3 domain of p150 c~b¢ is xPxxPPPzxP, where x can be any amino acid and z can be any hydrophobic amino acid (for reviews see Fry et al., 1993; Pawson and Schlessinger, 1993; Cohen et al., 1995). Such sequences appear to mediate binding of SH3 domains to a range of proteins, including Sos (a guanine nucleotide exchange factor), dynamin (a GTPase), 3BP-1 (a GTPase-activating protein), the formins and

555

Signalling by the p60 "-'r' family of protein-tyrosine kinases one subtype of the muscarinic acetylcholine receptor (for review see Fry et al., 1993). As with SH2 domains, particular SH3 domains may have specificity for particular proline-rich sequences. Recently, the three-dimensional structures of several SH2 and SH3 domains, with and without complexed ligands, have been determined in solution by N M R spectroscopy or X-ray crystallography (for reviews see Pawson and Schlessinger, 1993; Cohen et el., 1995). These structures document the modular nature of the SH2 and SH3 domains and have defined important features of the specific interactions between these domains and their ligands. BIOLOGICAL ROLES OF THE c-src PROTEIN-TYROSINE KINASES

Expression p a t t e r n s

Members of the c-src gene family can be placed into two groups on the basis of their expression patterns (for review see Brickell, 1992). The f y n , c-src, c - y e s and y r k genes are expressed in a broad range of tissues and cell types, whilst expression of the blk, c-fgr, hck, lck and lyn genes is restricted to particular haematopoietic cell lineages, as shown in Table 2. Expression of the f y n gene has been found in fibroblasts, endothelial cells, lymphocytes, monocytes, T-lymphocytes, platelets and neurons in specific regions of the central nervous system, including the hippocampus. T-lymphocytes contain the p59 ~:~'~ splice variant, whilst the brain contains the p59 ~'n{B~splice variant. The c-src gene is widely expressed, but attention has focussed particularly on its expression in neurons of the central nervous system, in platelets and in osteoclasts (Horne et al., 1992). The splice variants of p60 ~-s" have different distributions in the central nervous system. The c - y e s gene is expressed in many tissues, including liver, lung, placenta, platelets, keratinocytes, parts of the brain, proximal tubule epithelial cells in the kidney and spermatid acrosomes.

Expression of the y r k gene has been observed in haematopoietic and neural tissue (Sudol et el., 1993). Knowledge of these expression patterns has prompted a great deal of speculation concerning the probably biological roles of the p60 ...... family members, and they have been proposed to participate in the regulation of diverse bilogical processes in diverse cell types. A common theme in the patterns of expression of all c-src gene family members is that high level expression is largely restricted to terminally differentiated, post-mitotic cells. It therefore seems unlikely that these proteins are primarily involved in signalling pathways that regulate cell division. Rather, they seem to be involved in signalling pathways that regulate the function of terminally differentiated cells. In a number of cases, roles in regulating the changes in cytoskeletal structure that accompany exocytosis, phagocytosis, the regulation of endosomal membranes (Kaplan et al., 1992) and other cellular activities have been proposed. T a r g e t e d disruption

Further clues to the function of the members of the c-src gene family have recently been obtained from their targeted disruption ("knock-out") in vivo using transgenic mice. Soriano et el. (1991) produced transgenic mice in which the c-src gene was nonfunctional. Mice homozygous for this mutation developed normally but died soon after birth. Surprisingly, in view of the amount of attention previously given to the expression of c-src in neurons and platelets, there were no obvious abnormalities in these cell types, although it is possible that subtle structural and/or functional deficits went undetected. However, the mice had severe osteopetrosis, in which a decreased rate of bone resorption leads to skeletal abnormalities. Bone was perhaps the one tissue that had not been previously associated with c-src, but it was subsequently shown that the e-src gene is normally expressed in osteoclasts (Horne et el., 1992). Further work showed that osteoclasts were present in the c - s r e ( - ) transgenic mice

Table 2. Members of the c-src gene family whose expression is restricted to hematopoietic lineages platelet monocyte granulocyte macrophage T-lymphocyte B-lymphocyte blk c -fgr hck lck l)'n

+ + ~

+ + +

-~ + +

+

+ +

+

+

(+) +

556

P. Kefalas et al.

but that they failed to form ruffled membrane borders, which are characteristic of actively resorbing osteoclasts, and failed to form normal resorption pits on dentine slices in culture (Boyce et al., 1992). These data suggest that p60 'st" may be involved in regulating cytoskeletal structure in osteoclasts, perhaps by phosphorylating cytoskeletal proteins (Boyce et al., 1992). The apparent normality of platelets from c - s r c ( - ) mice may be explained by the fact that a number of other family members, including 59~'n and p62 <''e', are also expressed in platelets. It is possible that these proteins can substitute functionally for the loss of p60 c-'' in platelets. In contrast, the J),n, lyn and c-yes genes appear to be unable to substitute functionally for the loss of c-src in osteoclasts, even though they are expressed in these cells (Home et aL, 1992). Expression studies and biochemical analyses had implicated p59 ~'"~n and p5& k in signalling during T-lymphocyte maturation and activation (see below). It was therefore gratifying that transgenic mice homozygous for disrupted fyn or lck genes had abnormal T-lymphocytes. In mice that lacked both p59 ~v"(~and p59 ~'"~s~(Stein et al., 1992) or that lacked p59 fy"(n but retained p59 cyn(B) (Appleby et al., 1992), the ability of T-lymphocytes to respond to a signal delivered to the T-cell antigen receptor (TCR was impaired, although the impairment was less marked in mature T-lymphocytes. This indicates that p 5 ~ ~"~n has a role in TCR signalling that is particularly crucial in immature cells. The l c k ( - ) mice had a different defect, in which T-lymphocytes were unable to mature beyond a specific stage in the thymus (Molina et al., 1992). A similar defect was seen in transgenic mice expressing high levels of a dominantnegative mutant of p56 ~c*, which lacked P T K activity (Levin et al., 1993). The loss of p56 t~k function in these two model systems appeared to arrest T-lymphocyte development at a point where thymocytes normally undergo a series of mitotic divisions that result in expansion and maturation, and p56 tCkmay normally have a role in controlling these divisions (Levin et al., 1993). These data indicate that p56 fyn(73and p56 ~Ckhave specific roles in T-lymphocytes that cannot be compensated for by each other, or by p62 'y~, which is also present in T-lymphocytes. The p59fy"(s~ P T K was known to be expressed by neurons in specific regions of the central nervous system, including the hippocampus, but there were no gross physical abnormalities of

the central nervous system in mice that lacked both p59 fyn(B)and p59 ty"(~, and no obvious functional abnormalities when the mice were left to themselves (Stein et al., 1992). However, further investigation revealed three subtle defects (Grant et al., 1992). First, the f y n ( - ) mice displayed impaired long-term potentiation (LTP). This is a process by which the strength of synaptic connections can be increased by a particular pattern of synaptic activity. Second, they had impaired spatial learning, as revealed by their inability to learn the solution to a water maze. Third, they had a subtle defect in the structure of the hippocampus, which is involved in the long-term storage of memories. On the basis of these results, Grant et al. (1992) proposed that the fyn gene is important in the development of the hippocampus and also has a role in the mature synapse, related to the induction of long term potentiation. The data also support the idea that the phenomenon of LTP is linked to learning and memory. Disruption of the c-src and c-yes genes, which are also expressed in the hippocampus, had no discernible effect on LTP, spatial learning or the structure of the hippocampus (Grant et al., 1992). These studies are instructive not only because they identify a novel role for fyn and illuminate understanding of learning and memory, but also because they illustrate the point that deficits that are inconsequential, and therefore hard to detect, in mice living in a laboratory might nevertheless be devastating in the wild. They demonstrate that subtle assays will often have to be applied to transgenic "knock-out" mice if an accurate picture of gene function is to be obtained. More recently, Yagi et al., (1993) generated another strain o f f y n ( - ) mice that lacked both p59 ~'"(s) and p59Iynl~. They found that whilst the homozygous offspring of heterozygous parents survived to adulthood, the homozygous offspring of homozygous parents died because of an inability to suckle. Interestingly, homozygous mothers were able to nurse homozygous pups if lactation was first activated by the suckling of heterozygous pups from another litter. Yagi et al., (1993) suggested that the homozygous f y n ( - ) mice were unable to perceive the initial pheromone stimulus for suckling and could only suckle once lactation had been stimulated by other pups. In support of this, they found that the f y n ( - ) mice had an abnormality of the modified glomerular complex in the olfactory bulb, which acts in

Signalling by the p60 '~" family of protein-tyrosine kinases

transmission of the suckling stimulus to the hippocampus. These mice also had significantly reduced amounts of myelin in their brains, compared to normal mice (Umemori et al., 1994), although the numbers of neurons in the brain appeared to be normal. Interestingly, p59¢y~B) was found to be physically associated with the large myelin-associated glycoprotein, which is thought to be involved in signal transduction during the initial stages of myelination (Umemori et al., 1994). It is not clear why t h e f y n ( - ) mice studied by Grant et al., (1992) and Yagi et al., (1993) have different phenotypes, although the two groups did use different strategies to disrupt the f y n gene. These "knock-out" experiments have shown that despite their wide tissue distribution, the f y n and c-src genes seem to have a crucial function in only a very limited range of cell types. It may be that the function they fulfill in other cell types cannot be detected by existing assays or that their expression in these cells is completely gratuitous (Erickson, 1993). Alternatively, the loss of their function in these cell types could be compensated for by other c-src family members. In this regard, it will be important to breed the different "knock-out" mice together and so to examine the phenotypes of mice who have lost different combinations of c-src family members. In any case, it is important to be clear that both t h e f y n and c-src genes appear to be crucial to the well-being of the organism, and this presumably accounts for their evolutionary conservation. SIGNALLING PATHWAYS INVOLVING PROTEIN-TYROSINE KINASES

c-src

The transduction of signals from cellsurface receptors involves extensive phosphorylation of intracellular proteins, particularly on their tyrosine residues. It is becoming clear that the c-src PTKs have multiple and overlapping roles in transducing signals in a wide variety of biological situations. It seems that family members are able to take part in several quite different types of interaction with regulatory molecules, and that this allows them to process signals registered by a number of different classes of cell-surface receptor. Moreover, family members appear to be able to activate a number of different signalling pathways in response to the signals that they receive.

557

In recent years, considerable progress has been made in identifying cell-surface receptors with which c-src PTKs interact. Such interactions fall into two classes. First, c-src PTKs can bind via their SH2 domains to phosphotyrosine residues on receptor PTKs, including the receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF), colony stimulating factor-I (CSF-1), insulin, and insulin-like growth factor-I (for review see Brickell, 1992). Second, c-src PTKs can associate with transmembrane proteins that lack PTK activity. Such associations do not appear to involve SH2 and SH3 domains. These two classes of interaction will now be discussed in detail. Binding o f p 6 0 ..... f a m i l y members to receptor P T K s by SH2-phosphotyrosine interactions

The p60''", p62 ''ye~ and p59 t~" PTKs are coexpressed in fibroblasts, where it has been proposed that they may have a role in regulating fibroblast growth and division by means of a functional interaction with the P D G F /% receptor. Binding of P D G F to the P D G F fl-receptor results in dimerization of the receptor, activation of its cytoplasmic PTK domain and autophosphorylation of a number of tyrosine residues within the cytoplasmic region of the receptor (for review see Pawson and Schlessinger, 1993). These events are accompanied by a small increase in the PTK activities of p59/;'", p60 "-st"and p62 c--''e"and by the physical association of a small proportion of the cellular pools of these proteins with the activated PDGF/~-receptor (Kypta et al., 1990). As shown in Fig. 4, this association results from binding of the SH2 domain of p59~v", p60 c.... or p62 '~'e~ to phosphorylated Tyr-579 or Tyr-581 of the P D G F /~-receptor (Mori et al., 1993). The association is transient, possibly because the complexes are rapidly internalized and degraded. There is evidence that it is the activated forms of p59/y", p60 ...... and p62 cye~ that are associated with the P D G F fl-receptor, and indeed it can be proposed that activation of p5913°, p60 C~" and p62 'ye~ is a direct result of their physical association with the P D G F /~receptor. Thus, intermolecular binding of the p60 ...... SH2 domain to phosphorylated Tyr-579 or Tyr-581 of the P D G F /~-receptor would disrupt intramolecular binding of the p60 ...... SH2 domain to phosphorylated Tyr-527 of 60 c src. This would result in unfolding of the 60 ..... molecule and activation of its PTK domain, as

558

P. Kefalas et al.

~DGFR

P-Tyr-579-P-Tyr-581

active~

i~e/

~ Tyr-S79-P ~Tyr-581-P

I

~--P-Tyr~527

Fig. 4. Model for the interaction of p60"-" PTKs with the PDGF fl-receptor. discussed above. In this context, activation of p60 ..... would occur without the need for dephosphorylation of Tyr-527 by a PTPase. If this model is correct, it might be predicted that the p60 ....... SH2 domain would have a higher affinity for the phosphotyrosine-containing binding sites on the P D G F / ~ - r e c e p t o r than for its own phosphorylated Tyr-527. This indeed turns out to be the case (Roussel et aL, 1991; Liu et aL, 1993). The binding of p60 C~", or p59 j~'~ to the activated P D G F / ~ - r e c e p t o r could serve to recruit these enzymes to sites containing their substrates. In support of this idea a number of other signalling molecules are recruited to the activated P D G F /C-receptor by SH2 d o m a i n phospbotyrosine interactions. These include Pl3-kinase, PLC-71 and ras G A P (for reviews see Fry et al., 1993; Mayer and Baltimore, 1993; Pawson and Schlessinger, 1993). It has also been shown recently that interactions involving SH2 and SH3 domains provide a means of coupling receptor PTKs to Ras proteins (for reviews see Egan and Weinberg, 1993; McCormick, 1993; Marshall, 1995). The rapid recruitment of a range of signalling molecules and the formation of signal transduction complexes at the inner surface of the plasma membrane is thought to extend the range of substrates that are modified by P D G F stimulation beyond those that can be modified by the P D G F / % r e c e p t o r alone, There is evidence that at least some of the signalling proteins found in these complexes can be phosphorylated by c-src PTKs (Brott et al., 1991), although it is far from clear whether this

is functionally significant in vivo. The role of p59 t~", p60 ....... and p62 cye, in signal transduction complexes following P D G F stimulation of fibroblasts therefore remains unclear, and to understand the significance of their presence in such complexes represents an important goal for the future. Binding o f p6OCSr~family members to transmembrane proteins that lack P T K activity

The first such interaction to be discovered was the binding of the unique amino-terminus of p56 tck to the cytoplasmic tails of the Tlymphocyte surface membrane glycoproteins CD4 and CD8~ (for reviews see Mustelin and Burn, 1993; Brickell, 1992). Unlike the interaction of p60 ....... family PTKs with receptor PTKs, the association of p56 zckwith C D 4 / C D 8 e occurs in both resting and activated cells, and involves a high proportion of the p56 t'k molecules in the cell. CD4 and CD8e are important accessory molecules in the activation of Tlymphocytes through the T C R and their association with p 5 & k appears to be required for efficient activation of T-lymphocytes following stimulation of the T C R (Glaichenhaus et al., 1991; Z a m o y s k a et aL, 1989). It is not known how p56 ~ contributes to T-lymphocyte activation, but it is clear that stimulation of the T C R results in an increase in p56 ~ck P T K activity, and there is evidence that PLC-7I and PI3-kinase may be substrates for p56 zck in

CD4 f ~ CD45

~ ~ pS6 lck

CD4

p/Pasedomain

T,

;acrave

p50csk Fig. 5. Model for the regulation of p56~'k PTK activity in T-lymphocytes. The amino-terminus of p56t'k binds to the carboxyl terminus of CD4. Depfiosphorylation of Tyr-505 of p56~'k by CD45 results in unfolding of the molecule and activation of its catalytic domain. Phosphorylation of Tyr-505 by p50"k, or a related PTK, returns p56t~k to the inactive state. This is probably an oversimplification,since the p56~kSH2 domain canbind to phosphotyrosine residues on the TCR complex and on CD45, the latter having been phosphorylated by p50"k.

Signalling by the p60 < ' ' family of protein-tyrosine kinases

activated T-lymphocytes (Weber et al., 1992; Autero et al., 1994). Interestingly, PI3-kinase binds to the SH3 domain of p56 ~'k in Tlymphocytes (Vogel and Fujita, 1993). There is also evidence for a PTK-independent role for p56 t
..-.,.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

is responsible for the activation of p56 ~'k PTK activity in T-lymphocytes (for reviews see Mustelin and Burn, 1992; Brickell, 1993). It is also thought that p50 "k, or a related PTK, is responsible for the subsequent down regulation of p56 ~<~ PTK activity (Fig. 5). In an interesting twist to this story, Autero et al., (1994) have recently shown that p50 <~k can also phosphorylate CD45, activating its PTPase activity and generating a phosphotyrosine residue that can bind to the SH2 domain of p56 ~'k. This suggests that the regulatory circuit involving p50 <~, CD45 and p56 ~<~is far more complicated than was imagined. The details of these interactions have not yet been worked out.

....,.

: : Antigen presenting cell i :. : • . - . ' . ' . • ...,.,

-

•

'."

-

, . . . . . , . . . , ,

•' - ' - . . .

•

'

-.-.,

" . ' . . . ' . , . . . ' . ' . . , ' . . . . .

.

. . . . . . . , . . . . .

.'.MHCclassiI..

CD45

"~

-. •

<,1 10

CO3

[~

CD4

I I ~i

i + I>5o .

l

i P56'cI"xO\/ -

PI 3-kinase

PLC~,

e

--{::]Il-

pl20/130 [

Cellular activation ( ~ Activation

T-lymphocyte

p5OCSk

Zap 70 p50 csk

CD45

['t

"iI

1

Down regulation

["-1 SH2 domain ~ 1

559

PTK d o m a i n [~ Tyrosine Activation Motif (TAM)

Fig. 6. Putative pathways of signalling by PTKs in T-lymphocytes. For clarity, SH3 domains have been omitted from p50 csk, p59 D" and p56 t'k.

560

P. Kefalas et al.

Efficient activation of T-lymphocytes through the TCR also requires p59~'"~n, as evidenced by the phenotype of ~v,(_) mice as well as by in vitro studies. Like that of p56 t
TCR ( subunit (Weiss, 1993). The complex interactions between p56 tck, p59 ry"cn and the TCR are illustrated in Fig. 6. Since the discovery that p56 ~ckand p59 ry"~r)are physically and functionally associated with CD4/CD8a and the TCR, respectively, a large number of similar interactions between p60 ..... family members and transmembrane proteins have been identified (Table 2). This list serves to illustrate once again the extraordinary diversity of biological phenomena in which this family of proteins appears to participate. In a growing number of cases, the details of how these proteins bind to each other have been determined. The binding of p56 z
Table 3. Physical association of c-src PTKs with transmembrane proteins that lack PTK activity Family member

Transmembrane protein

Cell type(s) B-lymphocyte

Possible biological role(s)

p56b/k

BCR"

p58 '~tg~

IgG Fc receptor IIA (F%RIIA), IgG Fc receptor llC (Fc:RIIC) b

P59t~l~

TCR (7,~,~,r/ subunits)

p59t'"~B)

Large myelin-associated glycoprotein (large MAG) c

p59f~"

CD36 d

p59s'"

Interleukin-7 receptor (IL-7R) ¢

p59r'"

BCR d

p59r'"

IgE Fc receptor II (Fc, RII) f

p 5 6 t'k

CD4, CD8c~

T-lymphocyte

T-lymphocyte activation T-lymphocyte maturation

p56 t'k

Interleukin-2 receptor fl chain (IL-2Rfl)g

T-lymphocyte

T-lymphocyte activation

p53 ~v" P56 #v.

BCR a'h

B-lymphocyte

B-lymphocyte activation

p56 (v"

IgE Fc receptor I (Fc
Basophil

Basophil activation

p53 ~v° p56/~'o

CD36 ¢

Platelets

Platelet adhesion Platelet activation

p62 <-'e~

CD36 ~

Platelets

Platelet adhesion Platelet activation

p62 <-'~

IgE Fc receptor I (Fc, RI)'

Mast cell

Mast cell activation

Neutrophil

T-lymphocyte Oligodendrocyte Platelets Pre-B-lymphocyte B-lymphocyte Lymphocyte

B-lymphocyte activation Phagocytosis, antibody-dependent cellular cytotoxicity, generation of reactive oxygen intermediates, release of lysosomal enzymes T-lymphocyte activation Myelination Platelet adhesion Platelet activation B-lymphocyte maturation B-lymphocyte activation Lymphocyte activation

In most cases it has also been demonstrated that binding of ligand or crosslinking of the transmembrane proteins with specific antibodies stimulates the PTK activity of the associated c - s r c PTK. ~Burkhardt et al. (1993), bHamada et al. (1993), cUmemori et al. (1994), ~Huang et al. (1991), ~Ventikaraman and Cowling (1992), fSugie et al. (1991), gHatakeyama et al. (1991), hyamanashi et al. (1992), iEiseman and Bolen (1992).

Signalling by the p60~-" family of protein-tyrosine kinases

subunit involves the 10 amino-terminal amino acids of p59~,'"~nand the 41 membrane-proximal amino acids of the cytoplasmic domain of (Timson Gauen et al., 1992). The latter includes the so-called antigen receptor homology 1 (ARH1) motif, which has the consensus sequence D/ExxxxxxD/ExxYxxLxxxxxxxYxxL/I (Reth, 1989). This motif also appears in the cytoplasmic regions of the ),, 6, ~ and r/ subunits of the TCR, of the Ig-~ and Ig-fl subunits of the B-lymphocyte antigen receptor (BCR) and of the Fc receptors Fc,RI/~, Fc,RIT, F%RIIIA?, Fc;.RIIA and FcrRIIC. By analogy with the binding of p5~ to the TCR ff subunit, it has been suggested that p58 ~:rg~may bind to F%RIIA and Fc~RIIC via their ARH1 motifs (Hamada et al., 1993). In support of this idea, p58 ~/g~protein, since 7 of these are shared with p59/y". The presence of the tyrosine-containing region of the ARH1 motif (YxxLxxxxxxxYxxL/I) in components of the TCR, BCR and Fc receptors is essential for cellular activation through these receptors and this region has been therefore named the tyrosine activation motif, or TAM (Flaswinkel and Reth, 1994). The Tam of the BCR Ig-~ subunit is required for activation of PTKs during signal induction and the first tyrosine residue in the TAM is itself a target for phosphorylation by p59ty". This phosphorylated tyrosine may bind proteins with SH2domains, just as the TCR ff subunit TAM binds the SH2 domain of Zap-70 (Flaswinkel and Reth, 1994). The other associations listed in Table 2 presumably involve different kinds of interaction, since the transmembrane proteins involved lack ARHI motifs. Moreover, the associations may not always be mediated by the amino-terminal residues of the p60 ~~r' family PTK. For example, p56 ~ckbinds to IL-2R//via a portion of its SH1 domain (Hatakeyama et al., 1991). A similar mechanism might account for the binding of p59~'" to IL-7R (Venkitaraman and Cowling, 1992). It has become obvious from the research described above, that the members of the p60 ..... family of PTKs play multiple roles in transducing signals in a wide variety of biological situations. Until recently there was an almost complete lack of information concerning the signalling pathways in which these proteins participated. Now there is an almost embarrassing wealth of possible interactions, and an important task in the coming years will TM

561

be to discern which of these are biologically relevant. Acknowledgements--We are grateful to the Leukaemia Research Fund (P.K.) and the Arthritis and Rheumatism Council (T.R.P.B.) for supporting our research on the c-fgr protooncogene. REFERENCES

Appleby M. W., Gross J. A., Cooke M. P., Levin S. D., Qian X. and Perlmutter R. M. (1992) Defective cell receptor signaling in mice lacking the thymic isoform of p59I~. Cell 70, 751-763. Autero M., Saharinen J., Pessa-Morikawa T., SoulaRothhut M., Oetken C., Gassmann M., Bergman M., Alitalo K., Burn P., Gahmberg C. G. and Mustelin T. (1994) Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50 'sk kinase creates a binding site for p56 ~ck tyrosine kinase and activates the phosphatase. Molec. Cell. Biol. 14, 1308-1321. Bergman M., Mustelin T., Oetken C., Partanen J., Flint N. A., Amrein K. E., Autero M., Burn P. and Alitalo K. (1992) The human p50 "k tyrosine kinase phosphorylates p56 t'k at Tyr-505 and down regulates its catalytic activity. E M B O J. ll, 2919-2924. Boyce B. F., Yoneda T., Lowe C., Soriano P. and Mundy G. R. (1992) Requirements of pp60 '-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J. Clin. Invest. 90, 1622-1627. Brickell P. M. (1992) The p60~-~" family of protein-tyrosine kinases: structure, regulation and function. Crit. Rev. Oncogen. 3, 401~446. Brott B. K., Decker S., Sharer J., Gibbs J. B. and Jove R. (1991) GTPase-activating protein interactions with the viral and cellular src kinases. Proc. Natn. Acad. Sci. U.S.A. 88, 755-759. Burkhardt A. L., Brunswick M., Bolen J. B. and Mond J. J. (1991) Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc. Natn. Acad. Sei. U.S.A. 88, 7410-7414. Chan A. C., Iwashima M., Turck C. W. and Weiss A. (1992) ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR ff chain. Cell 71, 649~i62. Cichetti P., Mayer B. J., Thiel G. and Baltimore D. (1992) Identification of a protein that binds to the SH3 region of Abi and is similar to Bcr and GAP-rho. Science 257, 803-806. Cohen G. B., Ren R. and Baltimore D. (1995) Modular Binding Domains in Signal Transduction Proteins. Cell 80, 237 248. Cooke M. P. and Perlmutter R. M. (1989) Expression of a novel form of the f y n proto-oncogene in hematopoietic cells. New Biol. 1, 66-74. da Silva A. J., Janssen O. and Rudd C. E. (1993) T cell receptor ~/CD3-p59~cr)-associated p120/p130 binds to the SH2 domain of p59l*"cr).J. Exp. Med. 178, 2107~113. Egan S. E. and Weinberg R. A. (1993) The pathway to signal achievement. Nature 365, 781-783. Eiseman E. and Blen J. B. 91992) Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases. Nature 355, 78 80. Erickson H. P. (1993) Gene knockouts of c-src, transforming growth factor/~I, and tenascin suggest superfluous, nonfunctional expression of proteins. J. Cell Biol. 120, 107%1081.

562

P. Kefalas et al.

Flaswinkel H. and Reth M. (1994) Dual role of the tyrosine activation motif of the lg-~ protein during signal transduction via the B cell antigen receptor. EMBO J. 13, 83-89. Fry M. J,, Panayotou G., Booker G. W. and Waterfield M. D. (1993) New insights into protein tyrosine kinase receptor signaling complexes. Prot. Sci. 2, 1785 1797. Glaichenhaus N., Shastri N., Littman D. R. and Turner J. M. (1991) Reguirement for association of p56 ~'k with CD4 in antigen-specific signal transduction in T cells. Cell 64, 511-520. Gordon J. I,, Duronio R. J., Rudnick D. A., Adams S. P. and Gokel G. W. (1991) Protein N-myristoylation. J. biol. Chem. 266, 8647-8662, Gout I., Dhand R., Hiles 1. D., Fry M. J., Panayotou G., Das P., Truong O., Totty N. F., Hsuan J., Booker G. W., Campbell I. D. and Waterfield M. D. (1993) The GTPase protein dynamin binds to and is activated by a subset of SH3 domains. Cell 75, 25-36. Grant S. G. N., O'Dell T. J., Karl K. A., Stein P. L., Soriano P. and Kandel E. R. (1992) Impaired long-term potentiation, spatial learning and hippocampal development in fyn mutant mice. Science 258, 1903-1910. Hamada F., Aoki M., Akiyama T, and Toyoshima K, (1993) Association of immunoglobulin G Fc receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils, Proc. Nam. Acad. Sci. U.S.A. 90, 6305~5309. Hanks S. K., Quinn A. M. and Hunter T. (1988) The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science 241, 45-52. Hatakeyama M., Kono T., Kobayashi N., Kawahara A., Levin S. D., Perlmutter R. M. and Taniguchi T. (1991) Interaction of the IL-2 receptor with the src-family kinase p56~'k: identification of novel intermolecular association. Science 252, 1523 1528. Horne W. C., Neff L., Chatterjee D., Lomri A., Levy J. B. and Baron R. (1992) Osteoclasts express high levels of pp60 '-s" in association with intracellular membranes. J. Cell Biol. 119, 1003 1013. Hunter T. (1987) A tail of two src's: mutatis mutandis. Cell 49, 1-4. Jove R. and Hanafusa H. (1987) Cell transformation by the viral src oncogene. Ann. Rev. Cell Biol. 3, 31-56. Kaplan K. B., Swedlow J. R., Varmus H. E. and Morgan D. O. (1992) Association of p60 ..... with endosomal membranes in mammalian fibroblasts. J. Cell Biol. 118, 321-333. Kypta R. M., Goldberg Y., Ulug E. T. and Courtneidge S. A. (1990) Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell 62, 481-492. Levin S. D., Anderson S. J., Forbush K. A. and Perlmutter R. M. (1993) A dominant-negative transgene defines a role for p56 hk in thymopoiesis. EMBO J. 12, 1671-1680. Liu X,, Brodeur S. R., Gish G., Songyang Z., Cantley L. C., Laudano A. P. and Pawson T. (1993) Regulation of c-src tyrosine kinase activity by the src SH2 domain, Oncogene 8, 1119-1126. Marshall C. J. (1995) Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signalregulated kinase activation. Cell 80, 179-185. Matsuda M., Mayer B. J., Fukui Y. and Hanafusa H. (1990) Binding of transforming protein, p47 g~-~'k, to a broad range of phosphotyrosine-containing proteins. Science 2,48, 1537 1539.

Mayer B. J., Jackson P. K. and Baltimore D. (1991) The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine -phosphorylated cellular proteins with high affinity. Proc. Natn. Acad. Sci. U.S.A. 88, 627~3 l. Mayer B. J. and Baltimore D. (1993) Signalling through SH2 and SH3 domains. Trends Cell. Biol. 3, 8 13. McCormick F. (1993) How receptors turn Ras on. Nature 363, 15-16. Molina T. J., Kishihara K., Siderovski D. P., van Ewijk W., Narendran A., Timms E., Wakeham A., Paige C. J., Hartman K.-U, Veillette A., Davidson D. and Mak T. W. (1992) Profound block in thymocyte development in mice lacking p56t~k. Nature 357, 161-164. Mori S., R6nnstrand L., Yokote K., Courtneidge S. A., Claesson-Welsh L. and Heldin C.-H. (1993) Identification of two juxtamembrane autphosphorylation sites in the PDGF fl-receptor: involvement in the interaction with Src family tyrosine kinases. E M B O J. 12, 2257-2264. Mustelin T. and Burn P. (1993) Regulation of src family tyrosine kinases in lymphocytes. Trends Biochem. 18, 2 l 5-220. Nada S., Okada M., MacAuley A., Cooper J. A. and Nakagawa H. (1991) Cloning of a complementary DNA for a protei~tyrosine kinase that specifically phosphorylates a negative regulating site of p60<-'r'. Nature 351, 659-72. O'Brien M. C., Fukui Y. and Hanafusa H. (1990) Activation of the proto-oncogene p60...... by point mutations in the SH2 domain. Molec. Cell. Biol. 10, 2855-2862. Okada M., Nada S., Yamanishi Y., Yamamoto T. and Nakagawa H. (1991) CSK: a protein-tyrosine kinase involved in regulation ofsrc family kinases. J. biol. Chem. 266, 24249 24252. Pawson T. and Schlessinger J. (1993) SH2 and SH3 domains. Curr. Biol. 3, 434M-42. Pyper J. M. and Bolen J. B. (1990) Identification of a novel neuronal c-src exon expressed in human brain. Molec. Cell. Biol. 10, 2035-2040. Resh M. D. (1990) Membrane interactions of pp60"-sr<: a model for myristylated tyrosine protein kinases. Oncogene 5, 1437-1444. Reth M. (1989) Antigen receptor tail clue. Nature 338, 383 384. Roussel R. R., Brodur S. R., Shalloway D. and Laudano A. P. (1991) Selective binding of activated pp60 ..... by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60 '-st<, Proc. Natn. Aead. Sci. U.S.A. 88, 1069610700. Shiroo M., Goff L., Biffen M., Shivnan E. and Alexander D. (1992) CD45 tyrosine phosphatase-activated p59rv" couples the T cell antigen receptor to pathways of diacylglycerol production, protein kinase C activation and calcium influx. EMBO J. 11, 4887-4897. Soriano P., Montgomery C., Geske R. and Bradley A. (1991) Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell 64, 693-702. Stanley E., Ralph S., McEwen S., Boulet I., Holtzman D. A., Lock P. and Dunn A. R. (1991) Alternatively spliced murine lyn mRNAs code distinct proteins. Molec, Cell. Biol. 11, 3399-3406. Stein P. L., Lee H.-M., Rich S. and Soriano P. (1992) pp59~" mutant mice display differential signaling in thymocytes and peripheral T cells. Cell 70, 741 750. Sudol M., Greulich H., Newman L., Sarkar A., Sukegawa J. and Yamamoto T. (1993) A novel Yes-related kinase,

Signalling by the p60' ~,r, family of protein- tyrosine kinases Yrk, is expressed at elevated levels in neural and hematopoietic tissues. Oncogene 8, 823 831. Sugie K., Kawakami T., Maeda Y., Kawabe Y,, Uchida A. and Yodoi J. (1991)fyn tyrosine kinase associated with Fc, RI1/CD23: possible multiple roles in lymphocyte activation. Proc. Nam. Acad. Sci. U.S.A. 88, 9132 9135. Timson Gauen L. K., Kong A.-N. T., Samelson L. E. and Shaw A., S. (1992) p59~i" tyrosine kinase associates with multiple T-cell receptor subunits through its unique amino-terminal domain. Molec. Cell. Biol. 12, 5438-5446. Umemori H., Sato S., Yagi T., Aizawa S. and Yamamoto T. (1994) Initial events of myelination involve Fvn tyrosine kinase signalling. Nature 367, 572 576. Venkitaraman A. R. and Cowling R. J. (1992) Interleujkin 7 receptor functions by recruiting the tyrosine kinase p59~v" through a segment of its cytoplasmic tail. Proc. Natn. Acad. Sci. U.S.A. 89, 12083 12087. Vogel L. B. and Fujita D. J. (1993) The SH3 domain of p56 ~'k is involved in binding to phosphatidylinositol 3"kinase from T lymphocytes. Molec. Cell. Biol. 13, 7408 7417. Weber J. R., Bell G. M., Han M. Y., Pawson T. and Imboden J. B. (1992) Association of the tyrosine kinase Lek with phospholipase-C)'l after stimulation of the T cell antigen receptor. J. Exp. Med. 176, 373 379.

563

Weiss A. (1993) T cell antigen receptor signal transduction: a tale of tails and cytoplasmic protein tyrosine kinases. Cell 73, 209 212. Wilks A. F. (1990) Structure and function of the proteintyrosine kinases. Prog. Growth Fact. Res. 2, 97-111. Xu H. and Littman D. R. (1993) A kinase-independent function of Lck in potentiating antigen-specific T cell activation. Cell 74, 633~543. Yagi T., Aizawa S., Tokunaga T., Shigetani Y., Takeda N. and Ikawa Y. (1993) A role for Fyn tyrosine kinase in the suckling behaviour of neonatal mice. Nature 366, 741 745. Yamanashi Y.. Fukui Y., Wongsasant B., Kinoshita Y., Ichimori Y., Toyoshima K. and Yamamoto T. (1992) Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling. Proc. Natn. Acad. Sci. U.S.A. 89, 1118-I122. Zamoyska R., Derham P., Gorman S. D., Von Hoegen P., Bolen J. B., Veillette A. and Parnes J. R. (1989) Inability of CD8" polypeptides to associate with p5& k correlates with impaired function in vitro and lack of expression in vivo, Nature 342, 278-281. Zheng X. M., Wang Y. and Pallen C. J. (1992) Cell transformation and activation of pp60 '-~rc by overexpression of a protein-tyrosine phosphatase. Nature 359, 336 339.