src-related protein tyrosine kinases and their surface receptors

239

Biochimica et Biophysica Acta, 1155 (1993) 239-266 © 1993 Elsevier Science Publishers B.V. All rights reserved 0304-419X/93/$06.00

BBACAN 87273

src-related protein tyrosine kinases and their surface receptors Christopher E. Rudd a,b Ottmar Janssen a,b, K.V.S. Prasad a,b Monika Raab a,b Antonio da Silva a,b, Janice C. Teller a,b and Masahiro Y a m a m o t o a,b a Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA (USA) and b Department of Pathology, Harvard Medical School, Boston, MA (USA) (Received 18 May 1993)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239

II.

Src family of kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

241

IIIi p56 lck and p59 frn binding to T-cell receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p56 tCk binding to CD4 and CD8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Binding site within p56 tck . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Regulation of p56 lck kinase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. CD4-p56tck-associated intracellular molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. The TcR~/CD3-p59 fyn connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. CD4-p56 lck and TcR~'/CD3-p59 fyn association in a multimolecular complex . . . . . . . . . . . . . G. Biological functions of p56/ok and p59 fyn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. T-cell signalling.', activation/anergy/apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Thymic differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Receptor endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

243 243 245 245 247 249 251 251 251 254 256

IV.

Other kinase-receptor interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. p56 lck and the 4-1BB antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. p56 tck, p59 fyn and the IL-2 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. p56 tck, p59 fyn and the CD2 antigen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Src family and surface immunoglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Src family and GPI-linked surface proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. p59 fyn and the F c ~ I I / C D 2 3 receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Src family and P D G F receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

256 256 256 257 257 258 258 258

V.

Alternate mechanisms governing receptor-kinase binding

.............................

259

VI.

Other potential substrates in the phosphorylation cascade . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

259

VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

260

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

260

I. Introduction Protein-tyrosine kinases can be broadly categorised into two groups, so-called receptor and non-receptor kinases. Receptor kinases are represented by type-I

Correspondence to: C.E. Rudd, Division of Tumor Immunology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

transmembrane growth factor receptors such as receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and colony stimulating factor (CSF-1). Members of this category possess a transmembrane region which divides the ligand binding domain from the intracellular kinase domain(s). Structurally related tyrosine kinases such as ret, eck, etc., encode surface receptor with unknown ligands (for a review, see Refs. 1, 2). Non-receptor kinases which

240

Myristic acid

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260

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Fig. 1. The structure of Src-related protein-tyrosine kinases that associate with surface receptors. Structure of p60 src and other members of the family of non-receptor kinases (p56 lck, p59 fyn(T) and (B), p55b/k, p56/53lyn), p6OSrc serves as the prototype for other src-related kinases which bind to surface receptors. Each kinase is modified by the addition of a myristic acid moiety to the N-terminus allowing an interaction with the lipid bilayer of the plasma membrane. The N-terminal region (70-82 residues) contains sequences largely unique to each of the src-related kinases. Sites for serine phosphorylation include a protein kinase C site (Ser-12) and cAMP-dependent kinase site (Ser-17) in p60 src. Phosphorylation sites of p56 tck, Ser-42 and Ser-59 may act as substrates for protein kinase C and MAP-2 kinase, respectively. The site of interaction of p56 lck with CD4 and CD8 has been mapped within residues 10-30 of the N-terminal domain. Likewise, p59 fyn(T) binding to CD33,,8,~ and TcR~ is mediated by the first 10 residues. The N-terminal domain is followed by SH3 and SH2 domains which share moderate homology between src-related kinases. Similar domains have been identified in a variety of non-catalytic intracellular molecules. SH3 domains bind to proline-rich motifs, while the SH2 domains bind to phosphotyrosine residues located next to hyrophobic residues. Next is the kinase domain (SHI) which is highly conserved and possesses an autophosphorylation (Tyr-416 for pp6OSrC; Tyr-394 for p56 lck. The C-terminal portion of each kinase possesses a negative regulatory site (Tyr-527 for p60~rc; Tyr-505 for p56tck; Tyr-531 for p59fYn; Tyr-495 for p55 btk and Tyr-508 for p55/531yn). Isoforms of p59 fy" (T and B) and p53/56 ty" can be expressed by splicing mechanisms.

241 molecules, such as phosphotidylinositol 3'-kinase, thereby recruiting potential components of an intracellular cascade. This review will focus on our current knowledge of src-related kinases, their receptors and their roles in receptor-mediated signal transduction.

lack the transmembrane or extracellular regions can be further subdivided into the well-characterised src-like kinases typified by pp60 src, and other intracellular kinases such as syk, c-abl and c-fps. Src-related kinases are intracellular enzymes of 505 to 543 residues that attach to the inner face of the plasma membrane by a N-terminal fatty-acid moiety. As typified by pp60 src, they were first identified on the basis of their oncogenic potential. Oncogenic forms of c-src, c-yes and c-fgr have been detected as part of the Rous Sarcoma Virus (v-src), Yamaguchi 73 avian sarcoma virus (v-yes) and Garder-Rasheed feline sarcoma virus (v-fgr) [3-6]. Likewise, p56 ~ck is over-expressed in the murine thymic lymph0ma LSTRA [7,8]. Cellular homologues have been isolated by cross-hybridization and include a family of eight well-characterised members, p60 src, p59 fyn, p59 hck, p62 TM, p56 tek, p56 lrn, p55 fgr and p55 btk. Distinct but overlapping patterns of cellular expression have been observed, p60 ~rc is expressed at high levels in neurons and platelets, p59 hck is expressed in myeloid cells and p56 lck expression is restricted to lymphoid cells and primarily to T cells. While conserved structural motifs are shared within the kinase domains, each kinase possesses an unique N-terminal region. A role for src family members in intracellular signalling was first provided by the finding that p56 t~k physically associates with the cytoplasmic tail of the T-cell antigens CD4 and CD8 [9-12]. Other src-related kinases p59/yn, p59 btk and p56 lrn have recently been found associated with other surface receptors [13-17]. These receptor complexes in turn bind to intracellular

II. Src family of kinases

The structure of the src family of kinases has been defined by a combination of cDNA-cloning studies, radiolabelling analysis and by protein purification. Crystal structures of the overall kinase has yet to be determined. However, mutagenic and domain-swapping studies have allowed the dissection of key sites within each kinase. Each of the src kinases possesses a highly-conserved protein-tyrosine kinase domain of about 300 amino acids. The protein-tyrosine kinase domain is a feature shared by a related family of receptor tyrosine kinases such as the platelet-derived growth factor receptor (PDGF-R), the fibroblast growth factor receptor (FGF-R), the cloning-stimulating factor receptor (CSF-1-R), the insulin receptor and others. After the shared kinase domain, the structure of src-related kinases and receptor-tyrosine kinases diverge substantially. Features of the src family that are not found in receptor-tyrosine kinase receptors include an N-terminal sequence for the attachment of myristic acid, a receptor binding region, two globular src homology 2 and 3 domains (SH2 and SH3) and a carboxyl regulatory region. Fig. 1 illustrates the basic organization of the src-related kinases (p56 tck, p59 fyn, p59 tyn

A. NH2p60src p561ck p59fyn p561yn nR.qhlk

GSNKSKPKDASQRRRSLEPAENVH G~V~S N P E D DWM E N I D V~E N~H Y G~V Q~K D K E A A K L T E E R D G S L N N Q G~I KSKRKDN L N DD EV DS KTQPV R G L LSS KRQV S E KGKGWS PV K I RTQ

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Fig. 2. N and C-terminal sequence alignment of representative src-related kinases. (A) N-terminal unique sequences of src-related kinases reported to bind to cell-surface receptors. There is little similarity between the N-terminal 70 amino-acid regions of the tyrosine kinases. The first two cysteines within p56 tck mediate associations with the plasma membrane. Cysteines at sites 20 and 23 of the p56 tck sequence mediate the interaction of the kinase with CD4 and CD8. p59 fynO3 binding to CD3y,8,~ and TcR~" is mediated by the first 10 residues. Other cysteine residues are noted. (B) Extensive homology can be observed in the C-terminal sequences of the src-related kinases. The C-terminal Tyr residue lies within a conserved motif A / S T E P / G Q Y Q / E .

242 and p59 btk) that have been reported to associate with surface receptors. The N-terminus begins with the di-peptide Met-Gly from which the Met residue is cleaved. The amino group of Gly then becomes linked to a myristic group, a modification that is required for membrane attachment and localization [18-19]. Non-src-related proteins with myristic groups also possess N-terminal glycines. The fatty acid may attach to the membrane on the basis of hydrophobicity. Morphological transformation by v-src requires membrane localization via the fatty acid [18-19], suggesting that either the lipid bilayer acts as a substrate for src kinases, or that the localization is required for the phosphorylation of other target substrates. The small N-terminal region is followed by some 70-82 amino acids which vary considerably between p56 tck, p60 ~C and other src-family members (Fig. 2A). Depending on the kinase, the N-terminal region includes sites that associate with the surface receptors CD4 and CD8 [21-23], sites of serine phosphorylation by protein kinase C (Ser-12 for pp60 '~C) and cAMP-dependent kinases (Ser-17 for pp60 '~) [24-27]. Ser-17 serves as a major serine phosphorylation site in pp60 ~c although its function is unclear [25,26]. Deletion of this site is without effect [27]. Two serine phosphorylation sites within p56 tCk have been mapped to positions 42 and 59 by site-directed mutagenesis [28]. Phosphorylation of peptides corresponding to these sites suggests that Ser-42 and Ser-59 act as substrates for protein kinase C and MAP-2 kinase, respectively [28]. p59 fyn can also be phosphorylated at serine residues, although the sites of phosphorylation are unknown [29]. It is unclear whether blk and isoforms of lyn are serine or threonine phosphorylated [30]. In the case of p56 lck, the N-terminal region also possesses cysteine residues at positions 3 and 5 that influence the localization of the kinase to the plasma membrane [22]. Next from the N-terminal region resides the conserved SH2 and SH3 domains (Fig. 1). Both domains are found in other proteins, either in non-enzymatic structures (termed adaptors), or together with an adjacent enzymatic domain (reviewed in Ref. 31). SH2 domains of about 100 amino acids are found as part of a collection of intracellular polypeptides, including phospholipase C 7 (PLCy) [32,33], rasGAP (GTPaseactivating protein) [34], the regulatory subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) [35-37], p47 gag-~rk [38], tyrosine phosphatases [39,40], tensin [41], SHC [42] and others (reviewed in Ref. 31). SH2 domains as adaptors appear designed to facilitate the binding of non-SH2 carrying phosphotyrosylated structures to receptors. The domain binds to phosphotyrosine and adjacent hydrophobic sequences within the cytoplasmic tails of growth factor receptors (i.e., PDGF-R, CSF-1 R) and to pp60 ~C [44-47]. Nuclear

Magnetic Resonance (NMR) analysis of abl-SH2 and crystallographic analysis of the SH2 region of pp60 src shows a spherical domain with a hydrophobic pocket with residues capable of binding to one peptide at a time [48-51]. The overall structure is formed by two anti-parallel sheets surrounded by two alpha helices. The phosphotyrosine extends into the pocket interacting with a key conserved Arg residue (src Arg-175) which forms an ion pair with the phosphate. Other residues stablise the interaction by hydrogen bonding and amino-aromatic group interactions. Cantley and colleagues have defined the optimal binding sequences for different SH2 domains illustrating the importance of adjacent residues (pTyr + 1-3) in defining the specificity of binding [52]. SH2 domains of src-related kinases bind optimally to the pTyr-GluGlu-IIe (pTyr + 1-3 = YEEI), with lesser affinities of binding to substituted residues at pY + 1 = D,T,Q; pY + 2 = N,Y,D,Q; pY + 3 = M,L,V. Each of the src-related kinases (Src,Fyn,Lck) differ slightly in their affinities for individual residues. The dissociation constant of binding of the lck-SH2 domain to a phosphopeptide containing the YEEI motif (EPQpYEEIPIYL) is in the 1 nM range [53]. Crystal structure of this SH2-peptide high-affinity complex reveals a second conserved pocket within the SH2 domain for the pY + 3 residue towards the C-terminus [50,51]. Other SH2 domains of phosphatidylinositol 3-kinase (PI 3-kinase) (YDHP) and PLC-y (N-terminal = YLEL, C-terminal = YVIP) exhibit different specificities for phosphotyrosine containing sequences. Consistent with this, phosphotyrosine sites within the PDGF-R such as Tyr-751 binds to PI 3-kinase, but poorly if at all to ras GAP or PLCy [54-57]. In the case of src-kinases, deletion or mutations within this region of pp60 ~rc alters its association with intracellular proteins [58-60]. The SH2 domain (but not the SH3 domain) is required for oncogenic transformation by constitutively active p56 lck (F505) [61]. The SH3 motif constitutes a small domain of some 50 amino acids found in various proteins including PLCy [32,33], p47 gagcrk [38], a-spectrin [62], myosinIB, fodrin and a yeast actin-binding protein [63,64]. SH3 domains seem to function as independent units, being situated in various locations within polypeptides, and often together with SH2 domains. The crystal structure of this domain shows a compact /3-barrel of five anti-parallel /3-strands that forms a hydrophobic pocket [65,66]. Cicchetti and colleagues (1992) have identified an Abl-SH3-binding protein 3BP-1 with a proline-rich hydrophobic-binding motif (APTMPPPLPPVPP) [67]. A consensus sequence XPXXPPPXXP is found in other SH3-binding proteins such as the murine limb deformity (ld) proteins, formins and a subtype of the muscarinic acetylcholine receptor [68]. SH3 interactions have been implicated in the regulation of a

243 number of events including src-kinase binding to PI 3-kinase [69], and the regulation of Ras guanine nucleotide exchange [70,71]. Prasad and co-workers (1993) have shown binding of the fyn-SH3 domain to the p85 subunit of PI 3-kinase [69]. In another connection, the SH3-SH2-SH3 carrying drk protein in Drosophilia binds to the tyrosine kinase Sevenless, thereby coupling it the Ras guanine nucleotide-releasing protein (GNRP), Son ofsevenless, SOS [70,71]. The SH2 domain of drk binds the receptor-kinase, while its SH3 domain can bind to the C-terminal tail of SOS, a region that contains proline-rich motifs [70-72]. This interactive mechanism provides a means to couple protein-tyrosine k~nases to p21 ras activation. SH3 domains also been implicated with the cytoskeleton, potentially with proteins such as dynamin [64,73]. Deletion of the SH3 domain of c-abl prevents the kinase from binding actin, an event linked to transformation [74]. Another possible connection involves 3BP1 homology (outside SH3: binding motif) with a family of proteins with GTP-ase activity towards the R h o / R a c GTP-binding proteins [66]. Rho and Rac proteins are linked to events such as membrane ruffling and the formation of focal contacts [75]. Towards the C-terminus from the SH2/SH3 domains is situated the highly-conserved kinase domain of about 300 amino acids (also called the Src homology domain 1 (SH1)) that exhibits some 80% homology between c-src, c-yes, c-fgr, fyn, lyn, Ick, hck and tkl. This region has been defined by homologies with other kinases, mutational effects [76-78] and by the fact that proteolytic fragments of this region possess kinase activity [79]. The region has a glycine-rich ATP-binding site that includes a key lysine residue that interacts with ATP (src K-295; lck K-273) [80]. Included in the kinase domain is the autophosphorylation site, the primary site of in vitro phosphorylation [81-83]. Although wild-type kinase is not readily phosphorylated at this site in vivo, constitutively active, transforming versions of the kinase (i.e., pp60 v.... ) are phosphorylated at this site [82,84]. CD4 receptor aggregation induces a limited degree of in vivo p56 tck autophosphorylation at this site [85,86]. Additional sites such as a conserved leucine (Leu-516 in pp60 src) are also essential for kinase activity [87]. Next at the C-terminus is located a conserved tyrosine residue in a consensus motif that is involved in the regulation of kinase activity (Fig. 2B). Tyr-527 of pp60 c~rc and Tyr-505 in p56/ck serve as the principle of tyrosine phosphorylation in vivo [88]. pp60 ..... lacks this terminal tyrosine, and mutation of this residue induces constitutive kinase activity [89-92]. Phosphorylation of this residue inhibits catalytic activity, while dephosphorylation stimulates kinase activity. The interaction of middle t antigen of polyoma virus with the src

kinases pp60 csr¢, p59 fyn and pp60 cyes activates the kinase by preventing phosphorylation at this site [93,94]. Phosphorylation is mediated by the cytosolic tyrosine kinase Csk (C-terminal src kinase) [95,96], while dephosphorylation may be mediated by protein-tyrosine phosphatases (PTPases) such as CD45 [97-99]. Regulation by the C-terminal tyrosine is also influenced by the presence of the autophosphorylation site. For example, the autophosphorylation site in src at Y-416 is required for the full transforming potential of mutations at Y-527 [100]. Current models designed to account for the regulatory features of these regions postulate the folding of the kinase by the binding of the phosphorylated Cterminal tyrosine to the SH2 region of the kinase (i.e., either cis or trans) [101] (for a review, see Ref. 102). This auto-inhibitory model would create a fold that restrains the kinase in a repressed conformation. Dephosphorylation of the tyrosine would release these internal constraints, facilitating catalytic activity and the binding of phosphotyrosine-labelled proteins with the kinase. Consistent with this, phosphopeptides corresponding to the C-terminal motif will bind to the kinase within the SH2 region [103]. p47 gagcrk activates src kinases by competing for the carboxy phosphotyrosine, preventing an association with the internal src SH2 domain [104]. Alternative splicing within the src gene family has been noted to generate different isoforms of the src, fyn and lyn kinases (see Fig. 1) [105-110]. Murine c-src gene can encode three proteins, two of which are expressed in neural cells and a third expressed ubiquitously [105-107]. These forms differ within the SH3 region of the kinase [97]. The fyn gene can encode proteins p59 fy"(T) and p59 fyn(m by mutual exclusive splicing. The p59 f~(T) isoform is expressed exclusively in lymphoid cells, while p59 fyn(B) is found in other cell types, especially brain [108]. In the case of the lyn kinase, alternative splicing produces two isoforms (p53 tyn and p56 tyn) which differ in the N-terminal region [109]. Unlike p59 fyn, both isoforms of lyn are concordantly expressed in cells. Isoforms of another src kinase hck (expressed in myeloid and B-lymphoid cells) also differ at the N-terminal region (p56 hck and p59 hck) by utilizing alternative initiation codons [110]. These different forms have likely evolved to mediate interactions with intracellular molecules or surface receptors. III. p56/ck and p59 fyn binding to T-cell receptors I l i A . p56 lck binding to CD4 and CD8 Src kinases lack transmembrane and extracellular regions, making them unable to interact directly with the extracellular environment. Their role in signal

244 transduction and growth control became apparent with the finding that p56 tck could interact with the cellular receptors CD4 and CD8 [9-12]. To briefly review, the CD4 and CD8 antigens are expressed on T cells within the lymphoid system and play roles in the recognition of foreign antigen as presented by major histocompatibility complex (MHC) antigens, an event that induces T-cell proliferation (reviewed in Refs. 111, 112). T lymphocytes recognize antigen in the form of peptide bound to the polymorphic cleft of major histocompatibility (MHC) class I or II antigens (reviewed in Ref. 113). MHC-bearing cells present peptide recognised by the highly-variable a n t i g e n - r e c e p t o r c o m p l e x (TcR~'/CD3). The peripheral T-cell population is comprised largely of two mutually exclusive subsets, T c R / C D 3 ÷ CD4 ÷ C D 8 - and T c R / C D 3 ÷ CD4- CD8 + cells (i.e., 'CD4 or CD8 single positive cells'). CD4 facilitates the process by interacting with invariant regions of MHC class-II antigens, while CD8 reacts with MHC class I antigens [114,115]. CD4 (a single chain at 52 kDa) and CD8 (an a chain of 34 kDa and a 32-kDa /3 chain) are members of the immunoglobulin gene superfamily. The structure of two N-terminal domains of CD4 have been determined by X-ray crystallography [116,117]. CD4 possesses a lateral protusion from the N-terminal immunoglobulin domain, derived from an extra /3-pleated sheet structure. Overall, CD4 appears to form an extended rod-like structure. Binding to MHC class II involves an extended region on the face of the outer first two domains of CD4 [118,119]. By contrast, CD8 has a single N-terminal immunoglobulin-like domain [120]. The N-terminal domain is followed by a unique region which possesses a cysteine capable of binding to a second immunoglobulin-like/3 chain. CD8 can exist as either an a / a homodimer or an a//3 heterodimer. CD8 binds to a region within the o~3 membrane proximal domain of MHC class-I antigens [121,122]. The T c R ~ ' / C D 3 complex and C D 4 / C D 8 antigens presumably bind to the same MHC molecules during the presentation process. High-affinity binding of the T c R ~ ' / C D 3 complex to a n t i g e n / M H C does not always require CD4 and CD8 expression. The accessory function played by CD4 and CD8 enhances proliferation signals in cases of low-affinity binding to antigen, or low antigen abundance. Co-clustering of CD4 or CD8 with TcR~'/CD3 dramatically potentiates T-cell proliferation [123,124]. While the expression of the CD8a chain is sufficient to enhance proliferation, the presence of the /3 subunit further potentiates interleukin-2 production [125]. CD4 and CD8 designate functional T-cell subsets (helper, cytotoxic, suppressor-inducer functions) within the peripheral T cell compartment [112,126]. The interaction of p56 l~k with CD4 and CD8 provided a molecular basis for the signalling function of CD4 and CD8, as well as providing a role for the

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3 ~ io 2o ~a u MG~V~SSHPIEDDWMEN [OV~EN~ HYPJVPLOSJKJ~LPJRNG~V~P RRVCKCPRPVV KS E;® ® ® ®

Fig. 3. Comparisonof CD4 and CD8 cytoplasmicdomains(p56tc~-binding motif) and the N-terminalregion of p56tck. (I) CD4 and CD8ct cytoplasmic regions share a sequence of limited homologywhich includes the CXCP binding motif for p56tck. A larger consensus region is designatedby the CD4 residues (KTCQCPHRFQKT)and the CD8a sequences (RRVCKCPRPVVKS), providing the consensus sequence K/RK/RXCXCPXXXXKT/S. The CD8b chain, which lacks this sequence, does not bind to p56tc~. (II) CD4 and CD8 cysteine residues within the binding site are flanked by positively-charged amino acids. In contrast, the p56tck cysteines are flanked by negatively-chargedamino acids, suggestingan ionic interaction between CD4, CD8 and p56tc~. src-related kinases in signal transduction. The interaction was first shown by co-precipitation and in vitro kinase labelling [9-11]. Detergents vary in their extraction of the catalytically active complex, the octyl ether Brij 96 being most efficient [127]. Between 30 to 90% of p56 tck is stably associated with CD4 or CD8, depending on the T cell examined. CD4 and CD8 cytoplasmic regions share a region of limited similarity encoded by the CD4 sequence K K T C Q C P H R F Q K T and the CD8 sequence RRVCKCPRPVVKS designating a 13-residue motif ( K / R K / R X C X C P X X X X K T / S ) (Fig. 31). This sequence is located mid-way in the cytoplasmic domain of CD4 (residues 419-431) and proximal to the lipid bilayer in the CD8a chain (residues 190-203). Within this sequence is a conserved CXCP motif, as well as five basic amino acids that alternate with hydrophobic residues on either side of a possible alpha helix (Fig. 31I). Although the exact molecular basis of the interaction is unknown, mutation of the cysteines disrupts p56 tck binding [21-23]. The cysteine requirement may differ slightly for CD4 and CD8. Mutation of either cysteine eliminated complex formation of p56 to* with CD4. CD8a chain may require the mutation of both cysteines in order to disrupt the complex [22]. Cysteine residues do not undergo covalent bonding [10]. Although still somewhat unclear, other amino acids within the cytoplasmic sequence may directly or indirectly stabilise the interaction. This can been inferred from the observation that the deletion of C-terminal residues following the CXCP resulted in diminished levels of precipitable p56 tck [22]. In addition, while the

245 transfer of the CD8a VCKCPR motif to the VSV G viral protein associated with p56 t~k, the analogous sequence from CD4 was unable to support complex formation [23]. Another protein 4-1BB that is expressed after T-cell activation possesses the CKCP motif and has recently been reported to bind to p56 ~ck [128]. The MHC-like CDla antigen possessing the C X C sequence (RKRCFC), as does a putative receptor on natural killer cells (NK) NKI.1/NKRP1 which possesses the C X C P in a reverse orientation [129]. III-B. Binding site within p56 tck The N-terminal region of p56 lck differs from that of other src-family members (Fig. 2A). Residues 10-32 within the N-terminal region of p56 lck bind to the cytoplasmic sequences of CD4 and CD8 [21,22] (Fig. 311). Within this sequence, mutation of cysteines at positions 20 and 23 abrogates the binding to CD4 and CD8 [22]. Whether these cysteines mediate direct interactions, or play an indirect role on the conformational orientation of the kinase has not been established. Crystallographic analysis should resolve this issue. A direct interaction involving cysteine residues in both the receptor and kinase argue for the possible role of metal ions in the interaction. C-XX-C sequences are found in several proteins which undergo tetrahedral formation with metals. These include the metallothionin protein which binds Zn 2÷ [130], and the Tat protein of the Human Immunodeficiency Virus which binds Cd 2÷ and Zn 2÷ [131]. This region is also rich in negatively-charged amino acids which could interact ionically with alternating basic residues within the CD4 and CD8 binding sequence (Fig. 311). Ionic interactions do not appear to be required for complex formation, but could play roles in regulating kinase activity. III-C. Regulation of CD4 / CD8-p56 I~g kinase activity Conventional protein tyrosine kinase receptors, such as the PDGF-R and EGF-R, are stimulated by the combined action of ligand binding and receptor crosslinking (reviewed in Ref. 132). Insufficient information is available to drawn firm rules regarding the regulation of catalytic activity within the CD4-p56 l~k and CD8-p56 l~k complexes. Nevertheless, several regulatory mechanisms appear to operate including the binding of p56 lck to the CD4/CD8 cytoplasmic domain, receptor-ligation and dimerization, an interaction with adjacent receptors such as CD45, or with intraceUular kinases such as Csk [12]. During antigen-presentation by MHC-bearing adherent cells, CD4, CD8, T c R ¢ / CD3, CD45 and other receptors are likely to undergo clustering and aggregation. Clustering may be induced by aggregated MHC antigens, or by later events such as

patching or receptor co-capping. Antibody-induced CD4 cross-linking increases the level of precipitable in vitro p56 lck kinase activity as assessed by autophosphorylation, or the phosphorylation of the substrate enolase [85,86,133]. Autophosphorylation occurs at Tyr394, a site analogous to Tyr-416 in pp60 src. However, although suggestive of activation, it is questionable whether in vitro assays mimic receptor-mediated in vivo events. The difficulty concerns the fact that another kinase, pS0 csk which regulates p56 tck activity, fails to co-purify with in vitro purified p56 tck [134]. pS0 csk is SH2 and SH3-carrying tyrosine kinase that inhibits p56 tck activity by phosphorylating the kinase at Tyr-505 [95,96]. Without p50 csk, in vitro kinase labelling is characterised by a predominance of autophosphorylation at Tyr-394, a site weakly phosphorylated in vivo. In vivo labelling experiments have, therefore, been crucial to the interpretation of cross-linking events. Under this regime, anti-CD4 cross-linking exhibits less dramatic effects on the phosphorylation status of p56 to*, which in turn varies with different T-cells [85,86,133]. In one murine hybridoma, anti-CD4 induced some phosphorylation at Tyr-394 with the overall majority of labelling at Tyr-505 [85,133]. Tyr-505 was the principal site of phosphorylation in untreated cells. Tyr-505 phosphorylation is expected to be antagonistic to kinase activation. Whether Tyr-505 phosphorylation occurs on the same, or on different kinase molecules that are phosphorylated at Tyr-394 is unknown. Possibly only a subset of CD4-p56 tck complexes are induced to undergo Tyr-394 phosphorylation, and these are distinct from Tyr-505 phosphorylated complexes. The effect of dual phosphorylation on lck activity is unclear. In the case of human T-cell clones, anti-CD4 induced a significantly greater degree of phosphorylation at Tyr394, an effect that varied between T-cell clones [86]. Induction of Tyr-394 phosphorylation is strongly suggestive of enzymatic stimulation; however, the consequences of potential dual phosphorylation remain unclear. Anti-CD4 cross-linking augments tyrosine phosphorylation of substrates in the T-cell, an event that further argues for stimulation and targeting of the kinase [85,135]. Further information is required to determine whether the conventional wisdom regarding the effects of Tyr-505/Tyr-394 phosphorylation on unbound lck can be applied to the kinase when associated with CD4. Demonstrating an effect of anti-CD8 cross-linking on p56 lck activity has proven more difficult, possibly due the fact that this receptor can exist as a hetero (or//3) and homodimer ( a / a ) [136,137]. p56 Ick binds to the a, not the/3 chain [138]. This structural difference points to potential differences between the CD4 and CD8 receptors and their function. Generally, less p56 tck activity has been found to co-precipitate with CD8

246 than CD4. This is unlikely to be due to the binding affinity of the cytoplasmic sequence for the kinase. In fact, the avidity of p56 ~k for the CD8 motif may be greater than the CD4 motif, as judged by motif transfer experiments [23]. The consequences of homodimer and heterodimer formation on p56 tc~ activity remain unclear. The a - p 5 6 t ~ k / a - p 5 6 t~k receptor is presumably in a state of constitutive dimerization. CD8a can function in the absence of CD8fl, and is the only form of the receptor found on natural killer cells (NK) [136,139]. Both homodimers and heterodimers are expressed on the surface of peripheral T cells [136]. The influence of the/3 chain is unknown, although in certain instances, its presence increases the level of IL-2 expression [125]. Antibody-induced receptor cross-linking effects may be limited by the interference of the /3 chain on interkinase interactions. Similarly, constitutive a - p 5 6 t c k / a p56 t~ dimerization may be refractory to further crosslinking with antibody. Differences in molecular configuration of CD8 and its p56 tck binding motif may provide a basis for different CD8 and CD4 functions. In addition to receptor-p56 t~k cross-linking, p56 t~k activity is also activated by the mere binding of the kinase to the CD4/CD8 cytoplasmic tail. Reconstitu-

A,

tion studies of CD4 and p56 tck in baculoviral expression system shows that p56 tck binding to CD4 induces a 10-20-fold increase in activity relative to unbound kinase (Raab, M. and Rudd, C.E., data not shown). This is accompanied by a marked increase in in vitro kinase activity and in vivo labelling of p56 tck at the autophosphorylation site Tyr-394. The in vitro activity was observed in cell lysates containing p50 Csk. In addition, peptides corresponding to cytoplasmic tail of CD4 have been reported to induce a 20-fold increase in catalytic activity [140]. The phosphorylation induced by peptides occurred at sites Tyr-394 and Tyr-505. Activation occurred by increasing the Vmax without an apparent effect on the K m for substrate. Moreover, peptides with substituted cysteine sites functioned equally as well as peptides with cysteines. Limited binding with a high dissociation constant of 4.4/xM occurred, despite the substitution of cysteines. Further, polycations such as polylysine and polyarginine induced activation of p56 tCk, indicating an effect of ionic interactions on p56 t~g activity. This may correlate with the presence of positively-charged amino acids in the CD4 and CD8 cytoplasmic consensus sequence (Fig. 311). Inter-chain regulation of src kinases has been previously observed

B,

),

C.

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505

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~

~'~

Act!vity

PI3K. i1

PI3K

1

Activity

¥ 505

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G CSK

Fig. 4. Regulation of the CD4-p56 tck complex. (A) Receptor-unbound p56 tck may be restrained in a repressed state by the binding of the C-terminal phosphotyrosine (Tyr-505) to the SH2 domain within the same molecule. (B) Receptor binding induces a partial activation of the kinase (10-20-fold increase in activity), together with the association of certain intracellular molecules such as PI 3-kinase. Whether this involves the unfolding of the kinase is unknown. It may also be accompanied by the dephosphosphorylation by protein-tyrosine phosphatases such as CD45. Activation may be inhibited by p50 Csk tyrosine kinase which phosphorylates the C-terminal tyrosine residue. (C) Co-aggregation of assembled CD4-p561ok complexes with associated PI 3-kinase leads to a further moderate increase in p56 tck activity (2-5-fold increase in activity), accompanied by intermolecular tram phosphorylation at the autophosphorylation site (Tyr-394). The level of precipitable PI 3-kinase also increases by cross-linking of receptor complexes.

247 between pp60 src and middle-T antigen of Polyoma virus and p47 gag'crk [93,94,101]. Together these observations suggest a two-stage model for the regulation of the CD4-p56 ~k complex (Fig. 4A-C). In the first stage, the mere binding of p56 tck to residues within the cytoplasmic tail of CD4 or CD8 causes a 10-15-fold stimulation of the kinase (Fig. 4A, B). The molecular basis of this mode of stimulation is unclear, but may include charge interactions and conformational changes. Kinase-receptor binding requires the C-X-C-P motif. Putative ionic interactions between negatively-charged residues within the kinase and positively-charged residues within the CD4/CD8 cytoplasmic sequences could regulate activity. Accompanying events may involve conformational changes within p56 lck. Consistent with this model, CD4-p56 tck binding appears to be accompanied by the association of intracellular molecules with the complex (Fig. 4B). These include p110, p72 ~af and PI 3-kinase, each of which has been reported to associate with CD4-p56 tck as a complex, but not with free CD4 or p56 tck [69,141146]. In the second stage, ligand-induced dimerisation of CD4-p56 t~k may further stimulate p56 lck and associated enzymes (Fig. 4C). CD4-p56 tCk cross-linking may increase the catalytic activity of p56 lck, by either cis and trans phosphorylation. Similarly, receptor crosslinking modulates the level of precipitable PI 3-kinase activity [69,142]. Cross-linking may also act to target substrates for phosphorylation, as shown by anti-CD4induced phosphorylation of intracellular substrates [28,135]. Although our understanding of the mechanism of inter-chain regulation and recruitment is still preliminary, the emerging model illustrates a fundamental difference between the regulation of CD4-p56 t~k and receptor-tyrosine kinase receptors. In the case of the PDGF-R, stimulation of the kinase domain depends entirely on ligation by growth factor. Autophosphorylation is then acccompanied by the recruitment of associated proteins such as rasGAP, PI-3-kinase, PLCy, NcK and SHC. Inter-chain regulation of CD4-p56 ~cg would create an unconventional situation where unligated receptor-kinase complexes exist in a kind of primed, partially-activated state. The functional rationale for this two-phase regulation of p56 t~g is unclear, but may involve a requirement for partially-activated receptors (with associated intracellular proteins such as PI 3-kinase) in order to attain a threshold of signalling in low-afflnity interactions involving CD4 and MHC antigens. The activity of ligated and unligated CD4-p56 tog complexes may be further influenced by an antagonistic interplay between the receptor-phosphatase (PTP'ase) CD45 and p50 Csk (Fig. 4B,C). Other PTPases may include T-cell PTPase [147] and SH2-bearing

PTPases (SH-PTP1) [39,40,97-99,147-156]. Certain isoforms of CD45 may physically associate with CD4p56 lck [151,152]. CD45-negative T cells show an increase in Tyr-505 phosphorylation, as well as a concordant reduction of p56 tck activity [97,98]. Related p59 fyn(T) is also activated by dephosphorylation at Cterminal Tyr-531, an event that may be achieved by a physical association between CD45 and the TcR~'/CD3 complex [153-156]. Conversely, CD45 may be regulated by transient phosphorylation on tyrosine residues, possibly by lck or fyn [157]. Since the vast majority of in vivo phosphorylation of p56 tck has been observed at the carboxyl Tyr, it is unclear from these studies whether CD45 has a specific affinity for Tyr-505. Arguing against this, the co-clustering CD4 and CD45 on the cell surface caused the complete in vitro de-phosphorylation of p56 tck at both Tyr-505 and Tyr-394 [99]. Further information is required on the specificity of CD45 in vivo, and whether the targeting of certain residues is related to enzymatic specificity, or is controlled sterically by the positioning of CD45 within a multi-subunit complex. The dominant effect of CD45 implies that other PTPases may play less dramatic regulatory roles on p56 t~k and p59 £yn(T) activity. III-D. CD4-p56tCk-associated intracellular molecules Downstream signals mediated by CD4- or CD8p56 tck cross-linking are likely to be mediated by intracellular molecules that bind to the receptor complex, or act as substrates for p56 tck. Proteins may bind to the SH2 and SH3 domains of p56 lck or SH2-carrying proteins may bind to autophosphorylation sites with p56 t~k. To date, several intracellular proteins and enzymatic activities have been described that co-purify with CD4p56 lck or free, unbound p56 tck. These include a putative p32 GTP-binding protein [141], lipid kinases such as phosphatidylinositol 3-kinase [69,142-144], phosphatidylinositol 4-kinase [144], the serine/threonine kinase p72 raf [145] and a serine/threonine phosphorylated Raf-related p l l 0 molecule [146]. The identification of these interactions attests to the oligomeric nature of the CD4 antigen. The p32 protein was recognised by an antisera to the consensus GTP-binding region (GlyXa-Gly-Lys) of the heterotrimeric G proteins. GTP binding and hydrolytic activity are associated with the CD4 complex and [a-p32]GTP covalently labels p32 in UV-mediated cross-linking experiments [141]. Further studies will be required to determine the structure of p32 and whether p32 is an actual G protein capable of hydrolyzing GTP. Heterotrimeric G proteins mediate signalling by receptors such as the /3-adrenergic and muscarinic receptors [158]. Based on molecular weight, CD4-p56 tck associated p32 does not appear to fall within this category of GTP-binding protein, and may instead be related to the intermediate-molecular-mass

248 class of GTP-binding proteins [159]. Alternatively, p32 may simply bind GTP-binding proteins within a larger unrelated structure, but may not itself hydrolyse the nucleotide. For example, the TcR~" chain has been reported to bind to GTP [160]. Of the currently characterized SH2/SH3-carrying proteins, PI 3-kinase co-purifies with CD4-p56 t~k and CD8-p56 t~k [142-144]. Others failed to detect PI 3kinase using an anti-lck antiserum [161], a result that may be related to the fact that PI 3-kinase preferentially associates with CD4-p56 ~ck as a complex [142,144]. PI 3-kinase is comprised of a SH2-carrying p85 regulatory subunit coupled to a p110 catalytic subunit [35-37,162]. Various isoforms of both the p85 and p110 subunits exist. The enzyme is not a part of the classical pathway of lipid turnover, instead phosphorylating the D-3 position of the inositol ring of phosphotidylinositol [163]. This results in the generation of the phosphoinositides PI (3)P, PI (3,4)P 2 and PI (3,4,5)P 3. Each of these phosphoinositides are resistant to cleavage by established phospholipases. The targets of these metabolites are unknown; however, its importance has been underlined by the fact that the interaction between the enzyme and the PDGF-R required for signalling by ligand [164,165]. Anti-CD4 cross-linking increased precipitable activity of the enzyme by 10-20-fold. Consistent with this, T-cell activation generates the PI (3,4)P 2 and PI (3,4,5)P 3 [166]. The use of fusion proteins including lck-SH2, IckSH3 and Ick-SH2/SH3 showed that the CD4-p56 t~g complex utilises a unique mechanism to recruit PI 3-kinase (Ref. 69, 144 and Prasad, K.V.S. et al, data not shown). Unlike receptor-tyrosine kinases which recruit PI 3-kinase by the binding of the SH2 domain of p85 to autophosphorylation sites within the cytoplasmic domain, p56 leg uses its own SH3 domain to bind to residues within PI 3-kinase. Recognition is further enhanced by the presence of the SH2 region, although little if any direct binding to lck-SH2 domain was noted. The lck-SH3 interaction with PI 3-kinase represents a distinct mechanism by which src-related kinases bind and regulate activity. Importantly, it introduces the possibility that recruitment of PI 3-kinase by CD4lck can occur in a tyrosine kinase/phosphorylation independent manner. This further underlines the difference between conventional receptor tyrosine kinases and the CD4-p56 tCk system. In the case of the PDGF-R, PI 3-kinase is entirely dependent of ligand binding and autophosphorylation to create binding sites. SH3 binding does not appear to involve phosphotyrosine residues. Instead, SH3 domains to bind to a proline-rich consensus motif within the 3BP-1, 3BP-2, formin, SOS and the rat m4 muscarinic acetylcholine receptor [67,68,70,71]. Two potential proline-rich binding sites exist within the p85 subunit, but not within the p110 subunit. Whether different p85 subunits are involved in

binding CD4-p56 tck and receptor-tyrosine kinases remains to be determined. PI 3-kinase, like the drk-SOS interaction [70,71], represents a case of a SH3 interaction with a key signalling protein, underlining the importance of the SH3 domain in CD4-p56 lc~ signalling. It remains to be determined whether other mechanisms exist to recruit PI 3-kinase. Controversy exists as to the degree of tyrosine phosphorylation of PI 3-kinase under physiological conditions [167-169]. Besides PI 3-kinase, CD4-p56 tck was also found to co-precipitate PI 4-kinase activity, at levels as great as PI 3-kinase (Ref. 144 and Prasad, K.V.S. et al, data not shown). PI 4-kinase is part of the classical PI pathway with the generation of PI 4-P and PI 4,5-P 2 for PI turnover. PI 4-P and PI 4,5-P 2 can be acted upon by PI 3-kinase to generate PI 3,4-P 2 and PI 3,4,5-P 3. To date, the EGF-R is the only other receptor to have been reported to associate with PI 4-kinase [170]. As in the case of PI 3-kinase, antibody-induced CD4 cross-linking caused an increase in CD4 precipitable activity [144]. Further work is required to determine whether the enzyme associates with the kinase, or with the CD4 cytoplasmic domain. The connection between CD4-p56 tck and inositol turnover is further evident with the finding of phospholipase C3, (PLC~/) associated with p56 tCk [171]. PLC~/ catalyzes the hydrolysis of PI 4,5-P 2 to generate inositol 1,4,5-triphosphate (I P3) and diacylglycerol (DAG), thus mobilizing intracellular Ca 2÷ and stimulating protein kinase C [172]. The SH2-binding region of PLCy binds to phosphotyrosine residues within autophosphorylated EGF-R and PDGF-R [173,174]. Indeed, T-cell activation leads to the tyrosine phosphorylation and activation of PLCy [175,176]. Using T r p E / P L C y l fusion proteins, Weber et al. [171] provide evidence that the N-terminal SH2 domain of PLCy binds to p56 tck [171]. TcR~'/CD3 engagement promoted the association of PLCy with CD4-p56 tCk providing an intracellular link between the CD4 and TcR~'/CD3 complexes. AntiPLC3, co-precipitated several bands at 36, 38, 58 and 63 kDa, several of which appeared to co-migrate with anti-lck precipitated bands. A role of these associated proteins in mediating the binding between PLCy and the kinase is unclear. Aside from an indirect influence on serine/ threonine phosphorylation due to a connection between inositol turnover and PKC, CD4-p56 ~ck has been found associated with p72 raf and a related/associated Raf-related p110 [145,146]. p72 raf is serine/threonine kinase first identified as the transforming gene of the murine sarcoma virus 3611 [177,178]. p72 raf can be tyrosine phosphoryated and may associate in low amounts with the PDGF-R and other receptors [178,179]. p21 ras and pp60 v'sr~ appear to operate synergistically in the activation of downstream Raf-1 [180]. Precipitation and in vitro kinase labelling of Raf-1

249 III-E. The TcR~ / CD3-p59 fy~ connection

precipitates reveals the presence of a prominent p110115 polypeptide. In the case of CD4, both p72 ~ f and p l l 0 bind preferentially to CD4-p56 t~k as an assembled complex with little if any binding was detected to receptor-free lck. [145,146]. Although the primary structure of p l l 0 is unknown, it may be physically complexed with p72 r~f and conceivably act as a substrate for p72 Raf. Intriguingly, it also possesses an epitope recognized by some antiserum to the Cterminus of p72 Raf [120,121]. In fact, some antisera preferentially react with p l l 0 over p72 raf (data not shown). Given that the antisera reacts outside of the Raf kinase domain, its is unclear whether p l l 0 itself possesses serine/threonine kinase activity. Attempts to detect catalytic activity by labeling with ATP have been unsuccessful (data not shown). Anti-CD4 ligation induced an increase in p72 Raf activity, and a small degree of tyrosine phosphorylation [145]. By contrast, the phosphorylation of p l l 0 was detectable only on serine residues [146]. p72 Raf and p l l 0 may serve as a bridge between the CD4-p56 t~k complex and the downstream pathways of T-cell activation. In the aforementioned studies, the GST-SH2 fusion protein of GAP (GTPase-activating protein) failed to precipitate lck, while in other studies the same reagents readily co-precipitated the EGF-R and PDGF-R. In vitro reconstitution experiments have shown that ras GAP can be phosphorylated by p56 ~k and that some 1% of the molecule can be found associated with the kinase [181]. Phosphorylated rasGAP has also been detected in fibroblasts over-expressing the activated form of p56 t~k (p5@ kFS°5) [182]. However, to date, studies have failed to detect significant GAP-lck complexes in T cells. This is in keeping with the finding that there is variablility in the ability of different receptors to bind to intracellular proteins. For example, CSF-1-R binds to PI 3-kinase but not GAP [183,184].

Human CD3~' Human CD38 Human CD3£ Human ~ chain (1) Human ~,chain (2) Human ~ chain (3)

MouseMB1 MouseB29 Rat Fc~ychaln Rat Fc¢ p chain Human CD5

Lck l"yr394 Fyn Tyr 420

P R V G P G

N N P O Q H

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T cells express three src-related protein-tyrosine kinases, p56 zck, p59 ~('r) and p60 TM. p59 fyn of 59 kDa is expressed in most cell types; however, as a result of mutually exclusive splicing, p59 yyn exists as two isoforms [108]. The more prevalent form, p59 fyn(B) is highly expressed in brain and other cell types, while p59 fr'O3 is expressed in T cells. The splicing occurs within exon 7 in a region at the beginning of the kinase domain (Fig. 1). Samelson and co-workers first showed that p59 fy'°) can co-precipitate with the TcRff/CD3 complex [14,185]. The TcR~'/CD3 complex itself is comprised of the TcRa//3 chains, the CD3 % ~ and E chains and the ff/r//Fc3, chains [186]. Associated p59 fyÈ has been identified in only a limited number of detergents (such as digitonin or Brij 96) that appear to maintain the integrity of weak protein-protein interactions. Chemical cross-linking and co-capping studies have verified the existence of the complex [187,188]. Some 20% of fyn can be detected in association with the receptor, depending on the conditions of isolation. A surprisingly small 2-4% of total TcR~'/CD3 is fynassociated. In a few cell lines, an additional form of fyn at 72 kDa also associates with the complex and differs from p59 fy'(T) by the presence of additional sites of serine/threonine phosphorylation [189]. As with p56 tCk binding to CD4, the N-terminal region of p59 fyÈ mediates binding to the receptor [190]. The first 10 amino acids of fyn are sufficient to mediate binding to the TcR¢ chain, a region which is normally associated with myristoylation and membrane localisation. The molecular mechanism regulating the association remains unclear. As seen in Fig. 2A, this region is unrelated to other receptor-associated src-related kinases such as p60 ~r~, p56 l~k, p56 ty" or p55 blk. Consistent with this, neither p56/ck nor pp60 ~ bound

D

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Fig. 5. Conserved Y-Xn-Y-XXL motifs within the cytoplasmic domains of the CD3y,8,~, TcR~', MB1, B29 and CD5 antigens. The CD3y,8,E, TcR~', MB1, B29 and CD5 antigens share the Y-Xlt-Y-XXL activation motif. Each of these antigens are phosphorylated on tyrosine reidues as a result of receptor iigation. The Y-XX-L sequence is of particular importance for CD3, function. In the case of CD5, the Y-XX-L sub-motif has been replaced by the Y-XX-P sub-motif. CD5 possesses a set of residues next to the first Y residue (DNEY) which is homologous to the autophosphorylation site within p56 lck and p59 fyn.

250

CD5

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CD3-COMPLEX E 8

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P I 3,4,5-P 3

ZAP-70

Pathway B

Tyrosine Phosphorylation

Ca++ DAG PKC

MAP-2 KINASE

Fig. 6. Signalling cascade mediated by CD4-p56 tck and TcR~/CD3-p59 fynf'r), protein-tyrosine and lipid kinase mediated pathways. The CD4, CD5 and T c R ( / C D 3 structures, their associated kinases (p56 tck, p59 fyn(T), ZAP-70) and SH2/SH3-binding proteins (p120/130 and PI 3-kinase) are depicted. One signalling pathway is mediated by the protein-tyrosine kinase domain of p56 tCk and p59 fynO3 and the phosphorylation of TcR~, CD5 and possibly the CD3 subunits. Co-localisation and ligation of CD4-p56 tCk, CD5, TcR~/CD3-p59 fyncr) and CD45 (not shown) stimulates the kinase activity leading to the tyrosine phosphorylation of substrates. TcR~" phosphorylation at the activation motif Y-XX-L-X7. 8Y-XXL/I leads to the recruitment of SH2-carrying proteins such as ZAP-70. Similarly, CD5 possesses a version of the Y-Xll-Y-XXL activation motif with residues next to the first Y residue (DNEY) that are homologous to the autophosphorylation site with p56 tck and p59 fyn. CD4-p56tCk-mediated tyrosine kinase activity appears correlated to the activation of MAP-2 kinase (ERK) (pathway B) An alternate pathway mediated by PI 3- and PI 4-kinases leading to PI turnover (Ca 2 + release, DAG and PKC activation) as well as the generation of PI 3-P, PI 3,4-Pz, PI 3,4,5-P3 (pathway A). Pathways A and B may function in conjunction with each other. Alternatively, the demonstration that Lck and Fyn SH3 domains mediate binding to PI 3-kinase introduces the possibility that PI 3-kinase binding and function may Operate independent of the kinase domain.

251 TcR~" or CD3 subunits, although the transfer of the fyn N-terminal motif to src conferred binding to TcR~'. The binding sequence possesses two Cys residues, the proximal Cys being conserved in p56 tck and p56 ty", but not pp60 src or p55 blk. In the case of p56/ck, the first Cys has been implicated in membrane localisation [22]. p59 fyn(T) associates with the various CD3 y, 6 and • chains as well as the phosphorylated TcR~" [190]. As first pointed out by Reth [191], each of these chains share a common motif defined by the sequence Y-XXL-X7_8-Y-XXL/I [191] (Fig. 5). It is unclear as to whether the binding of p59 fyn to these various chains requires phosphorylation of the tyrosine residues. Although historically, tyrosine phosphorylation of the CD3 7, 3 and • chains has been difficult to detect, a low level of tyrosine phosphorylation has recently been reported [192]. Although the stoichiometry of the fyn(T)-TcR~'/ CD3 interaction appears quite low, antibody-induced TcR~'/CD3 stimulation of p59 fy~(T) activity can be detected [193,194]. By contrast, p56 t~k was not consistently activated, although this may vary between cell lines [85,193,194]. p59 fy"°') stimulation was accompanied by a dramatic increase in the phosphorylation of p59fY"(T)-associated p120/130 molecule [194]. Transfection of p59 frncr) in fibroblasts has indicated that p120/130 interacts directly with p59 ry", and therefore associates indirectly with the TcR~'/CD3 complex. The Fyn-SH2 domain binds to the p120/130 polypeptides (da Silva, A. et al., data not shown). Although the identity of p120/130 is unestablished, it appears to associate with p59 fyn and not p56 lck (da Silva, A. et al., data not shown). As in the case of p56 lck, the use of GST fusion proteins has shown that p59 f y ~ utilises its own intrinsic SH3 domain to bind to PI 3-kinase [69]. Although a low level of GST fyn-SH2 binding to PI 3-kinase was observed in T cells, the level of GST fyn-SH3 binding was some 50-times higher than GST fyn-SH2 binding. Further, fyn-SH3 binds directly to the p85 subunit of PI 3-kinase, a chain that possesses proline-rich domains. As seen in Fig. 6, receptor-associated p59 Zy~(T) is organised in discrete functional casettes that regulate both binding to the receptor complex and to the downstream targets. While the N-terminal region of fyn binds to the CD3 y, /~, • chains and the TcR~" chain, the p59fyncr)-SH2 can bind p120/130, and p59fy"(T)-SH3 binds PI 3-kinase. Both PI 3-kinase and p120/130 co-purify with receptor-free fyn and the T c R ~ / C D 3 complex, p59 fynfr) may therefore introduce PI 3-kinase and p120/130 into the TcR~'/CD3 prior to receptor ligation. As in the case of CD4-p56 tCk, the fact that fyn-SH3 mediates binding to PI 3-kinase within TcR~'/CD3-p59 fyncl3 leads to the important prediction that PI 3-kinase is recuited independent of fyn tyrosine kinase activity and phospho-

rylation. Signals derived from the SH3-PI 3-kinase interaction may be therefore be distinct from signals generated by the protein-tyrosine kinase domain. Consistent with this, anti-CD3 induced stimulation of PI 3-kinase has been reported to occur independent of tyrosine phosphorylation [166]. By contrast, PLC7 may associate with the TcRff/CD3 complex and as such be regulated by tyrosine phosphorylation [195].

III-F. CD4-p56 tck and TcR~ / CD3-p59 fy~ association in a multimolecular complex CD4, CD8 and the TcR~'/CD3 complex synergise in the potentiation of T-cell growth. Antibody-induced co-clustering of CD4-p56 lck and the TcR~'/CD3 complex dramatically increases DNA synthesis and IL-2 release [123,124]. Such effects are consistent with the formation of a multimolecular complex induced by antigen-presentation. Antibody-induced modulation of TcR~'/CD3 from the cell surface is accompanied by CD4 co-modulation [196,197]. Although potentially explained by modulatory effect of protein kinase C activation on CD4 [198], these studies are also consistent with the notion of a physical association between CD4 and T c R / C D 3 [199,200]. Direct evidence of a multimolecular complex formed by these receptors was demonstrated by co-precipitation analysis. Burgess et al. [201] co-precipitated various CD3 subunits in antiCD4 and anti-lck precipitates as detected by the sensitivity of in vitro phosphotransferase labelling techniques [201]. Anti-CD4 preferentially co-precipitated the CD3e chain, making this chain a potential candidate to mediate the interaction. Energy transfer FRET analysis further suggests that lck is required for this interaction [202]. A functional correlation also exists between between the mitogenicity of anti-CD3 antibodies, their ability to induce co-modulation and the phosphorylation of the TcR~" chain [203]. In recent years, other components have been reported within a multimeric complex that include CD2 [204], CD5 [204,205] and CD45 [151-156]. Co-capping of CD4, p56 tck, TcR~'/CD3, p59 fyn and specific CD45 isoforms have also been reported in murine T-helper- l(Th 1) and T-helper-2 (Th 2) clones [206].

III-G. Biological functions of p56 lck and p59 fyn(rj III-G. 1. T-cell signalling: activation / anergy / apoptosis As previously outlined, CD4, CD8 and TcR~/CD3 regulate T-cell growth by the recognition of antigen in the form of peptide presented by major histocompatibility (MHC) class I or II antigens (reviewed in Refs. 111, 113). A functional linkage between the TcR~'/CD3 complex and p59 fyncr) has been demonstrated most aptly in transgenic mice. Overexpression of p59 fyn is correlated with enchanced Ca 2+ influx and prolifera-

252 tion [207]. By contrast, expression of the dominant negative form of kinase-inactive p59 fyn inhibited these events. Similarly, mice generated by homologous recombination that lack either p59 fyn(T~ [208], or p59 fy~T+a~ [209] are partially defective in TcR~'/ CD3-mediated signalling. The signalling defect was most pronounced in thymocytes, although peripheral T cells also showed a decrease in Ca2÷-mobilization and proliferation (> 50%) to anti-CD3 plus phorbol ester. p59fY~T~ may therefore be most important in signalling during development in a population of thymocytes. In mature splenic T cells, the response to alloantigen was either only partially affected, or unaffected by the loss of p59 fyn [208,209]. In terms of proliferation per se, p59 fyn appears to be partially required in T-cell signalling. Redundancy may exist in the ability of p56 tck to compensate for p59 fyn. However, individual T cells may differ in the dependence on distinct src-related kinases and the nature of effector functions linked to individual kinases may be found to differ. A differential role for the p59 fy~ff~ isoform has been suggested by the use of constitutively-active forms of the kinase in the transfection of T-cell clones. The transforming form of p59 fyn~T~,but not p59 fyn(B), has been reported to potentiate IL-2 production [210]. Constitutively-activated forms of p56 tog (F505) can also augment TcR-induced tyrosine phosphorylation and IL-2 production, even in the absence of CD4 and CD8 [211]. Caron and colleagues [212] found that the SH2 domain, but not the SH3 domain, was essential for TcR-induced tyrosine phosphorylation; however, both were essential for TcR-induced lymphokine production [212]. Slightly different results were obtained by Luo and Sefton [213]. T-cells transfected with the constitutively-activated form of lck (F505) produce significantly higher amounts of IL-2. However, in this case, the .lck SH2 domain, but not the Ick SH3 domain, appeared to be necessary for increased lymphokine production [213]. Although clearly of potential importance, the use of transforming versions of src-related kinases in this kind of analysis needs to be treated with certain degree of caution. Substrate specificity and regulatory feedback mechanisms may differ under conditions of constitutive phosphorylation compared to the transient activity and phosphorylation of the wild-type kinase. Transforming forms of pp60 src potentiate T-cell responses, including IL-2 expression, despite the fact that src is not normally expressed in T cells [214]. The one instance where the transfection of wild type p56 tCk factilitated T-cell function involved a requirement for the kinase in T-cell-mediated cytolytic responses [215]. An obvious role for the CD4-p56 t~k and CD8-p56 tck complexes during antigen-presentation would be to introduce p56 t~k to the TcR~'/CD3 complex. Within a multimolecular complex, TcR~', CD5, p59 fyn and PLCy

could serve as potential substrates of the kinase [185,195,205,216,217]. Co-receptor function of CD4 and CD8 requires associated p56 lck [218-222]. Forms of CD4 that lack the cytoplasmic tail, or that lack cysteines within the lck-binding motif are profoundly defective in co-stimulatory function. In certain cases, the requirement for p56 tCk can be overcome by increasing the density of antigen, or CD4, CD8 on the cell surface [218,219]. An enhanced response to low antigen density is of importance given the fact that physiological concentrations of antigen are likely to be quite limited [223]. Using a CD4-dependent T-cell hybridoma, Glaichenhaus and co-workers demonstrated that the CD4-p56 tC~ interaction potentiated responses to antigen by 50-100-fold [222]. The contribution of the SH2, SH3 or kinase domain of p56 ~ck in this system remains to be resolved. A smaller contribution was made by CD4-MHC adhesion, independent of p56 ~k. p56 ~k is therefore necessary in antigen-stimulation which requires CD4/CD8 co-receptor function. Peripheral T cells from lck-negative transgenic mice are also profoundly defective in T-cell signalling by antigen. Although these mice exhibit a block in thymic differentiation, there exists a small population of T cells in the periphery [224]. Given the caveat that aberrant differentiation may alter signalling, p56 t~k appears necessary for signalling by antigen. Interestingly, the same cells could be stimulated by anti-CD3 ligation, suggesting that antibody may be capable of stimulating cells via an alternative pathway, perhaps employing p59 fyn(T). Further support for an essential role of p56 l~k in the activation of certain T cells has come from the study of a mutant of the Jurkat T-cell line. Straus and Weiss [225] have analysed a mutant T-cell tumor (JCaM1) which lacks p56 tck expression and is defective in response to TcR~'/CD3 ligation. Both the induction of tyrosine phosphorylation and Ca 2+ release was dramatically impaired [225]. Expression of p56 lCk restored the defect. The signalling defect did not involve, in obvious way, T c R ( / C D 3 ligation in the context of the CD4-p56 lck complex. The cells did not express CD8 and expressed very low levels of CD4. Although low levels of CD4-p56 t~k might constitutively associate with TcR~'/CD3, as found in other cells [201], this study introduced the possibility that p56 t~k may function independent of CD4. p56 t~k and p59 fyn(T) could generate intracellular signals via the protein-tyrosine kinase domain, by SH3 binding to PI 3-kinase, or by SH2 binding to phosphotyrosine-labelled substrates (Fig. 6). In a scenario involving the tyrosine kinase domain, TcR~" phosphorylation may act as the first step in a multistep process that recruits downstream molecules. One candidate is the SH2 carrying, non-myristoylated tyrosine kinase ZAP70 which is related to the syk family of tyrosine kinases [226]. Unlike p56 t~k and p59 fyn(T~, ZAP-70 associates

253 with the TcR~ chain only after receptor cross-linking. Further, the association in COS cells requires the co-expression of either p59 fyn or p56 t~k. TcRff possesses three repeats of a Y-XX-L-X7_8-Y-XXL/I motif found in a variety of receptor phosphoproteins (Fig. 5). p56t~k/p59fY" are likely to directly phosphorylate the Y-XX-L-XT_8-Y-XXL/I motif, which in turn recruits SH2-carrying proteins such as ZAP-70. TcR ff dimerization alone is capable of generating intracellular signals [227,228]. ZAP-70 binding to the Y-XX-L-X7_8-YX X L / I motif accounts for ff function as measured by Ca 2÷ mobilization and tyrosine phosphorylation [229]. The C-terminal motif is essential. Expression of multiple repeats of the motif in chimeric constructs results in enhanced signalling, suggesting that the redundancy of the motif within the TcRff chain may act to amplify the signal. Both SH2 domains within ZAP-70 are required suggesting tandem binding to the two tyrosine residues within the Y-XX-L-X7_8-Y-XXL/I motif. In this sense, TcR~'-ZAP70 binding is somewhat analogous to recruitment of SH2 binding proteins by the EGR-R and PDGF-R. The major difference is that the initial phosphorylation event is mediated by a separate kinase, rather than by autophosphorylation. It is likely that the CD3y,3,e chains utilise a similar mechanism to recruit downstream targets. These chains appear to be capable of generating distinct kinase-mediated signals [230,231], which may lead to the phosphorylation of a distinct, but overlapping spectrum of proteins [231]. Situations of abundant ligand, or high affinity binding between antigen and the TcRa//3 chains, may suffice to directly activate p59 fy" and thereby phosphorylate TcR~'. Under conditions where the amount of ligand is limiting, or where the binding affinity of the TcR~'/CD3 complex for ligand is low, engagement may be insufficient to allow stimulation of p59 fy"~T),thereby necessitating the co-ligation of CD4-p56 t~k. Alternatively, since only a small% of TcR~'/CD3 complexes bind to p59 fy~O3, CD4-p56 ~ck may be required to phosphorylate TcR~" in p59fYnO~-negative complexes. On a basic level, p56 l~k could be conceived as a back-up system for p59 fyn. However, the system will almost certainly involve subtle differences in the affinity of p56 lck and p59 fy"~T) for different tyrosine residues within the TcR~" and CD3 chains. Different affinities of p56 t~k and p59 fyn for the various sites should allow for the selection of different substrates. By analogy to the EGF-R and FGF-R, intracellular substrates such as PLCy and PI 3-kinase have varying affinities for separate Tyr-P sites [50-53]. Other T-cell antigens and receptors may also impinge on this process. The stimulatory properties of CD2, Thy-1 and Ly-6 in T cells are known to depend on TcR~ expression [232-234]. These antigens can induce TcRff phosphorylation and may associate with

src-related kinases [17,233-235]. CD5, a member of the macrophage scavenger receptor family [236], shares a number of characteristics with the TcR~ chain. Both structures can be co-purified with the TcR/CD3 complex in mild detergents and act as a substrates for a protein-tyrosine kinase [204,205,217]. As seen in Fig. 5, CD5 possesses a version of the Y-Xn-Y-XXL activation motif. However, the Y-XX-L sub-motif found in the TcRff, CD3, MB1, B29 and FcE chains has been replaced by the Y-XX-P sub-motif. Mutational analysis has shown the Y-XX-L sequence to be of particular function importance in CD3~ [231]. This suggests the recruitment of a distinct intracellular mediator by CD5. CD5 also possesses an intriguing set of residues next to the first Y residue (DNEY) which is homologous to the autophosphorylation site with p56 lck and p59 fyn (see Fig. 5). CD5 therefore appears to be tailor-made to act as a substrate for these src kinases. Not unexpectedly, CD5 is amongst the most rapidly phosphorylated substrates within the T cell (ta/2 = 20 S [205], see also Fig. 6). Further, co-expression of CD5 and p56 tck in the baculoviral expression system resulted in CD5 phosphorylation (Raab, M. and Rudd, C.E., data not shown). Hence, CD5 is likely to recruit downstream molecules in a manner analogous to TcR~" and receptor-tyrosine kinases. This signalling event may be tied to its ability to bind to the B-cell antigen CD72 [237]. Indirect evidence implicating a function for the tyrosine kinase domain in the activation process has involved studies on CD45 negative mutants. These cells are defective in T-cell signalling and in anti-CD4 induction of substrate phosphorylation [238-240]. The defect in tyrosine phosphorylation may be linked to the depressed activity of p56 tCk and possibly p59 fyn in these cells [154-156,175,176]. Tyrosine kinase inhibitors also ablate T-cell function [241,242]. The requirement may be restricted to early events of activation, since transfection with the muscarinic receptor, a G-protein-dependent receptor, can by-pass the defect and induce activation related events [239]. Another way of by-passsing the absence of CD45 is to co-ligate CD4-p56 l~k and the TcR~'/CD3 complex, an event that caused the phosphorylation of PLCy [243]. Trans phosphorylation between fyn and lck may be sufficient to activate one or both kinases, and to overcome the low basal level of activity imposed by the absence of the phosphatase. Aside from the tyrosine kinase domain, p56 ~ck and p59 £yn possess SH2 and SH3 domains which generate signals by recruiting downstream molecules. The finding that the SH3 domain of these kinases binds to PI 3-kinase introduces the possibility of an alternate pathway of src-ldnase signalling [69] (Fig. 6, pathway A). Fyn-SH3 domain mediates binding to the p85 subunit of PI 3-kinase [69]. Since the SH3 domain binds to proline-rich motifs, SH3 binding to PI 3-kinase does

254 not appear to involve phosphotyrosine residues and may thus occur independent of the tyrosine-kinase domain (pathway B). In support of an involvment in T-cell activation, the level of Fyn-SH3-precipitated PI kinase activity increased significantly with ligation of the TcR~'/CD3 complex [69]. Receptor ligation has also been reported to generate PI (3,4)P 2 and PI (3,4,5)P 3 [166]. We propose that the Lck/Fyn-SH3 interaction with PI 3-kinase may provide the alternate signalling mechanism in co-receptor function. In this sense, it is intriguing that the co-receptor function of CD4-p56 tCk can be retained with the elimination of the kinase domain (Littman, D., personal communication). PI 3-kinase has been found crucial for mitogenesis initiated by the platelet-derived growth factor (PDGF) [164,165]. Paradoxically, in contrast to co-ligation, ligation of CD4 with soluble antibody prior to TcR~'/CD3 ligation inhibits T-cell activation, and in certain cases, induces apoptosis (i.e., programmed cell death). This is marked contrast to the potentiating effect of co-immobilized anti-CD4 and anti-CD3 antibody [123,124]. Inhibition occurs independent of CD4 recognition of MHC class-II antigens [244,245]. Further, antibodytitration studies show that the majority of receptor complexes need to be occupied before an inhibitory effect is observed [246]. Preligated CD4 may therefore prevent co-clustering and an association between the CD4 and TcR~'/CD3 complexes. Alternatively, preclustering of CD4-1ck complexes may actively generate negative signals in the T cell. Julius and co-workers have shown that inhibition requires p56 zck association with CD4 [247,248]. Further, the mere expression of CD4-p56 tck may inhibit activation via anti-TcR, but not by anti-CD3E antibodies. One interpretation is that CD4 may sequester p56 t~k, thereby preventing an interaction with TcR~'/CD3. This may account for the fact that antibodies to CD8, a receptor which appears to associate more poorly with p56 t~k, fails to exert this effect [249]. Alternatively, CD4-p56l~-CD4-p56 tCk pre-ligation may change the substrate specificity/occupancy of the kinase and induce the recuitment of alternate intracellular components (i.e., such as p32, p110, PI 3-kinase or others). For example, increased amounts of p59 frn and reduced p56 tc~ may be correlated with the induction of non-responsiveness/anergy in T cells [250]. The differential effects of anti-TcR and anti-CD3 antibodies may relate to the differential use of the TcR( and CD3 pathways within the TCR~'/CD3 complex. Antigen-mediated, but not anti-CD3-mediated, signalling is defective with truncated forms of TcR( [251]. Since both antigen and anti-TcR bind to the T c R a / f l heterotrimer, signalling via TcRa/fl may preferentially use TcR~" and thus implicate CD4-p56 tck. Direct engagement of CD3 subunits with their own

Y-XX-L-7.8-Y-XXL/I motifs may be capable of bypassing the TcR~" pathway. CD4-p56 tck and TcR~'/CD3-p59 fyn complexes will induce numerous downstream events. Growth factor activation of receptor-tyrosine kinases induces increased GTP-occupancy on p21ras, the activation of Raf-1, MAP kinase kinase, MAP-2 kinase (ERKs), $6 kinase and others [94,252-254]. Anti-TcRff/CD3 cross-linking and constitutively active pp60 .... ¢ will activate p21 ras [254-256]. Receptor-associated lck and fyn could theoretically regulate p21 ras by a mechanism similar to the receptor-kinase mediated drk-SOS connection [70,71]. Further downstream, anti-CD3 activates the serine kinase MAP-2 kinase, an event that is augmented by CD4 co-ligation, and inhibited by antiCD4 pre-ligation [257]. MAP-2 kinase activity is regulated by the combined effects of serine and tyrosine phosphorylation. In T-cell clones requiring CD4 expression for a functional response, the loss of CD4 is correlated with the loss of the phosphorylation and activation of MAP-2 kinase [258].

III-G.2. Thymic differentiation Thymic differentiation is profoundly dependent on the expression of p56 tck. At least part of this requirement is likely to involve signalling of the CD4-p56 tck complex. Although the complexities of T-cell differentiation are beyond the scope of this review, precursor T cells leave the bone marrow and migrate to the thymus where they undergo a complicated series of differentiation steps that involves positive or negative selection of self-reactive T cells (for a review, see Ref. 259). Processes such as clonal deletion, functional non-responsiveness and suppression may play roles in the elimination or control of auto-reactive cells [260]. Each of these functions could be influenced by p56 tc~ signalling. Both CD4 and CD8 play active roles in the selection process, as shown by antibody-infusion studies and in transgenic models [261-263]. Newly seeded C D 4 - / C D 8 - cells progress to CD4+/CD8 ÷ cells and then to mature CD4÷CD8 - or CD4-CD8 ÷ subsets. Self-reactive thymic cells are deleted at the boundary from double (CD4÷/CD8 +) to single positive T cells [264]. This developmental progression is sensitive to alterations in p56 tck expression. Over-expression of p56 tck in transgenic model systems results in a blockage and accumulation of CD3-CD81° or CD3-CD8cells and incomplete TcR/3 rearrangement [265,266]. Conversely, thymi of lck-negative transgenic mice undergo atrophy and a dramatic reduction in the presence of double-positive thymocytes [224]. Mature single-positive thymocytes were not detectable. Both positive and negative selection for the male (H-Y) antigen has been reported to depend on associated p56 tck [267]. Over-expression of CD4 altered the positive se-

255 lection of a class-l-directed TcR, possibly by sequestration of p56 lck. Although not the subject of this review, a major debate in field concerns the mechanism by which T cells become committed to the single CD4 or CD8 + lineage (for a review, see Ref. 268). The instructive model postulates that the binding of the TcRa//3 to MHC antigens generates the signal that instructs the cells to become a CD4 or CD8-positive cell. The stochastic/selective model argues that corn-

mitment to either lineage occurs irrespective of the receptor specificity. Co-receptor function of CD4 or CD8 with the TcRa//3 would function to rescue committed cells from apoptosis [269]. Neither model has been formally excluded, however, evidence in support of a stochastic model has come from studies showing that the consitutive over-expression of CD4 rescues a population of mismatched class-II-specific TcRs with CD8 [270]. Despite this, rescue experiments involving

TABLE I

Src-related kinase-receptor interactions Receptor

Kinase

Location

Kinase activation

Kinasemediated events

Associated molecules

References

CD4

p56 tck

T cells

+

substratesP

p32 p110

9, 10, 21-23 85, 86, 112, 127, 133, 141-146

p72 raf

PI-3-kinase PI-4-kinase

+

CD8

p56 lck

+

4-1BB TcR~/CD3

p56 tck p59 fyn(T)

? -

PI-3-kinase p56 tck p59 frn p56/ck

+ +

IL-2R/3 CD2

+

p59 fyn mlgM

mlgD

FcERII FcERI Fc y RIIIA CD59 CD55 CD48 Thy-1 (GPIlinked) PDGF-R

p53/56 ty"

B cells

+ +

p59 fyn

+

p56 blk p56 lck

+ +

PI-3-kinase p53/56 ty" p59 fy~

+ + +

p56 blk p56 lck

+ +

p59 fyn p56/yn p62 yes unknown

YT cells basophils mast cells NK cells

p56 tc*

T cells

p59 fyn

fibroblasts

p60 src

p62 T M PI-3-kinase

+ + -

p32 ? TcR~-P substratesP

zap-70 PLCyl

15, 296 235, 303

substratesP substratesP

MB1 (mlgM app32) B29 (Ig/3 pp37) Igy pp34

13, 16, 309-311,350

Synk

MB1 B29 Igy pp34

16,310

318 349 TcR~'-P

TcR~',rt FcERIy

substratesP (ras GAP Nck PLC-y)

214 17, 314

substratesP

+

10, 11, 21-23, 141 284-287 14, 69, 142, 185, 187-189 190, 193-195

ras GAP Nck PLC-y

320, 321

256 transgenic expression of CD8 have failed. A better understanding of the difference in the nature of signals generated by co-aggregation the two different co-receptors with the TcR will be important. Others have found that an instructive signal that commits a CD4+CD8 ÷ progenitor to the single positive CD4 lineage resides in the transmembrane/intracellular domain of CD4 [271]. CD4-p56 tCk signalling may further influence the differentiation of thymocytes by altering TcR~'/CD3 expression [272,273]. In contrast to p56 t~k, neither of the other src-related kinases in T cells, p59 fyn or p60 yes influence the phenotype of the thymus [208,209]. The major defect is a reduced mitogenic response of p59fY~ff)-negative thymocytes. CD4+CD8 ÷ thymocytes undergoing positive selection to the H-Y display higher p59 &n and p56 tck catalytic activities than unselected cells [274].

III-G.3. Receptor endocytosis The kinase domain of conventional transmembrane tyrosine kinase receptors is essential for the internalization [275]. p56 tck also influences CD4 endocytosis from the cell surface. CD4-p56 lck (but not CD8-p56 t¢g) complexes are modulated from the cell surface by phorbol ester [112,276,277] and gangliosides [278], an event accompanied by the dissociation of p56 t~k. The kinase influences both the rate and the pathway of endocytosis. CD4-p56 tck complexes are modulated from the cell surface more slowly than the kinase-free CD4 [279,280]. Consistent with this, endocytosis of CD4 in resting lymphocytes occurs more slowly than in nonlymphoid cells. Furthermore, in resting T ceils, CD4 endocytosis does not occur via coated pits, a pathway used in fibroblasts [279]. Transfection of fibroblasts with lck both slowed the rate of endocytosis and resuited in the exclusion of CD4 from coated pits [281]. Whether this is due to a direct effect of kinase or an Ick associated molecule is unclear. Possible SH3 binding of p56 t~, or its association with the cytoskeleton [282] may influence modulation. Serine phosphorylation may also regulate the association of kinase with its receptor. Mutation of cytoplasmic residue Leu-413 prevents endocytosis induced by phorbol ester [283]. Interesting, even though endocytosis was prevented, serine phosphorylation of CD4 was accompanied by the dissociation of p56 tck. Serine phosphorylation of CD4 would appear to be crucial in this system since CD8-p56 t~k, a non-phosphorylated receptor is unaffected by phorbol ester treatment [112,276, 277]. IV. Other kinase-receptor interactions

With the identification of the CD4/CD8-p56 l~k interaction, a variety of other receptor interactions with src family members have been described [12-16,235].

Src-related kinases may therefore play a variety of functional roles in receptor-signalling systems. Several of these interactions have been confirmed, while others still must be verified. Reported interactions include associations between the 4-1BB antigen and p56 tck, interleukin-2 receptor (IL-2R) and p561~k/p59fYn~X), CD2 and p56tCk/p59 frn~T), B-cell surface immunoglobulin and p53tY"/p561~k/p59fYn/p59 btk, phosphatidylinositol (GPI)-anchored proteins and p56t~k/other kinases, the Fc receptor and p59 fy~°') and the PDGF-R and p60Sr~/p59fY"~B)/p62yes (Table I). While the 4lBB-p56 tck, CD4-p56 t~k, CD8-p56 lck and TcR~'/CD3p59 fyn(T) exhibit binding specificity, the other receptorkinase interactions show a greater degree of promiscuity in binding to multiple src family members. Several of the associations such as mlg-p53tYn/p561ck/ p59fY"/p59 blk require extraction with non-disruptive detergents to ensure the integrity of the complex, similar to TcR(/CD3-p59 fy"~T). Other interactions, such as CD2-p56 t~k and IL-2R-p56 t~k, have been reported to withstand extraction in harsher detergents, similar to CD4-p56 tck. The biological role of these other receptor-kinase interactions in intracellular signalling remains to be clarified.

IV-A. p56 tck and the 4-1BB antigen 4-1BB is a T-cell-specific antigen that is structurally related to members of the nerve growth factor superfamily [284,285]. Members of this superfamily possess distinct cytoplasmic regions suggesting different signalling functions. Significantly, like CD4 and CD8, the 4-1BB antigen possesses the C-X-C-P motif in its cytoplasmic tail. An association with p56 tck has been demonstrated by co-precipitation [287]. 4-1BB expression is induced on CD4 ÷ and CD8 ÷ subsets by TcR~'/CD3 ligation and various mitogens [286]. Antibody to 4-1BB has been reported to enhance CD3mediated proliferation, an event probably mediated by p56 lck [286]. Its expression following anti-CD3 ligation suggests that p56 tck may play a role in regulating later events in the activation/effector pathway.

IV-B. p56 tck, p59 fyn and the IL-2 receptor Although the initial binding of antigen to the TcRff/CD3 receptor results in T-cell proliferation, a second receptor system, the interleukin-2 receptor (IL2R) serves as an obligatory second signal allowing progression to DNA synthesis [288]. The lyrnphokine interleukin 2 (IL-2) binds its receptor in an autocrine loop. The IL-2R is comprised of a IL-2Ra chain (p55) and /3 chain (p70-75) that bind IL-2 with low and intermediate affinities (K d 10 nM and 1 nM, respectively). A recently cloned third chain 7 (p64) associates with the /3 chain and participates in the formation of

257 high and intermediate-affinity IL-2 receptors [289]. Signalling can occur by the 0-chain-possessing intermediate and high-affinity receptors, but not the low-affinity homodimer [289,290]. The a and /3 chains are integral type-1 proteins with cytoplasmic tails which possess no homology with kinases [291,292]. The cytoplasmic tail of the IL-2/3 possesses some 286 residues, while the IL-2a chain has only 13 amino-acid residues. The /3 chain cytoplasmic region can be divided into a 'serinerich' region and an 'acidic region'. Deletion analysis has shown the 'serine-rich' region (but not the 'acidic region') to be key to IL-2-mediated growth control [293]. The 3' chain also lacks a kinase domain, but possesses an intriguingly region of homology with the last two subdomains of the SH2 domain [289]. IL-2 binding induces tyrosine phosphorylation, thereby suggesting linkage to an intracellular kinase [294]. Hatakeyama et al. have described an interaction between p56 lck and the IL-2/3 chain [15]. The reported association of 0.5-1% of intracellular lck with receptor (assuming 1 : 1 stoichiometry) could occupy as many as 10-30% of the IL-2 receptors. Mapping studies reveal that the presence of the 'acidic region' is crucial to complex formation [15]. Within this region reside two Tyr residues (Tyr-355 and Tyr-358) that may serve as substrates for the kinase. Conversely, the N-terminal half of the kinase domain o f p56 t~k was needed for an association with IL-2R/3. In other cells, p59 fy" has also been found to associate with the receptor (Hatakeyama, M., personal communication). Although p56 tck and p59 yy~ may not be required for signalling by the IL-2R [295], the mystery over the assignment of kinase binding to a seemingly non-essential region of IL-2/3 may be resolved with the demonstration that there exist two pathways, one leading to the induction of c-jun and c-fos, and another involving the induction of c-myc. Abrogation of p56 tck binding interferes with c-jun and c-fos, but not c-myc induction [296].

IV-C. p56 tck, p59 fy~ and the CD2 antigen CD2 is a pan T-cell antigen which binds to a structurally-related antigen (LFA-3) and plays a key role in a variety of T-cell functions, including activation-related lymphokine release and cytotoxicity [297,298]. Antibodies to distinct epitopes on human CD2 can activate T cells, an event dependent on the expression of the TcR~" chain [297,299]. The binding of the natural ligand for CD2 fails to directly stimulate, rather serving as a co-stimulatory antigen [300]. A proline-histidine rich region within the cytoplasmic tail is required for signalling via this receptor [301,302]. Imboden and coworkers have reported that both p56 ICk and p59 fyn can be co-purified with rat CD2 in a variety of detergents including NP-40 [235]. Specificity was shown by the fact

that the association could only be detected in the CD53 ÷ subset of thymocytes, even though CD2 and p56 lck are expressed at equivalent levels in both the CD53 ÷ and CD53- subsets. The interaction therefore appears to be developmentally regulated by unknown mechanisms. Furthermore, the association is resistant to alkylating agents, arguing that the molecular mechanism of association differs from the CD4-p56 tck interaction. CD2 ligation has al.so been reported to stimulate p56tck activity [303]. Whether p56 ~Ck can account for the stimulatory function of CD2 remains unclear, given the difficulty in demonstrating the interaction in mature peripheral human T cells.

IV-D. Src family and surface immunoglobulin B cells express membrane-bound immunoglobulin (mlg) on their cell surface which act to recognize specific foreign antigen. The complex is comprised of Ig heavy and light chain associated with at least three other polypeptide chains, MB-I(IgM-a) at 32-34 kDa, B29(Ig-/3) at 37 to 39 kDa and Ig-y at 35 kDa [304,305]. MB-1 forms a disulphide-linked heterodimer with either B29 or Ig-y. Cross-linking of mlgs can activate B cells to enter G 1 of the cell cycle, at which time they become induced to proliferate by helper T-cells. Alternatively, cross-linking can induce tolerance in immature and mature B cells [306]. mlg engagement also induces the rapid tyrosine phosphorylation of an array of intracellular substrates [307] as well a turnover of phosphatidylinositol and Ca 2+ mobilization [308]. Each of the mlgM-associated proteins may act as substrates for tyrosine kinases. Several groups have reported the detection of src-related kinases with mlg. Initially, Yamanashi and coworkers first reported the co-precipitation of the tyrosine kinase p53/56 tyn with mlgM [309]. Burkhardt et al. further identified blk, fyn and lyn associated with IgD and IgM [16]. Campbell and Sefton further identified lck in association with the receptor [310]. Only a small% (1-2%) of total lyn was found associated with the receptor, and as in the case of the TcR~'/ CD3:p59 fyn interaction, the binding appears somewhat weak as shown by its dissociation by detergents such as NP-40. The nature of the subunit(s) that interact with the kinase is unclear, although the associated MB1 and B29 subunits are likely targets. Significantly, both subunits possess a Y-XX-L-XT-Y-XXL/I activation motif and are tyrosine phosphorylated in response to Ig ligation (Fig. 5). As in the case of ZAP-70 binding to the TcR~ chain, src-related kinases may regulate the association of the SH2 carrying kinase Syk to the MB1 or B29 subunits [311]. Alternatively, PI 3-kinase has been reported to associate with the Lyn kinase and can be stimulated by mIgM cross-linking [350].

258 IV-E. Src family and GPI-linked surface proteins Many surface receptors are anchored to the cell surface by glycophosphotidylinositol (GPI). These include the Thy-1, Ly-6, CD14, CD24, CD48, CD55 and CD59 antigens. Several of these receptors have been implicated in cell adhesion and complement fixation; however, the biological roles of many are unknown. Antibodies to Thy-1 and Ly-6 either directly stimulate, or potentiate T-cell proliferation [312,313]. Given that these structures fail to extend into the cytoplasm of the cell, it has been a puzzle as to the molecular basis of their role in signal transduction. Stefanova et al. have reported that various members of the src family associate with GPI-linked molecules [17]. In T-cells, CD48, CD55, CD59 and Thy-1 were found to associate in NP-40 lysates with p56 ~ck. CD24 from B-CLL (Bchronic lymphoblastic leukemia) cells and CD48, CD55 and CD59 from a variety of cell types precipitated putative 55-~i0-kDa src-like kinases of unknown identities. Their involvement in signalling was further demonstrated by antibody-induced phosphorylation of numerous substrates. In the case of Thy-1, another interaction with p60 fy" has also been reported [314]. This molecular basis of this most fascinating interaction has yet to be determined. Another common intermediate (i.e., a docking receptor) may interact with

GPI-linked proteins and kinases. Alternatively, the hydrophobic lipid tails of GPI-linked proteins may form aggregates in solution. It has been a general finding that GPI-linked proteins tend to cluster and co-precipitate each other in solution. IV-F. p59 fy" and the FceRII / CD23 receptor The FceRII/CD23 antigen serves as a low-affinity Fc receptor for IgE, regulates IgE production and also generates activation signals in B cells and natural killer (NK) cells [315,316]. Antibodies to CD23 have been reported to induce a rise in intracellular Ca 2+ and inositol phosphate turnover [317]. Sugie et al. have reported the physical association of p 5 9 / r n with CD23 in an NK-like cell termed YT [318]. Depletion analysis showed that anti-p59 fyn, but not anti-p56 tck, depleted CD23 reactive material, thereby suggesting specificity for fyn in the interaction. No data presently exist on whether CD23 binding can regulate the activity, and whether other src-family members bind to the receptor in B cells. IV-G. Src family and PDGF receptor The PDGF receptor possesses its own intrinsic kinase activity, an event required for signal transduction B.

A.

CD4 (orCD8)

TcRr.=/CD3

C.

IL2-R~ ~

p56

I I fyn

p59

PDGF-R 1

I

qg~fi~gggg~lgg~gggpggggggggggggggg~ggg~g~g

~

{and other

~

SH2 proteins) B

~'l/.,

~1~ p56

?

,ok

......

, _

Klnases

I~ I

--

Fig. 7. Different mechanisms of Src-related k~nase binding with surface receptors. (A) Binding of p56lck and p59fy" to the cytoplasmie tails of CD4/CD8 or TcR~/CD3 receptors, respectively, is mediated by sequenceswithin the N-terminal region of the kinases. Cysteines at sites 20 and 23 of the p56lCk sequence mediate the interaction of the kinase with C-X-C-P motif within CD4 and CD8a. p59/ynG) binding to CD37,8,~ and TcR~" is mediated by the first 10 residues. Phosphoryiation of TcR~" leads to the binding of 7__~P-70 kinase to the Y - ~ - L - X T . s - Y - X X L / I activation motif. (B) Binding of p56 tc~ to the high-affinity receptor for interleukin-2 (IL2-Rfl chain) is mediated by sequences in the N-terminal portion of the kinase domain. The binding site within the IL2-R has been mapped to the acidic domain (shaded area) of the p75 subunit. (C) The platelet-derived growth factor receptor (PDGF-R) is a receptor protein-tyrosine kinase capable of binding to multiple SH2-carrying proteins. The enhanced catalytic activity of the PDGF-R leads to autophosphorylation at the tyrosine 857 which lies within the kinase domain, as well as other sites. Binding of pp60 s'c, p59/y" and p62 T M to the PDGF-R is mediated by the SH2 domain of the kinases.

259 in this system [50-53]. Autophosphorylation allows the association of numerous SH2-bearing intracellular proteins including PLCy, GAP, Nck and PI 3-kinase to specific phosphotyrosine sites. Other SH2-carrying proteins, such as SHC, do not appear to bind the PDGF-R, but do bind to the EGF-R. Both specificity and redundancy exist between various receptor systems [135]. Small amounts of the serine/threonine kinase p72 Raf may also associate with the receptor [319]. Given this importance of associated intracellular molecules in signalling, it is of interest that at least three src-family members, pp60 src, p59 fyn and pp62 c'ye~ also associate with the PDGF-R [320]. Binding is mediated by the SH2 domains of the src-related kinases [321]. Mutational analysis has shown that the major autophosphorylation site at 857 within the catalytic domain is required for efficient association [321]. Whether this is related to a overall affect of the mutation on kinase activity, or is specific site of association is unclear. This site is distinct from other autophosphorylation sites at Tyr-740, 751 and 771 in the kinase-insert region and Tyr-977 and 989 at the carboxy-tail which mediate binding to PI 3-kinase, Nck, rasGAP and PLC3,. Significantly, the activities of each src-related kinase were increased with PDGF binding. Some 5% of PDGF-R bound to the kinases, while 5-10% of the pool of src-related kinases bound the receptor. The response to PDGF, therefore, appears to involve a complicated network of lipid kinases, SH2-adaptor proteins, GTPase-activating proteins, serine/threonine kinase and src-family members. V. Different mechanisms govern receptor-kinase binding

From the above data, it is clear that different mechanisms govern the manner by which src-family members associate with surface receptors (Fig. 7A-C). Three broad classes of interaction can be defined. The first class would include the CD4-p56 lck, CD8-p56 tck and the TcRff/CD3 receptor-kinase interaction (Fig. 7A). While the N-terminal 10 to 32 amino acids of p56 tck mediate binding to CD4 and CD8, the first ten amino acids of p59 fy~ bind to the CD3y,~,e and TcR~ chains. Within this grouping, the CD4/CD8-p56 lck binding is characterized by C-X-C-P interactions, a high level of stoichiometry and stability in different detergents. By contrast, the TcR~'/CD3 occurs with lower stoichiometry and is disrupted by certain detergents. A second class is represented by the IL2R-p56 t~k and p59 fyn complex, an interaction involving the Nterminal region of the p56 lck domain and the IL-2R/3 chain (Fig. 7B). A third class as represented by the interaction between the PDGF-R-src kinases is mediated by an interaction between the SH2 domain of the src-related kinase and phosphotyrosine residues within

the PDGF-R (Fig. 7C). Intrinsic PDGF-R autophosphorylation probably represents the first event, thereby creating sites of recognition by src-ldnase SH2 domains. The molecular basis of other receptor-kinase interactions such as the mlg-p53 ly~ has yet to be defined. The mechanism of GPI-linked proteins with src kinase will probably involve unique mechanisms. Another possible mechanism of binding that has yet to be described would involve the binding of a phosphotyrosine labelled intracellular substrate to its receptor, a process that would recuit other intracellular proteins by phosphotyrosine-SH2 binding. For example, in the case of the insulin receptor, a major phosphotyrosine substrate termed the p185 IRS protein binds PI 3kinase and recruits the enzyme to the receptor [322]. VI. Other potential substrates in the receptor-kinase cascade

Receptor ligation on T and B cells induces the phosphorylation of an array of substrates at 16 kDa, 30-34 kDa, 40-42 kDa, 55-62 kDa, 68-72 kDa, 81 kDa, 110-120 kDa, 130 kDa, 135 kDa, 140 kDa and 150 kDa. Intracellular candidates include PLC-3, (130150 kDa), p85/pl10 subunits of PI 3-kinase, p120 rasGAP and associated p62 and pl90K, SHC proteins (46, 66 kDa), car (96 kDa) and others [3237,42,43,94,184,323,324]. However, to date, surprisingly few substrates of wild-type src-related kinases have been definitively identified. Given the emergence of new families of cytosolic SH2-carrying protein-tyrosine kinases such as Tec, Syk, Tsk, ZAP-70, Jak 1 and Jak 2, which may bind to src-kinases or their receptor substrates, it has been increasingly diffficult to uncover a direct link src kinases and substrates [325-328]. Nevertheless, from the point of view of intracellular localization, transmembrane surface receptors would appear to constitute an important group of substrates for src-kinases. As mentioned, these would include a variety of receptors (TcR~', CD5, CD3 subunits, B29 and MB1) that possess the Y-XI1-Y-XXL/I target motif (Fig. 5). Each may be phosphorylated by ligation of receptors that associate with src-related kinases. Other plasma-membrane-associated proteins include fyn-associated p120/130 (p130) [158]. Likewise, platelet Fyn has been reported to bind pl2OrasGAP, a substrate of receptor-tyrosine kinases [329]. Another approach has been to define intracellular proteins which undergo pronounced phosphorylation in cells transformed with the constitutively active form of pp60 sr¢. These include lipocortin-calpactin [330,331], ezrin [332] and MAP (ERK) serine/threonine kinases [333,334]. A ezrin-related protein (p81) is also rapidly phosphorylated by TcR~'/CD3 ligation, potentially implicating lck or fyn [335]. pp60 src forms stable complexes with two tyrosine phosphorylated proteins p l l 0

260 and p130 [59,336]. For the association of p130/110 with p60 src, both the intrinsic tyrosine kinase activity and the integrity of SH2 and SH3 domains appear to be required [59]. The SH3 domain is required for the association of pll0, and the SH2 for the formation of a tertiary complex of p130, p l l 0 and p60 sr~ [59]. pp60 v'src transformation leads to biochemical redistribution of p130 from the nucleus to cellular membranes and pll0, which is normally localized with cytoskeletal elements, becomes concentrated in adhesion plaques (podosomes). The p120 observed in mammalian cells in association with p60 ~ corresponds to pp125 FAK (focal adhesion associated protein tyrosine kinase). The possible regulation of pp125 FA~ by pp60 ~c, or vice versa is the subject of much investigation [337]. Other targets of src-transformation have also been described [338]. Another potential target of CD4-p56 ~Ck and TcR~'/ CD3-p59 fyn is the ray protein. This intriguing protein, first identified as a human oncogene in hematopoietic cells, possesses two SH3 domains, a SH2 domain, putative nuclear localization signals, putative leucine zipper and helix-loop-helix domains, and a homology region with a GDP-GTP exchange factor, although DNA-binding properties of this protein have been questioned [323]. Vav contains no sequences with homology to known enzymes. Tyrosine phosphorylation of p95 va~ has been noted in T cells by TcR~/CD3 ligation alone or CD4-TcR~'/CD3 co-ligation [339-340]. In the process, ray the guanine nucleotide exchange activity may be activated [341]. Other potential substrates include SH2/SH3 adaptor proteins such as crk [38,342] Nck [343-345], GRB2 / s e m - 5 [43,346347] and the SHC proteins [42]. p47 gag-~rk, the transforming protein of the avian retrovirus CT10, is a fusion protein derived from viral Gap and a SH2 and SH3 domain of cellular c-Crk [342]. p35 cc~k is itself comprised of the SH2 and SH3 domains, together with an additional SH3 domain [342]. Although binding of c-Crk to receptor-kinase complexes has not been demonstrated, a potential regulatory role is shown by the fact that Crk SH2 can protect tyrosine-phosphorylated proteins from de-phosphorylation in vitro by tyrosine phosphatases. Another oncogenie protein p47 Nck possesses three SH3 domains followed by an SH2 domain [348]. Nck is tyrosine-phosphorylated by EGF stimulation and v-src induced cellular transformation [343-305]. Another potential target is the SHC protein(s) which possesses a SH2 region and a region of homology with collagen [42]. SHC acts as a tyrosine-phosphorylated substrate and binds to pp60 s~C [42].

VII. Summary The CD4-p56 zCk and CDS-p56 lck complexes have served as a paradym for an expanding number of

interactions between src-family members (p56 tck, p59 fyn, p56 tyn, p55 btk) and surface receptors. These interactions implicate src-related kinases in the regulation of a variety of intracellular events, from lymphokine production and cytotoxicity to the expression of specific nuclear binding proteins. Different molecular mechanisms appear to have evolved to facilitate the receptor-kinase interactions, including the use of Nterminal regions, SH2 regions and kinase domains. Variation exists in stoichiometry, affinity and the nature of signals generated by these complexes in cells. The CD4-p56 ~ck complex differs from receptor-tyrosine kinases in a number of important ways, including mechanisms of kinase domain regulation and recruitment of substrates such as PI 3-kinase. Furthermore, they may have a special affinity for receptor-substrates such as the TcR~', MB1/B29 or CD5 receptors, and act to recruit other SH2-carrying proteins, such as ZAP-70 to the receptor complexes. Receptor-src kinase interactions represent the first step in a cascade of intracellular events within the protein-tyrosine kinase/phosphatase cascade.

Acknowledgements This work was supported by National Institutes of Health Grant R01 12069 (C.E.R.). C.E.R. is a Scholar of the Leukemia Society of America. A.d.S. is the recipient of a fellowship from the National Cancer Center, New York. J.C.T. is the recipient of the Ryan Fellowship and the Markey Fellowship in Developmental Biology, Harvard University, Boston.

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