Oncogene (1998) 17, 1353 ± 1364  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Antigen receptor signaling: integration of functions

Idan Tamir1 and John C Cambier1

1Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, Colorado 80206, USA

Antigen receptors on T and B cells function to processing and presentation to T cells, and to transduce signals leading to a variety of biologic transduce signals that lead to multiple, often alter- responses minimally including antigen receptor editing, native responses. This receptor, termed the B cell apoptotic death, developmental progression, cell activa- antigen receptor (BCR), belongs to the family of tion, proliferation and survival. The response to antigen Multichain Immune Recognition Receptors (MIRR), depends upon antigen anity and valence, involvement which includes the T cell receptor (TCR) and receptors of coreceptors in signaling and di€erentiative stage of for the Fc portions of IgG (FcgRI, FcgRIIA, FcgRIIC, the responding cell. The requirement that these FcgRIIIA) and IgE (FceRI). Common to this family of receptors integrate signals that drive an array of receptors is an oligomeric structure which uses di€erent responses may explain their evolved structural complex- membrane-spanning subunits for the purpose of ity. Antigen receptors are composed of multiple subunits antigen/Ig recognition vs signal transduction. The compartmentalized to provide antigen recognition and BCR is comprised of a membrane-associated form of signal transduction function. In lieu of on-board immunoglobulin (mIg), noncovalently associated with enzymatic activity these receptors rely on associated a disul®de linked CD79a/CD79b heterodimer (Figure Protein Tyrosine Kinases (PTKs) for their signaling 1) (for review see Cambier, 1995a; Gold and function. By aggregating the receptors, and hence their DeFranco, 1994). Immunoglobulins of any isotype appended PTKs, antigens induce PTK transphosphor- can function as the mIg component of the BCR. ylation, activating them to phosphorylate the receptor While immature B cells express only mIgM-containing within conserved motifs termed Immunoreceptor Tyr- receptors, mature B cells co-express mIgM and mIgD, osine-based Activation Motifs (ITAMs) found in and memory B cells express the isotype, i.e., IgG, IgA transducer subunits. The tyrosyl phosphorylated ITAMs or IgE, that their daughter cells will secrete. then interact with Src Homology 2 (SH2) domains Membrane-associated immunoglobulins di€er from within the PTKs leading to their further activation. As their secreted counterparts in containing a short receptor phosphorylation is ampli®ed, other e€ectors, spacer sequence at the normal heavy chain C- such as Shc, dock by virtue of SH2 binding, and serve, terminus, a single transmembrane spanning region in-turn, as substrates for these PTKs. This sequence of and a short [3 (mIgM and mIgD) to 28 (mIgM) events not only provides a signal ampli®cation mechan- residues] cytoplasmic tail. Although the mIg tail ism by combining multiple consecutive steps with contributes to signaling (Pleiman et al., 1994b; Weiser positive feedback, but also allows for signal diversi®ca- et al., 1997), CD79a and CD79b function as the tion by di€erential recruitment of e€ectors that provide receptor's primary signal transducers (Cambier, 1995a; access to distinct parallel downstream signaling path- Gold and DeFranco, 1994; Pao et al., 1997). They are ways. The subject of antigen receptor signaling has been members of the immunoglobulin superfamily, contain- recently reviewed in depth (DeFranco, 1997; Kurosaki, ing a single extracellular immunoglobulin-like domain, 1997). Here we discuss the biochemical basis of antigen a single transmembrane spanning region and cytoplas- receptor signal transduction, using the B cell receptor mic tails of 48 (CD79b) and 61 (CD79a) amino acid (BCR) as a paradigm, with speci®c emphasis on the residues. The cytoplasmic domains of both CD79a and involved PTKs. We review several speci®c mechanisms CD79b contain the sequence motif YX2LX7YX2L, by which responses through these receptors are termed the Immunoreceptor Tyrosine-Based Activa- propagated and modi®ed by accessory molecules, and tion Motif or ITAM (Cambier, 1995b), that functions discuss how signal ampli®cation and diversi®cation are as the receptor's interface with cytoplasmic e€ectors. achieved. This motif is highly conserved among the cytoplasmic tails of MIRR and has been shown to be both Keywords: signal transduction; antigen receptors; sucient and necessary for signal transduction. Both protein tyrosine kinases the residue spacing (9 ± 11) between the two tyrosines in this motif and the presence of hydrophobic residues (leucine or isoleucine) at position +3 to both tyrosyl residues are required for proper ITAM function. As Antigen receptors-the MIRR family will be discussed below, these structural constraints are important for both ITAM phosphorylation and signal As pre-B cells transit into the immature stage they propagation by e€ector binding. begin to express speci®c receptors that function to recognize and internalize antigen for subsequent Antigen receptor activation of Src family PTKs

The resting BCR associates with protein tyrosine Correspondence: JC Cambier kinases (PTKs) of the Src-family by virtue of an Signal transduction in lymphocytes I Tamir and JC Cambier 1354 interaction involving the receptor's ITAM and a these domains in Src-family PTK activation is domain found in the kinases' N-terminus (Clark et discussed below. al., 1992; Pleiman et al., 1994a; Yamanashi et al., 1991) The resting BCR-associated Src-family PTKs exist (Figure 1). The kinases bind preferentially to the in a dynamic equilibrium between inactive and CD79a ITAM via a site which involves the DCSM partially active states. The transition between these sequence found in CD79a, but not in CD79b (Clark et states has been shown to correlate with the phosphor- al., 1994). This low anity interaction of non- ylation of a single tyrosyl residue found at the kinase's phosphorylated ITAM with the kinase N-terminal C-terminal tail (Cooper and Howell, 1993; Pao and region is stabilized in vivo by concurrent kinase Cambier, 1998). The phosphorylation of this residue, association with the adjacent plasma membrane via which is conserved among Src-family members, is its covalent N-terminal myristylation/palmitylation mediated by the cytosolic PTK Csk (for C-terminal (Resh, 1993, 1994). In the absence of these mem- Src-family kinase) and results in an enzymatically- brane-docking modi®cations, the association of these inactive state (Hata et al., 1994). Dephosphorylation kinases with the plasma membrane and thus their of this residue, which is mediated by the transmem- ability to respond to receptor aggregation are inhibited branal protein tyrosine phosphatase (PTP) CD45, (Kabouridis et al., 1997; Timson-Gauen et al., 1996). leads to partial activation of the kinase (Mayer, The N-terminal sequence of Src-family PTKs, which is 1997; Thomas, 1989). The steady-state equilibrium of responsible for their association with the resting Src-family kinases in resting lymphocytes between the receptor, di€ers extensively among Src-family mem- C-terminally phosphorylated and non-phosphorylated bers and is thus termed `unique'. In addition to this forms favors the latter, probably due to the higher domain, Src-family members share four structurally- intrinsic activity of and di€erent localization of CD45 homologous domains, namely the catalytic domain (membrane) compared to Csk (cytosolic). Hence, most (termed SH1 ± for src-homology 1), the SH2, SH3 (for of the resting receptor-associated Src-family PTK Src-Homology 2 and 3, respectively) and a C-terminal molecules are in the partially active state that allows tail containing a conserved tyrosyl residue (Brown and them to rapidly respond to receptor aggregation. Cooper, 1996). SH2 and SH3 domains are found in Several studies have suggested that kinase inactivity many and are involved in mediating protein- results from an intramolecular interaction of the C- protein interactions. SH2 domains bind inter- or intra- terminal phosphotyrosyl residue with the kinase's SH2 molecularly to phosphotyrosyl residues with speci®city domain (Brown and Cooper, 1996). The recent three- which is governed by the phosphotyrosyl ¯anking dimensional structures of two Src-family kinases, residues. SH3 domains interact with proline-rich namely c-Src and Hck, solved by X-ray crystal- domains found within many adaptor proteins (Cohen lography (Sicheri et al., 1997; Xu et al., 1997), have and Baltimore, 1995; Pawson, 1995). The role(s) of actually demonstrated such interaction.

Figure 1 Schematic representation of early signaling events activated upon BCR aggregation Signal transduction in lymphocytes I Tamir and JC Cambier 1355 These crystallographic studies have also shed light studies using B cells from Lyn knock-out mice (Chan on another facet of Src regulation involving its SH3 et al., 1997) have shown delayed, yet normal tyrosyl domain. It has been known for some time that the phosphorylation of CD79a, Syk and Shc upon BCR kinase's SH3 domain plays some role in regulating it's aggregation. Taken together, these data suggest that activity (Pleiman et al., 1994c; Superti-Fuga et al., Src-family members are the most proximal element 1993), however the mechanism was obscure since Src- activated upon antigen receptor aggregation and have family kinases lack a proline-rich domain which, by a critical role in its phosphorylation. However, the binding its own SH3, could be responsible for this functional redundancy of members of this family of e€ect. The three-dimensional structure reveals, how- PTKs varies between B- and T-cells (see also below in ever, an unexpected intramolecular interaction between the section dealing with di€erences in Syk and ZAP-70 Src's SH3 and the back of its catalytic domain, largely activation). mediated by the linker segment connecting the SH2 As will be discussed later, the expression of CD45 is and catalytic domains. The linker segment, which does required for e€ective receptor aggregation-induced not contain the consensus SH3-binding PXXP motif, tyrosyl phosphorylation of Src-family kinases in B assumes a left-handed polyproline-II helical conforma- cells, suggesting that transphosphorylation is depen- tion that occupies the SH3 ligand-. Thus, dent on dephosphorylation of the C-terminal inhibitory via intramolecular interactions both the SH2 and SH3 motif (Pao and Cambier, 1997). However, the domains contribute to the stabilization of a closed normally-observed tyrosyl phosphorylation of many conformation which apparently does not allow the other intracellular substrates, including receptor sub- catalytic domain to assume an enzymatically-active units as well as activation of Syk and Btk, upon BCR state. Destabilization of this conformation by either C- aggregation was not compromised, suggesting either terminal dephosphorylation or competitor binding to that other PTKs may compensate for Src-family Src's SH2 and/or SH3 domains should result with an kinases' inactivity or that the activity of C-terminally increase in catalytic activity. Consistent with this phosphorylated Src-family kinases towards the ITAM hypothesis a recent study has shown a 50-fold increase is intact. It is noteworthy that even the C-terminal in the activity of Hck, a Src-family member, upon its tyrosyl phosphorylated, and thus repressed, kinase was interaction with the HIV Nef protein, which is a high shown to be capable of in vitro transphosphorylation, anity ligand of Hck's SH3 domain (Moare® et al., suggesting that Src-family kinase activity is not 1997). completely inhibited by this covalent modi®cation Additional regulation of the activity of Src-family (Moare® et al., 1997). Alternatively, receptor aggrega- kinases is achieved by phosphorylation of another tion may in this case lead to the direct activation of conserved tyrosyl residue (Y416 for c-Src) found within receptor-associated Syk, further leading to the the activation segment of the catalytic domain. phosphorylation of receptor subunits by this kinase Phosphorylation of this tyrosine is believed to result (Hutchcroft et al., 1991). However, in vitro studies in a conformational change in the catalytic domain indicate that BCR ITAMs are not e€ective Syk leading to its activation (Johnson et al., 1996). substrates (Famiglietti and Cambier, unpublished Speci®cally, this event is believed to involve the observation). interaction of the phosphotyrosyl residue with posi- Tyrosyl phosphorylation of Src-family PTKs upon tively charged residues buried within the catalytic BCR aggregation is accompanied by the phosphoryla- domain. By relieving steric hindrance, this interaction tion of conserved BCR ITAM tyrosines (Cambier, is thought to allow a glutamate residue within the 1995a; Pao et al., 1998). These tyrosyl residues are amino-terminal lobe of the catalytic domain to contact within the optimal context, with respect to their and reposition a lysine residue which is involved in ¯anking residues, for phosphorylation by Src-family coordinating the phosphate group of ATP (Yamaguchi kinases (Schmitz et al., 1996; Songyang et al., 1994a, and Hendrickson, 1996). The phosphorylation at Y416 1995), however, only one of them, speci®cally the N- was shown to follow ®rst order kinetics with respect to terminal ITAM tyrosyl residue (Y182) of CD79a, fully the kinase, and is thus believed to be mediated by Src- meets Lyn and Blk substrate recognition requirements family PTKs themselves. Therefore, the close apposi- determined in vitro (Schmitz et al., 1996). Consistent tion of receptor-associated Src-family molecules during with this, it has been shown that the presence of this receptor aggregation should allow for their ecient residue within the BCR is critical for the tyrosyl transphosphorylation (Moare® et al., 1997). phosphorylation of both CD79a and CD79b upon Consistent with the above, BCR aggregation leads to receptor aggregation (Flaswinkel and Reth, 1994; Pao rapid tyrosyl phosphorylation of several Src-family et al., 1998) and is the major in vivo tyrosyl PTK members, including Lyn, Fyn, , Blk and Fgr phosphorylation site within the BCR. Studies employ- (Burkhardt et al., 1991; Gold et al., 1994; Wechsler and ing baculovirus-expressed Src-family kinases and Monroe, 1995), of which at least some have been bacterially-expressed CD79a and CD79b con®rmed shown to associate with the resting receptor (Campbell this observation, showing about fourfold higher in and Sefton, 1992; Lin and Justement, 1992; Yamanashi vitro phosphorylation of CD79a at position Y182 than et al., 1991). This phosphorylation putatively occurs at at Y193 (Pao et al., 1998). the activation segment of the kinases, accounting in Although not formally examined to date, a similar part for the observed increase in their enzymatic receptor tyrosyl phosphorylation hierarchy may exist in activity. While studies with Lck-negative T cell lines the TCR, where the associated z chain contains three have shown an almost complete dependence of ITAMs and thus six possible tyrosyl phosphorylation receptor-mediated tyrosyl phosphorylation events (of sites. Interestingly, z is di€erentially phosphorylated in the z chain and practically all other cellular substrates) response to activating vs z anergizing stimuli on the activity of this kinase (Straus and Weiss, 1992), (Madrenas et al., 1995; Sloan-Lancaster et al., 1994), Signal transduction in lymphocytes I Tamir and JC Cambier 1356 suggesting that the extent and perhaps site of ITAM tandem SH2 domains binding to the two, properly tyrosyl phosphorylation is dependent on stimulus spaced, phosphotyrosyl residues of the ppITAM (Chan intensity. The latter is probably a function of receptor and Shaw, 1995; Hatada et al., 1995; Kurosaki et al., cluster lifetime and size, which are respectively 1995). Mutational analysis within the cytoplasmic tails dependent on the receptor's anity and ligand valence. of both CD79a and CD79b demonstrate a strict Since most MIRR contain at least two di€erent requirement for both conserved ITAM tyrosyl residues transducer chains, each carrying a unique ITAM(s), it for the BCR-induced tyrosyl phosphorylation of Syk is likely that the subunits di€er in their signaling roles (Pao et al., 1998), indicating that the binding of Syk to as a function of the relative phosphorylation of their ppITAM is a crucial step in its activation. The binding tyrosines and the ability of these tyrosines, once of Syk and its T-cell homologue ZAP-70 to ppITAM phosphorylated, to bind distinct e€ectors. The latter displays a high apparent anity (10-20 nM) (Bu et al., is determined by the amino acid sequence ¯anking the 1995; Isakov et al., 1995), which is dramatically tyrosyl residue. Such a di€erential role has been reduced for the binding of a single SH2 domain of observed for the case of the high anity immunoglo- ZAP-70 to ppITAM. Crystal structure data for ZAP-

bulin E receptor (FceRI) which is mainly expressed in 70 double SH2 domains complexed to a ppITAM mast cells (Johnson et al., 1995; Jouvin et al., 1994). In provides the rationale for this observation (Hatada et this system, the receptor's b chain was shown to al., 1995). In this complex each of ZAP-70 SH2 preferentially interact with the Src-family kinase Lyn, domains binds to a single speci®c phosphotyrosyl

while the FceRI g chain speci®cally interacts with Syk. residue within the ppITAM. Of the possible two This PTK segregation leads to signal ampli®cation by a orientations for such an interaction only one was mechanism which probably involves initial Lyn- observed, in which the N-terminal SH2 domain of induced b chain phosphorylation on one of it's ITAM ZAP-70 binds the C-terminal phosphotyrosyl residue tyrosyl residues, thus allowing Lyn's association, via its of the ppITAM. The N-terminal phosphotyrosyl SH2 domain, with that residue. This results in both the residue of the ppITAM is however bound by a stable association of Lyn with the receptor and its domain created mainly from the incomplete C- activation, leading to ecient tyrosyl phosphorylation terminal SH2 domain of ZAP-70, complemented by of the associated g chain on both of its ITAM tyrosyl residues from the N-terminal SH2 domain. The residues, further leading to the recruitment and tyrosyl internal ITAM sequence (i.e., that between the two phosphorylation of Syk. There is evidence that a phosphotyrosyl residues) forms a coiled a helix that similar division of labor is operative in BCR and interacts with the C-terminal SH2 domain thus further TCR (Isakov et al., 1995; Johnson et al., 1995). stabilizing the complex. However, at least in the case of TCR signaling, The high anity of Syk/ZAP-70 interaction with the mutational analyses have revealed signi®cant redun- ppITAM, resulting from both fast association and slow dancy and overlapping function of the three z and dissociation rates (Bu et al., 1995) may provide a single CD3 e ITAMs (Shinkai et al., 1995; Shores et al., rationale for the asymmetrical nature of ITAM 1994). Another interpretation of these results is that phosphorylation. It is possible that this asymmetric although di€erent ITAMs display varying tyrosyl phosphorylation pattern is required to allow lower phosphorylation susceptibilities and varying e€ector anity interactions between the tyrosyl phosphorylated speci®cities, which in multi-ITAM receptors results in ITAM and single SH2-containing e€ectors molecules PTK (e€ector) segregation, they have sucient such as Shc (Osman et al., 1995). Assuming a molar redundancy to allow any ITAM to function indepen- excess of Syk/ZAP-70 expression over ppITAM, a dently both as its own `ampli®er' and as an e€ector symmetrical pattern of ITAM phosphorylation would recruiter. This interpretation further suggests that the probably limit the spectrum of recruited molecules to presence of multiple di€erent ITAMs within a given only those which can either directly associate with Syk MIRR serves to amplify and diversify the initial signal (ZAP-70) or can compete with its binding to the by the mechanism of e€ector segregation, while ppITAM. providing sucient functional redundancy to allow Binding of the ppITAM to Syk was shown to signal transduction even in cases of a partially increase its speci®c activity (Rowley et al., 1995; Shiue compromised receptor. et al., 1995), suggesting that Syk is subject to activation following receptor phosphorylation by dimerization, probably involving transphosphorylation, or an allos- Syk and ZAP-70 recruitment and activation teric mechanism. An additional possibility is supported by the fact that Syk is phosphorylated to some extent Upon their phosphorylation, ITAM tyrosines have in response to BCR aggregation in Lyn-de®cient DT40 been shown to act as a sca€old, directly recruiting B cells (Takata et al., 1994) and in the CD45-de®cient e€ectors such as Shc, Syk and additional Src-family lymphoma cell line J558Lmm3 (Pao and Cambier, kinases to the receptor via their SH2 domains (Clark et 1997) in which Src-family kinases' recruitment to and al., 1994; D'Ambrosio et al., 1996; Law et al., 1993). activation by the BCR are inhibited. Furthermore, Syk Binding of Src-family kinases to the tyrosyl-phos- has been shown to co-precipitate with the resting BCR phorylated ITAM further increases their speci®c (Hutchcroft et al., 1992), providing a possible activity, as discussed earlier, and this may account in mechanism for a Syk-mediated phosphorylation of part for induced phosphorylation of additional ITAM tyrosyl residues upon BCR aggregation substrates such as Syk (El-Hillal et al., 1997; Johnson (Richards et al., 1996). The ability of Syk to tyrosyl et al., 1995; Kurosaki et al., 1995, 1994). Syk phosphorylate both a z-derived ITAM-bearing peptide association with the doubly tyrosyl-phosphorylated in-vitro as well as a z-bearing chimera in transiently ITAM (ppITAM) was shown to require both of its transfected Cos-1 cells has been demonstrated (Latour Signal transduction in lymphocytes I Tamir and JC Cambier 1357 et al., 1997). Taken together, it is possible that some the BCR and its localization to the cytosol upon BCR level of ITAM phosphorylation and activation of aggregation (Pao and Cambier, 1997; Peters et al., downstream signaling pathways may be mediated by 1996). Syk, independent of Src-family kinases. It should be In contrast to the behaviour of Syk, the tyrosyl however emphasized that loss of Src-family kinases, phosphorylation of ZAP-70 has been shown to such as Lyn in the B cell DT40, greatly diminishes the critically depend on the presence of Src-family kinases BCR-induced activation of Syk, indicating a prominent (Kolanus et al., 1993; Straus and Weiss, 1992), role of Src-family kinases in this process (El-Hillal et suggesting that these kinases may function as direct al., 1997; Kurosaki et al., 1994). regulators of ZAP-70 activation. The importance of Autophosphorylation of Syk occurs in vitro on this dichotomy in regulation is underscored by the role several tyrosyl residues (Furlong et al., 1997; Keshvara ZAP-70 plays in TCR signaling. In apparent contrast et al., 1997), at least two of which ± Y518 and Y519, to the low phosphotyrosyl content of the resting BCR, located within the activation loop of the catalytic the TCR z chain was shown to be constitutively domain have been identi®ed as in vivo phosphorylation phosphorylated and to associate with ZAP-70 even in sites (Couture et al., 1996). Although mutation of quiescent T cells (Van Oers et al., 1995). This either or both these sites to phenylalanine has limited association is, however, insucient for ZAP-70 e€ect on the catalytic activity of Syk in vitro, it blocks activation, which requires its tyrosyl phosphorylation the normally observed tyrosyl phosphorylation of by the Src-family kinase Lck (Chan et al., 1995; Wange many cellular proteins (Couture et al., 1997). Since et al., 1995), mediated by the latter's recruitment (via the phosphorylation of these sites in Syk is also CD4 or CD8) to the TCR (Carrera et al., 1992; Garcia required for the activation of Lck, by its phosphoryla- et al., 1996; Itano et al., 1996; Killeen and Littman, tion at Y192 within its SH2 domain, it is possible that 1993). The inability of ZAP-70, as opposed to Syk, to Syk is also involved in the positive regulation of Src- autophosphorylate may be due to its 100-fold lower family kinases (Couture et al., 1994b). This further intrinsic kinase activity (Latour et al., 1996). Syk and suggests that the observed association between Lck and ZAP-70 di€er also in their roles with respect to T cell Syk in stimulated cells (Couture et al., 1994b) and the development. While thymocytes from mice lacking binding of SH2 domains of various members of the both ZAP-70 and Syk are arrested at the CD47 Src-family of kinases to tyrosyl phosphorylated Syk in CD87 (DN) stage and completely fail to progress to vitro (Aoki et al., 1995; Couture et al., 1994a) is the CD4+CD8+ (DP) one (Cheng et al., 1997), the lack mediated by Syk's phosphorylation at Y518/519. The of either one of these kinases alone has no apparent corresponding tyrosyl residues within the activation e€ect on this progression (Cheng et al., 1995; Negishi loop of ZAP-70 ± Y492 and Y493 have also been et al., 1995; Turner et al., 1995). However, thymocytes shown to undergo phosphorylation following TCR from mice lacking ZAP-70 fail to progress from the aggregation, and to be essential for the signaling TCRlo DP stage to the TCRhi one, and thus fail to function of ZAP-70 (Chan et al., 1995; Wange et al., further develop into either of the single-positive (SP) 1995). Consistent with this observation, mutation of CD4+ or CD8+ stages (Negishi et al., 1995). The same both these residues to alanine in a membrane- phenotype is observed in mice having an inactivating recruitable chimeric ZAP-70 molecule or co-transfec- point mutation within a highly conserved motif in the tion of a dominant negative form of Lck (which is kinase domain of ZAP-70 (Wiest et al., 1997). required for ZAP-70 activation) blocked the otherwise Seemingly inconsistent with the unique function of observed transcription of a reporter in a ZAP-70, the block in thymocyte development in ZAP- transfected T cell line (Graef et al., 1997). 70-de®cient mice can be overcome by expression of Tyrosyl phosphorylation of Syk may also serve human Syk which restores both development and regulatory functions that are unrelated to enzymatic function (Gong et al., 1997). Thus, apparent differ- activity. Phosphorylation of Y130, within Syk's inter- ences in ZAP-70 and Syk biological functions may SH2 domain region was shown to be one of the earliest re¯ect di€erences in expression or compartmentaliza- in vitro Syk phosphorylation events (Keshvara et al., tion of these molecules. Mutations in the human ZAP- 1997). Mutation of this residue to phenylalanine 70 gene are associated with a failure in normal (Y130F) inhibits the normally observed activation of signaling by T cells in peripheral lymphoid organs Syk in anti-IgM treated DT40 B cells, while its and skewed development of T cell sub-populations mutation to glutamic acid (Y130E) increases the basal (Arpaia et al., 1994; Chan et al., 1994; Elder et al., activity of Syk in resting cells. In contrast, the ability 1994). In B cells, which do not express ZAP-70, the of Syk to associate with the aggregated BCR is reduced loss of Syk results in their arrest at the transition from in the Y130E mutant, while that of the Y130F is pro- to pre-B stage (Cheng et al., 1995; Turner et al., increased. These results are consistent with a model in 1995). which Syk phosphorylation at Y130 is responsible for reducing its binding to the tyrosyl phosphorylated B- cell receptor (Keshvara et al., 1997). In addition, the Btk activation and role in BCR function Y130E mutation reduces the spectrum of tyrosyl phosphorylated proteins observed upon BCR aggrega- The Tec family of protein tyrosine kinases is tion, suggesting that Syk departure from the receptor represented in T- and B-cells by the molecules Itk complex is essential for its access to substrates, or (Siliciano et al., 1992) and Btk (Tsukada et al., 1993; alternatively, essential for recruitment of other sub- Vetrie et al., 1993), respectively. Mutations in Btk strates to the phosphorylated ITAM. This may provide result in the B-cell immunode®ciencies X-linked a mechanism for the observed dissociation of the agammaglobulinemia (XLA) in humans and X-linked activated and tyrosyl phosphorylated form of Syk from immunode®ciency (xid) in mice. The role of Btk in B Signal transduction in lymphocytes I Tamir and JC Cambier 1358 cell development and signaling will be discussed only PLC-g and phosphoinositide hydrolysis brie¯y here since it has been recently reviewed by Desiderio (1997). The translocation of Btk to the PLCg1 was shown to be recruited to the antigen plasma membrane is both essential (Kawakami et al., receptor by the binding of its SH2 domains to tyrosyl 1994; Li et al., 1995) and sucient for its activation (Li phosphorylated Syk, which involves the phosphotyr- et al., 1997a). A pleckstrin homology domain located osyl residues Y348 and/or Y352 in the linker region of at the amino-terminal region of all members of the murine Syk (Law et al., 1996a). The tyrosyl phosphor- Tec-family of kinases has been implicated in their ylation of PLCg2, the more abundant form of this membrane association (Hemmings, 1997). Mutations in B cells, was shown to be dependent on Syk within the PH domain of Btk which result in XLA and expression (Takata et al., 1994) and the presence of its xid correlate with abolished binding of Btk to SH2 domains and Y518/Y519 autophosphorylation

phosphoinositides PI(3,4,5)P3 (PIP3), IP4,IP5 and IP6 sites (Kurosaki et al., 1995). Since the phosphorylation (Fukuda et al., 1996; Salim et al., 1996), The of these sites was shown to induce the association of association of Btk's PH domain with the plasma Syk with Src-family kinases (Couture et al., 1996) it membrane may be mediated by other, membrane- also suggests a role for the latter in the tyrosyl associating proteins such as PKC (Yao et al., 1994) phosphorylation of PLCg2. However, studies with the and the bg subunits of heterotrimeric G proteins DT40 B cell line lacking the Src-family kinase Lyn (Langhans ± Rajasekaran et al., 1995; Tsukada et al., (which is reportedly the only member of this family 1994). As discussed below in the context of CD19 expressed in this cell line) have shown that both the

accessory functions, considerable evidence indicates tyrosyl phosphorylation of PLCg2 and inositol 1,4,5-P3

that BCR-mediated translocation of Btk to the plasma (IP3) generation in response to BCR aggregation are membrane and subsequent activation is consequent to independent of Lyn's expression in this cell line

PI3-K activation and PIP3 generation. (Takata et al., 1994). In contrast, calcium mobilization

Antigen receptor aggregation results in the tyrosyl was delayed in these cells, suggesting that IP3 phosphorylation and activation of Itk (August et al., generation alone is insucient for inducing the rapid 1994) in T-cells and Btk (Aoki et al., 1994; de Weers rise in the intracellular concentration of free calcium 2+ et al., 1994; Saouaf et al., 1994) in B-cells by Src- ions ([Ca ]i) normally observed upon BCR aggrega- (Mahajan et al., 1995; Rawlings et al., 1996) and/or tion. As discussed below, this may re¯ect in part a Syk-family PTKs (Kurosaki and Kurosaki, 1997). In requirement for CD19 phosphorylation and subsequent B-cells this phosphorylation was shown to occur activation of PI3-K and Btk for e€ective calcium initially at Btk's Y551, within the activation loop of mobilization. the kinase, and to be mediated by Src-family kinases The recruitment of PLCg isoforms to the plasma (Rawlings et al., 1996). This phosphorylation results in membrane and their tyrosyl phosphorylation leads to an increase in Btk enzymatic activity further leading both their co-localization with their substrates and to autophosphorylation at Y223 within the SH3 their activation, which results in the hydrolysis of

domain (Park et al., 1996; Wahl et al., 1997). phosphatidylinositol-4,5-P2 (PIP2) producing two solu-

Crystallographic analysis of Itk shows an intramole- ble products, namely ± IP3 and diacylglycerol (Bijster- cular interaction between its SH3 domain and an bosch et al., 1985). These soluble second messengers adjacent N-terminal proline-rich region (Andreotti et have been implicated in calcium ion mobilization and al., 1997). Thus by extrapolation, phosphorylation of C (PKC) activation, respectively. Btk at Y223 may disrupt this intramolecular Deletion of PLCg2 in DT40 B cells results in complete

interaction allowing interaction with other signaling blockage of both IP3 production and calcium mobiliza- molecules. One such molecule may be a member of tion, highlighting its critical role in this process (Takata the Src-family of kinases, whose SH3 domains have et al., 1995). Of the several PKC isoforms expressed in been shown to bind in vitro to the proline-rich domain B cells, PKCm has been shown to associate with Syk, of Btk (Cheng et al., 1994; Yang et al., 1995). This PLCg1 and PLCg2 in B cells (Sidorenko et al., 1995) may provide a mechanism for Btk association with and to undergo a PLCg2-dependent activation upon Src-family kinases. BCR aggregation (Sidorenko et al., 1996). This PKC isoform has also been implicated with the phosphor- ylation of Syk, which may serve to inhibit the latter's Tertiary signal transduction components ability to phosphorylate PLCg1 (Sidorenko et al., 1996). Available evidence arising from in vitro binding and co- precipitation studies indicate that the activated BCR, The Ras pathway with its associated Syk- and Src-family kinases is able to recruit an additional `layer' of e€ectors to the Analysis of phosphotyrosyl peptide sequence require- receptor (Pleiman et al., 1994c; Sidorenko et al., 1995; ments for recognition by di€erent SH2-containing Sillman and Monroe, 1995). These include phospholi- proteins revealed that phosphorylation of CD79a at pase Cg1 (PLCg1), the Shc-Grb2-Sos-Ras complex and Y182 and CD79b at Y195 likely create a binding site for phosphatidylinositol 3-kinase (PI3-K). Due to space the SH2 domain of the adaptor protein Shc, while limitations we will address here only the known phosphorylation of CD79a at Y193 (or CD79b at Y206) mechanisms by which these e€ectors are recruited is optimal for Src-family kinase SH2 binding (Songyang and activated by the antigen receptor. The reader is et al., 1993, 1994b). Consistent with this, Shc has been referred to the excellent recent reviews by DeFranco, shown to bind in vitro via its SH2 domain to the (1997) and Kurosaki, (1997) for a more comprehensive phosphorylated ITAMs of CD79a/CD79b and to discussion of the signaling functions of these e€ectors. undergo tyrosyl phosphorylation in response to BCR Signal transduction in lymphocytes I Tamir and JC Cambier 1359 aggregation (D'Ambrosio et al., 1996; Saxton et al., In mature B cells, CD19 occurs in a hetero-oligomeric 1994; Smit et al., 1994). The recruitment of Shc to the complex with CD81, a tetraspan molecule, CD21, a tyrosyl-phosphorylated receptor suggests a link between C3dg complement fragment-binding molecule and BCR activation and the p21ras signaling pathway Leu13. This complex constitutes the type 2 comple- (Harwood and Cambier, 1993; Lazarus et al., 1993). ment receptor (CR2). It is noteworthy that CD21 is Both Src- (Nagai et al., 1995) and Syk-family (Jabril- expressed only in mature B cells, thus in immature B Cuenod et al., 1996) kinases have been shown to be cells CD19 function is independent of CD21. involved in antigen receptor-induced tyrosyl phosphor- Available evidence indicates that CD19 functions as ylation of Shc, which serves to recruit the adapter protein a signal transducer in three distinct circumstances. It is Grb2, via its SH2 domain, to the receptor. This was required for normal B cell development and when shown to result in further recruitment of the protein Sos, aggregated in pre-B cells triggers the activation of Src- via its proline-rich region interaction with the SH3 family kinases (Chalupny et al., 1993; Krop et al., domains of Grb2, to the receptor (Ravichandran et al., 1996; Roifman and Ke, 1993; Sato et al., 1997c; Uckun 1993; Saxton et al., 1994). Sos has been shown to activate et al., 1993). Importantly, CD19 function in develop- GDP-GTP exchange in Ras, which in-turn activates the ment requires its cytoplasmic tail supporting its MAPK pathway (Smit et al., 1994). function as a signal transducer (Sato et al., 1997b). Thus, CD19 ligation by some as yet unde®ned ligand may lead pre-B cells to transverse a checkpoint in The PI3-K pathway development. The presumptive ligand may at one BCR aggregation results in the tyrosyl phosphorylation extreme be a molecule expressed on some stromal and activation of PI3-K (Gold et al., 1992) following element, or, at the other, it may be the pre-BCR or its recruitment to the antigen receptor. This recruit- CD79a/CD79b dimers (Nagata et al., 1997). In mature ment possibly occurs by multiple mechanisms; one is B cells, C3dg-antigen complexes induce co-aggregation by the binding of the SH3 domain of active Src-family of CR2 with BCR which may enhance the immune kinases to a proline-rich sequence in the PI3-K p85 response to antigen as much as 10 000-fold (Dempsey subunit. This interaction may also lead to additional et al., 1996). Underlying this synergy is increased Vav activation of Src-family PTKs by ligation of their SH3 phosphorylation, calcium mobilization, and activation domains, as discussed previously. Conversely, the of the MAPK kinase pathway following BCR-CD19 binding of PI3-K to Src-family SH3 domains was co-aggregation (Dempsey et al., 1996; Li et al., 1997b; shown to activate its enzymatic activity (Pleiman et al., Tooze et al., 1997). CD19 also plays an important role 1994c). PI3-K recruitment to the BCR is also mediated in BCR signaling in mature B cells independent of its by its association with phosphotyrosyl residues in co-aggregation with the antigen receptor. B cells from ITAMs (Pleiman et al., 1994a) and the BCR-accessory CD197/7 knockout mice fail to proliferate in response transmembranal molecule CD19 (Hippen et al., 1997; to BCR aggregation (Engel et al., 1995). CD19 Tuveson et al., 1993). Its activation following BCR expression is required for BCR-mediated PI3-kinase aggregation is inhibited by disruption of Src-family activation, phosphoinositide hydrolysis and Ca2+ kinase SH3 domain interactions using proline-rich mobilization (Buhl et al., 1997). This accessory peptides as well as by obviating CD19 function. Thus function of CD19 may depend on its association with PI3-K may be regulated cooperatively via Src-family the BCR, as CD19 has been reported to co-cap and co- kinases and CD19 association. immunoprecipitate with the BCR, which appears to be mediated by the interaction of a 17 amino acid juxtamembrane sequence in CD19 with the BCR Molecular accessories in antigen receptor signaling (Carter et al., 1997; Pesando et al., 1989). It is unclear whether CD19 is pre-associated with the BCR, or Signal transduction by the B lymphocyte antigen becomes associated following BCR signaling. In the receptor is not an autonomous process. At least three latter case, activated e€ectors may provide an additional integral membrane glycoproteins, CD19, intracellular bridge from the BCR to CD19. CD45 and CD22, participate in BCR signaling, each The molecular mechanisms underlying CD19 providing distinct accessory functions. Provision of accessory function are beginning to become clear. these functions may require physical association of BCR ligation leads to CD19 tyrosyl phosphorylation these molecules with BCR (Brown et al., 1994; Carter on two residues, Y484 and Y515 (Buhl et al., 1997; et al., 1997; Peaker and Neuberger, 1993). However, Chalupny et al., 1993; Roifman and Ke, 1993). These such associations have proven much more dicult to tyrosines occur within YXXM motifs, which are document than those involving mIg and CD79a/ known to bind the SH2 domain of the p85 subunit CD79b dimers, thus they generally are not considered of PI3-K. Indeed, these phosphotyrosyl residues have components of the BCR complex. In at least one been shown to mediate PI3-K (p85) association with instance, that involving CD22, accessory interaction CD19, and to be required for BCR-mediated with the BCR appears to be regulated; changing as a activation of PI3-K (Buhl et al., 1997; Tuveson et function of cell development and activation (Cyster al., 1993). PI3-K is activated by binding to and Goodnow, 1997; Razi and Varki, 1997). phosphorylated CD19 (Hippen et al., 1997). One of

CD19 is a *95 kDa B-lymphocyte lineage-speci®c the targets of PI3-K is phosphatidylinositol-4,5-P2 integral membrane protein expressed from the pro-B to (PIP2), which is also the substrate for PLCg the plasma cell stage (Tedder and Isaacs, 1989). It isozymes. PI3-K phosphorylates PIP2 producing contains 2 extracellular Ig-like domains and a 242 phosphatidylinositol-3,4,5-P3 (PIP3) (Whitman et al., amino acid tail with nine potential tyrosyl phosphor- 1988). This phospholipid, as well as other inositol 3- ylation sites (for review see Fearon and Carter, 1995). phosphates, binds with high anity to the PH domain Signal transduction in lymphocytes I Tamir and JC Cambier 1360 of many proteins, including Tec family kinases, in and the activation of p21ras (Pao et al., 1997). These vitro (Fukuda et al., 1996; Rameh et al., 1997; Salim ®ndings indicate that CD45, presumably via its role in et al., 1996). Src-family kinase activation, is required for some ras The CD19 expression requirement for IP3 produc- function, possibly involving p21 , that is downstream

tion and calcium mobilization appears to re¯ect a from PLCg phosphorylation and required for PIP2

requirement for PIP3-dependent Btk activation. B cells hydrolysis. Given the earlier discussion, one obvious from xid mice and Btk7/7 DT40 cells are defective in possibility is that CD45 expression is required for

the generation of either IP3 or a prolonged calcium activation of the CD19-PI3-K-Btk pathway, a possibi- mobilization response following BCR aggregation lity which has not yet been explored. (Kurosaki and Kurosaki, 1997; Lindsberg et al., Finally, CD22 may play an important accessory role 1991; Rigley et al., 1989; Takata and Kurosaki, 1996; in BCR signal transduction. CD22 is a B lineage- Yamada et al., 1993). Although there is some restricted cell surface protein that is expressed at high disagreement in this regard, Btk participation in this levels only in mature B cells and functions as a lectin, signaling step does not appear to involve PLCg binding certain a2,6-sialoglycoproteins via a site in the phosphorylation, raising the possibility that Btk two distal of its seven extracellular immunoglobulin- functions primarily to provide a linker that is critical like loops (Cyster and Goodnow, 1997; Law et al.,

for PIP2 hydrolysis. In addition to its PH domain, Btk 1994; Tedder et al., 1997). CD22 binds secreted IgM contains proline-rich sequences known to interact with via this domain, and thus may bind BCR by virtue of Src-family kinases as well as SH2 and SH3 domains its mIg component (Hanasaki et al., 1995). This may (Desiderio, 1997). PLCg isoforms contain one full and provide the basis of its association with BCR and two partial PH domains, as well as two SH2 and one consequently, its regulation of BCR function. Interest- SH3 domains and unique regions that could mediate ingly, the lectin-binding activity of CD22 is regulated interactions with Btk (Rhee and Bae, 1997). The by its own glycosylation (Braesch ± Andersen and

consequence of PLCg-Btk-PIP3- Stamenkovic, 1994; Sgroi et al., 1996). While inactive

complex formation is the e€ective hydrolysis of PIP2, in resting B cells, CD22 on cycling cells binds to

generating IP3. The latter mediates the release of exogenous a2,6-sialoglycoproteins. Thus, CD22 regula- calcium ions from intracellular stores, which leads to tion of BCR function depends not only on expression capacitative calcium entry. BCR-activated capacitative levels, which change during development, but also on calcium entry, and to a lesser but signi®cant extent the activation state of cells on which it is expressed. intracellular calcium release, is dependent on CD19 BCR aggregation leads to the tyrosyl phosphoryla- expression and function. tion of CD22 on multiple cytoplasmic tyrosyl residues. Cells of the hematopoietic lineage express, as Upon its tyrosyl phosphorylation CD22 becomes predominant membrane proteins, alternately spliced associated with multiple signal transduction e€ectors, forms of the protein tyrosine phosphatase CD45 including Lyn, Syk, PLCg1, PI3-K and SHP-1 a protein (Justement, 1997). Alternate splicing determines varied tyrosine phosphatase (Doody et al., 1995; Law et al., expression of three extracellular domain exons. B 1996b; Tuscano et al., 1996). Several studies using lineage cells express only the full length 220 kDa CD22 knock-out mice have demonstrated hyper- from. While variations in extracellular structure responsiveness of CD227/7 B cells to sub-optimal suggests the existence of ligand binding site in this BCR aggregation, as judged by calcium mobilization portion of the molecule, no speci®c CD45 ligand has and proliferative responses (Nitschke et al., 1997; been described to date. However, some evidence O'Keefe et al., 1996; Otipoby et al., 1996; Sato et al., suggests that CD45 can associate with BCR (Brown 1996). In addition, these animals express an unusually et al., 1994), though the basis of this association is high level of serum immunoglobulins. In view of the unclear. phenotypic similarity between CD227/7 and SHP-17/7 Studies of B cells from CD45 knock-out mice (motheaten) mice it has been suggested that CD22 indicate that CD45 expression is required for BCR- functions as a negative regulator of BCR signaling via mediated activation of B cell proliferation and recruitment and activation of SHP-1 (Cyster and generation of antibody responses (Benatar et al., Goodnow, 1997). Consistent with this, CD22 functions 1996; Byth et al., 1996; Kishihara et al., 1993). This to reduce BCR-CD19 co-aggregation-mediated Vav appears to re¯ect a requirement that CD45 depho- phosphorylation (Sato et al., 1997a; Tooze et al., sphorylate regulatory phosphotyrosyl residues found at 1997). While these data indicate that CD22 functions the C-terminus of Src-family tyrosine kinases in order in an inhibitory capacity, it appears to be a positive for these PTK to function in BCR signaling (Cooper regulator of certain B cell responses. CD22 expression is and Howell, 1993). In CD45-negative B cells these required for colony formation by B lymphoid cell lines kinases are phosphorylated at their C-terminus and and for antibody responses to T-cell-independent BCR ligation does not trigger their activation (Pao and antigens (Kiesel et al., 1988; Nitschke et al., 1997; Cambier, 1997). Similarly, neither phosphoinositide O'Keefe et al., 1996; Otipoby et al., 1996; Sato et al., hydrolysis nor calcium mobilization are seen in 1996). It is tempting to speculate that these functions CD45-negative B cells (Justement et al., 1991; Pao et involve CD22 association with Syk, PLCg and/or PI3- al., 1997). Despite these de®cits, many BCR aggrega- K. Clearly elucidation of the BCR accessory functions tion-mediated tyrosine phosphorylation events, such as of CD22 will require considerable additional study. that of CD79a, CD79b, PLCg1 and PLCg2, as well as The lesson to be learned from the preceding the activation of Syk, appear normal in these cells paragraphs is that antigen receptor signaling, and (Kawauchi et al., 1994; Pao et al., 1997). Conspicu- hence its biologic output, are both positively and ously absent in CD45-negative B cells are the tyrosyl negatively modulated by other membrane molecules phosphorylation of Shc, a linker to the ras pathway, with which it interacts. These interactions, and hence Signal transduction in lymphocytes I Tamir and JC Cambier 1361 their regulatory outcome, can be determined by promote binding to the SH3 and SH2 domains of other changes in expression of accessory molecules during proteins. Thus, Cbl seems to play a role of an adapter development, e.g., CD45 and CD19 expression protein serving to promote protein-protein interactions gradually increase during B cell development and and/or translocate e€ectors to the plasma membrane. CD22 expression rises precipitously during B cell Among the proteins reported to interact with Cbl are transition from immature to mature phenotype. Src- and Syk-family kinases (Fitzer-Attas et al., 1997; Changes in the anity of the molecules on the cell Lupher et al., 1996; Ota et al., 1996; Ota and surface for each other may provide another mechanism Samelson, 1997; Panchamoorthy et al., 1996). Src- for modulating their interaction, e.g. CD22 interaction family kinase have also been shown to be required for with BCR is a likely function of its a2,6-sialic acid the tyrosyl phosphorylation of Cbl (Tezuka et al., binding anity, which changes following cell activa- 1996), while association with Cbl has been shown to tion. Finally, complex ligands may induce unique negatively-regulate Syk's activity (Ota and Samelson, responses by co-aggregating membrane receptors that 1997). Other proteins that were shown to interact with function cooperatively, e.g. BCR and CR2 co- Cbl are the p85 subunit of PI3-K (Hartley et al., 1995; aggregation by complement-coupled antigens. Kim et al., 1995; Meisner et al., 1995; Panchamoorthy et al., 1996), Btk (Cory et al., 1995) and Grb2 (Buday et al., 1996; Meisner et al., 1995; Panchamoorthy et al., Future challenges 1996). Finally, SLP-76 is an adapter molecule, shown to Although many gaps remain, the details of the play important roles in T cell signal transduction signaling pathways initiated by antigen receptors, (recently reviewed in Koretzky, 1997). It contains an which are downstream to the action of receptor- N-terminal acidic domain, that upon its tyrosyl associated PTKs, are beginning to emerge. In addition phosphorylation, probably by ZAP-70 (Wardenburg to the above-described PTK targets, several others et al., 1996), was shown to interact with Vav (Wu et have been discovered. Among these targets are proteins al., 1996), a proline-rich domain which may provide a which supposedly play a role of adaptors, while others binding site for Grb2 (Motto et al., 1996) and a C- have a catalytic function(s) which is still poorly terminal SH2 domain. All these domains have been de®ned. The following provides a brief review of the shown to be required for the function of SLP-76 in T most prominent of these proteins and their known cells (Musci et al., 1997). SLP-76 has been shown to be associations with other signaling molecules. Future involved in TCR-stimulated IL-2 promoter activity, research should clarify the roles these proteins play in suggesting that it acts to link proximal TCR signaling antigen receptor signaling and properly place them in events to distal ones in T-cell activation. the signal transduction cascade. Vav is a 95 kDa protein which becomes tyrosyl phosphorylated upon aggregation of antigen receptors. ZAP-70 has been shown to be upstream to the tyrosyl Abbreviations phosphorylation of Vav in T cells, since the expression 2+ of a dominant negative ZAP-70 mutant blocks this BCR, B cell receptor; [Ca ]i; intracellular concentration of free calcium ions; DN, double negative; DP, double process (Qian et al., 1996). Furthermore, ZAP-70 was positive; Ig, immunoglobulin; IP3, inositol 1,4,5-triphos- shown to recruit Vav in T cells (Wu et al., 1996) via the phate; IP , inositol 1,3,4,5-tetrakisphosphate; IP , inositol latter's SH2 domain and Y315 phosphorylation of 4 5 1,3,4,5,6-pentakisphosphat; P6, inositol 1,2,3,4,5,6-hexakis- ZAP-70 (Wu et al., 1997). Similarly, tyrosyl phosphor- phosphate; ITAM, immunoreceptor tyrosine-based acti- ylation of Syk at Y341 was shown to allow its vation motif; m, membrane; MAPK, mitogen-activated association with Vav, resulting in its tyrosyl phosphor- protein kinase; MIRR, multichain immune recognition ylation (Deckert et al., 1996). Thus Vav and PLCg1 receptor; PH, pleckstrin homology; PI, phosphatidy- compete for the same binding site in Syk. The role of linositol; PI3-K, phosphatidylinositol 3-kinase; PIP2, Vav in B- and T-cell development has been recently phosphatidylinositol 4,5-diphosphate; PIP3, phosphatidy- reviewed (Bonnefory-Berard et al., 1996). linositol 3,4,5-triphosphate; PKC, protein kinase C; PLC, phospholipase C; ppITAM, doubly-phosphorylated One of the proteins that undergoes massive tyrosyl immunoreceptor tyrosine-based activation motif; PTK, phosphorylation upon BCR and TCR aggregation is protein tyrosine kinase; PTP, protein tyrosine phos- the 120 kDa proto-oncogene product Cbl (Cory et al., phatase; SH, Src homology; SHP, SH2 domain-containing 1995; Panchamoorthy et al., 1996). This protein is protein tyrosine phosphatase; SP, single positive; TCR, T composed of several proline-rich regions and multiple cell receptor; xid, X-linked immunode®ciency in mouse; tyrosyl phosphorylation sites, which could respectively XLA, X-linked agammaglobulinemia.

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