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Adaptor proteins in lymphocyte antigen-receptor signaling
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Adaptor proteins in lymphocyte antigen-receptor signaling Erik J Peterson*, James L Clementst, Nan Fangs and Gary A Koretzky§ Adaptor molecules, proteins that possess no intrinsic enzymatic function, but which mediate protein-protein interactions, have a critical role in integrating signal transduction pathways following engagement of cell-surface receptors. Several newly described adaptor molecules have been shown to serve important functions in the regulation of signaling events initiated by lymphocyte antigen receptors. Understanding how these adaptor proteins function to modulate signaling cascades will provide important insights into the complex biology of lymphocyte activation.
Addresses
*t§Department of Internal Medicine, $§lnterdisciplinary Graduate Program in Immunology and §Department of Physiology and Biophysics, Universityof Iowa College of Medicine, Iowa City, Iowa 52242, USA *e-maih [email protected] Correspondence: Gary A Koretzky Current Opinion in Immunology 1998, 10:337-344
http:l/biomednet.comlelecref1095279150100033? © Current Biology Ltd ISSN 0952-7915 Abbreviations BCR B-cell receptor ITAM immunoreceptortyrosine-based activation motif NFAT nuclearfactor of activated T cells PH pleckstrinhomology
PLC
phospholipaseC
PTB PTK SH
phosphotyrosinebinding proteintyrosine kinase
Src homology
but which serve critical functions in signal transduction by mediating protein-protein interactions. Adaptor proteins are characterized by a modular domain structure and/or specific amino-acid residues responsible for binding other proteins [10°']. These domains include Src homology 2 (SH2) [11] and phosphotyrosine-binding (PTB) [12] domains which mediate interactions with tyrosine-phosphorylated proteins; Src homology 3 (SH3) domains which display affinity for proteins containing proline-rich sequences [11]; pleckstrin homology (PH) domains which bind phospholipids [13]; WW domains which mediate interactions with proteins containing consensus proline/tyrosine or proline/leucine motifs [14]; and PDZ domains which bind short motifs containing carboxy-terminal hydrophobic residues [15]. Many adaptor proteins contain proline-rich regions that mediate binding to other proteins with SH3 domains. Some possess tyrosines which, when phosphorylated, permit interactions with SH2 or PTB domains of other adaptor or effector molecules. Through these interactions, adaptor proteins mediate the formation of a diverse array of signaling complexes following antigen-receptor ligation. In the past several years, a number of newly described adaptor proteins, some expressed ubiquitously and others expressed exclusively in hematopoietic cells, have been shown to play important roles as regulators of antigen-receptor signaling. In this review, several of these proteins are described and their possible roles in lymphocyte activation are discussed (Figure 1). Grb2 and She
Introduction
Much has been learned in recent years about the signal transduction events that follow the engagement of antigen receptors on lymphocytes and lead to cytokine production and cellular proliferation or to apoptosis. Initial studies focused on the receptors themselves and the enzymes that are stimulated following receptor ligation. This work elucidated a number of second messenger pathways that are activated following binding of antigen to either the T-cell receptor (TCR; [1]) or the B-cell receptor (BCR; [2]). The earliest known antigen-receptor-induced biochemical event is activation of cytosolic protein tyrosine kinases (PTKs) [3-5]. PTK activity is required for antigen receptor-dependent phosphorylation of several key intermediate enzymes, including phospholipase Cy1 (PLCy1) [6], phospholipase Cy2 (PLCy2) [7], ZAP-70 [8] and Syk [9]. It is, however, clear that other substrates of the receptor-stimulated PTKs also have major roles in the integration of signaling pathways. One class of such substrates are the adaptor molecules, proteins without known enzymatic or transcriptional activation capacities,
Growth factor receptor binding protein 2 (Grb2) is a small, ubiquitously expressed protein comprising a central SH2 domain flanked by two SH3 domains [16]. In unstimulated cells, Grb2 associates with Sos, a guanine nucleotide exchange factor for Ras, via an interaction between proline rich regions of Sos and the SH3 domains of Grb2 [17]. A role for Grb2 in the integration of signal transduction pathways was first described for growth factor receptors. Grb2 binds directly or indirectly to activated receptors, bringing Sos to the plasma membrane where it can facilitate Ras activation [17,18]. Recent work suggests that Grb2 is also important for coupling antigen receptors to downstream signaling events. In one study, transfection of the Grb2 SH2 domain into Jurkat T cells resulted in inhibition of TCR-induced activation of nuclear factor of activated T cells (NFAT) and AP-1function [19°1. However, the mechanisms whereby Grb2 couples antigen-receptor engagement with downstream signals are not well defined. The Grb2/Sos association represents one potential link. As in non-hematopoietic cells,
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Lymphocyte activation and effector functions
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Schematic of the adaptor proteins and their structural features discussed in this review. Grb2, growth factor receptor binding protein 2; LAT, linker for activation of T cells; SKAP-55, Src-kinase-associated protein of 55 kDa; SLP-76, SH2-containing leukocyte phosphoprotein of 76 kDa; SLAP-130, SLP-76-associated protein of 130 kDa. Other proteins are described in the text. LZ, leucine zipper; PH, pleckstrin homology domain; Pro-rich, sequences rich in proline residues; PTB, phosphotyrosine binding domains. P-Y sites, putative phosphorylation sites on tyrosine residues; SH2, Src homology 2 domain; SH3, Src homology 3 domain. TM, putative hydrophobic transmembrane domain. Numbers refer to amino acids in the sequences.
Grb2 associates with Sos in the cytosol of lymphocytes [20,21], suggesting a means for coupling antigen-receptor ligation to Ras activation. One study showed that, in contrast to growth factor receptor systems, association of Grb2 with Sos appears to be inducible following T C R ligation [22]. However, whereas ligation of growth factor receptors leads to eventual phosphorylation-dependent dissociation of Sos from Grb2 [23,24] and downregulation of Ras activity, the Grb2/Sos complex in T cells is more stable [22,25]. T h e TCR-inducible association of Grb2 with Sos may be mediated by Shc, an adaptor protein that contains a single carboxy-terminal SH2 domain [26] and an amino-terminal PTB domain [27]. Following T C R ligation, Shc has been shown to be recruited to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within the T C R complex ~ chain [28]. Additionally, tyrosine phosphorylation of Shc after T C R engagement and subsequent binding to the SH2 domain of Grb2 has been reported to enhance the association between Grb2 and Sos [22]. To date, however, there is little direct evidence that a Shc/Grb2/Sos complex functions to recruit Sos to the T-cell membrane [29]. Following BCR engagement, Shc is rapidly phosphorylated on tyrosines as a consequence of the kinases Lyn and Syk [30]. Formation of a stable Shc/Grb2/Sos complex in B cells requires two Grb2-binding sites in Shc, suggesting that a single Shc molecule may simultaneously
bind two Grb2 molecules [31°]. While a Shc/Grb2/Sos complex may promote positive BCR signaling through Ras activation, a number of additional molecules with potential negative signaling capacity which associate with a Shc/Grb2 complex or with Shc alone have been described, including Cbl ([32]; see below) and inositol polyphosphate 5-phosphatase (SHIP; [33°,34]). The Shc/SHIP association occurs, and Shc/Grb2 binding is simultaneously impaired, when the BCR is co-crosslinked with IgG receptor FcyRII, suggesting a potential mechanism for the negative signaling effects mediated by the receptor FcyRII [34,35°]. SLP-76,
SLAP-130
(Fyb), and SKAP55
SLP-76 (SI-12 domain-containing leukocyte phosphoprotein of 76 kDa) is a protein of 533 amino acids expressed exclusively in cells of hematopoietic origin. SLP-76 was cloned initially as a Grb2-associated substrate of the TCR-stimulated PTKs [36]. SLP-76 contains an amino-terminal region with tyrosine phosphorylation sites, a central proline-rich region responsible for its interaction with Grb2 via the Grb2 SH3 domains [37°°], and a carboxy-terminal SH2 domain [36]. SLP-76 expression has been reported in virtually all T-cell lines tested and in monocytic, natural killer (NK), and basophilic cell lines [36,38]. SLP-76 expression in B ceils remains controversial [39-41]. SLP-76 overexpression in Jurkat T cells results in significant augmentation of NFAT or IL-2 promoter activity following T C R engagement [37°°]. SLP-76 overexpression
Adaptor proteins in antigen-receptor signaling Peterson et aL
does not affect TCR-mediated calcium mobilization, but does enhance activation of mitogen-activated protein kinase (MAPK) and AP-1 activity [42"]. SLP-76-mediated enhancement of T C R signaling requires phosphorylation of SLP-76 in its amino-terminal region [43",44"]. Additionally, overexpression of SLP-76 mutants lacking the central proline-rich region or a functional SH2 domain fail to augment TCR-mediated activation events, suggesting that all three domains of SLP-76 are required for its optimal function [37"',42"]. Since SLP-76 itself has no enzymatic function, efforts have been made to identify proteins with which SLP-76 interacts in an attempt to understand how SLP-76 augments T C R signaling. Several groups have found that following T C R engagement, tyrosine-phosphorylated SLP-76 inducibly associates with the SH2 domain of Vav [45"-47"]. Interestingly, in overexpression studies, SLP-76 and Vav have a synergistic effect on TCR-mediated activation of the IL-2 promoter, suggesting that signaling pathways augmented by both molecules overlap [47"]. However, it has been reported that TCR-dependent IL-2 production in a murine T-cell hybridoma does not require Vav-SLP-76 interactions [48], suggesting that SLP-76 and Vav need not directly interact to promote signaling. T h e SH2 domain of SLP-76 binds, directly or indirectly, to two other phosphotyrosine-containing proteins following T C R engagement. One of these, a molecule of 62 kDa, remains unidentified while the cDNA encoding the second has been cloned. This phosphoprotein was purified both as a protein that binds the SLP-76 SH2 domain (therefore designated SLP-76-associated phosphoprotein of 130kDa, or SLAP-130) [49"'] as well as a binder of a fusion protein containing the SH2 domain of the Fyn PTK (thus named Fyn-binding protein, or Fyb) [50",51]. SLAP-130/Fyb is a protein of 783 amino acids which possesses a region rich in proline residues, another region rich in tyrosine residues, possible nuclear localization sequences and an SH3-1ike domain [49"',50"]. T h e role of SLAP-130/Fyb in T-cell activation remains unclear. In Jurkat T cells, overexpression of SLAP130/Fyb results in a decrease in TCR-mediated signaling as measured by NFAT activity [49"']. Furthermore, cooverexpression of SLAP-130/Fyb with SLP-76 abrogates the positive effect of SLP-76 on reporter gene activity [49"']. In contrast, however, when overexpressed in the DC27.10 T-cell hybridoma, SLAP-130/Fyb augments IL-2 production [50"']. Further studies of the effects of overexpression and lack of expression of SLAP-130/Fyb and an analysis of structural features contributing to its signaling function are needed. In addition to SLP-76 and Fyn, SLAP-130/Fyb associates with another phosphoprotein in T cells. SKAP-55 (for Src-kinasc-associated phosphoprotein of 55 kDa) is also a lymphocyte-specific adaptor protein possessing an SH3 do-
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main, a PH domain and tyrosines within motifs predicted to bind Src family P T K SH2 domains [52"']. In support of this notion, SKAP-55 binds to the Fyn SH2 domain in T cells [52"']. The interaction between SLAP-130/Fyb and SKAP-55 is mediated by the SKAP-55 SH3 domain and the central proline-rich region of SLAP-130/Fyb (AM Cardine, LR Hendricks-Taylor, NJ Boerth, H Zhao, B Schraven, GK Koretsky, unpublished data). At present, SKAP-55 signaling function remains unresolved. LAT ( p p 3 6 ) Western blotting analysis of phosphotyrosine-containing proteins within whole-cell lysates of activated T cells reveals a major species that migrates at approximately 36 kDa. When detected by tyrosine phosphorylation, this protein is associated predominantly with the plasma membrane, pp36 has been shown to associate with the SH2 domains of Grb2 and PLCy1, suggesting a role in coupling the T C R with both the phosphatidylinositol and Ras signaling pathways [21,53]. Further evidence for the importance of pp36 in TCR-mediated signal transduction came from studies of cells where pp36 was selectively dephosphorylated by transfection of a chimeric phosphatase. While the most proximal signaling events remained intact, T C R engagement on the transfectant failed to result in the generation of soluble inositol phosphate and an intracellular calcium flux [54]. More evidence for the role of pp36 came from examination of hyporesponsive T cells in non-obese diabetic (NOD) mice. In NOD T cells, decreased phosphorylation of pp36 correlates with decreased membrane targeting of the Grb2/Sos complex and impaired Ras activation [55"].
Recently, the cDNA for pp36 was cloned. T h e protein product, termed LAT, for linker for activation of T ceils, consists of 233 amino acids with a putative transmembrane domain at the amino terminus [56"']. LAT has no obvious enzymatic function according to its sequence. Although it contains no regions homologous to SH2, SH3 or PTB domains, LAT possesses several tyrosines within motifs predicted to mediate interactions with the SH2 domains of Grb2 and PLCy1. Northern analysis suggests that LAT is expressed exclusively in hematopoietic cells, most prominently in T cells, NK cells and mast cells, but not in B cells. In activated T cells, LAT associates directly or indirectly with many critical signaling molecules including Grb2, the 85kDa subunit of phosphoinositide 3-kinase (PI3K), PLCy1, SLP-76, Cbl and Sos [56"'] (Figure 2). Overexpressed tyrosine-to-phenylalanine LAT mutants in Jurkat cells exert a dominant inhibitory effect on TCR-induced NFAT activity, indicating the importance of this molecule as an integrator of T-cell activation signals. Cbl a n d C r k In the past few years it has become clear that in addition to acting as positive regulators of signaling events, adaptor proteins also have a critical role as inhibitors of signal transduction. Recent evidence suggests
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Current Opinion in Immunology
Adaptor protein complexes that promote T-cell antigen receptor signaling. Following TCR engagement, ZAP-70 is recruited to tyrosine-phosphorylated ITAMs within the CD3 chains. Activated ZAP-70 then phosphorylates a number of substrates, including the adaptor proteins LAT and SLP-76 (Y*). Tyrosine phosphorylation of LAT may promote the assembly of several distinct signaling complexes. 1 One model proposes recruitment of Grb2/Sos to tyrosine-phosphorylated LAT with consequent Ras activation. 2 PLCy1 is also recruited to LAT, permitting phosphatidylinositol hydrolysis and engagement of a calcium-dependent pathway leading to activation of gene transcription. 3 Tyrosine phosphorylation of SLP-76 results in the recruitment of Vav; however, the mechanisms by which SLP-76 interacts with the mitogen-activated protein kinase (MAPK) cascade or Vav to promote activation are unknown. TCR-dependent tyrosine phosphorylation of SLAP-130/Fyb is required for recruitment to the SLP-76 SH2 domain (dark shading), but the physiological roles of SLAP-130/Fyb and its constitutive binding partner SKAP-55 in TCR signaling remain unclear. 4 An Shc/Grb2/Sos complex formed after TCR-induced tyrosine phosphorylation of Shc provides an alternative model for membrane recruitment of Sos with resultant Ras activation; the binding site for this complex at the plasma membrane has not been defined. SH3 domains are indicated by light shading. DAG, diacylglycerol; IP3, inositol trisphosphate; PIP2, phosphatidylinositol biphosphate; PKC, protein kinase C.
that Cbl, a ubiquitously expressed adaptor molecule of 906 amino acids, is one such negative regulator in lymphocytes [57°°]. Cbl contains an amino-terminal PTB domain [58], a ring finger domain, a proline-rich segment and a carboxy-terminal leucine zipper domain [59]. Cbl also possesses a number of tyrosine phosphorylation sites which dictate associations with a number of SH2 domain-containing proteins, including Vav [60], Crk [61], PI3K [62], Fyn [63] and Syk family PTKs (see below). Cbl is inducibly phosphorylated following antigen-receptor engagement in both T cells [64] and B cells [65°]. Ovcrexpression of Cbl in the Jurkat T-cell line impairs AP-1 activity after TCR ligation [66"]. One potential mechanism for the inhibitory function of Cbl may involve its association with Syk family PTKs. A recent study employing Cbl overexpression in rat basophilic leukemia cells demonstrated that Cbl can bind to Syk and prevent
Syk recruitment to the phosphorylated ITAMs of the activated high-affinity IgE receptor [67"]. The Cbl/Syk complex blunts Syk kinase activity and blocks signaling via the receptor. Cbl may inhibit antigen-receptor signals through a similar mechanism, as it also inducibly binds Syk in B cells [65"] and ZAP-70 in T cells [68"]. Another model for the inhibitory effects of Cbl was suggested by the observation that phosphorylated Cbl binds the SH2 domain of the CrkL adaptor protein in activated T cells [69] and in the Ramos B-cell line [70]. CrkL is a member of the Crk adaptor protein family; it contains an amino-terminal SH2 domain and tandem carboxy-terminal SH3 domains [71]. The Cbl/CrkL complex is thought to bring C3G, a guanine-nucleotide-exchange factor, into a membrane-proximal position where it can enhance GTP binding to the low-molecular-weight G protein Rapl [57",72°]. Rapl activation, in turn, leads to decreased
Adaptor proteins in antigen-receptor signaling Peterson et a/.
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Model for Cbl and CrkL adaptor-protein-mediated inhibition of T-cell activation. Following agoniststimulus of the TCR (right side of schematic), protein tyrosine kinases (PTKs) are recruited and activated. Ras and the MAPK cascade are then activated, leading to gene transcription and cellular activation. After partial agonist/antagonist engagement of the TCR (left side of the schematic), the Src family PTK Fyn is phosphorylated and activated. 1 Fyn tyrosine phosphorylates (Y*) and binds Cbl. 2 Phospho-Cbl associates with CrkL, which is bound to C3G. 3 Membrane localization of C3G enhances exchange of GTP for GDP on Rap1.4 Activated Rap1 binds Raf kinase. 5 Rap1 competes for Ras binding, and thus impairs Ras-directed MAPK activation. Light shading depicts SH3 domains and dark shading represents SH2 domains.
Ras function, perhaps as a consequence of competition for Raf, providing a means of uncoupling Ras from the MAPK cascade [57"]. These observations suggest a model wherein differential Rapl and Ras activation may determine whether the T cell becomes activated or remains unresponsive after T C R ligation. After agonist or 'productive' stimulation of the T C R (that is, in conjunction with CD28 co-stimulation), Ras activation predominates, leading to IL-2 gene upregulation. Under conditions of partial or 'antagonist' T C R engagement, as occurs during anergy induction, Rapl becomes persistently GTP-bound and blocks activating signaling events downstream of Ras ([57°°]; Figure 3).
studies in cell lines as well as experiments in genetically manipulated whole animals will be required to uncover the details of their biological actions.
Acknowledgements The authorsthankK Latinis,J Lee, P Myung,and B Schravenfor critical reviewof the manuscript. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • = of outstanding interest 1,
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Conclusions It is becoming increasingly clear that a complete appreciation of the biology of signal transduction will require understanding not only the receptors and effector molecules, but also the adaptor proteins that regulate and integrate signaling cascades. T h e past few years have seen dramatic progress in the identification of novel adaptor molecules in lymphoid cells. Much has been learned about the protein-protein associations mediated by many of these molecules, suggesting mechanisms by which they may act, both positively and negatively, in lymphocyte activation. While it is certain that adaptor proteins have critical roles in this process, further structure-function
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Fu C, Chan AC: Identification of two tyrosine phosphoproteins, pp70 and pp68, which interact with phospholipase Cgamma, Grb2, and Vav after B cell antigen receptor activation. J Biol Chem 1997, 272:27362-27368.
42. •
Musci MA, Motto DG, Ross SE, Fang N, Koretzky GA: Three domains of SLP-76 are required for its optimal function in a T cell line. J Immuno11997, 159:1639-1647. These experiments demonstrate that the Src homology-2 (SH2) domaincontaining phosphoprotein SLP-76 function in augmenting TCR-stimulated nuclear factor of activated T cells (NFAT) activation depends both upon TCR-mediated activation of tyrosine kinases and upon the presence of all three functional SLP-?6 domains: the SH2 domain, the Grb2-binding site, and amino-terminal phosphorylated tyrosine residues. SLP-76 augmentation of TCR-induced AP1 activity is also shown. 43. •
Wardenburg JB, Fu C, Jackman JK, Flotow H, Wilkinson SE, Williams DH, Johnson R, Kong G, Chan AC, Findell PR: Phosphorylation of SLP-76 by the ZAP-70 protein-tyrosine kinase is required for T-cell receptor function. J Bio/Chem 1996, 271:19641-19644.
Both this paper and Fang et al. [44"] use point mutagenesis to demonstrate that inducible tyrosine phosphorylation is required for the ability of the phosphoprotein SLP-76 to augment TCR-stimulated activation of the nuclear factor of activated T cells. Additional evidence is provided in Wardenburg et el. for the likelihood that SLP-76 serves as an in vivo substrate of ZAP-?0. Fang N, Motto DG, Ross SE, Koretzky GA: Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity. J Immuno/1996, 157:3769-3773. Both this paper and Wardenburg eta/. [43"] use point mutagenesis to demonstrate that inducible tyrosine phosphorylation is required for the ability of the phosphoprotein SLP-76 to augment TCR-stimulated activation of the nuclear factor of activated T cells (NFAT).
This study and Musci et el., 1997 [49"'] describe the purification and cloning of SLAP-130/Fyb. Musci et el. utilize the phosphoprotein SLP-76 Src homomlogy 2 (SH2) domain to isolate human SLAP-130/Fyb; da Silva et a/. took purified SLAP-130/Fyb through its association with the SH2 domain of Fyn. In contrast to Musci eta/., who find that SLAP-130/Fyb acts as an inhibitor of TCR-induced nuclear factor of activated T cells (NFA'I') activity, da Silva et el. find that when overexpressed in a T-cell hybridoma line, SLAP130/Fyb causes a modest (three-to-fourfold) increase in TCR-stimulated IL-2 production. 51.
Onodera H, Motto DG, Koretzky GA, Rothstein DM: Differential regulation of activation-induced tyrosine phosphorylation and recruitment of SLP-76 to Vav by distinct isoforms of the CD45 protein-tyrosine phosphatase. J Biol Chem 1996, 271:2222522230. This study examines tyrosine phosphorylation and physical association of SLP-76 and p95Vav in Jurkat clones expressing distinct isoforms of CD45 protein tyrosine phosphatase. After TCR stimulation, Jurkat cells expressing the highest molecular weight isoform of CD45 show enhanced SLP-?6 tyrosine phosphorylation and SLP-?6-Vav association compared to cells expressing the lowest molecular weight CD45 isoform. Results suggest that CD45 isoform expression correlates with TCR-mediated association between SLP-76 and Vav. 46. •
Tuosto L, Michel F, Acuto O: p95vav associates with tyrosinephosphorylated SLP-76 in antigen-stimulated T cells. J Exp Ivied 1996, 184:1161-1166. This paper shows that stimulation of a T cell hybridoma with antigen-presenting cells induces association of p95Vav with phosphorylated SLP-76. Further, the SLP-76-Vav association is dependent on Vav SH2. The work suggests that a physiological stimulus induces endogenous SLP-76/Vav association.
Da Silva AJ, Rosenfield JM, Mueller I, Bouton A, Hirai H, Rudd CE: Biochemical analysis of p120/130: a protein-tyrosine kinase substrate restricted to T and myeloid cells. J Immuno/1997, 158:2007-2016.
52. •.
Marie-Cardine A, Bruyns E, Eckerskorn C, Kirchgessner H, Meuer SC, Schraven B: Molecular cloning of SKAP55, a novel protein that associates with the protein tyrosine kinase p59fyn in human T-lymphocytes. J Bio/Chem 1997, 272:16077-16080. A description of SKAP-55 purification and cloning via its association with the Src homology-2 (SH2) domain of Fyn. GST fusion protein analysis suggests that SKAP-55 binds selectively through phosphotyrosine to SH2 domains of Src family, but not Syk family protein tyrosine kinases. SKAP may mediate Fyn signaling. 53.
Sieh M, Batzer A, Schlessinger J, Weiss A: GRB2 and phospholipase C-gamma 1 associate with a 36- to 38kilodalton phosphotyrosine protein after T-cell receptor stimulation. Mo/Cel/Biol 1994, 14:4435-4442.
54.
Motto DG, Musci MA, Ross SE, Koretzky GA: Tyrosine phosphorylation of Grb2-associated proteins correlates with phospholipase C-gamma-1 in T cells. Mo/Cel/Bio/1996, 16:2823-2829.
44. •
45. •
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55.
Salojin K, Zhang J, Cameron M, Gill B, Arreaza G, Ochi A, Delovitch TL: Impaired plasma membrane targeting of Grb2murine son of sevenless (mSOS) complex and differential activation of the Fyn-T cell receptor (TCR)-zeta-Cbl pathway mediate T cell hyporesponsiveness in autoimmune nonobese diabetic mice. J Exp Med 1997, 186:887-897. This paper examines T-cell hyporesponsiveness in non-obese diabetic mice. Decreased Ras pathway activity correlates with an enhanced Fyn/TCR-~Cbl complex, decreased ZAP-70 and Grb2/Sos membrane targeting, and depressed phosphorylation of pp36. These data support the idea that pp36 (LAT) tyrosine phosphorylation is important for transducing activating TCR signals. •
56. •.
Zhang W, Sloan-Lancaster J, Kitchen J, Trible RP, Samelson LE: LAT: The ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Ce//1997, 92:83-92. A description of the purification, cloning and initial characterization of I_AT. I_AT is tyrosine phosphorylated upon TCR stimulation and binds to Grb2, SLP-?6, phospholipase Cy1 and phosphatidylinositol 3-kinase. Overexpressed tyrosine point mutants of LAT act as dominant interfering molecules for TCR stimulation of the nuclear factor of activated T cells (NFAT).
Wu J, Motto DG, Koretzky GA, Weiss A: Vav and SLP-76 interact • and functionally cooperate in IL-2 gene activation./mmunity 1996, 4:593-602. This work demonstrates that when tyrosine phosphorylated, the phosphoprotein SLP-76 binds to the Vav Src homnology-2 (SH2) upon TCR stimulation (see also Onodera eta/. [45"] and Tuosto eta/. [46"]) in Jurkat T cells. Cooverexpression of SLP-76 and Vav synergistically auguments TCR-induced IL-2 promoter activity.
Boussiotis VA, Freeman G J, Berezovskaya A, Barber DL, Nadler LM: Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science 199?, 278:124-128. This study describes the association of T-cell anergy with altered membraneproximal signaling complexes. Experiments show hyperphosphorylation of Cbl and recruitment of CrkL and C3G to Cbl in anergic cells. Enhanced GTP-bound Rap1 and recruitment of Raf to Rap1 are also specific for anergic cells, suggesting a mechanism for Ras pathway inhibition in induction of anergy.
48.
58.
Lupher ML Jr, Reedquist KA, Miyake S, Langdon WY, Band H: A novel phosphotyrosine-binding domain in the N-terminal transforming region of Cbl interacts directly and selectively with ZAP-70 in T cells. J Bio/Chern 1996, 271:24063-24068.
59.
Blake TJ, Shapiro M, Morse HCD, Langdon WY: The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a prolinerich domain and a leucine zipper-like motif. Oncogene 1991, 6:653-657.
60.
Marengere LE, Mirtsos C, Kozieradzki I, Veillette A, Mak TW, Penninger JM: Proto-oncoprotein Vav interacts with c-Cbl in activated thymocytes and peripheral T cells. J Immuno/1997, 159:70-76.
61.
Sawasdikosol S, Chang JH, Pratt JC, Wolf G, Shoelson SE, Burakoff SJ: Tyrosine-phosphorylated Cbl binds to Crk after T cell activation. J Irnmunol 1996, 157:110-116.
62.
Meisner H, Conway BR, Hartley D and Czech MP: Interactions of Cbl with Grb2 and phosphatidylinositol 3'-kinase in activated Jurkat cells. Mol Ceil Bio/1995, 15:3571-3578.
47.
Raab M, da Silva AJ, Findell PR, Rudd CE: Regulation of VavSLP-76 binding by ZAP-70 and its relevance to TCR zeta/CD3 induction of interleukin-2./mrnunity 1997, 6:155-164.
49. ••
Musci M, Hendricks-Taylor L, Motto D, Paskind M, Kamens J, Turck C, Koretzky G: Molecular cloning of SLAP-130, an SLP-76associated substrate of the T cell antigen recepter-stimulated protein tyrosine kinases. J Bio/Chem 199?, 272:11674-11677. This study and that of da Silva eta/., 1997 [50 °°] describe the purification and cloning of SLAP-130/Fyb. Musciet el. utilize the phosphoprotein SLP-76 Src homology 2 (SH2) domain to isolate human SLAP-130/Fyb; da Silva eta/. took purified SLAP-130/Fyb through its association with the SH2 domain of Fyn. In contrast to Musci eta/., who find that SLAP-130/Fyb acts as an inhibitor of TCR-induced nuclear factor of activated T cells (NFAT) activity, da Silva eta/. find that, when overexpressed in a T-cell hybridoma line, SLAP-130/Fyb causes a modest (three to fourfold) increase in TCRstimulated IL-2 production. 50. ••
Da Silva A, Li Z, De Vera C, Canto E, Findell P, Rudd C: Cloning of a novel T-cell protein FYB that binds FYN and SH2-domaincontaining leukocyte protein 76 and modulates interleukin 2 production. Proc Nat/Acad Sci USA 1997, 94:7493-7498.
57. ,,•
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63.
Tsygankov AY, Mahajan S, Fincke JE, Bolen JB: Specific association of tyrosine-phosphorylated c-Cbl with Fyn tyrosine kinase in T cells, J Bio/Chem 1996, 271:27130-27137.
64.
Donovan JA, Wange RL, Langdon WY, Samelson LE: The protein product of the c-cbl protooncogene is the 120-kDa tyrosinephosphorylated protein in Jurkat cells activated via the T cell antigen receptor. J Biol Chem 1994, 269:22921-22924.
65. •
Panchamoorthy G, Fukazawa T, Miyake S, Soltoff S, Reedquist K, Druker B, Shoelson S, Cantley L, Band H: p120cbl is a major substrate of tyrosine phosphorylation upon B cell antigen receptor stimulation and interacts in vivo with Fyn and Syk tyrosine kinases, Grb2 and Shc adaptors, and the p85 subunit of phosphatidylinositol 3-kinase. J Biol Chem 1996, 271:31873194. This provides a demonstration of prominent B-cell receptor (BCR)-induced Cbl association with the protein tyrosine kinase Syk, and Cbl binding to Grb2, Shc, PI-3K, and Fyn. The data suggest that Cbl may affect signal transduction through the BCR. 66. •
Rellahan B, Graham L, Stoica B, DeBell K, Bonvini E: Cblmediated regulation of T cell receptor-induced AP1 activation. J Biol Chern 1997, 272:30806-30811. This paper reports that overexpression of Cbl in Jurkat T cells can inhibit AP1 activity following TCR stimulation. 67. •
Ota Y, Samelson LE: The product of the proto-oncogene c-cbh a negative regulator of the Syk tyrosine kinase. Science 1997, 276:418-420. This demonstrates that overexpression of Cbl in the RBL mast cell line inhibits Syk kinase activity and serotonin release after stimulation through the IgE receptor FcERI. The inhibition of Syk kinase activity is due to its binding to Cbl and subsequent dissociation from the immunoreceptor tyrosine-based activation motifs.
68.
Lupher ML Jr, Songyang Z, Shoelson SE, Cantley LC, Band H: The cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the tyr292 negative regulatory phosphorylation site of ZAP-70. J Bio/Chem 1997, 272:3314033144. The authors show that the Cbl PTB domain recognition sequence is a motif that is present in ZAP-70; moreover, the recognition site for the Cbl PTB domain within ZAP-70 includes the tyrosine residue associated with negative regulation of ZAP-70 function. Point mutation of residues within the ZAP-70 recognition site removes that ability of Cbl to bind to ZAP-70 in vivo, •
69.
Reedquist KA, Fukazawa T, Panchamoorthy G,/angdon WY, Shoelson SE, Druker BJ, Band H: Stimulation through the T cell receptor induces Cbl association with Crk proteins and the guanine nucleotide exchange protein C3G. J Bio/Chem 1996, 271:8435-8442.
70.
Ingham RJ, Krebs DL, Barbazuk SM, Turck CW, Hirai H, Matsuda M, Gold MR: B cell antigen receptor signaling induces the formation of complexes containing the Crk adaptor proteins. J Bio/Chem 1996, 271:32306-32314,
71.
Ten Hoeve J, Morris C, Heisterkamp N, Groffen J: Isolation and chromosomal localization of CRKL, a human crk-like gene. Oncogene 1993, 8:2469-2474.
72. •
Ichiba T, Kuraishi Y, Sakai O, Nagata S, Groffen J, Kurata T, Hattori S, Matsuda M: Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2. J B/o/Chem 199'7, 272:22215-22220. This provides an analysis of the Crk structural features required to enhance C3G guanine nucleotide exchange activity for Rap1. Both Src homology (SH)2 and SH3 domains of Crk are required, but membrane targeting of Crk can substitute for the SH2 domain, suggesting that recruitment of C3G to the cell membrane is a Crk function.
Adaptor proteins in lymphocyte antigen-receptor signaling Erik J Peterson*, James L Clementst, Nan Fangs and Gary A Koretzky§ Adaptor molecules, proteins that possess no intrinsic enzymatic function, but which mediate protein-protein interactions, have a critical role in integrating signal transduction pathways following engagement of cell-surface receptors. Several newly described adaptor molecules have been shown to serve important functions in the regulation of signaling events initiated by lymphocyte antigen receptors. Understanding how these adaptor proteins function to modulate signaling cascades will provide important insights into the complex biology of lymphocyte activation.
Addresses
*t§Department of Internal Medicine, $§lnterdisciplinary Graduate Program in Immunology and §Department of Physiology and Biophysics, Universityof Iowa College of Medicine, Iowa City, Iowa 52242, USA *e-maih [email protected] Correspondence: Gary A Koretzky Current Opinion in Immunology 1998, 10:337-344
http:l/biomednet.comlelecref1095279150100033? © Current Biology Ltd ISSN 0952-7915 Abbreviations BCR B-cell receptor ITAM immunoreceptortyrosine-based activation motif NFAT nuclearfactor of activated T cells PH pleckstrinhomology
PLC
phospholipaseC
PTB PTK SH
phosphotyrosinebinding proteintyrosine kinase
Src homology
but which serve critical functions in signal transduction by mediating protein-protein interactions. Adaptor proteins are characterized by a modular domain structure and/or specific amino-acid residues responsible for binding other proteins [10°']. These domains include Src homology 2 (SH2) [11] and phosphotyrosine-binding (PTB) [12] domains which mediate interactions with tyrosine-phosphorylated proteins; Src homology 3 (SH3) domains which display affinity for proteins containing proline-rich sequences [11]; pleckstrin homology (PH) domains which bind phospholipids [13]; WW domains which mediate interactions with proteins containing consensus proline/tyrosine or proline/leucine motifs [14]; and PDZ domains which bind short motifs containing carboxy-terminal hydrophobic residues [15]. Many adaptor proteins contain proline-rich regions that mediate binding to other proteins with SH3 domains. Some possess tyrosines which, when phosphorylated, permit interactions with SH2 or PTB domains of other adaptor or effector molecules. Through these interactions, adaptor proteins mediate the formation of a diverse array of signaling complexes following antigen-receptor ligation. In the past several years, a number of newly described adaptor proteins, some expressed ubiquitously and others expressed exclusively in hematopoietic cells, have been shown to play important roles as regulators of antigen-receptor signaling. In this review, several of these proteins are described and their possible roles in lymphocyte activation are discussed (Figure 1). Grb2 and She
Introduction
Much has been learned in recent years about the signal transduction events that follow the engagement of antigen receptors on lymphocytes and lead to cytokine production and cellular proliferation or to apoptosis. Initial studies focused on the receptors themselves and the enzymes that are stimulated following receptor ligation. This work elucidated a number of second messenger pathways that are activated following binding of antigen to either the T-cell receptor (TCR; [1]) or the B-cell receptor (BCR; [2]). The earliest known antigen-receptor-induced biochemical event is activation of cytosolic protein tyrosine kinases (PTKs) [3-5]. PTK activity is required for antigen receptor-dependent phosphorylation of several key intermediate enzymes, including phospholipase Cy1 (PLCy1) [6], phospholipase Cy2 (PLCy2) [7], ZAP-70 [8] and Syk [9]. It is, however, clear that other substrates of the receptor-stimulated PTKs also have major roles in the integration of signaling pathways. One class of such substrates are the adaptor molecules, proteins without known enzymatic or transcriptional activation capacities,
Growth factor receptor binding protein 2 (Grb2) is a small, ubiquitously expressed protein comprising a central SH2 domain flanked by two SH3 domains [16]. In unstimulated cells, Grb2 associates with Sos, a guanine nucleotide exchange factor for Ras, via an interaction between proline rich regions of Sos and the SH3 domains of Grb2 [17]. A role for Grb2 in the integration of signal transduction pathways was first described for growth factor receptors. Grb2 binds directly or indirectly to activated receptors, bringing Sos to the plasma membrane where it can facilitate Ras activation [17,18]. Recent work suggests that Grb2 is also important for coupling antigen receptors to downstream signaling events. In one study, transfection of the Grb2 SH2 domain into Jurkat T cells resulted in inhibition of TCR-induced activation of nuclear factor of activated T cells (NFAT) and AP-1function [19°1. However, the mechanisms whereby Grb2 couples antigen-receptor engagement with downstream signals are not well defined. The Grb2/Sos association represents one potential link. As in non-hematopoietic cells,
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Lymphocyte activation and effector functions
Figure 1
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CurrentOpinionin Immunology
Schematic of the adaptor proteins and their structural features discussed in this review. Grb2, growth factor receptor binding protein 2; LAT, linker for activation of T cells; SKAP-55, Src-kinase-associated protein of 55 kDa; SLP-76, SH2-containing leukocyte phosphoprotein of 76 kDa; SLAP-130, SLP-76-associated protein of 130 kDa. Other proteins are described in the text. LZ, leucine zipper; PH, pleckstrin homology domain; Pro-rich, sequences rich in proline residues; PTB, phosphotyrosine binding domains. P-Y sites, putative phosphorylation sites on tyrosine residues; SH2, Src homology 2 domain; SH3, Src homology 3 domain. TM, putative hydrophobic transmembrane domain. Numbers refer to amino acids in the sequences.
Grb2 associates with Sos in the cytosol of lymphocytes [20,21], suggesting a means for coupling antigen-receptor ligation to Ras activation. One study showed that, in contrast to growth factor receptor systems, association of Grb2 with Sos appears to be inducible following T C R ligation [22]. However, whereas ligation of growth factor receptors leads to eventual phosphorylation-dependent dissociation of Sos from Grb2 [23,24] and downregulation of Ras activity, the Grb2/Sos complex in T cells is more stable [22,25]. T h e TCR-inducible association of Grb2 with Sos may be mediated by Shc, an adaptor protein that contains a single carboxy-terminal SH2 domain [26] and an amino-terminal PTB domain [27]. Following T C R ligation, Shc has been shown to be recruited to phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within the T C R complex ~ chain [28]. Additionally, tyrosine phosphorylation of Shc after T C R engagement and subsequent binding to the SH2 domain of Grb2 has been reported to enhance the association between Grb2 and Sos [22]. To date, however, there is little direct evidence that a Shc/Grb2/Sos complex functions to recruit Sos to the T-cell membrane [29]. Following BCR engagement, Shc is rapidly phosphorylated on tyrosines as a consequence of the kinases Lyn and Syk [30]. Formation of a stable Shc/Grb2/Sos complex in B cells requires two Grb2-binding sites in Shc, suggesting that a single Shc molecule may simultaneously
bind two Grb2 molecules [31°]. While a Shc/Grb2/Sos complex may promote positive BCR signaling through Ras activation, a number of additional molecules with potential negative signaling capacity which associate with a Shc/Grb2 complex or with Shc alone have been described, including Cbl ([32]; see below) and inositol polyphosphate 5-phosphatase (SHIP; [33°,34]). The Shc/SHIP association occurs, and Shc/Grb2 binding is simultaneously impaired, when the BCR is co-crosslinked with IgG receptor FcyRII, suggesting a potential mechanism for the negative signaling effects mediated by the receptor FcyRII [34,35°]. SLP-76,
SLAP-130
(Fyb), and SKAP55
SLP-76 (SI-12 domain-containing leukocyte phosphoprotein of 76 kDa) is a protein of 533 amino acids expressed exclusively in cells of hematopoietic origin. SLP-76 was cloned initially as a Grb2-associated substrate of the TCR-stimulated PTKs [36]. SLP-76 contains an amino-terminal region with tyrosine phosphorylation sites, a central proline-rich region responsible for its interaction with Grb2 via the Grb2 SH3 domains [37°°], and a carboxy-terminal SH2 domain [36]. SLP-76 expression has been reported in virtually all T-cell lines tested and in monocytic, natural killer (NK), and basophilic cell lines [36,38]. SLP-76 expression in B ceils remains controversial [39-41]. SLP-76 overexpression in Jurkat T cells results in significant augmentation of NFAT or IL-2 promoter activity following T C R engagement [37°°]. SLP-76 overexpression
Adaptor proteins in antigen-receptor signaling Peterson et aL
does not affect TCR-mediated calcium mobilization, but does enhance activation of mitogen-activated protein kinase (MAPK) and AP-1 activity [42"]. SLP-76-mediated enhancement of T C R signaling requires phosphorylation of SLP-76 in its amino-terminal region [43",44"]. Additionally, overexpression of SLP-76 mutants lacking the central proline-rich region or a functional SH2 domain fail to augment TCR-mediated activation events, suggesting that all three domains of SLP-76 are required for its optimal function [37"',42"]. Since SLP-76 itself has no enzymatic function, efforts have been made to identify proteins with which SLP-76 interacts in an attempt to understand how SLP-76 augments T C R signaling. Several groups have found that following T C R engagement, tyrosine-phosphorylated SLP-76 inducibly associates with the SH2 domain of Vav [45"-47"]. Interestingly, in overexpression studies, SLP-76 and Vav have a synergistic effect on TCR-mediated activation of the IL-2 promoter, suggesting that signaling pathways augmented by both molecules overlap [47"]. However, it has been reported that TCR-dependent IL-2 production in a murine T-cell hybridoma does not require Vav-SLP-76 interactions [48], suggesting that SLP-76 and Vav need not directly interact to promote signaling. T h e SH2 domain of SLP-76 binds, directly or indirectly, to two other phosphotyrosine-containing proteins following T C R engagement. One of these, a molecule of 62 kDa, remains unidentified while the cDNA encoding the second has been cloned. This phosphoprotein was purified both as a protein that binds the SLP-76 SH2 domain (therefore designated SLP-76-associated phosphoprotein of 130kDa, or SLAP-130) [49"'] as well as a binder of a fusion protein containing the SH2 domain of the Fyn PTK (thus named Fyn-binding protein, or Fyb) [50",51]. SLAP-130/Fyb is a protein of 783 amino acids which possesses a region rich in proline residues, another region rich in tyrosine residues, possible nuclear localization sequences and an SH3-1ike domain [49"',50"]. T h e role of SLAP-130/Fyb in T-cell activation remains unclear. In Jurkat T cells, overexpression of SLAP130/Fyb results in a decrease in TCR-mediated signaling as measured by NFAT activity [49"']. Furthermore, cooverexpression of SLAP-130/Fyb with SLP-76 abrogates the positive effect of SLP-76 on reporter gene activity [49"']. In contrast, however, when overexpressed in the DC27.10 T-cell hybridoma, SLAP-130/Fyb augments IL-2 production [50"']. Further studies of the effects of overexpression and lack of expression of SLAP-130/Fyb and an analysis of structural features contributing to its signaling function are needed. In addition to SLP-76 and Fyn, SLAP-130/Fyb associates with another phosphoprotein in T cells. SKAP-55 (for Src-kinasc-associated phosphoprotein of 55 kDa) is also a lymphocyte-specific adaptor protein possessing an SH3 do-
339
main, a PH domain and tyrosines within motifs predicted to bind Src family P T K SH2 domains [52"']. In support of this notion, SKAP-55 binds to the Fyn SH2 domain in T cells [52"']. The interaction between SLAP-130/Fyb and SKAP-55 is mediated by the SKAP-55 SH3 domain and the central proline-rich region of SLAP-130/Fyb (AM Cardine, LR Hendricks-Taylor, NJ Boerth, H Zhao, B Schraven, GK Koretsky, unpublished data). At present, SKAP-55 signaling function remains unresolved. LAT ( p p 3 6 ) Western blotting analysis of phosphotyrosine-containing proteins within whole-cell lysates of activated T cells reveals a major species that migrates at approximately 36 kDa. When detected by tyrosine phosphorylation, this protein is associated predominantly with the plasma membrane, pp36 has been shown to associate with the SH2 domains of Grb2 and PLCy1, suggesting a role in coupling the T C R with both the phosphatidylinositol and Ras signaling pathways [21,53]. Further evidence for the importance of pp36 in TCR-mediated signal transduction came from studies of cells where pp36 was selectively dephosphorylated by transfection of a chimeric phosphatase. While the most proximal signaling events remained intact, T C R engagement on the transfectant failed to result in the generation of soluble inositol phosphate and an intracellular calcium flux [54]. More evidence for the role of pp36 came from examination of hyporesponsive T cells in non-obese diabetic (NOD) mice. In NOD T cells, decreased phosphorylation of pp36 correlates with decreased membrane targeting of the Grb2/Sos complex and impaired Ras activation [55"].
Recently, the cDNA for pp36 was cloned. T h e protein product, termed LAT, for linker for activation of T ceils, consists of 233 amino acids with a putative transmembrane domain at the amino terminus [56"']. LAT has no obvious enzymatic function according to its sequence. Although it contains no regions homologous to SH2, SH3 or PTB domains, LAT possesses several tyrosines within motifs predicted to mediate interactions with the SH2 domains of Grb2 and PLCy1. Northern analysis suggests that LAT is expressed exclusively in hematopoietic cells, most prominently in T cells, NK cells and mast cells, but not in B cells. In activated T cells, LAT associates directly or indirectly with many critical signaling molecules including Grb2, the 85kDa subunit of phosphoinositide 3-kinase (PI3K), PLCy1, SLP-76, Cbl and Sos [56"'] (Figure 2). Overexpressed tyrosine-to-phenylalanine LAT mutants in Jurkat cells exert a dominant inhibitory effect on TCR-induced NFAT activity, indicating the importance of this molecule as an integrator of T-cell activation signals. Cbl a n d C r k In the past few years it has become clear that in addition to acting as positive regulators of signaling events, adaptor proteins also have a critical role as inhibitors of signal transduction. Recent evidence suggests
340
Lymphocyteactivation and effector functions
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Current Opinion in Immunology
Adaptor protein complexes that promote T-cell antigen receptor signaling. Following TCR engagement, ZAP-70 is recruited to tyrosine-phosphorylated ITAMs within the CD3 chains. Activated ZAP-70 then phosphorylates a number of substrates, including the adaptor proteins LAT and SLP-76 (Y*). Tyrosine phosphorylation of LAT may promote the assembly of several distinct signaling complexes. 1 One model proposes recruitment of Grb2/Sos to tyrosine-phosphorylated LAT with consequent Ras activation. 2 PLCy1 is also recruited to LAT, permitting phosphatidylinositol hydrolysis and engagement of a calcium-dependent pathway leading to activation of gene transcription. 3 Tyrosine phosphorylation of SLP-76 results in the recruitment of Vav; however, the mechanisms by which SLP-76 interacts with the mitogen-activated protein kinase (MAPK) cascade or Vav to promote activation are unknown. TCR-dependent tyrosine phosphorylation of SLAP-130/Fyb is required for recruitment to the SLP-76 SH2 domain (dark shading), but the physiological roles of SLAP-130/Fyb and its constitutive binding partner SKAP-55 in TCR signaling remain unclear. 4 An Shc/Grb2/Sos complex formed after TCR-induced tyrosine phosphorylation of Shc provides an alternative model for membrane recruitment of Sos with resultant Ras activation; the binding site for this complex at the plasma membrane has not been defined. SH3 domains are indicated by light shading. DAG, diacylglycerol; IP3, inositol trisphosphate; PIP2, phosphatidylinositol biphosphate; PKC, protein kinase C.
that Cbl, a ubiquitously expressed adaptor molecule of 906 amino acids, is one such negative regulator in lymphocytes [57°°]. Cbl contains an amino-terminal PTB domain [58], a ring finger domain, a proline-rich segment and a carboxy-terminal leucine zipper domain [59]. Cbl also possesses a number of tyrosine phosphorylation sites which dictate associations with a number of SH2 domain-containing proteins, including Vav [60], Crk [61], PI3K [62], Fyn [63] and Syk family PTKs (see below). Cbl is inducibly phosphorylated following antigen-receptor engagement in both T cells [64] and B cells [65°]. Ovcrexpression of Cbl in the Jurkat T-cell line impairs AP-1 activity after TCR ligation [66"]. One potential mechanism for the inhibitory function of Cbl may involve its association with Syk family PTKs. A recent study employing Cbl overexpression in rat basophilic leukemia cells demonstrated that Cbl can bind to Syk and prevent
Syk recruitment to the phosphorylated ITAMs of the activated high-affinity IgE receptor [67"]. The Cbl/Syk complex blunts Syk kinase activity and blocks signaling via the receptor. Cbl may inhibit antigen-receptor signals through a similar mechanism, as it also inducibly binds Syk in B cells [65"] and ZAP-70 in T cells [68"]. Another model for the inhibitory effects of Cbl was suggested by the observation that phosphorylated Cbl binds the SH2 domain of the CrkL adaptor protein in activated T cells [69] and in the Ramos B-cell line [70]. CrkL is a member of the Crk adaptor protein family; it contains an amino-terminal SH2 domain and tandem carboxy-terminal SH3 domains [71]. The Cbl/CrkL complex is thought to bring C3G, a guanine-nucleotide-exchange factor, into a membrane-proximal position where it can enhance GTP binding to the low-molecular-weight G protein Rapl [57",72°]. Rapl activation, in turn, leads to decreased
Adaptor proteins in antigen-receptor signaling Peterson et a/.
341
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Model for Cbl and CrkL adaptor-protein-mediated inhibition of T-cell activation. Following agoniststimulus of the TCR (right side of schematic), protein tyrosine kinases (PTKs) are recruited and activated. Ras and the MAPK cascade are then activated, leading to gene transcription and cellular activation. After partial agonist/antagonist engagement of the TCR (left side of the schematic), the Src family PTK Fyn is phosphorylated and activated. 1 Fyn tyrosine phosphorylates (Y*) and binds Cbl. 2 Phospho-Cbl associates with CrkL, which is bound to C3G. 3 Membrane localization of C3G enhances exchange of GTP for GDP on Rap1.4 Activated Rap1 binds Raf kinase. 5 Rap1 competes for Ras binding, and thus impairs Ras-directed MAPK activation. Light shading depicts SH3 domains and dark shading represents SH2 domains.
Ras function, perhaps as a consequence of competition for Raf, providing a means of uncoupling Ras from the MAPK cascade [57"]. These observations suggest a model wherein differential Rapl and Ras activation may determine whether the T cell becomes activated or remains unresponsive after T C R ligation. After agonist or 'productive' stimulation of the T C R (that is, in conjunction with CD28 co-stimulation), Ras activation predominates, leading to IL-2 gene upregulation. Under conditions of partial or 'antagonist' T C R engagement, as occurs during anergy induction, Rapl becomes persistently GTP-bound and blocks activating signaling events downstream of Ras ([57°°]; Figure 3).
studies in cell lines as well as experiments in genetically manipulated whole animals will be required to uncover the details of their biological actions.
Acknowledgements The authorsthankK Latinis,J Lee, P Myung,and B Schravenfor critical reviewof the manuscript. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • = of outstanding interest 1,
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