TY - JOUR
AU - Koretzky, Gary A.
AB - Introduction Intestinal inflammation is caused by activation of infiltrating and resident cytotoxic and helper T lymphocytes, neutrophils, and macrophages (1,2). These effector cells produce tissue damage either directly through physical contact or indirectly through the release of soluble factors such as reactive oxygen and nitrogen metabolites, lytic enzymes, and cytokines. Therefore, it has become clear that an increased appreciation of the signaling events that regulate immune cell activation is required to understand the biology of intestinal inflammation. The past decade has seen significant advances in our understanding of the molecular mechanisms of T cell activation. This has involved the identification and characterization of second messenger cascades that couple antigen recognition to downstream effector functions. Much effort has been expended to define the enzymes responsible for initiating signal transduction events and, more recently, the nonenzymatic modulators of these signaling cascades. By understanding better the molecules that act as either positive or negative regulators of T cell signaling, insight will be gained into how the diverse signaling pathways involved are coordinately regulated leading to physiological and possibly pathological responses. Activation of T lymphocytes is initiated by engagement of the T cell antigen receptor (TCR). The TCR is composed of a multimeric protein complex consisting of a disulfide-linked α/β heterodimer associated noncovalently with the CD3 γ, δ, ε, and ζ chains (3,4). While the α and β proteins contain all of the information necessary for antigen recognition, the CD3 molecules are responsible for transducing signals. The most proximal signaling event known to occur following TCR engagement is the activation of Fyn and Lck, two members of the src family of protein tyrosine kinases (PTKs) (5,–10). Substrates of these PTKs include specialized domains, immunoreceptor tyrosine-based activation motifs (ITAMs), found as a single copy within each of the CD3 chains and in triplicate within the ζ chains (11). Within each ITAM are two tyrosine residues organized in a specific motif (YXXI/L spacer YXXL, where Y represents tyrosine, I is isoleucine, L is leucine, and X denotes any amino acid), which are inducibly phosphorylated following antigen recognition. The phosphorylated tyrosines become docking sites for the src homology 2 (SH2) domains of ZAP-70, a member of the Syk family of PTKs (12). Association of ZAP-70 with the TCR allows for subsequent phosphorylation and activation of ZAP-70 by the Src family PTKs (13,14). The importance of the association of the TCR with an active PTK has been underscored by the demonstration that all downstream activation events in T cells require the recruitment and activation of ZAP-70 (15,16). One of the best-studied markers of T cell activation is the production of interleukin 2 (IL2). Over the past decade, it has become clear that transcriptional activation of the IL2 gene requires the coordinated activation of a number of signal transduction pathways (17). These include production of phosphoinositide-derived second messengers and increases in cytosolic-free calcium, activation of protein kinase C (PKC), stimulation of lipid kinases, activation of Ras, Rac, Rho, and possibly other small GTP binding proteins, and stimulation of multiple members of the mitogen-activated protein kinase (MAPK) family of enzymes. The outcome of these signaling cascades leads to the activation of a number of transcription factors including AP-1, NF-κB and the nuclear factor of activated T cells (NFAT) (18). These transcription factors work in concert to initiate transcription of numerous early response genes and play a critical role in production of cytokines such as IL2. While many of the signaling pathways required for optimal activation have now been identified, it remains less clear how the various second messenger cascades are integrated to elicit the appropriate biological outcome. One strategy to explore how the different TCR-mediated signaling pathways are integrated has been to identify and characterize other substrates of the TCR-associated PTKs. One such substrate identified a number of years ago is phospholipase Cγ1 (PLCγl) (19), the enzyme responsible for converting membrane-associated phosphatidylinositol 4,5 bis-phosphate (PIP2) into the two second messengers, inositol 1, 4, 5 tris-phosphate (IP3) and 1,2 diacylglycerol (DAG) (20). IP3 is a soluble sugar that interacts with its receptor on endoplasmic reticulum resulting in the release of calcium from these intracellular stores. DAG plays an important role as a physiological activator of PKC. Since PLCγ1 tyrosine phosphorylation is important for its enzymatic activity (19), it became clear how PTK function could link cell surface receptor engagement to initiation of calcium transients and PKC activation. More recently, additional substrates of the TCR-activated PTKs have been identified. However, in contrast to proteins such as PLCγ1, several of these molecules have no intrinsic enzymatic function. Instead these proteins, now designated adapter molecules, contain discreet modules responsible for mediating protein-protein interactions. Adapter Proteins Contain Discreet Interaction Domains Over the past few years, several modular domains have been characterized that are responsible for proteinprotein interactions (21) (Table 1). The first adapter module identified was described by its resemblance to domains found in all Src PTKs and accordingly was named the Src homology 2 (SH2) domain (22,23). SH2 domains are approximately 100 amino acids in length and bind to other proteins containing phosphotyrosine residues in specific motifs. The SH2 domain binding specificity is conferred by three or four amino acids carboxyl terminal to the phosphorylated tyrosine residue (24,25). Since the discovery of SH2 domains, other motifs that promote intermolecular interactions have been identified. One of these, the phosphotyrosine binding (PTB) domain, also allows for interactions with phosphotyrosine-containing proteins (26,–28). In contrast to SH2 domains, the specificity of PTB modules is determined by an hydrophobic amino acid located amino-terminal to the phosphorylated tyrosine (25,28,29). Table 1. Adapter domains important for protein-protein interactions Open in new tab Table 1. Adapter domains important for protein-protein interactions Open in new tab In addition to domains that require tyrosine phosphorylation to promote protein interactions, other modules have been identified that are responsible for mediating the association between molecules based on specific amino acid sequences and/or specialized structural features. Src homology 3 (SH3) domains mediate binding with proteins that contain short proline rich sequences (30,–32). SH3 domains typically interact with proteins containing a consensus PXXP (where P denotes proline), which form a left-handed type II helix (33,34). Like SH3 domains, WW domains (named for the two tryptophans [W] located in the protein binding site) are shorter than SH2 domains (including only 35-40 amino acids) and bind proline rich motifs (35). In the case of WW domains, these proline sequences (PPXY or PPLP) generally are found near phosphorylated serine or threonine residues (36,37). PDZ domains contain amino acid residues that form an antiparallel β pleated sheet and recognize proteins containing a stretch of amino acids with a carboxyl terminal hydrophobic residue near an amino acid with a free carboxyl side chain similar to the ES/TDV (E-glutamic acid, S-serine, T-threonine, D-aspartic acid, V-valine) motif found in several ion channels (38,39). In addition to domains that mediate proteinprotein interactions, modules have been described that promote binding between proteins and phospholipids. For example, plextrin homology (PH) domains bridge proteins to the charged head groups of phosphoinositides (40,41). PH domains can therefore regulate the subcellular targeting of signaling proteins to regions of the plasma membrane. The biology of adapter protein domains is being studied extensively in numerous laboratories (42). One of the major challenges is to understand how the localization of molecules by these various domains regulates signaling events in the cell. It is becoming increasingly clear that protein-protein complexes organized by these adapter proteins play a critical role by providing a scaffold that permits the initiation of and crosstalk among diverse signaling pathways. Initial insight into how adapter proteins could potentially link signal transduction cascades to PTK activation was provided by experiments examining signaling events following engagement of the epidermal growth factor receptor (EGFR), a cell surface receptor PTK (43). Early studies demonstrated that ligation of the EGFR results in an increase in its enzymatic activity. Additional work showed that EGFR engagement also results in the exchange of GTP for GDP on Ras in a PTK-dependent fashion (44). The link between these two signal transduction pathways was discovered by the cloning and characterization of growth factor receptor binding protein 2 (Grb2) (45). Grb2 is a cytosolic protein comprised of a single SH2 domain flanked by two SH3 domains. It associates constitutively with Son of Sevenless (Sos) (46,–49), the guanine nucleotide exchange factor responsible for exchanging GTP for GDP on Ras. When the EGFR is bound by EGF the receptor dimerizes and transphosphorylates critical tyrosine residues. The phosphorylated EGFR tail becomes a docking site for the Grb2/Sos complex via the Grb2 SH2 domain, thus bringing Sos to the plasma membrane where it facilitates Ras activation (50,–53). Based on this paradigm, experiments were designed to examine possible roles for Grb2 and other adapter proteins in TCR signaling. Although a Grb2/Sos interaction in T cells has been described by several investigators (54), there is no conclusive evidence that TCR-stimulated Ras activation depends upon recruitment of the Grb2/Sos complex to the TCR itself. Thus, a number of laboratories have attempted to identify other Grb2 binding proteins in T cells in an effort to provide additional insight into how signaling pathways are coupled following TCR engagement. These studies have led to the description of several novel hematopoietic-specific adapter proteins (Fig. 1) that appear to play critical roles as either positive (Fig. 2) or negative (Fig. 3) regulators of T cell development and activation. Fig. 1. Open in new tabDownload slide Adapter proteins involved in TCR signaling discussed in this review. The domain organization for each molecule is illustrated. SH2, Src homology 2 domain; SH3, Src homology 3 domain; TM, transmembrane domain; P-Tyr sites, tyrosine phosphorylation site; PH, plextrin homology domain; PTB, phosphotyrosine binding domain. Fig. 2. Open in new tabDownload slide Model for LAT and SLP-76 function as regulators of TCR signaling. Following TCR engagement the Src PTKs, Lck and Fyn, are activated leading to CD3 phosphorylation. Activated ZAP-70 phosphorylates LAT and SLP-76 initiating Ras/MAPK and PLCγ1 signaling cascades. Tyrosine-phosphorylated LAT binds to the Gads SH2 domain. Since Gads constitutively associates with SLP-76, the SLP-76/Gads complex may be recruited to LAT thus promoting Ras/MAPK activation. Tyrosine-phosphorylated LAT also recruits PLCγ1 to the membrane, placing PLCγ1 in close proximity with its substrate, PIP2 (phophatidylinositol 4,5 bis-phosphate). PIP2 is hydrolyzed IP3 (inositol 3,4,5 tris-phosphate) and DAG (diacylglycerol), which leads to increases of cytosolic-free calcium and activation of protein kinase C (PKC). Tyrosine phosphorylated SLP-76 also associates with Vav thus promoting the formation of a SLP-76/Vav/Nck/Pak complex, which may be important for the regulation of cytoskeletal rearrangements. SLP-76 function may also be regulated by its association with SLAP-130/FYB or other molecules. Y-P denotes phosphorylated tyrosine residues. Fig. 3. Open in new tabDownload slide Schematic illustration of signals thought to inhibit TCR-induced signaling. In resting T cells, Cbl associates with Grb2 possibly maintaining Ras in an inactive state. Following TCR ligation, ZAP-70 phosphorylates Cbl thus promoting the association of Cbl with target proteins such as CrkL and Syk family PTKs. Association of Cbl with the Syk family PTKs leads to the degradation of the kinases thereby blocking further signal transduction. Formation of the Cbl/CrkL/C3G complex promotes the activation of Rap-1. Induction of Rap-1 activity results in sequestration of Raf-1 and inhibits MAPK activation resulting in anergy. Y-P denotes phosphorylated tyrosine residues. Adapter Proteins that Positively Impact TCR Signaling LAT One of the most predominant substrates of the TCRstimulated PTKs is a molecule with a relative molecular mass of 36-38 kDa that associates with the SH2 domain of Grb2 (54). The cDNA encoding this protein was obtained recently confirming earlier studies that the linker for activation of T cells (LAT) is a transmembrane protein that is phosphorylated early after TCR engagement (55). The primary sequence of LAT suggests that this protein acts as an adapter molecule following its tyrosine phosphorylation by associating with other proteins possessing SH2 domains. Thus in addition to binding Grb2 (and other Grb2 family members, see below), LAT inducibly associates with PLCγ1 and the 85 kDa subunit of phosphatidylinositol 3′ kinase (PI-3′ kinase). Prior to its cloning, experiments were designed to address the potential role of LAT in the regulation of TCR signaling. These studies made use of a chimeric tyrosine phosphatase engineered to selectively dephosphorylate LAT (56). In cells expressing this phosphatase, TCR engagement fails to lead to the production of phosphatidylinositolderived second messengers suggesting that LAT could potentially play a role in coupling the TCR with PLCγ1 function. Subsequent to its cloning, more definitive experiments were performed demonstrating the requirement of LAT for TCR signal transduction. These experiments involved transfection of wild type and mutant forms of LAT into the Jurkat T cell leukemic line and analysis of a Jurkat variant, J.CaM2, which is defective in TCR-mediated signaling (57). Ligation of the TCR on J.CaM2 results in activation of PTKs, but fails to result in stimulation of either the Ras or phosphoinositide-derived second messenger cascades. Analysis of J.CaM2 revealed that LAT is not expressed in this mutant line. Reconstitution of LAT by gene transfer results in a rescue of the signaling phenotype. Further studies documented the requirement for LAT phosphorylation in its function. A mutant form of LAT with two tyrosines changed to phenylalanine fails to bind to Grb2 and other associated molecules. Expression of this mutant inhibits TCR-induced NFAT and AP-1 activation in wild type Jurkat and fails to rescue the signaling defect in J.CaM2. Further transfection studies demonstrated that, in addition to its tyrosine phosphorylation, LAT must be localized to the plasma membrane for its function (58). Interestingly, it appears that membrane association is not sufficient for LAT function since mutants that fail to localize to glycolipidenriched microdomains (GEMs) are poorly tyrosinephosphorylated following TCR engagement. In addition to LAT, PLCγ1 and other signaling molecules are present in GEMs suggesting that targeting of molecules to these microdomains is important for optimal induction of TCR-induced signals. SLP-76 SLP-76 is another newly described adapter protein that is important for T cell activation (59). This molecule was isolated originally in a screen for novel substrates of the TCR-stimulated PTKs. The SLP-76 primary sequence shows several distinct domains including an amino-terminal acidic region with tyrosines within motifs predicted to bind SH2 domains, a proline rich central region, and a carboxyl-terminal SH2 domain. Like LAT, SLP-76 is expressed exclusively in hematopoietic cells including T cells, mast cells, natural killer cells, macrophages, and platelets, but not B cells. Expression of SLP-76 appears to be regulated as its levels increase in T cells following antigen receptor engagement and in memory T cells (60). A potential role for SLP-76 in T cell activation was suggested by initial studies with SLP-76 demonstrating that transient overexpression of SLP-76 in Jurkat cells augments TCR-activation of AP-1 and NFAT function (61,62). Analysis of more proximal signals showed that overexpression of SLP-76 enhances TCR coupled MAPK activation (63). The importance of SLP-76 in integrating TCR-mediated signals was confirmed by the generation of a SLP-76-deficient Jurkat T cell line whose phenotype is similar to that of J.CaM2 (64). Engagement of the TCR on the SLP-76-deficient cells activates ZAP-70, but fails to result in PLCγ1 tyrosine phosphorylation, calcium flux, or MAPK activation. Reconstitution of SLP-76 expression rescues the signaling deficiency in these cells. Together, these data strongly support the idea that SLP-76 plays an important role in mediating both MAPK activation and the release of intracellular calcium stores following TCR ligation. In addition to these studies in transformed cell lines indicating that SLP-76 plays a critical role in promoting TCR signaling in mature T cells, other recent work demonstrates that SLP-76 is also essential for T cell development (65,66). These studies have made use of mice made deficient in SLP-76 expression by targeted gene disruption. These mice have a striking phenotype in that there is a complete block in appearance of mature T cells in the periphery. Examination of the thymi of SLP-76-deficient mice reveals a block in T cell development at the CD25+/CD44- (pro-T3) stage. Analysis of these thymocytes reveals normal rearrangement of the TCRβ locus and expression of pre-TCRa mRNA suggesting that the developmental arrest is due to a failure of signal transduction and not receptor expression. Collectively, the studies in cell lines and mice indicate that both SLP-76 and LAT play critical roles in coupling activation of ZAP-70 to more downstream signals in mature and developing T cells. Studies of platelets provides further evidence that SLP-76 may couple signaling events mediated by receptors that rely on Syk family PTKs. Stimulation of the collagen receptor on platelets results in activation of Syk and subsequent phosphorylation of SLP-76 and PLCγ2 (67,68). Interestingly, collagen-dependent aggregation and granule release is defective in platelets from SLP-76-deficient mice despite normal activation of Syk. This observation suggests that SLP-76 is a required component of collagen-induced signaling pathways in platelets and lies downstream of Syk, but upstream of PLCγ2. Although, SLP-76 is also expressed and inducibly phosphorylated in macrophages and natural killer T cells, studies to date using these cell types reveal no functional defect (Myung et al., unpublished observations). Therefore, the role of SLP-76 in regulating signaling remains complicated. Ongoing studies involve the search for SLP-76 homologues in other cell types that may couple the Syk PTKs to downstream signaling events. This may be a promising strategy since B cells express a SLP-76 homologue, B cell linker protein (BLNK), which is a positive regulator of B cell receptor signaling (69). Since SLP-76 serves as an adapter molecule, it becomes important to identify SLP-76-associated proteins in an effort to understand better how SLP-76 couples signaling events. Because mutation of the acidic aminoterminus, the central proline rich region, or the SH2 domain of SLP-76 each abrogates the ability of SLP-76 to augment TCR-dependent NFAT activation, it seems likely that proteins that associate with SLP-76 via these domains may be important in promoting the effect of SLP-76 on T cell signaling (63). Studies from several laboratories have now identified proteins that interact with each of the SLP-76 domains. Some of these proteins have been characterized and have known roles as regulators of signal transduction events, while others of the SLP-76 binding partners are novel molecules whose function remain unclear. Several groups have reported the inducible association of SLP-76 with the SH2 domain of Vav (a guanine nucleotide exchange factor for the small molecular weight GTP binding proteins, Rac/Rho) following tyrosine phosphorylation of SLP-76 (70,–73). Two tyrosine residues (Y113, Y128) in a repeated DYESP motif are required for the SLP-76/Vav interaction (59,73). Interestingly, overexpression of either SLP-76 or Vav augments activation of the IL2 gene following TCR ligation, and co-overexpression of SLP-76 with Vav has a synergistic effect on this downstream activation event. Since mutation of either tyrosine in SLP-76 alone does not significantly reduce NFAT activation, while Vav binding is markedly diminished, the SLP-76/Vav association appears not to be required for augmentation of NFAT activation. It is still likely, however, that the association between SLP-76 and Vav is important for some T cell functions. In support of this notion, it was shown that following TCR ligation, SLP-76 associates inducibly with the SH2 domain of Nck, another SH2/SH3 domain-containing adapter protein (74). Nck also associates with the p21-activated kinase (Pak) and the Wiskott-Aldrich syndrome protein (WASP), two proteins that bind to activated Rho-GTPases (75,76). Since Vav catalyzes the exchange of GTP for GDP on Rho, SLP-76 may serve to colocalize Vav with Rho via an association with Nck. Supporting this idea, mutations in either SLP-76, Nck, or Vav that prevent assembly of the trimolecular complex diminish TCR-dependent Pak activation. Furthermore, these same mutations inhibit actin polymerization following TCR stimulation, implicating the SLP-76/Nck/V Vav complex in the regulation of cytoskeletal reorganization in response to TCR ligation. Since actin polymerization and cytoskeletal reorganization are required for some downstream signals, receptor internalization, and T cell motility, the role of SLP-76 and its associated proteins in the regulation of these processes in lymphocytes becomes an important area of study. In a further attempt to understand how SLP-76 may function to promote T cell signaling, other molecules that bind to SLP-76 are being studied. Following engagement of the TCR, two phosphoproteins of apparent molecular weights of 130 kDa and 62 kDa associate inducibly with the SLP-76 SH2 domain. Although the identity of pp62 remains elusive, the cDNA encoding the 130 kDa protein has been cloned. Interestingly, this protein (named SLAP-130 for SLP-76 associated protein of 130 kDa) was identified also in an independent study examining proteins that bind to the SH2 domain of the Src family PTK, Fyn (77,78). Sequence analysis of SLAP-130/FYB (Fyn binding protein) reveals that this protein is, itself, an adapter molecule with several recognizable domains including a central proline-rich region, several potential tyrosine phosphorylation sites, and two putative nuclear localization sequences, which may mediate its interaction with other targets. One of these proteins, Src kinase associated protein of 55 kDa (SKAP-55), has now been identified and shown to associate constitutively to SLAP-130/FYB via the proline rich region of SLAP-130 and an SH3 domain of SKAP-55 (79,80). To make matters even more complex, it has now been shown that SKAP-55 belongs to a larger family of adapter molecules, some of which may compete for binding to SLAP-130/FYB (81,82). The function of SLAP-130/FYB and SKAP-55 in T cell signaling remains controversial. In some model systems, SLAP-130/FYB appears to be a negative regulator of SLP-76 function while in others it seems to play a positive role. Defining the precise role of these proteins as regulators of signal transduction will require further characterization of the larger molecular complexes formed under various experimental conditions. In addition to inducible binding to other proteins with its amino-terminal phosphorylation sites and carboxylterminal SH2 domain, SLP-76 associates constitutively with the Grb2 family member Gads, via the SLP-76 central proline-rich region and the Gads SH3 domains (83). Gads was identified initially as part of a protein complex in fibroblasts. Recent studies demonstrated that Gads binds to SLP-76 in T cells and apparently precludes an association between SLP-76 and Grb2. The potential importance of the Gads/SLP-76 interaction is suggested by the observation that Gads also binds inducibly to tyrosine phosphorylated LAT. Thus, it is possible that Gads bridges SLP-76 to LAT allowing these two critical adapter proteins to function together to promote T cell activation. 3BP2 and She In addition to the molecules described above, several other adapter proteins have been identified that appear to play important roles as positive regulators of TCRsignaling events. For example, a recent study making use of the yeast two hybrid screen to isolate Syk PTK interacting proteins in B cells resulted in the identification of 3BP2 (84). Sequence analysis of 3BP2 reveals that it contains an amino-terminal PH domain, a central proline rich motif, and a carboxyl-terminal SH2 domain. In addition to Syk, the 3BP2 SH2 domain associates with ZAP-70, LAT, Grb2, PLCγ1, and Cbl in activated T cells. Overexpression of 3BP2 induces transcriptional activation of the IL-2 promoter and its NFAT or AP-1 elements (84). Optimal activity of 3BP2 is dependent on its SH2 and PH domains and requires functional Syk kinases, Ras, and calcineurin. These studies thus suggest that 3BP2 may be an adapter capable of coupling activated ZAP-70 or Syk to a LAT-containing signaling complex important for TCR-mediated gene transcription. In addition to the inducible association with LAT and SLP-76, Grb2 has also been shown to bind the adapter protein Shc in activated T cells (85,86). This Grb2/Shc complex has been found associated with CD3ζ following TCR engagement (87). Shc consists of a single carboxylterminal SH2 domain, a central proline/glycine rich collagen homology domain, and an amino-terminal PTB domain. Following TCR ligation, Shc is recruited to the phosphorylated ITAMs found in ζ via the Shc SH2 domain. Shc is then tyrosine-phosphorylated and mediates an association with Grb2 through the Grb2 SH2 domain (88). Although binding of Shc to Grb2 has been shown to enhance the inducible association of Grb2 with Sos in T cells, the effect of this complex on TCR-dependent Ras activation has not been determined. Even though a ζ-Shc-Grb2 complex has been shown, overexpression of Shc fails to promote TCR-inducible Ras activation. Experimental evidence suggests, however, that Shc may play a more important role in signaling events initiated by ligation of CD4 and the IL-2 receptor (89). Additional studies are required to elucidate more definitively the roles of 3BP2 and Shc in T cell activation. Lymphocyte Adapter Molecules that Inhibit TCR Signaling Cbl, Crk, and CasL In addition to promoting T cell activation, several adapter proteins have been shown to inhibit TCR signaling (Fig. 3). In addition to associating with positive regulators, Grb2 has been shown to bind a 116 kDa protein, which is inducibly phosphorylated following TCR engagement and recently shown to be Cbl (90,91). Cbl was initially identified as the cellular homologue of the Cas NS-1 murine retroviral oncogene, v-Cbl. The sequence of Cbl reveals an amino-terminal PTB domain, a zinc RING finger motif followed by a proline-rich region, a carboxyl terminal ubiquitination recognition sequence, a putative leucine zipper, and several tyrosine residues that are phosphorylated following TCR ligation. Early evidence suggesting that Cbl plays a negative regulatory role in cell signaling was provided by the observation that the Cbl homologue (Sli-1) in C. elegans inhibits Ras activation following LET-23 receptor PTK ligation (92). In resting T cells, Cbl may serve to maintain Ras in its inactive state. This idea is supported by studies showing that Cbl overexpression inhibits AP-1 activity following TCR ligation. One model to explain the mechanism by which Cbl functions invokes the observation that this adapter associates with the amino-terminal SH3 domain of Grb2 under basal conditions (93). This intermolecular interaction may prevent the association of Grb2 with Sos (94), thus preventing Sos translocation to the plasma membrane and activation of Ras/MAPK (95). Following TCR ligation, Cbl becomes tyrosine-phosphorylated and dissociates from Grb2. This then allows Grb2 to bind Sos leading to activation of Ras. Compelling data indicate, however, that Cbl serves a more complex role in T cell signaling. In this regard, it has been shown that Cbl binds to other proteins including ZAP-70 and Syk via the Cbl PTB domain (96,97), and to the SH2 domains of Vav, the p85 subunit of PI-3' kinase and CrkL (an SH2 and SH3 domain-containing adapter protein) via Cbl phosphotyrosines (93,94,98,–100). The interaction between Cbl and Syk family kinases appears to be very important as experimental evidence suggests that the association between Cbl and the PTKs promotes the degradation of the kinases (101,102). This would then lead to dampening of the activation response due to the loss of critical upstream effector molecules (95). Consistent with this notion, mice made deficient in Cbl expression by targeted gene disruption exhibit enlarged spleens and thymi in which ZAP-70, SLP-76, and LAT are hyperphosphorylated. These mice also demonstrate hyperresponsive T cells, presumably due to the loss of Cbl as an inhibitor of TCR signaling (103,104). Other studies have provided alternative, but not mutually exclusive models, for a mechanism by which Cbl interferes with activation events. In the absence of costimulation, T cells stimulated through their TCR become unresponsive (anergic) and fail to produce cytokines or proliferate (105). Recently, it has been shown that in anergic T cell clones, engagement of the TCR fails to result in activation of the Ras signaling pathway (106,107). A possible connection to Cbl is based on the observation that in anergic T cells, Cbl associates preferentially with CrkL (108). The Cbl/CrkL complex recruits C3G, an exchange factor for the small molecular weight GTP binding protein, Rap-1 (109,–111). Activation of Rap-1 is postulated to cause sequestration of Raf-1, a critical kinase activated following loading of GTP onto Ras. Thus, in the presence of the Cbl/CrkL/C3G complex, Ras-stimulated activation events are precluded due to the functional loss of Raf-1, the effector immediately downstream of Ras. The CrkL-associated adapter protein Cas-L has also been implicated as a potential regulator of signaling pathways in T cells (112). Cas-L was initially identified as a 105 kDa protein that is phosphorylated following β 1 integrin cross-linking in T cells (113). Cas-L binds to the carboxyl-terminal domain of focal adhesion kinase (Fak) via the Cas-L amino-terminal SH3 domain, and is a substrate of Fak and Src family tyrosine kinases following β1 integrin cross-linking (112,114). Thus, Cas-L may function to promote signals generated during T cell adhesion and migration. Following TCR/CD3 ligation, CasL is tyrosine-phosphorylated and binds to the SH2 domain of CrkL (115). A C3G/CrkL/Cas-L complex can also be found and may also regulate Rap-1 activity via C3G (112). Since Cas-L appears to be involved in both TCR-mediated signaling as well as extracellular matrixmediated signaling, it may therefore provide an important link that promotes crosstalk between signaling events coupling these two important signaling events. SAP Adapter proteins may also regulate T cell signaling by interfering with the formation of activating complexes. The cell surface receptor CDw150 (also known as SLAM for signaling lymphocyte-activation molecule) is present on the surface of both B and T cells (116). A SLAM-associated protein (SAP) has recently been identified. SAP expression is restricted to T cells. The primary sequence of SAP reveals a single SH2 domain followed by a short carboxyl-terminal tail. Following coligation of SLAM and the TCR, SAP is recruited to phosphorylated tyrosine residues within the cytoplasmic tail of SLAM via the SAP SH2 domain. The binding of SAP to SLAM appears to block the recruitment of SHP-2 (a protein tyrosine phosphatase that plays a critical role in regulation of numerous signaling pathways) to the SLAM cytoplasmic domain in activated T cells. Although the biochemical consequences of this remain uncertain, the importance of SAP has been underscored by the finding that mutations in SAP lead to X-linked lymphoproliferative disease (XLP), a disorder in which patients fail to control B-cell proliferation in response to Epstein-Barr virus infections leading to a lymphoproliferative phenotype. Thus, the absence of functional SAP appears to result in uncontrolled lymphocyte responses, presumably due to abnormal regulation of signaling complex formation. Conclusions Over the past several decades a great deal has been learned about the molecular biology and biochemistry of signal transduction events leading to T cell activation. Recognizing the central role that PTK activation plays in initiating and propagating TCR-mediated signals, numerous laboratories have identified and characterized substrates of the TCR-stimulated PTKs. Recently, in addition to the TCR complex itself and key enzymes, a new class of substrates has been appreciated. These proteins, collectively known as adapter molecules, regulate signal transduction events due to their ability to promote protein-protein interactions. In the past few years the importance of adapter molecules has become evident using cell lines, genetically manipulated mice, and analysis of patients with immune cell dysfunction. It is now appreciated that numerous adapter proteins play critical functions in both the initiation and termination of immune responses. Under basal conditions, these proteins are found both in microdomains at the cell surface (for example, LAT) as well as in the cytosol (for example SLP-76). Upon receptor engagement, adapter proteins form larger complexes and recruit important effector molecules. Thus adapters, by their ability to form a molecular scaffold, integrate various signaling cascades resulting in the appropriate biologic response following initial triggering of cell surface receptors. There are numerous challenges for the future to understand better how adapter proteins serve these critical functions. In addition to identifying and characterizing additional adapter molecules, experiments are underway evaluating the molecular and cell biology of protein complex formation, both temporally and spatially. Results of these studies will hopefully provide further insight into the complex biology of lymphocyte activation. References 1. Baumgart DC, McVay LD, Carding SR. Mechanisms of immune cell-mediated tissue injury in inflammatory bowel disease. Int J Mol Med 1998;1:315-32. 2. Kolios G, Petoumenos C, Nakos A. Mediators of inflammation: production and implication in inflammatory bowel disease. 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TI - Adapter Molecules in T Cell Receptor Signaling
JF - Inflammatory Bowel Diseases
DO - 10.1097/00054725-199905000-00007
DA - 1999-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/adapter-molecules-in-t-cell-receptor-signaling-mJsT0f5Pib
SP - 107
EP - 118
VL - 5
IS - 2
DP - DeepDyve
ER -