T cell receptor-induced activation of phospholipase C-gamma1 depends on a sequence-independent function of the P-I region of SLP-76

SLP-76 forms part of a hematopoietic-specific adaptor protein complex, and is absolutely required for T cell development and activation. T cell receptor (TCR)-induced activation of phospholipase C-gamma1 (PLC-gamma1) depends on three features of SLP-76: the N-terminal tyrosine phosphorylation sites, the Gads-binding site, and an intervening sequence, denoted the P-I region, which binds to the SH3 domain of PLC-gamma1 (SH3(PLC)) via a low affinity interaction. Despite extensive research, the mechanism whereby SLP-76 regulates PLC-gamma1 remains uncertain. In this study, we uncover and explore an apparent paradox: whereas the P-I region as a whole is essential for TCR-induced activation of PLC-gamma1 and nuclear factor of activated T cells (NFAT), no particular part of this region is absolutely required. To better understand the contribution of the P-I region to PLC-gamma1 activation, we mapped the PLC-gamma1-binding site within the region, and created a SLP-76 mutant that fails to bind SH3(PLC), but is fully functional, mediating TCR-induced phosphorylation of PLC-gamma1 at tyrosine 783, calcium flux, and nuclear factor of activated T cells activation. Unexpectedly, full functionality of this mutant was maintained even under less than optimal stimulation conditions, such as a low concentration of the anti-TCR antibody. Another SLP-76 mutant, in which the P-I region was scrambled to abolish any sequence-dependent protein-binding motifs, also retained significant functionality. Our results demonstrate that SLP-76 need not interact with SH3(PLC) to activate PLC-gamma1, and further suggest that the P-I region of SLP-76 serves a structural role that is sequence-independent and is not directly related to protein-protein interactions.     

Identification of a phospholipase C-gamma1 (PLC-gamma1) SH3 domain-binding site in SLP-76 required for T-cell receptor-mediated activation of PLC-gamma1 and NFAT

SLP-76 is an adapter protein required for T-cell receptor (TCR) signaling. In particular, TCR-induced tyrosine phosphorylation and activation of phospholipase C-gamma1 (PLC-gamma1), and the resultant TCR-inducible gene expression, depend on SLP-76. Nonetheless, the mechanisms by which SLP-76 mediates PLC-gamma1 activation are not well understood. We now demonstrate that SLP-76 directly interacts with the Src homology 3 (SH3) domain of PLC-gamma1. Structure-function analysis of SLP-76 revealed that each of the previously defined protein-protein interaction domains can be individually deleted without completely disrupting SLP-76 function. Additional deletion mutations revealed a new, 67-amino-acid functional domain within the proline-rich region of SLP-76, which we have termed the P-1 domain. The P-1 domain mediates a constitutive interaction of SLP-76 with the SH3 domain of PLC-gamma1 and is required for TCR-mediated activation of Erk, PLC-gamma1, and NFAT (nuclear factor of activated T cells). The adjacent Gads-binding domain of SLP-76, also within the proline-rich region, mediates inducible recruitment of SLP-76 to a PLC-gamma1-containing complex via the recruitment of both PLC-gamma1 and Gads to another cell-type-specific adapter, LAT. Thus, TCR-induced activation of PLC-gamma1 entails the binding of PLC-gamma1 to both LAT and SLP-76, a finding that may underlie the requirement for both LAT and SLP-76 to mediate the optimal activation of PLC-gamma1.     

Cbl enforces Vav1 dependence and a restricted pathway of T cell development

Extensive studies of pre-TCR- and TCR-dependent signaling have led to characterization of a pathway deemed essential for efficient T cell development, and comprised of a cascade of sequential events involving phosphorylation of Lck and ZAP-70, followed by phosphorylation of LAT and SLP-76, and subsequent additional downstream events. Of interest, however, reports from our lab as well as others have indicated that the requirements for ZAP-70, LAT, and SLP-76 are partially reversed by inactivation of c-Cbl (Cbl), an E3 ubiquitin ligase that targets multiple molecules for ubiquitination and degradation. Analysis of signaling events in these Cbl knockout models, including the recently reported analysis of SLP-76 transgenes defective in interaction with Vav1, suggested that activation of Vav1 might be a critical event in alternative pathways of T cell development. To extend the analysis of signaling requirements for thymic development, we have therefore assessed the effect of Cbl inactivation on the T cell developmental defects that occur in Vav1-deficient mice. The defects in Vav1-deficient thymic development, including a marked defect in DN3-DN4 transition, were completely reversed by Cbl inactivation, accompanied by enhanced phosphorylation of PLC-γ1 and ERKs in response to pre-TCR/TCR cross-linking of Vav1^-/-Cbl^-/- DP thymocytes. Taken together, these results suggest a substantially modified paradigm for pre-TCR/TCR signaling and T cell development. The observed consensus pathways of T cell development, including requirements for ZAP-70, LAT, SLP-76, and Vav1, appear to reflect the restriction by Cbl of an otherwise much broader set of molecular pathways capable of mediating T cell development.     

Evidence that phospholipase C-gamma2 interacts with SLP-76, Syk, Lyn, LAT and the Fc receptor gamma-chain after stimulation of the collagen receptor glycoprotein VI in human platelets.

Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor gamma-chain (FcR gamma-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-gamma2 (PLC-gamma2). In this study tyrosine-phosphorylated proteins that associate with PLC-gamma2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-gamma2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-gamma2. The majority of the tyrosine-phosphorylated proteins that associate with PLC-gamma2 bind to its C-terminal SH2 domain. These were found to include PLC-gamma2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR gamma-chain. Direct association was detected between PLC-gamma2 and SLP-76, and between PLC-gamma2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR gamma-chain and Lyn were found to co-immunoprecipitate with PLC-gamma2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-gamma2 in platelets is in contrast with that for PLC-gamma1 and Syk in B cells. The in vivo function of PLC-gamma2 SH2 domains was examined through measurement of Ca2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-gamma2. These data indicate that the C-terminal SH2 domain of PLC-gamma2 is important for PLC-gamma2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR gamma-chain.     

Identification of the minimal tyrosine residues required for linker for activation of T cell function

The linker for activation of T cells (LAT) is essential for signaling through the T cell receptor (TCR). Following TCR stimulation, LAT becomes tyrosine-phosphorylated, creating docking sites for other signaling proteins such as phospholipase C-gamma(1) (PLC-gamma(1)), Grb2, and Gads. In this study, we have attempted to identify the critical tyrosine residues in LAT that mediate TCR activation-induced mobilization of intracellular Ca(2+) and activation of the MAP kinase Erk2. By using the LAT-deficient Jurkat derivative, J.CaM2, stable cell lines were established expressing various tyrosine mutants of LAT. We show that three specific tyrosine residues (Tyr(132), Tyr(171), and Tyr(191)) are necessary and sufficient to achieve a Ca(2+) flux following TCR stimulation. These tyrosine residues function by reconstituting PLC-gamma(1) phosphorylation and recruitment to LAT. However, these same tyrosines can only partially reconstitute Erk activation. Full reconstitution of Erk requires two additional tyrosine residues (Tyr(110) and Tyr(226)), both of which have the Grb2-binding motif YXN. This reconstitution of Erk activation requires that the critical tyrosine residues be on the same molecule of LAT, suggesting that a single LAT molecule nucleates multiple protein-protein interactions required for optimal signal transduction.     

Tyrosine phosphorylation of SLP-76 is downstream of Syk following stimulation of the collagen receptor in platelets

Collagen-related peptide (CRP), a collagen homologue, induces platelet activation through a tyrosine kinase-dependent pathway, leading to sequential tyrosine phosphorylation of Fc receptor (FcR) gamma-chain, Syk, and phospholipase C-gamma2. Here we report that CRP and the platelet low affinity immune receptor FcgammaRIIA stimulate tyrosine phosphorylation of the T cell adapter SLP-76, whereas the G protein-coupled receptor agonist thrombin induces only minor tyrosine phosphorylation. This suggests that SLP-76 has a specific role downstream of receptors that signal via an immunoreceptor tyrosine-based activation motif. Immunoprecipitation studies demonstrate association of SLP-76 with SLAP-130, Vav, Fyn, Lyn, and the FcR gamma-chain in CRP-stimulated platelets. Several of these proteins, including SLP-76, undergo tyrosine phosphorylation in in vitro kinase assays performed on SLP-76 immunoprecipitates. Tyrosine phosphorylation of all of these proteins in the in vitro kinase assay was abrogated by the Src family kinase inhibitor PP1, suggesting that it is mediated by either Fyn or Lyn. The physiological significance of this is uncertain, however, since tyrosine phosphorylation of SLP-76 in vivo is not altered in either Fyn- or Lyn-deficient platelets. CRP stimulation of Syk-deficient platelets demonstrated that in vivo tyrosine phosphorylation of SLP-76 is downstream of Syk. The absence of Syk in the SLP-76 immunoprecipitates raises the possibility that another protein is responsible for bringing SLP-76 to Syk. Candidates for this include those proteins that co-immunoprecipitate with SLP-76, including the FcR gamma-chain. Tyrosine phosphorylation of PLC-gamma2 and Ca2+ mobilization is markedly attenuated in SLP-76-deficient platelets following CRP stimulation, suggesting that the adapter plays a critical role in the regulation of the phospholipase. The increase in tyrosine phosphorylation of SLAP-130 in response to CRP is also inhibited in SLP-76-deficient platelets, placing it downstream of SLP-76. This work identifies SLP-76 as an important adapter molecule that is regulated by Syk and lies upstream of SLAP-130 and PLC-gamma2 in CRP-stimulated platelets.     

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP-76, Gads and LAT. Three well-characterized SLP-76 tyrosine phosphorylation sites recruit key components, including a Tec-family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP-76 phosphorylation site, Y173, which was phosphorylated upon T-cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor-induced phosphorylation of phospholipase C-γ1 (PLC-γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL-2 promoter in T cells, and degranulation and IL-6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP-70-targeted tyrosines at the N-terminus of SLP-76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP-70-dependent priming of SLP-76 at three N-terminal sites triggers reciprocal regulatory interactions between Itk and SLP-76, which are ultimately required to couple active Itk to its substrate, PLC-γ1.     

Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa and phospholipase C gamma 1 are required for NF-kappa B activation and lipid raft recruitment of protein kinase C theta induced by T cell costimulation

The NF-kappaB activation pathway induced by T cell costimulation uses various molecules including Vav1 and protein kinase C (PKC)theta. Because Vav1 inducibly associates with further proteins including phospholipase C (PLC)gamma1 and Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), we investigated their role for NF-kappaB activation in Jurkat leukemia T cell lines deficient for expression of these two proteins. Cells lacking SLP-76 or PLCgamma1 failed to activate NF-kappaB in response to T cell costimulation. In contrast, replenishment of SLP-76 or PLCgamma1 expression restored CD3/CD28-induced IkappaB kinase (IKK) activity as well as NF-kappaB DNA binding and transactivation. PKCtheta activated NF-kappaB in SLP-76- and PLCgamma1-deficient cells, showing that PKCtheta is acting further downstream. In contrast, Vav1-induced NF-kappaB activation was normal in SLP-76(-) cells, but absent in PLCgamma1(-) cells. CD3/CD28-stimulated recruitment of PKCtheta and IKKgamma to lipid rafts was lost in SLP-76- or PLCgamma1-negative cells, while translocation of Vav1 remained unaffected. Accordingly, recruitment of PKCtheta to the immunological synapse strictly relied on the presence of SLP-76 and PLCgamma1, but synapse translocation of Vav1 identified in this study was independent from both proteins. These results show the importance of SLP-76 and PLCgamma1 for NF-kappaB activation and raft translocation of PKCtheta and IKKgamma.     

Vav1 transduces T cell receptor signals to the activation of the Ras/ERK pathway via LAT, Sos, and RasGRP1

Vav1 is a signaling protein required for both positive and negative selection of CD4(+)CD8(+) double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase Cgamma1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase Cgamma1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells.     

Distinct role of ZAP-70 and Src homology 2 domain-containing leukocyte protein of 76 kDa in the prolonged activation of extracellular signal-regulated protein kinase by the stromal cell-derived factor-1 alpha/CXCL12 chemokine

Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1alpha is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special functions of SDF-1alpha that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly understood. In this study we show that SDF-1alpha stimulation of prolonged ERK activation in Jurkat T cells requires both the ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role of LAT in SDF-1alpha-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor LAT, itself are required for SDF-1alpha to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results describe the distinct mechanism by which SDF-1alpha stimulates prolonged ERK activation in T cells and indicate that this pathway is specific for cells expressing both ZAP-70 and SLP-76.     

Cutting edge: SLP-76 cooperativity with FYB/FYN-T in the Up-regulation of TCR-driven IL-2 transcription requires SLP-76 binding to FYB at Tyr595 and Tyr651

SLP-76 (Src homology (SH) 2-domain-containing leukocyte protein of 76 kDa) and FYB/SLAP (FYN-T-binding protein/SLP-76-associated protein) are two hemopoietic cell-specific adaptor proteins downstream of TCR-activated protein tyrosine kinases. SLP-76 has been implicated as an essential component in T cell signaling. FYB is selectively phosphorylated by FYN-T, providing a template for the recruitment of FYN-T and SLP-76 SH2 domains. Coexpression of FYN-T, FYB, and SLP-76 can synergistically up-regulate IL-2 production in T cells upon TCR ligation. In this report, we show that two tyrosines, Tyr595 and Tyr651, of FYB are major sites of phosphorylation by FYN-T and mediate binding to SLP-76 in Jurkat T cells. Furthermore, the synergistic up-regulation of IL-2 promoter activity in the FYN-T-FYB-SLP-76 pathway is contingent upon the interaction between FYB and SLP-76, but not the interaction between FYB and FYN-T. These observations define a pathway by which SLP-76 interacts with downstream components in the up-regulation of T cell cytokine production.     

Role of two adaptor molecules SLP-76 and LAT in the PI3K signaling pathway in activated T cells

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). We also demonstrated that tyrosine phosphorylation either at the 113 and/or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LAT adaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation.     

Independent and Cooperative Roles of Adaptor Molecules in Proximal Signaling during Fc?RI-Mediated Mast Cell Activation

Activation through FcɛRI, a high-affinity IgE-binding receptor, is critical for mast cell function during allergy. The formation of a multimolecular proximal signaling complex nucleated by the adaptor molecules SLP-76 and LAT1 is required for activation through this receptor. Based on previous T-cell studies, current dogma dictates that LAT1 is required for plasma membrane recruitment and function of SLP-76. Unexpectedly, we found that the recruitment and phosphorylation of SLP-76 were preserved in LAT1^−/− mast cells and that SLP-76^−/− and LAT1^−/− mast cells harbored distinct functional and biochemical defects. The LAT1-like molecule LAT2 was responsible for the preserved membrane localization and phosphorylation of SLP-76 in LAT1^−/− mast cells. Although LAT2 supported SLP-76 phosphorylation and recruitment to the plasma membrane, LAT2 only partially compensated for LAT1-mediated cell signaling due to its decreased ability to stabilize interactions with phospholipase Cγ (PLCγ). Comparison of SLP-76^−/− LAT1^−/− and SLP-76^−/− mast cells revealed that some functions of LAT1 could occur independently of SLP-76. We propose that while SLP-76 and LAT1 depend on each other for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions also mediate a positive signaling pathway downstream of FcɛRI in mast cells.Mast cell activation during allergic inflammation is mediated by the high-affinity immunoglobulin E (IgE)-binding receptor FcɛRI. Cross-linking of FcɛRI on mast cells by IgE/cognate antigen complexes results in the rapid release of a wide array of inflammatory mediators, including vasoactive amines and cytokines/chemokines that give rise to allergic symptoms, ranging in severity from simple urticaria to anaphylactic shock and death (14). As allergy affects ∼30% of the population in developed countries (13), much attention has been placed on studying the signal transduction mechanisms involved in mast cell activation downstream of FcɛRI in hopes of finding novel targets for therapeutic intervention.Signal transduction downstream of FcɛRI is initiated by the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) contained in the signaling components (β and γ chains) of the FcɛRI complex (30, 37). Once phosphorylated, these chains serve as docking sites for several protein tyrosine kinases (PTKs), including Lyn and spleen tyrosine kinase (Syk) (9, 19, 34). Recruitment of Syk to the membrane by FcɛRI results in the phosphorylation of scaffold proteins known as adaptor molecules. Adaptor proteins lack enzymatic activity but instead contain protein-binding domains that are critical for the formation of a multimolecular complex, which orchestrates downstream signaling in a temporal and spatial manner. The adaptor molecules Src homology 2 (SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) and linker of activated T cells 1 (LAT1) organize the assembly of a proximal signaling complex downstream of FcɛRI. Failure to form this complex is detrimental to FcɛRI-mediated mast cell function, as demonstrated by the finding that both SLP-76-deficient (22, 29, 41) and LAT1-deficient (25, 31, 32) mast cells display severely diminished degranulation and cytokine/chemokine production following FcɛRI ligation.Similar proximal signaling complexes are formed downstream of several different ITAM-containing receptors. Much of our understanding of the role of adaptor molecules in signal transduction has come from identification of phosphoproteins during T-cell receptor (TCR)-mediated activation of the human Jurkat T-cell line (1, 33). These studies eventually led to a paradigm describing the sequence of events in the formation of the SLP-76/LAT1 signaling complex. According to this model, SLP-76 is found constitutively bound to Grb2-related adaptor downstream of Shc (GADS) (24) and resides in the cytosol. Upon TCR activation, the tyrosines of membrane-resident LAT1 are phosphorylated and become attachment sites for proteins such as phospholipase Cγ (PLCγ) and GADS (43, 45). SLP-76 is drawn to the membrane through a GADS/LAT1 interaction, which then permits Syk family PTKs to maximally phosphorylate the N-terminal tyrosines of SLP-76 (5, 10). Several lines of evidence support this model whereby a LAT1/SLP-76 module organizes TCR signaling. First, both SLP-76- and LAT1-deficient Jurkat T cells display similar biochemical defects, such as diminished PLCγ and extracellular signal-regulated kinase (ERK) activation (10, 42). Second, T cells in SLP-76^−/− and LAT1^−/− mice are blocked at the same stage of development (7, 44). Third, SLP-76 can be coimmunoprecipitated with LAT1 but not with LAT1 harboring tyrosine-to-phenylalanine mutations (45). Finally, expression of a fusion protein comprised of the membrane-localizing domain of LAT1 and SLP-76 that forces localization of SLP-76 to the plasma membrane rescues the TCR-induced functional defects of both SLP-76- and LAT1-deficient Jurkat T cells (3). This model implies a mutually dependent relationship between SLP-76 and LAT1, where SLP-76 and LAT1 rely on each other to carry out their roles.One might suspect that this model for LAT1/SLP-76 function would operate in all other cells that utilize these adaptor molecules for ITAM-containing receptor-mediated signaling. However, the published defects of LAT1-deficient mast cells in FcɛRI-mediated signaling appeared milder than those of SLP-76-deficient mast cells, although a direct comparison has never been reported. In the present study, we show that LAT1-deficient mast cells display distinct functional and biochemical defects compared to SLP-76-deficient mast cells, implying that unlike in T cells, SLP-76 may not depend entirely on LAT1 for its function in mast cells. Surprisingly, the membrane recruitment and phosphorylation of SLP-76 were also preserved in LAT1^−/− mast cells. We show that LAT2 (also known as non-T-cell activation linker [NTAL] or linker for activation of B cells [LAB]), which is not expressed in naïve T cells but is expressed in mast cells (15), is responsible for phosphorylation and plasma membrane recruitment of SLP-76 in the absence of LAT1. However, LAT2 cannot support all LAT1/SLP-76-associated functions, such as sustained Ca²⁺ flux, likely due to decreased stability of the LAT2/SLP-76/PLCγ complex. Comparison of SLP-76^−/− LAT1^−/− and SLP-76^−/− mast cells also revealed that some functions of LAT1 could occur independently of SLP-76. We propose that although SLP-76 and LAT1 are interdependent for many of their functions, LAT2/SLP-76 interactions and SLP-76-independent LAT1 functions mediate positive signaling downstream of FcɛRI in mast cells.     

Cross-linking of 4-1BB activates TCR-signaling pathways in CD8+ T lymphocytes

Cross-linking of 4-1BB, a member of the TNFR family, increased tyrosine phosphorylation of TCR-signaling molecules such as CD3epsilon, CD3zeta, Lck, the linker for activation of T cells, and SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76). In addition, incubation of activated CD8+ T cells with p815 cells expressing 4-1BBL led to redistribution of the lipid raft domains and Lck, protein kinase C-theta;, SLP-76, and phospholipase C-gamma1 (PLC-gamma1) on the T cell membranes to the areas of contact with the p815 cells and recruitment of 4-1BB, TNFR-associated factor 2, and phospho-tyrosine proteins to the raft domains. 4-1BB ligation also caused translocation of TNFR-associated factor 2, protein kinase C-theta;, PLC-gamma1, and SLP-76 to detergent-insoluble compartments in the CD8+ T cells, and cross-linking of 4-1BB increased intracellular Ca2+ levels apparently by activating PLC-gamma1. The redistribution of lipid rafts and Lck, as well as translocation of PLC-gamma1, and degradation of IkappaB-alpha in response to 4-1BB were inhibited by disrupting the formation of lipid rafts with methyl-beta-cyclodextrin. These findings demonstrate that 4-1BB is a T cell costimulatory receptor that activates TCR-signaling pathways in CD8+ T cells.     

Minimal requirement of tyrosine residues of linker for activation of T cells in TCR signaling and thymocyte development

Linker for activation of T cells (LAT) is a membrane-associated adaptor protein that is phosphorylated on multiple tyrosines upon TCR cross-linking. Previous studies show that LAT is essential for TCR-mediated signaling and thymocyte development. In this study, we expressed a series of LAT Tyr to Phe mutants in LAT-deficient J.CaM2.5 cells and examined their tyrosine phosphorylation; association with Grb2, Gads, and phospholipase C (PLC)-gamma1; and function in T cell activation. Our results showed that the five membrane-distal tyrosines were phosphorylated upon T cell activation. Grb2, Gads, and PLC-gamma1 associated with LAT preferentially via different sets of tyrosine residues; however, they failed to interact with LAT mutants containing only one tyrosine. We also determined the minimal requirement of LAT tyrosine residues in T cell activation and thymocyte development. Our results showed that a minimum of three tyrosines is required for LAT to function in T cell activation and thymocyte development. LAT mutants that were capable of binding Grb2 and PLC-gamma1 could reconstitute T cell activation in LAT-deficient cells and thymocyte development in LAT-deficient mice.     

Association of Grb2, Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on T cell angigen receptor-mediated signaling

The linker for activation of T cells (LAT) is a critical adaptor molecule required for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on tyrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma1 bind LAT via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR engagement fails to induce ERK activation, Ca(2+) flux, and activation of AP-1 and NF-AT. We mapped the tyrosine residues in LAT responsible for interaction with these specific signaling molecules by expressing LAT mutants with tyrosine to phenylalanine mutations in LAT-deficient cells. Our results showed that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are responsible for Grb2-binding; Tyr(171), and Tyr(191), but not Tyr(226), are necessary for Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma1 binding. Mutation of all three distal tyrosines also abolished PLC-gamma1 binding, suggesting there might be multiple binding sites for PLC-gamma1. Mutation of Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Since Grb2 binding is not affected by this mutation, these results strongly suggest that PLC-gamma activation regulates Ras activation in these cells. Mutation of individual Grb2 binding sites had no functional effect, but mutation of two or three of these sites, in combination, also affected Erk and NF-AT activation.     

TCR and CD28 are coupled via ZAP-70 to the activation of the Vav/Rac-1-/PAK-1/p38 MAPK signaling pathway.

CD28 costimulation amplifies TCR-dependent signaling in activated T cells, however, the biochemical mechanism(s) by which this occurs is not precisely understood. The small GTPase Rac-1 controls the catalytic activity of the mitogen-activated protein kinases (MAPKs) and cell cycle progression through G1. Rac-1 activation requires the phospho-tyrosine (p-Tyr)-dependent recruitment of the Vav GDP releasing factor (GRF) to the plasma membrane and assembly of GTPase/GRF complexes, an event critical for Ag receptor-triggered T cell activation. Here, we show that TCR/CD28 costimulation synergistically induces Rac-1 GDP/GTP exchange. Our findings, obtained by using ZAP-70-negative Jurkat T cells, indicate that CD28 costimulation augments TCR-mediated T cell activation by increasing the ZAP-70-mediated Tyr phosphorylation of Vav. This event regulates the Rac-1-associated GTP/GDP exchange activity of Vav and downstream pathway(s) leading to PAK-1 and p38 MAPK activation. CD28 amplifies TCR-induced ZAP-70 activity and association of Vav with ZAP-70 and linker for activation of T cells (LAT). These results favor a model in which ZAP-70 regulates the intersection of the TCR and CD28 signaling pathways, which elicits the coupling of TCR and CD28 to the Rac-1, PAK-1, and p38 MAPK effector molecules.