Tyrosine Phosphorylation Of SLP-76 Research Articles

Association of the Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) with the p85 subunit of phosphoinositide 3-kinase

To investigate additional functions of the T cell adaptor, Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD (SLP-76), we performed a yeast two-hybrid assay using the N-terminal region of SLP-76 fused with the kinase domain of Syk. By screening a human leukemia cDNA library, we identified the p85 subunit of phosphoinositide 3-kinase (PI3K) as one of the interacting molecules. Unlike the SH2 domain of Vav or Nck, tyrosine phosphorylation of SLP-76 at position 113 or 128 was sufficient for it to associate with the N-terminal SH2 of p85. Collectively, these data suggest that SLP-76 may play a role in PI3K signaling pathways.

Evidence for a dual pathway of activation in CD43-stimulated Th2 cells: differential requirement for the Lck tyrosine kinase.

CD43 is the most abundant cell surface-expressed sialoglycoprotein on T lymphocytes. Despite evidence demonstrating the activation of some signaling components by CD43, the exact function of CD43 in T cell biology remains controversial. In this study, we demonstrate that the sole ligation of CD43 in cloned Th2 cells resulted in cytokine production, cellular proliferation, and upregulation of CD25 and CD69 activation markers. Similarly, cross-linking of CD43 on naive splenic T cells led to a significant proliferative response and an enhancement of the expression of CD25 and CD69 markers. These responses required no additional signals from other T cell molecules, including TCR. In Lck-deficient Th2 cells, however, CD43 ligation led to IL-4 production and an increase in the expression of CD25 and CD69 antigens but, surprisingly, no proliferation. Analysis of signaling pathway components revealed that CD43 associates with the adaptor protein SLP-76 within 30 s of activation. This induces the tyrosine phosphorylation of SLP-76 and promotes the recruitment and phosphorylation of another adaptor, Shc. The formation of this multi-component complex was strictly dependent on Lck. In contrast, comparison of tyrosine phosphorylated proteins in whole extracts of normal and Lck-deficient cells revealed a strikingly similar pattern of phosphorylation involving two major protein bands at 26 and 78 kDa. This suggests that tyrosine kinases other than Lck are activated by CD43 ligation. Taken together, the data support the notion that CD43 ligation may induce a dual pathway leading to the activation of different effector functions in Th2 lymphocytes.

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.

Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

LAT Is Essential for FcεRI-Mediated Mast Cell Activation

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow–derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of FcεRI, Syk, and Vav was intact in LAT-deficient BMMCs following FcεRI engagement, tyrosine phosphorylation of SLP-76, PLC-γ1, and PLC-γ2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after FcεRI cross-linking. These results show that LAT plays a critical role in FcεRI-mediated signaling in mast cells.

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.

SLP-76-Cbl-Grb2-Shc Interactions in FcγRI Signaling

SLP-76 and Cbl are complex adapter proteins that have the capacity to bind to smaller adapter proteins, such as Grb2, which subsequently binds the nucleotide exchange protein Sos in the transmission of intracellular signals. SLP-76, Cbl, Shc, and Grb2 have been implicated in immunoreceptor tyrosine-based activation motif (ITAM) signaling, leading to activation of Ras. However, their mechanism of action has not been determined. To date, there have been no reports of SLP-76 involvement in FcγRI-receptor signaling and no data exist for an interaction between Cbl, Shc, and SLP-76 in vivo. We provide evidence that SLP-76, Cbl, and Shc are tyrosine phosphorylated on FcγRI-receptor stimulation and are associated with the adapter protein Grb2 in γ-interferon–differentiated U937 cells (U937IF). The interactions between SLP-76 and Cbl and SLP-76 and Grb2 are present in resting U937IF cells. However, the interaction between SLP-76 and Grb2 becomes augmented twofold on FcγRI-receptor aggregation. Our results provide the first evidence for a phosphorylation-dependent interaction between SLP-76 and Shc, induced at least 10-fold on FcγRI receptor stimulation. Our data indicate that a significant portion of a multimolecular complex containing Cbl, SLP-76, Shc, and Grb2 is distinct from a trimolecular complex containing the Ras guanine nucleotide exchanger Sos, Shc, and Grb2. FcγRI-induced tyrosine phosphorylation of SLP-76, Cbl, Shc, and the highly induced SLP-76-Shc interaction provide the first evidence that SLP-76 and Cbl are involved in FcγRI signaling and suggest a functional significance for these interactions in FcγRI signal relay in the control of Ras in myeloid cells.© 1998 by The American Society of Hematology.

Tyrosines 113, 128, and 145 of SLP-76 are required for optimal augmentation of NFAT promoter activity.

SLP-76 (SH2 domain leukocyte protein of 76 kDa) is a recently identified substrate of the TCR-stimulated protein tyrosine kinases that functions in the signal transduction cascade linking the TCR with IL-2 gene expression. In this report, we demonstrate that engagement of the TCR results in tyrosine phosphorylation of SLP-76 in its amino-terminal acidic region. Two tyrosines (Y113 and Y128) fall within an identical five amino-acid motif and are shown to be phosphorylated upon TCR ligation. Although mutation of either Y113 and Y128 has a minimal effect on SLP-76 function, mutation of both residues decreases significantly the ability of SLP-76 to promote T cell activation. A third tyrosine within the amino-terminal region (Y145) appears to be the most important for optimal SLP-76 function, as altering it alone to phenylalanine has a potent impact on SLP-76 augmentation of NFAT promoter activity.