Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production.

Abstract

SummaryRecently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 to amino acids 224-244. We also demonstrate that upon T cell receptor (TCR) stimulation, SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62-and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production. L igation of the TCR results in the rapid activation of several protein tyrosine kinases (PTKs) with the subsequent phosphorylation of numerous cellular proteins (1-3), including components of the TCR itself (2, 3), Src and Syk family PTKs (Lck, Fyn, and ZAP-70 [for review see reference 4]), phospholipase C~/1 (5-7), GAP (8), ezrin (9), CD5 (10, 11), Shc (12), Vav (13,14), Cbl (15), and several cytoskeletal proteins (16). However, many substrates of the TCR-activated PTKs remain unidentified. Our laboratory has been interested in characterizing some of these molecules. As one strategy, we chose to investigate tyrosine phosphoproteins that associate with Grb2, an adapter molecule that has been shown to play a central role in coupling fignaling pathways in other systems.Using this approach, we and others have demonstrated that tyrosine phosphoproteins of 36, 76, and 116 kD associate with Grb2 in T cells (17)(18)(19)(20). The 116-kD protein has been identified recently as the product of the c-Cbl protooncogene (15); however, its function remains unclear. The molecular identity of the 36-kD protein remains uncertain, but recent data from our laboratory suggest that tyrosine phosphorylation of this molecule is required for coupling the TCK with the phosphatidylinositol second messenger pathway (Motto, D., M. Musci, S. Ross, and G. Koretzky, manuscript submitted for publication). Recently, in a collaborative effort (Paul Findell, Palo Alto, CA), we purified the 76-kD Grb2-associated phosphoprotein and cloned its cDNA, which encodes a novel hematopoietic cell-specific protein we have termed SLP-76, for S_H2 domain leukocyte Erotein o...

SLP-76 Coordinates Nck-Dependent Wiskott-Aldrich Syndrome Protein Recruitment with Vav-1/Cdc42-Dependent Wiskott-Aldrich Syndrome Protein Activation at the T Cell-APC Contact Site

“…In keeping with this finding, a recent study shows that Vav-1 interacts with linker for activation of T cells in a fashion that depends on phosphorylation by Itk (59), and we have recently found that Itk-deficient cells, like the SLP-76 mutants, are defective in Vav recruitment and localized Cdc42 activation. 4 Although SLP-76 has been shown to bind both Grb2 and Nck (13,60,61), we find that only Nck participates in WASP recruitment. Our results showing that the third SH3 domain of Nck is required for WASP recruitment are consistent with a previous study showing that this SH3 domain binds to the proline-rich domain of WASP in vitro (26).…”

SLP‐76 is recruited to CD22 and dephosphorylated by SHP‐1, thereby regulating B cell receptor‐induced c‐Jun N‐terminal kinase activation

“…WEHI-231 cells were transfected with flag-tagged WT or mutant forms of SLP-76 and immunoprecipitated SLP-76 was subjected to anti-CD22 immunoblotting. The following four mutants were used: SLP-76DN, SLP-76 lacking the minimal prolinerich region required for Grb2/Gads binding (SLP76DPro) [39], SLP-76 lacking the SH2 domain (SLP-76DSH2), and SLP-76 lacking both proline-rich region and the SH2 domain (SLP-76DProDSH2). Although the amount of all exogenously introduced SLP-76 mutants was comparable (Fig.…”

CD28 Utilizes Vav-1 to Enhance TCR-Proximal Signaling and NF-AT Activation

“…To this aim, we tested ZAP-70 and the adapter protein SLP-76, two key elements required for NF-AT-induced transcriptional activation (48). Similarly to Vav-1, both proteins are tyrosine phosphorylated following TCR ligation (49,50) and when overexpressed in Jurkat cells they cause an increase of basal (40,42) and Fig. 1, A and B, and Fig.…”