Molecular Cloning of SLP-76, a 76-kDa Tyrosine Phosphoprotein Associated with Grb2 in T Cells

Abstract

The activation of protein tyrosine kinases is a critical event in T cell antigen receptor (TCR)-mediated signaling. One substrate of the TCR-activated protein tyrosine kinase pathway is a 76-kDa protein (pp76) that associates with the adaptor protein Grb2. In this report we describe the purification of pp76 and the molecular cloning of its cDNA, which encodes a novel 533-amino acid protein with a single carboxyl-terminal Src homology 2 (SH2) domain. Although no recognizable motifs related to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential motifs recognized by SH2 and SH3 domains. A cDNA encoding the murine homologue of pp76 was also isolated and predicts a protein with 84% amino acid identity to human pp76. Northern analysis demonstrates that pp76 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines. In vitro translation of pp76 cDNA gives rise to a single product of 76 kDa that associates with a GST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion protein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum directed against phospholipase C-gamma 1 coprecipitates a tyrosine phosphoprotein with an electrophoretic mobility identical to that of pp76. These results demonstrate that this novel protein, which we term SLP-76 (SH2 domain-containing Leukocyte Protein of 76 kDa), is likely to play an important role in TCR-mediated intracellular signal transduction.

SCIMP, a Transmembrane Adaptor Protein Involved in Major Histocompatibility Complex Class II Signaling

“…It has been shown that both SLP65 and SLP76 interact via their proline-rich motifs with the SH3 domain of Grb2 (15,24), thereby forming a complex where both proteins have SH2 domains available for binding. We suggest that this "bivalent" mode of binding would substantially increase the avidity of the interaction between Grb2, SLP65/SLP76, and SCIMP.…”

Src Homology Region 2 (SH2) Domain-Containing Phosphatase-1 Dephosphorylates B Cell Linker Protein/SH2 Domain Leukocyte Protein of 65 kDa and Selectively Regulates c-Jun NH2-Terminal Kinase Activation in B Cells

“…In addition, we have previously demonstrated that SHP-1 is constitutively associated with SLP-76 in the mouse immature B cell line, WEHI-231 (27). SLP-76 is a leukocyte-specific cytoplasmic protein comprised of acidic region at the NH 2 terminus containing three tyrosine residues that can be phosphorylated by Syk family PTKs, followed by a proline-rich region and a COOH-terminal SH2 domain (28). This molecule is involved in the regulation of signaling events initiated by TCR (28 -33) and Fc⑀RI (34).…”

Negative Signaling in Fc Receptor Complexes

“…Besides its N-terminal SH2 domain, SLP-76 contains a central proline-rich region and multiple C-terminal tyrosines (Jackman et al, 1995). Once phosphorylated, its binds to a variety of molecules including the exchange factor Vav (Tuosto et al, 1996) and other adapters such as Gads, Nck and SLAP-130 (Boerth et al, 2000).…”