MOLECULAR-CLONING OF SLP-76, A 76-KDA TYROSINE PHOSPHOPROTEIN ASSOCIATED WITH GRB2 IN T-CELLS

The activation of protein tyrosine kinases is a critical event in T ce ll 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 descr ibe the purification of pp76 and the molecular cloning of its cDNA, wh ich encodes a novel 533-amino acid protein with a single carboxyl-term inal Src homology 2 (SH2) domain, Although no recognizable motifs rela ted to tyrosine, serine/threonine, or lipid kinase domains are present in the predicted amino acid sequence, it contains several potential m otifs recognized by SH2 and SH3 domains, A cDNA encoding the murine ho mologue of pp76 was also isolated and predicts a protein with 84% amin o acid identity to human pp76, Northern analysis demonstrates that pp7 6 mRNA is expressed solely in peripheral blood leukocytes, thymus, and spleen; and in human T cell, B cell and monocytic cell lines, In vitr o translation of pp76 cDNA gives rise to a single product of 76 kDa th at associates with a GrST/Grb2 fusion protein, demonstrating a direct association between these two molecules. Additionally, a GST fusion pr otein consisting of the predicted SH2 domain of pp76 precipitates two tyrosine phosphoproteins from Jurkat cell lysates, and antiserum direc ted against phospho lipase C-gamma 1 coprecipitates a tyrosine phospho protein with an electrophoretic mobility identical to that of pp76, Th ese 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.