Abstract
Antibodies to either CD3 or CD45 have been shown to induce dramatic changes in cell morphology, increased tyrosine phosphorylation of cellular proteins, and the association of a subset of these proteins with the tyrosine kinase Lck. The current study was initiated to determine the identity of the tyrosine-phosphorylated 70-80 kDa protein that becomes Lck-associated after stimulation with anti-CD45 or anti-CD3. We demonstrate that the cytoskeletal protein paxillin becomes tyrosine-phosphorylated when cells are plated on immobilized antibodies specific for CD45 or CD3. Only tyrosine-phosphorylated paxillin is associated with Lck, suggesting that the association is through the SH2 domain of Lck. Consistent with this we demonstrate that the SH2 domain of Lck binds tyrosine-phosphorylated paxillin. In contrast, the association of paxillin with the FAK-related kinase Pyk2 was found to be constitutive and not altered by the phosphorylation of either protein. Finally, we establish that the phosphorylation of paxillin is dependent on the expression of Lck. Taken together, these results demonstrate that paxillin is physically associated with kinases from two different families in T cells and suggest that paxillin may function as an adaptor protein linking cellular signals with cytoskeletal changes during T cell activation.
Highlights
T cells have been shown to undergo dramatic changes in cell morphology, coincident with cytoskeletal rearrangements, upon activation [1, 2]
Immobilized Antibodies to CD45 or CD3 Induce the Tyrosine Phosphorylation of a 70 – 80-kDa Cellular Protein and Its Association with Lck—When T cell clones are plated on immobilized anti-CD45 or anti-CD3, but not on anti-Class I MHC, the cells undergo dramatic changes in cellular morphology typified by extensive spreading [6]
We have provided the first demonstration that antibodies to CD45 or CD3 stimulate the phosphorylation of paxillin leading to its association with the SH2 domain of Lck
Summary
Cell Lines—The murine H-2b-specific CTL clones 11 and AB. have been described previously [15]. Antibodies and Reagents—The monoclonal antibodies 145-2C11 (2C11, anti-mouse CD3) and OKT3 (anti-human CD3) were obtained through the ATCC. Trowbridge, and PY-72 (anti-phosphotyrosine) was obtained from Dr B Sefton, both of whom are at The Salk Institute (La Jolla, CA). The monoclonal antibody specific for paxillin was obtained from Transduction Laboratories (Lexington, KY). Antiserum to the carboxyl terminus of Lck was generated in our laboratory using a bovine serum albumin-coupled peptide based on amino acids 476 –509 of human Lck [6]. Many of the experiments have been done with anti-Lck (carboxyl terminus) antiserum obtained from Upstate Biotechnology Inc. Anti-Pyk antiserum was generated in our laboratory as described previously [16]. If a monoclonal antibody was used, rabbit anti-mouse antiserum was added to facilitate immunoprecipitation. Blots were developed by chemiluminescence (NEN Life Science Products) as described in the product bulletin
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