A gene encoding a 35-kilodalton guanosine triphosphate (GTP)-binding protein, Gem, was cloned from mitogen-induced human peripheral blood T cells. Gem and Rad, the product of a gene overexpressed in skeletal muscle in individuals with Type II diabetes, constitute a new family of Ras-related GTP-binding proteins. The distinct structural features of this family include the G3 GTP-binding motif, extensive amino- and carboxyl-terminal extensions beyond the Ras-related domain, and a motif that determines membrane association. Gem was transiently expressed in human peripheral blood T cells in response to mitogenic stimulation; the protein was phosphorylated on tyrosine residues and localized to the cytosolic face of the plasma membrane. Deregulated Gem expression prevented proliferation of normal and transformed 3T3 cells. These results suggest that Gem is a regulatory protein, possibly participating in receptor-mediated signal transduction at the plasma membrane.
The beta 1 subfamily of integrins is thought to play an important role in both the adhesion/migration and proliferation/differentiation of T cells. beta 1 integrins can provide T cell costimulation through interaction of very late antigen (VLA) 4 (VLA-4) (alpha 4 beta 1) and VLA-5 (alpha 5 beta 1) with the extracellular matrix protein fibronectin (FN), or by VLA-4 binding to its cell surface ligand, vascular cell adhesion molecule (VCAM) 1. The mechanism by which beta 1 integrin members transduce T cell-costimulatory signals is poorly understood. Studies in non-T cells have demonstrated regulation of the tyrosine focal adhesion kinase pp125FAK by beta 1 integrin engagement and, most recently, indicate a role for pp125FAK in linking integrin- mediated signal transduction to the Ras pathway (Schaller, M. D., and J. T. Parsons, 1994, Curr. Opin. Cell. Biol. 6: 705-710; Schlaepfer, D. D., S. K. Hanks, T. Hunter, and P. Van der Geer. 1994. Nature (Lond.), 372:786-790). Although pp125FAK kinase occurs in T cells, there are no reports on its regulation in this cell type. The studies described in this article characterize novel regulation of pp125FAK by the T cell receptor (TCR)-CD3 antigen complex and beta 1 integrins, and provide the first account, in any cell type, of integrin alpha 4 beta 1- mediated pp125FAK tyrosine phosphorylation. We demonstrate a rapid and sustained synergistic increase in tyrosine phosphorylation of human pp125FAK in Jurkat T cells after simultaneous (a) triggering of the TCR- CD3 complex, and (b) alpha 4 beta 1 and alpha 5 beta 1 integrin- mediated binding of these cells to immobilized FN or alpha 4 beta 1 integrin-mediated binding to immobilized VCAM-1. Studies with normal peripheral blood-derived CD4+ human T blasts confirm the synergistic action of a TCR-CD3 complex-mediated costimulus with a FN- or VCAM-1- dependent signal in the induction of T cell pp125FAK tyrosine phosphorylation. In vitro kinase assays performed on pp125FAK immunoprecipitates isolated from Jurkat cells and normal CD4+ T cells identified a coprecipitating 57-kD tyrosine-phosphorylated protein (pp57), distinct from pp59fyn or pp56lck. These results indicate, for the first time, the involvement of a specific kinase, pp125FAK, in alpha 4 beta 1- and alpha 5 beta 1-mediated T cell-costimulatory signaling pathways. In addition, the data demonstrate novel regulation of pp125FAK tyrosine phosphorylation by the TCR-CD3 complex.
Expression of the multicomponent T-cell antigen receptor (TCR) complex on the surface of thymocytes is developmentally controlled. Most immature CD4-CD8- 'double negative' and CD4+CD8+ 'double positive' thymocytes express either no or few TCR on their surface, and maturation to CD4+CD8- or CD4-CD8+ 'single positive' thymocytes is accompanied by a dramatic increase in the number of surface TCR complexes. Although the initial appearance of TCR during differentiation results from rearrangement and initiation of transcription of TCR genes in the thymus, the mechanisms regulating the quantitative changes in TCR expression during intrathymic differentiation are unknown. Surface TCR levels in T-hybridoma cells can be quantitatively regulated by a series of post-translational processes, including sorting to alternative intracellular compartments and degradation, which ensure that only fully and correctly assembled receptor complexes are efficiently transported to the cell surface. Quantitative increases in TCR expression on the surface of CD4+CD8+ thymocytes occur in vivo in response to anti-CD4 antibody treatment. Here we present evidence that immature CD4+CD8+ thymocytes normally retain and degrade in the endoplasmic reticulum greater than 90% of some endogenously synthesized TCR chains, and that the increased surface TCR expression on immature CD4+CD8+ thymocytes induced by anti-CD4 is due to an increase in the escape of newly synthesized receptor chains from the endoplasmic reticulum, and is not due to increases in RNA levels, translation, or assembly. Post-translational mechanisms therefore control the levels of TCR complexes on CD4+CD8+ thymocytes, and these mechanisms can be modulated by signalling through CD4 surface molecules.
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