MODULATION OF THE GRB2-ASSOCIATED PROTEIN COMPLEX IN HUMAN CD4(-CELLSBY RECEPTOR ACTIVATION() T)

A panel of human CD4(+) T cell clones was utilized to dissect and anal yze the biochemical consequences of activation of CD3 or CD28. To mole cularly characterize receptor-activated proximal signaling events, tyr osine-phosphorylated proteins co-precipitating with a Grb2 fusion prot ein after receptor activation were analyzed. Ligation of CD28, but not other costimulatory molecules, induced the tyrosine phosphorylation o f two previously identified Grb2 binding proteins (pp76 and pp116). A third Grb2 binding protein (pp36) was extensively tyrosine phosphophor ylated in response to combined CD3 and CD28 activation, but not in res ponse to ligation of either receptor alone. cAMP and co-ligation of CD 45 affected the receptor-activated tyrosine phosphorylation of Grb2-as sociated proteins. Furthermore, we demonstrated that two signaling mol ecules, Vav and phosphatidylinositol 3'-kinase (PI(3)K), also interact ed with the Grb2 protein complex. The activity of PI(3)K was required for T cell activation, because wortmannin, a PI(3)K inhibitor, blocked T cell proliferation and cytokine production induced by ligation of C D3 and CD28. In conclusion, we demonstrate that in activated human T c ell clones, the composition of Grb2 protein complex is modulated by co stimulatory signals and cAMP, which may be important for the regulatio n of intracellular signal transduction.