The Role of p21^ras in CD28 Signal Transduction: Triggering of CD28 with Antibodies, but Not the Ligand B7-1, Activates p21^ras

CD28 is a 44-kD homodimer expressed on the surface of the majority of human T cells that provides an important costimulus for T cell activation. The biochemical basis of the CD28 accessory signals is poorly understood. Triggering of the T cell antigen receptor (TCR) activates the p21^ras proteins. Here we show that ligation of CD28 by a monoclonal antibody (mAb) also stimulates p21^ras and induces Ras-dependent events such as stimulation of the microtubule-associated protein (MAP) kinase ERK2 and hyperphosphorylation of Raf-1. One physiological ligand for CD28 is the molecule B7-1. In contrast to the effect of CD28 mAb, the present studies show that interactions between CD28 and B7-1 do not stimulate p21 r-s signaling pathways. Two substrates for TCR-regulated protein tyrosine kinases (PTKs) have been implicated in p21^ras activation in T cells: p95^Vav and a 36-kD protein that associates with a complex of Grb2 and the Ras exchange protein Sos. Triggering CD28 with both antibodies and B7-1 activates cellular PTKs, and we have exploited the differences between antibodies and B7-1 for p21 -'s activation in an attempt to identify critical PTK-controlled events for Ras activation in T cells. The data show that antibodies against TCR or CD28 induce tyrosine phosporylation of both Vav and p36. B7-1 also induces Vav tyrosine phosphorylation but has no apparent effect on tyrosine phosphorylation of the Grb2- associated p36 protein. The intensity of the Vav tyrosine phosphorylation is greater in B7-1 than in TCR-stimulated cells. Moreover the kinetics of Vav tyrosine phosphorylation is prolonged in the B7-1-stimulated cells. These studies show that for CD28 signaling, the activation of p21^ras correlates more closely with p36 tyrosine phosphorylation than with Vav tyrosine phosphorylation. However, the experiments demonstrate that Vav is a major substrate for B7-activated PTKs and hence could be important in CD28 signal transduction pathway.