DIFFERENTIAL PHOSPHORYLATION OF THE T-LYMPHOCYTE COSTIMULATORY RECEPTOR CD28 - ACTIVATION-DEPENDENT CHANGES AND REGULATION BY PROTEIN-KINASE-C

Treatment of T lymphocytes with phorbol ester and anti-CD28 monoclonal antibody (mAb) can induce proliferation and interleukin 2 production by triggering still undefined intracellular signaling pathways. We hav e developed a deglycosylation procedure that allows the precise identi fication of a distinct CD28 protein band, facilitating the analysis of activation-dependent changes in the phosphorylation state of CD28. Ph orbol 12-myristate 13-acetate (PMA) treatment induced the in vitro pho sphorylation of CD28 on threonine as detected in immune complex kinase assays. This effect of PMA was (i) rapid, preceding a PMA-induced inc rease in CD28 surface expression; (ii) occurred using kinase buffer co ntaining either manganese or magnesium; and cells, and in a Jurkat sub clone, J. Cam1, that is deficient in Lek tyrosine kinase activity. In contrast, anti-CD28 monoclonal antibody stimulation led to in vitro ph osphorylation of CD28 on tyrosine that was manganese-dependent and req uired Lek tyrosine kinase activity, as it was undetectable in J. Cam1 cells. Importantly, CD28 was phosphotyrosine antibodies after stimulat ion with anti-CD28 monoclonal antibody. The in vivo tyrosine phosphory lation of Cd28 was inhibited by PMA treatment and was absent in J. Cam 1 cells. Thus, the CD28 coreceptor can trigger different intracellular signaling pathways, depending upon the nature of the initial co-stimu latory signal.