ACTIVATION-DEPENDENT PHOSPHORYLATION OF THE T-LYMPHOCYTE SURFACE-RECEPTOR CD28 AND ASSOCIATED PROTEINS

CD28 is a costimulatory receptor that can provide the second signal ne cessary for T-cell activation and function in response to stimulation through the T-cell antigen receptor/CD3 complex. We found that a disti nct array of proteins was phosphorylated on tyrosine following stimula tion with anti-CD28 monoclonal antibody, as detected by immune-complex kinase assays. Anti-CD28 stimulation of in vitro kinase activity was detergent-dependent, occurring in immune complexes prepared with Brij 96 but not Nonidet P-40. Pretreatment of cells with low concentrations of phorbol ester increased the activation-independent phosphorylation of proteins in CD28 immune complexes. Reimmunoprecipitation studies i ndicated that the cytoplasmic protein-tyrosine kinases Lck and Fyn wer e associated with CD28. CD28 itself was phosphorylated both in vitro a nd in vivo in an activation-dependent manner, as detected by nonreduci ng/reducing SDS/PAGE analyses. The activation-stimulated phosphorylati on of CD28 may play a key role in signaling through this receptor.