Activation-dependent phosphorylation of the T-lymphocyte surface receptor CD28 and associated proteins.
CD28 is a costimulatory receptor that can provide the second signal necessary for T-cell activation and function in response to stimulation through the T-cell antigen receptor/CD3 complex. We found that a distinct array of proteins was phosphorylated on tyrosine following stimulation 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 indicated that the cytoplasmic protein-tyrosine kinases Lck and Fyn were associated with CD28. CD28 itself was phosphorylated both in vitro and in vivo in an activation-dependent manner, as detected by nonreducing/reducing SDS/PAGE analyses. The activation-stimulated phosphorylation of CD28 may play a key role in signaling through this receptor.
Hutchcroft
J E
JE
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115.
Bierer
B E
BE
D000936:Antigen-Antibody Complex; D018106:CD28 Antigens / Q000378:metabolism*; D003902:Detergents; D006801:Humans; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D046911:Macromolecular Substances; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D013601:T-Lymphocytes / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D000936:Antigen-Antibody Complex; D018106:CD28 Antigens; D003902:Detergents; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051076:Proto-Oncogene Proteins c-fyn; D013755:Tetradecanoylphorbol Acetate