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 have developed a deglycosylation procedure that allows the precise identification of a distinct CD28 protein band, facilitating the analysis of activation-dependent changes in the phosphorylation state of CD28. Phorbol 12-myristate 13-acetate (PMA) treatment induced the in vitro phosphorylation of CD28 on threonine as detected in immune complex kinase assays. This effect of PMA was (i) rapid, preceding a PMA-induced increase in CD28 surface expression; (ii) occurred using kinase buffer containing either manganese or magnesium; and (iii) was found in human peripheral T cells, Jurkat T cells, and in a Jurkat subclone, J.Cam1, that is deficient in Lck tyrosine kinase activity. In contrast, anti-CD28 monoclonal antibody stimulation led to in vitro phosphorylation of CD28 on tyrosine that was manganese-dependent and required Lck tyrosine kinase activity, as it was undetectable in J.Cam1 cells. Importantly, CD28 was phosphorylated on tyrosine in vivo as detected with anti-phosphotyrosine antibodies after stimulation with anti-CD28 monoclonal antibody. The in vivo tyrosine phosphorylation of CD28 was inhibited by PMA treatment and was absent in J.Cam1 cells. Thus, the CD28 coreceptor can trigger different intracellular signaling pathways, depending upon the nature of the initial costimulatory signal.
Hutchcroft
J E
JE
Division of Pediatric Oncology, Dana-Farber Cancer Institute, Hematology-Oncology Division, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Tsai
B
B
Bierer
B E
BE
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D018106:CD28 Antigens / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008213:Lymphocyte Activation; D008970:Molecular Weight; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D013912:Threonine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate