Analysis of the T-cell activation signaling pathway mediated by tyrosine kinases, protein kinase C, and Ras protein, which is modulated by intracellular cyclic AMP.
T-cell receptor (TCR) triggering by an anti-CD3 antibody or phytohemagglutinin (PHA) as well as the treatment with phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), induces activation of Ras in T-lymphocytes (Downward, J. et al. (1990)) Nature 364, 719-723). In this paper, we studied the role of Ras in the process of TCR-mediated T-cell activation using a human lymphomic Jurkat cell line. The stimulatory effect of TCR cross-linking on Ras activation was inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinases (PTKs), whereas PMA-induced Ras activation was not affected. On the other hand, calphostin C, a specific inhibitor of PKC, blocked not only PMA-induced, but also TCR-mediated formation of Ras.GTP. Furthermore, down-regulation of PMA-sensitive PKC severely impaired the activation of Ras in response to TCR-stimulation. Tyrosine-phosphorylation and translocation to the particulate fraction of phospholipase C-gamma 1 (PLC-gamma 1) were observed upon T-cell activation. Subcellular localization of PKC was also changed when the cells were stimulated with an anti-CD3 antibody or PMA. While TCR-stimulated translocation of PKC was observed only transiently, PMA-induced translocation of PKC was more sustained. These results suggest that the activation of PLC-gamma 1 by PTK and subsequent activation of PKC are important for TCR-mediated Ras activation in Jurkat cells. An activated form of Ras enhanced the activation of interleukin 2 (IL-2) promoter by TCR stimulation or PMA treatment, although the activated Ras by itself was insufficient for IL-2 promoter activation. On the other hand, a dominant-inhibitory Ras diminished almost completely the activation of IL-2 promoter induced by PMA plus calcium ionophore, indicating that Ras is essential for transduction of T-cell activation signals. Cholera toxin (CTX), which directly activates Gs alpha, is shown to inhibit the activation of IL-2 promoter. TCR-mediated Ras activation, tyrosine phosphorylation and translocation of cellular proteins including ZAP-70, PLC-gamma 1 , and PKC. An activated Gs alpha mutant as well as dibutylyl cAMP (dBcAMP) also showed similar inhibitory effects.
Ohtsuka
T
T
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan.
Kaziro
Y
Y
Satoh
T
T
D002451:Cell Compartmentation; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D008213:Lymphocyte Activation; D019000:Phosphotyrosine / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000502:physiology*; D014158:Transcription, Genetic; D010738:Type C Phospholipases / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
D004791:Enzyme Inhibitors; D007376:Interleukin-2; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D006153:Guanosine Diphosphate; D019000:Phosphotyrosine; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D010738:Type C Phospholipases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)