Activity of both Raf and Ras is necessary for activation of transcription of the human T cell receptor beta gene by protein kinase C, Ras plays multiple roles.
Transcription of the T cell receptor beta gene is up-regulated during T cell activation. We have previously defined the elements within the TcR beta gene enhancer responsible for increased transcription in response to phorbol esters, which mimic part of the pathway for T cell activation. Using a reporter construct (beta E2 x 4) containing four copies of this inducible element, activation was achieved by the addition of phytohemagglutinin or phorbol esters. Activation was observed after 2 h of treatment, was maximal by 8 h, and could be blocked by the protein synthesis inhibitor cycloheximide. Coexpression of v-Ha-Ras-, v-Raf-, and v-Src-activated beta E2 x 4, and constitutively active mutants of protein kinase C also increased transcription from this construct. Calcium ionophore generated signals which synergized with both Ras and protein kinase C. Expression of a truncated Raf protein which has been shown to act in a dominant negative manner, was able to inhibit activation of beta E2 x 4, demonstrating that Raf plays an important role in T cell signaling. A dominant negative mutant of v-Ha-Ras inhibited all methods of activation tested, including transfection with v-Raf. Thus, Ras activity appears to be necessary at more than one point in the transduction of signals from T cell receptor to nucleus.
Wotton
D
D
Laboratory of Lymphocyte Molecular Biology, Imperial Cancer Research Fund, London, United Kingdom.
Ways
D K
DK
Parker
P J
PJ
Owen
M J
MJ
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D003513:Cycloheximide / Q000494:pharmacology; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation / Q000187:drug effects; D011905:Genes, ras; D016391:Genes, src; D006801:Humans; D007700:Kinetics; D015459:Leukemia-Lymphoma, Adult T-Cell; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D015513:Oncogene Proteins / Q000096:biosynthesis*; D009857:Oncogenes; D011493:Protein Kinase C / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis; D019908:Proto-Oncogene Proteins c-raf; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D000199:Actins; D009838:Oligodeoxyribonucleotides; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D003513:Cycloheximide; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate