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.