Protein kinase C isoforms involved in the transcriptional activation of cyclin D1 by transforming Ha-Ras-1

Transcriptional activation of the cyclin D1 by oncogenic Ras appears to be mediated by several pathways leading to the activation of multiple transcri ption factors which interact with distinct elements of the cyclin D1 promot er. The present investigations revealed that cyclin D1 induction by transfo rming Ha-Ras is MEK and Rac-dependent and requires the PKC isotypes epsilon , lambda, and zeta, but not cPKC-alpha. This conclusion is based on observa tions indicating that cyclin D1 induction by transforming Ha-Ras was depres sed in a dose-dependent manner by PD98059, a selective inhibitor of the mit ogen-activated kinase kinase MEK-1, demonstrating that Ha-Ras employs extra cellular signal-regulated kinases (ERKs) for signal transmission to the cyc lin D1 promoter. Evidence is presented that PKC isotypes epsilon and zeta, but not lambda are required for the Ras-mediated activation of ERKs. Expres sion of kinase-defective, dominant negative (DN) mutants of nPKC-epsilon or aPKC-zeta inhibit ERK activation by constitutively active Raf-1. Phosphory lation within the TEY motif and subsequent activation of ERKs by constituti vely active MEK-1 was significantly inhibited by DN aPKC-zeta, indicating t hat aPKC-zeta functions downstream of MEK-1 in the pathway leading to cycli n D1 induction. In contrast, TEY phosphorylation induced by constitutively active MEK-1 was not effected by nPKC-epsilon, suggesting another position for this kinase within the cascade investigated. Transformation by oncogeni c Ras requires activation of several Ras effector pathways which may be PKC -dependent and converge on the cyclin D1 promoter. Therefore, we investigat ed a role for PKC isotypes in the Ras-Rac-mediated transcriptional regulati on of cyclin D1. We have been able to reveal that cyclin D1 induction by on cogenic Ha-Ras is Rac-dependent and requires the PKC isotypes epsilon, lamb da, and zeta, but not cPKC-alpha. Evidence is presented that aPKC-lambda ac ts upstream of Rac, between Ras and Rac, whereas the PKC isotypes epsilon a nd zeta act downstream of Rac and are required for the activation of ERKs.