Role of protein kinase C in ras-mediated fos-expression

Abstract

HC-11 mouse mammary epithelial cells stably transfected with a glucocorticoid-inducible Ha- ras construct encoding a transforming (val 12) p21 Ha- ras were cotransfected with a c- fos-CAT construct containing the human c- fos promoter up to position −711 and the CAT reporter gene. Expression of Ha- ras by dexamethasone leads to a transcriptional activation of the fos-CAT construct which was found to be sensitive to the PKC inhibitor ilmofosine (BM41440) and abrogated by PKC depletion following long-term exposure to TPA. The responsiveness to Ha- ras is retained if only the portion of the fos promoter covering the serum response element (SRE) and the adjacent fos AP-1 (FAP) site are put in front of a CAT gene linked to a thymidine kinase (TK) promoter. Further depletion of the FAP-site does not affect the inducibility by Ha- ras. Transcriptional activation of the SRE-FAP-TK-CAT as well as the SRE-TK-CAT constructs by Ha- ras is sensitive to the PKC-inhibitor ilmofosine (BM41440) and blocked by long-term exposure to TPA. Long-term exposure to TPA depletes cells of PKC α and significantly reduces the PKC ϵ levels. Long-term exposure in bryostatin 1 selectively depletes PKC α. Depletion of PKC α by bryostatin 1 does not reduce the transcriptional activation of the SRE-FAP-TK-CAT-construct by Ha- ras. It is concluded that (i) transforming Ha- ras induces c- fos in HC-11 cells via PKC (presumably ϵ), (ii) the signal is mediated to the serum response element (SRE) of the fos promoter and (iii) the fos AP-1 (FAP) site is not required for this mechanism.