A NOVEL 3',5'-CYCLIC ADENOSINE MONOPHOSPHATE-RESPONSIVE SEQUENCE IN THE BOVINE CYP17 GENE IS A TARGET OF NEGATIVE REGULATION BY PROTEIN-KINASE-C

The bovine 17-alpha-hydroxylase cytochrome P450 gene (CYP17) contains at least two cAMP-responsive sequences (CRS) within its 5'-flanking region. In this study it is demonstrated that one of the sequences, CRS1, is also a target for protein kinase C (PKC)-mediated regulation. Forskolin-induced, CRS1-dependent transcription of a heterologous minimal promoter/structural gene which had been transfected into the mouse adrenocortical tumor cell line Y1 was suppressed by activation of PKC by phorbol esters such as 12-O-tetradecanoyl phorbol-14-acetate and phorbol 12,13-didecanoate-beta (PDD-beta). Use of the active and inactive forms of PDD (PDD-alpha and PDD-beta) as well as down-regulation of PKC by prolonged treatment of the cells with 12-O-tetradecanoyl phorbol-14-acetate demonstrated that the effect of phorbol esters on transcription conferred by CRS1 was mediated through the PKC pathway and not a consequence of general toxicity to the cells. Analysis of the different steps in the signal transduction pathway between the adenylate cyclase and the CRS1 element suggests that phorbol esters do not exert their effect by altering the forskolin-induced cAMP production, activation of PKA, or the binding of nuclear proteins to CRS1. These results establish the CRS1 element as a target not only for PKA, but also for the PKC-mediated signal transduction pathway. They further suggest that PKC interferes with the transcriptional activation competence of factors bound to CRS1 and the minimal promoter.