Autoregulation of cloned human protein kinase C beta and gamma gene promoters in U937 cells

Abstract

Protein kinase C (PKC) serine/threonine kinases transduce cellular signals initiated by phospholipase C activation and diacylglycerol production. Human gene sequences from the beta and gamma isoforms were cloned and sequenced, and transcriptional regulation was studied. The major PKC beta transcription initiation site was identified by primer extension and S1 nuclease protection. Additional transcription initiation sites were located within a CpG-rich region proximal to the ATG. The transcription initiation site of the PKC gamma gene was identified by primed cDNA synthesis. In transfection experiments, the PKC gamma promoter was expressed at high level in U937 and HL60 cells but not in COS-1 cells. A sequence motif (AnAGATTCanAGAGnCa), reiterated over at least 1 kb, was located approximately 1.5 kb 5' of the PKC gamma translation initiation codon. This repetitive motif is abundant in run-on RNA in the hematopoietic and epithelial cell lines tested. Analysis of promoter deletion constructs by transient transcription assays in U937, IM9, and COS-1 cells showed negative regulation of the PKC beta promoter by sequences located between -3,000 and -690. although no homology between PKC beta and PKC-gamma 5'-flanking sequences was observed, both PKC beta and PKC gamma promoters were potently induced by 12-phorbol 13-myristate in transfected U937 cells.