Autoregulation of Cloned Human Protein Kinase C Î² and Î³ Gene Promoters in U937 Cells
AbstractProtein kinase C (PKC) serine/threonine kinases transduce cellular signals initiated by phospholipase C activation and diacylglycerol production. Human gene sequences from the Î² and Î³ isoforms were cloned and sequenced, and transcriptional regulation was studied. The major PKCÎ² transcription initiation site was identified by primer extension and S 1 nuclease protection. Additional transcription initiation sites were located within a CpG-rich region proximal to the ATG. The transcription initiation site of the PKCÎ³ gene was identified by primed cDNA synthesis. In transfection experiments, the PKCÎ³ 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 5â² of the PKCÎ³ 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 PKCÎ² promoter by sequences located between â3,000 and â690. Although no homology between PKCÎ² and PKC-Î³ 5â²-flanking sequences was observed, both PKCÎ² and PKCÎ³ promoters were potently induced by 12-phorbol 13-myristate in transfected U937 cells.