Autoregulation of Cloned Human Protein Kinase C β and γ 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 β and γ isoforms were cloned and sequenced, and transcriptional regulation was studied. The major PKCβ 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 PKCγ gene was identified by primed cDNA synthesis. In transfection experiments, the PKCγ 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 PKCγ 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 PKCβ promoter by sequences located between −3,000 and −690. Although no homology between PKCβ and PKC-γ 5′-flanking sequences was observed, both PKCβ and PKCγ promoters were potently induced by 12-phorbol 13-myristate in transfected U937 cells.