Ets and GATA Transcription Factors Play a Critical Role in PMA-Mediated Repression of the ck[beta] Promoter via the Protein Kinase C Signaling Pathway

Choline kinase is the most upstream enzyme in the CDP-choline pathway. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg.sup.2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ck[alpha] and ck[beta], which produce three isoforms, CK[alpha]1, CK[alpha]2, and CK[beta]. Previous studies have associated ck[beta] with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ck[beta] has never been elucidated. In this report, the distal promoter region of the ck[beta] gene was characterized. Mutational analysis of the promoter sequence and electrophoretic mobility shift assays (EMSA) showed that Ets and GATA transcription factors were essential for the repression of ck[beta] promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ck[beta] promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ck[beta] promoter activity through Ets and GATA elements. PMA also decreased the ck[beta] mRNA and protein levels about 12 hours after the promoter activity was down-regulated. EMSA further revealed that PMA treatment increased the binding of both Ets and GATA transcription factors to their respective DNA elements. The PMA-mediated repressive effect was abolished by chronic PMA treatment and by treatment with the PKC inhibitor PKC412, but not the PKC inhibitor Go 6983, suggesting PKC[epsilon] or PKC[eta] as the PKC isozyme involved in the PMA-mediated repression of ck[beta] promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKC[epsilon] as the isozyme that mediated the PMA repression of ck[beta] promoter. These results demonstrate the participation of the PKC signaling pathway in the regulation of ck[beta] gene transcription by Ets and GATA transcription factors.