Abstract
BackgroundCholine 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 Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. In humans, choline kinase (CK) is encoded by two separate genes, ckα and ckβ, which produce three isoforms, CKα1, CKα2, and CKβ. Previous studies have associated ckβ with muscle development; however, the molecular mechanism underlying the transcriptional regulation of ckβ has never been elucidated.Methodology/Principal FindingsIn this report, the distal promoter region of the ckβ 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 ckβ promoter activity. Supershift and chromatin immunoprecipitation (ChIP) assays further identified that GATA3 but not GATA2 was bound to the GATA site of ckβ promoter. In addition, phorbol-12-myristate-13-acetate (PMA) decreased ckβ promoter activity through Ets and GATA elements. PMA also decreased the ckβ 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 PKCε or PKCη as the PKC isozyme involved in the PMA-mediated repression of ckβ promoter. Further confirmation by using PKC isozyme specific inhibitors identified PKCε as the isozyme that mediated the PMA repression of ckβ promoter.Conclusion/SignificanceThese results demonstrate the participation of the PKC signaling pathway in the regulation of ckβ gene transcription by Ets and GATA transcription factors.
Highlights
Choline kinase (CK) (EC 2.7.1.32) uses Mg2+ as a cofactor to catalyze the ATPdependent phosphorylation of choline, yielding phosphocholine (PCho) and ADP
The analysis showed that the ckb promoter contains neither a CAAT box nor a recognizable consensus TATA box in close proximity to the transcription start site, which is typical of GC-rich promoters
We have previously shown that a promoter region located 2000 bp upstream of the ckb start codon was transcriptionally active in MCF-7 cells [17]
Summary
Choline kinase (CK) (EC 2.7.1.32) uses Mg2+ as a cofactor to catalyze the ATPdependent phosphorylation of choline, yielding phosphocholine (PCho) and ADP. CKs are encoded by two separate genes, cka and ckb Based on their high sequence homology, cka and ckb might have appeared after genetic duplication from a common ancestor [7]. It catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP and Mg2+ during the biosynthesis of phosphatidylcholine, the major phospholipid in eukaryotic cell membranes. Choline kinase (CK) is encoded by two separate genes, cka and ckb, which produce three isoforms, CKa1, CKa2, and CKb. Previous studies have associated ckb with muscle development; the molecular mechanism underlying the transcriptional regulation of ckb has never been elucidated. Phorbol-12-myristate-13-acetate (PMA) decreased ckb promoter activity through Ets and GATA elements. Further confirmation by using PKC isozyme specific inhibitors identified PKCe as the isozyme that mediated the PMA repression of ckb promoter
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