Cloning and characterization of the murine PKC alpha promoter: identification of a retinoic acid response element.
Protein kinase C (PKC) is a family which consists of multiple isoforms whose distinct physiological roles within the cell are unknown. We have previously demonstrated that levels of PKC alpha mRNA, protein, and enzyme activity in B16 melanoma cells can be modulated by retinoic acid. We investigated this regulation by cloning and characterizing the promoter region of the murine PKC alpha gene. A 13 kb mouse genomic fragment containing the 5' flanking region, first exon, and first intron was isolated and sequenced. Two transcription initiation sites were identified at 919 and 925 bp upstream from the translation start site. The promoter region contained a TATA-like box at -93 bp upstream of the transcription start site, but no CAAT box. Promoter activity differed between cell lines and correlated with the levels of PKC alpha expressed in these cell lines. Reporter gene assays showed that the region between -179 and -452 bp likely contains a silencer element(s). The promoter activity of a -179 bp fragment in B16 cells was stimulated twofold by retinoic acid. Within this region (-93 to -65 bp) there is a retinoic acid response element. An oligonucleotide spanning this region specifically bound exogenous RAR-RXR heterodimers and endogenous RAR from B16 nuclear extracts. These results suggest that retinoic acid increases PKC alpha gene expression in B16 cells, at least in part, through direct transcriptional stimulation of its promoter.
Desai
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DS
Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa Ku, Yokohama, 236, Japan. desai.dinakar@mayo.edu
Hirai
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S
Karnes
W E
WE
Niles
R M
RM
Ohno
S
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D005809:Genes, Regulator / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics*; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011493:Protein Kinase C / Q000235:genetics*; D051571:Protein Kinase C-alpha; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007527:Isoenzymes; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014212:Tretinoin; D004247:DNA; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha