Involvement of protein kinase C in the transcriptional regulation of 12-O-tetradecanoylphorbol-13-acetate-inducible genes modulated by AP-1 or non-AP-1 transacting factors

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Abstract
The involvement of protein kinase C (PKC), a 12-O- tetradecanoylphorbol-13-acetate (TPA) receptor, in the transcriptional regulation of TPA-inducible genes was determined. Expression plasmids harboring full-length or kinase domain of PKCα and PKC§ (PKCαK and PKC§K) were constructed. Transient transfection of PKCαK and PKC§K into COS cells resulted in ∼20- and 16-fold increase in phospholipid-, calcium-independent protein kinase activity. To determine the effects of overexpression of PKCαK and PKC§K on the AP-1-mediated TPA-inducible genes, we transfected into COS cells the PKCαK or PKC§K expression plasmids with collagenase chloramphenicol acetyltransferase (CAT) reporter construct containing one TPA responsive element (TRE), or a construct containing five synthetic TRE linked to a thymidine kinase promoter. PKCαK or PKC§K overexpression resulted in a comparable increase (∼ 4-fold) in CAT activity. However, CAT activity was not increased after transfection of PKC constructs with non-TPA responsive thyroid hormone responsive elements CAT construct (ΔMTV TyRE-pCAT). We also found that deletion of the AP-1-like motif in the SV40 promoter abolished the PKCαK- or PKC§K-induced activity of luciferase (luc) reporter constructs. Overexpression of full-length PKC§ in COS cells also increased the activity of the CAT construct with TRE after TPA treatment. We determined the effects of overexpression of PKCαK and PKC§K on transcription of the ornithine decarboxylase (ODC) gene, which has a non-AP-1 TRE. Cotransfection of PKCαK or PKC§K expression plasmids with a TPA-inducible ODC Iuc construct (−72/+130-ODC-Iuc) into HeLa cells resulted in an increased Iuc activity. These results indicate that both PKCα (calcium dependent) and PKC§ (calcium independent) may mediate the transcription of TPA-inducible genes through both AP-1 and non-AP-1 sequences.

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