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.
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 PKC alpha and PKC delta (PKC alpha K and PKC delta K) were constructed. Transient transfection of PKC alpha K and PKC delta K into COS cells resulted in approximately 20- and 16-fold increase in phospholipid-, calcium-independent protein kinase activity. To determine the effects of overexpression of PKC alpha K and PKC delta K on the AP-1-mediated TPA-inducible genes, we transfected into COS cells the PKC alpha K or PKC delta 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. PKC alpha K or PKC delta K overexpression resulted in a comparable increase (approximately 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 (delta MTV-TyRE-pCAT). We also found that deletion of the AP-1-like motif in the SV40 promoter abolished the PKC alpha K or PKC delta K-induced activity of luciferase (luc) reporter constructs. Overexpression of full-length PKC delta in COS cells also increased the activity of the CAT construct with TRE after TPA treatment. We determined the effects of overexpression of PKC alpha K and PKC delta K on transcription of the ornithine decarboxylase (ODC) gene, which has a non-AP-1 TRE. Cotransfection of PKC alpha K or PKC delta K expression plasmids with a TPA-inducible ODC luc construct (-72/+130-ODC-luc) into HeLa cells resulted in an increased luc activity. These results indicate that both PKC alpha (calcium dependent) and PKC delta (calcium independent) may mediate the transcription of TPA-inducible genes through both AP-1 and non-AP-1 sequences.
Tseng
C P
CP
Department of Human Oncology, University of Wisconsin Comprehensive Cancer Center, Madison 53792.
Kim
Y J
YJ
Kumar
R
R
Verma
A K
AK
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000235:genetics; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D017931:DNA Primers; D007527:Isoenzymes; D016755:Proto-Oncogene Proteins c-jun; D011493:Protein Kinase C; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium