Modulation of the human protein kinase C alpha gene promoter by activator protein-2.
Protein kinase Calpha (PKCalpha) is a phospholipid-dependent protein-serine/threonine kinase that plays a major role in intracellular signaling pathways associated with transformation and tumor progression. Glioblastoma multiforme (GBM) and GBM cell lines exhibit increased levels of PKCalpha compared to normal brain tissue that relates to their proliferative and invasive potential. To investigate the transcriptional regulation of PKCalpha, the 5'-flanking sequence of the human PKCalpha gene was cloned and its promoter activity assessed in U-87 GBM cells. This sequence contained a TATA-less promoter region and a single transcription start site within an initiator sequence. Basal promoter activity was restricted to a region spanning -227 to +77 relative to the transcription start site. DNase I footprinting revealed multiple activator protein-2 (AP-2) binding sites and one Sp1 binding site within this region, and point mutations of two AP-2 elements resulted in a loss of DNA binding and transcriptional activation. Overexpression of Sp1 in either U-87 or insect cells increased transcription from the -227/+77 promoter region, whereas overexpression of AP-2 increased transcription only in insect cells. Cis activation of the promoter in U-87 cells was increased by phorbol esters but not by cyclic AMP or phosphatidylinositol 3-kinase inhibitors. These results provide evidence that cis activation of the basal promoter of the human PKCalpha gene occurs through an AP-2-dependent, phorbol ester-responsive pathway, which suggests an autoregulatory manner of transcription in GBM.
Clark
Joannah Hackenbruck
JH
Department of Pharmacology, Lombardi Cancer Center, Georgetown University School of Medicine, 3970 Reservoir Road NW, Washington, D.C. 20007, USA.
Haridasse
Vedanandam
V
Glazer
Robert I
RI
D020121:5' Untranslated Regions / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000502:physiology*; D004789:Enzyme Activation / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D005909:Glioblastoma / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011493:Protein Kinase C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D015290:Second Messenger Systems / Q000187:drug effects / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000502:physiology*; D024363:Transcription Initiation Site; D014162:Transfection; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D016329:Sp1 Transcription Factor; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D015240:Phorbol 12,13-Dibutyrate; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D013755:Tetradecanoylphorbol Acetate