Protein kinase Ciota enhances the transcriptional activity of the porcine P-450 side-chain cleavage insulin-like response element.
IGF-I enhances steroidogenesis in granulosa cells by stimulating the expression of the rate-limiting steroidogenic enzyme, cytochrome P-450 side-chain cleavage (P-450(scc)). This effect is mediated through an IGF response element (IGFRE) that binds polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF) and Sp1. Sp1 is essential for activation of the IGFRE, and PSF functions as a repressor. We investigated mechanisms of modulation of the IGFRE by the atypical protein kinase C (PKC)iota in a porcine stable granulosa cell line, JC-410. PKCiota was found in nuclear extracts, and levels were increased by IGF-I after 24 and 48 h of treatment. Immunoprecipitation experiments demonstrated that PSF and PKCiota associated with each other in nuclear extracts from JC-410 cells. Transient transfection with expression plasmids of kinase-active and kinase-deficient PKCiota isoforms enhanced transcriptional activity of the IGFRE regardless of kinase catalytic activity. Depletion of PKCiota protein by small interfering RNA suppressed basal IGFRE activity but did not prevent IGF-I stimulation of the IGFRE. We conclude that PKCiota enhances transcriptional activity of the porcine P-450(scc) IGFRE independently of kinase activity by a mechanism involving protein-protein interaction with PSF.
Urban
Randall J
RJ
Department of Internal Medicine, The University of Texas Medical Branch, Galveston, TX 77555-1060, USA. rurban@utmb.edu
Bodenburg
Yvonne H
YH
Jiang
Jie
J
Denner
Larry
L
Chedrese
Jorge
J
D000818:Animals; D002467:Cell Nucleus / Q000502:physiology; D002478:Cells, Cultured; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D006107:Granulosa Cells / Q000166:cytology / Q000502:physiology; D007527:Isoenzymes / Q000378:metabolism*; D010957:Plasmids; D011493:Protein Kinase C / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D020218:Response Elements / Q000502:physiology*; D013002:Somatomedins / Q000235:genetics; D034421:Sus scrofa; D015533:Transcriptional Activation / Q000502:physiology; D014162:Transfection; D000099293:Protein Kinase C-lambda
D002786:Cholesterol Side-Chain Cleavage Enzyme; D007527:Isoenzymes; D011493:Protein Kinase C; D034741:RNA, Small Interfering; D013002:Somatomedins; D000099293:Protein Kinase C-lambda