Hormonal activation of gene transcription in ras-transformed NIH3T3 cells overexpressing RII alpha and RII beta subunits of the cAMP-dependent protein kinase.
ras-Transformed NIH3T3 (R3T3) cells were transfected with expression vectors for the RII alpha and RII beta regulatory subunits of the type II isozyme of cAMP-dependent protein kinase, and the effects on gene activation by corticotropin-releasing factor (CRF) and prostaglandin E1 (PGE1) were analyzed. In RII alpha and RII beta-overexpressing cells, type II isozyme levels were increased, and type I isozyme levels were eliminated, demonstrating that both RII regulatory subunits compete efficiently with RI for catalytic subunit. The type II isozyme separated into three peaks on high performance liquid chromatography, referred to as A, B, and C. Western blot analysis strongly suggests that peak A and peak C correspond to holoenzymes containing RII beta and RII alpha, respectively. Overexpression of RII alpha resulted in the loss of peak A and a dramatic reduction in RII beta protein with no change in RII beta mRNA, indicating that the level of RII beta protein is controlled posttranscriptionally and that RII beta protein may become unstable when displaced from C. The role of type I and II kinases in transcriptional activation was investigated by comparing the response of control and RII expressing clones to site-selective cAMP analogs and the hormones, CRF and PGE1. The site-selective analogs demonstrated that either type I or type II kinase could activate the cAMP-responsive alpha-subunit promoter. The response to various concentrations of CRF or PGE1 was identical in control cells and transfected clones containing very little type I kinase. These experiments suggest that in the CRF and PGE1 response pathways leading to gene induction, the magnitude and sensitivity of the response are not influenced by the presence or absence of type I cAMP-dependent protein kinase.
Otten
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Department of Pharmacology, University of Washington, Seattle 98195.
Parenteau
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LA
Døskeland
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McKnight
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D016475:3T3 Cells; D000527:Alprostadil / Q000502:physiology; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002851:Chromatography, High Pressure Liquid; D003346:Corticotropin-Releasing Hormone / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D011905:Genes, ras; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D007527:Isoenzymes; D003346:Corticotropin-Releasing Hormone; D011494:Protein Kinases; D000527:Alprostadil