Regulation of gene expression by transfected subunits of cAMP-dependent protein kinase.
cAMP regulates the expression of several genes by activation of a promoter consensus sequence which functions as a cAMP-response element. Evidence indicated that this is accomplished via cAMP dissociation of cAMP-dependent protein kinase into its regulatory (R) and catalytic (C) subunits. Our investigations of the role of these two subunits in gene expression provide direct and quantitative evidence that the C subunit is required for cAMP stimulation of the cAMP-response element in the vasoactive-intestinal-peptide gene in rat pheochromocytoma cells. After cotransfection of a metallothionein-regulated C-subunit expression vector (pCEV) and a vasoactive-intestinal-peptide--chloramphenicol acetyltransferase construct containing a cAMP-response element, we could demonstrate expression of transfected C-alpha-subunit mRNA (truncated size 1.7 kb) by Northern blot and a concentration-dependent C subunit stimulation of chloramphenicol acetyltransferase activity. Basal activity was stimulated 12- and 50-fold by pCEV (30 micrograms), in the absence and presence, respectively, of Zn2+. Metallothionein-regulated expression of C was demonstrated by results that showed a 2-4-fold increase in chloramphenicol acetyltransferase activity in the presence versus the absence of 90 microM Zn2+. In contrast, overexpression of the R-II beta regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R-II beta transfected together with C (ratio 2:1 and 4:1) inhibited the stimulation by the C subunit 70% and 90% respectively. Our results indicate that transfection of cAMP-dependent protein kinase subunits results in functional expression of both C-alpha and R-II beta subunits. Expression of the C subunit mediated cAMP-regulated gene expression but this expression could be inhibited by cotransfected R-II beta subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP-dependent protein kinase.
Büchler
W
W
Department of Internal Medicine, University of Würzburg, Federal Republic of Germany.
Meinecke
M
M
Chakraborty
T
T
Jahnsen
T
T
Walter
U
U
Lohmann
S M
SM
D000818:Animals; D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D005822:Genetic Vectors; D006801:Humans; D008668:Metallothionein / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011494:Protein Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
D012333:RNA, Messenger; D014660:Vasoactive Intestinal Peptide; D008668:Metallothionein; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases; D015032:Zinc