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‐α‐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 μg), 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 μM Zn2+. In contrast, overexpression of the R‐IIβ regulatory subunit did not stimulate chloramphenicol acetyltransferase activity, and R‐IIβ 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‐α and R‐IIβ subunits. Expression of the C subunit mediated cAMP‐regulated gene expression but this expression could be inhibited by cotransfected R‐IIβ subunit, indicating intracellular reconstitution of the inactive holoenzyme of cAMP‐dependent protein kinase. Copyright © 1990, Wiley Blackwell. All rights reserved