Cyclic AMP-dependent protein kinase controls basal gene activity and steroidogenesis in Y1 adrenal tumor cells.
Transfection of mouse Y1 adrenal tumor cells with DNA encoding mutant type I regulatory subunit generated stable transformants in which the basal activity of cAMP-dependent protein kinase was repressed. As expected, steroidogenesis in these kinase-deficient cells was no longer stimulated by corticotropin or cAMP analogues, and the expression of three cAMP-regulated genes (ornithine decarboxylase, urokinase-type plasminogen activator, and P450 side-chain cleavage) could no longer be induced. However, in addition to the loss of hormone responsiveness, the basal level of steroidogenesis and the constitutive expression of these cAMP-inducible genes was also repressed in kinase-defective mutant clones. To verify that functional cA-PK would revert this repressed phenotype, we transfected a cA-PK defective subclone of Y1 cells, Kin 8, with DNA encoding the C alpha and C beta subunits of cAMP-dependent protein kinase. Basal levels of steroid production were restored to normal in stable transformants, and the elevation of kinase activity following induction of the C-subunit expression vectors elicited a steroidogenic response. Gene transcription was also shown to be regulated by either C alpha or C beta as measured by the induction of plasminogen activator and ornithine decarboxylase mRNA levels and transcription rates. The dominant role played by cAMP-dependent protein kinase in these adrenal cells was demonstrated by experiments showing the regulation of ornithine decarboxylase gene expression by protein kinase C requires basal cAMP-dependent protein kinase activity.
Clegg
C H
CH
Department of Pharmacology, University of Washington, Seattle 98195.
Abrahamsen
M S
MS
Degen
J L
JL
Morris
D R
DR
McKnight
G S
GS
D000302:Adrenal Cortex / Q000166:cytology / Q000502:physiology*; D000310:Adrenal Gland Neoplasms; D000818:Animals; D015152:Blotting, Northern; D002384:Catalysis; D002454:Cell Differentiation; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D015536:Down-Regulation; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D051379:Mice; D009955:Ornithine Decarboxylase / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013256:Steroids / Q000096:biosynthesis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics
D012333:RNA, Messenger; D013256:Steroids; D003577:Cytochrome P-450 Enzyme System; D011494:Protein Kinases; D011493:Protein Kinase C; D014568:Urokinase-Type Plasminogen Activator; D009955:Ornithine Decarboxylase; D013755:Tetradecanoylphorbol Acetate