Expression of cDNAs for two isoforms of the catalytic subunit of cAMP-dependent protein kinase.
We have previously reported the cloning of cDNAs coding for two isoforms (termed C alpha and C beta) of the catalytic (C) subunit of cAMP-dependent protein kinase (Uhler, M., Chrivia, J., and McKnight, G. S. (1986) J. Biol. Chem. 261, 15360-15363). We have constructed cDNA expression vectors for the C alpha and C beta proteins using the mouse metallothionein promoter so that mRNA transcripts from the expression vectors are inducible with Zn. In stable transformants of NIH 3T3 and AtT-20 cells, the induction of both C alpha and C beta mRNA with Zn increases the cAMP-dependent as well as the cAMP-independent kinase activity. The C alpha and C beta proteins synthesized from these expression vectors can be detected by Western blot analysis and have slightly different molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Interestingly, the levels of RI protein are also shown to increase in cells expressing high levels of the C alpha or C beta subunits although there is no change in RI mRNA. In contrast, the levels of RII protein are not significantly affected by increasing either C alpha or C beta expression in these cell lines. We conclude that cells can compensate for the increased levels of either C alpha or C beta subunits with a corresponding elevation of RI protein implying that both isoforms of C can interact with RI to form a holoenzyme. The relevance of these experimental results to the coordinate regulation of cAMP-dependent protein kinase subunits is discussed.
Uhler
M D
MD
Department of Pharmacology, University of Washington, Seattle 98195.
McKnight
G S
GS
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D005822:Genetic Vectors; D007527:Isoenzymes / Q000235:genetics*; D046911:Macromolecular Substances; D051379:Mice; D010903:Pituitary Gland, Anterior / Q000201:enzymology; D011494:Protein Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D007527:Isoenzymes; D046911:Macromolecular Substances; D012333:RNA, Messenger; D004247:DNA; D011494:Protein Kinases