The C gamma subunit is a unique isozyme of the cAMP-dependent protein kinase.
There are at least three isozymes (C alpha, C beta, and C gamma) of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase (PKA) (Beebe, S., Oyen, O., Sandberg, M., Froysa, A., Hansson, V., and Jahnsen, T. (1990) Mol. Endocrinol. 4, 465-475). To compare the C gamma and C alpha isozymes, the respective cDNAs were expressed in permanently transformed Kin-8 PKA-deficient Y1 adrenal cells using the mouse metallothionein promoter. The recombinant C subunits were characterized as immunoreactive, zinc-inducible, cAMP-dependent kinase activities. In contrast to C alpha, histone was a better substrate than Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) for C gamma. Furthermore, C gamma histone kinase activity was not inhibited by the protein kinase inhibitor peptide (5-24 amide), which has been widely used as a PKA-specific inhibitor. The major C gamma peak (type I) eluted from DEAE-Sepharose at a higher NaCl concentration (120 mM) than the C alpha type I eluted (70 mM). C gamma and C alpha type II eluted between 220 and 240 mM NaCl. C gamma required higher concentrations of cAMP than C alpha did for dissociation from the mutant type I holoenzyme. These differences provided a basis for the separation of the mutant RI-associated isozymes on DEAE-Sepharose. Both C alpha (41-42 kDa) and C gamma (39-40 kDa) were identified by a C subunit antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. Zinc induced the PKA-mediated rounding phenotype in C gamma and C alpha clones, thereby restoring the cells to the parent Y1 adrenal cell phenotype. Collectively, these data indicate that C gamma is an active PKA C subunit but suggest that C gamma and C alpha have different protein and peptide recognition determinants.
Beebe
S J
SJ
Department of Pediatrics, Eastern Virginia Medical School, Norfolk 23501.
Salomonsky
P
P
Jahnsen
T
T
Li
Y
Y
D000311:Adrenal Glands; D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000494:pharmacology; D004790:Enzyme Induction; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
D007527:Isoenzymes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008668:Metallothionein; D000242:Cyclic AMP; D011494:Protein Kinases; D015032:Zinc