mRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase.
The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.
D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D007525:Isoelectric Focusing; D046911:Macromolecular Substances; D051379:Mice; D009154:Mutation; D011061:Poly A / Q000378:metabolism; D011494:Protein Kinases / Q000096:biosynthesis*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*
D046911:Macromolecular Substances; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D000242:Cyclic AMP; D011494:Protein Kinases