Mutations in the catalytic subunit of cAMP-dependent protein kinase result in unregulated biological activity.
Mutations were identified in the catalytic subunit (C) of the cAMP-dependent protein kinase (EC 2.7.1.37) that block inactivation by regulatory subunit (R) without compromising catalytic activity. Randomly mutagenized mouse C expression vectors were screened functionally for clones that stimulated gene induction in the presence of excess R. Point mutations in the C coding sequence were identified that result in a His----Gln substitution at amino acid 87 (His87Gln) and a Trp----Arg change at amino acid 196 (Trp196Arg). In contrast to wild-type C, both mutants retained partial activity in the presence of excess R isoform RI alpha, although only Trp196Arg retained partial activity in the presence of excess R isoform RII alpha. A C expression vector that included both mutations was fully active in promoting gene induction and was virtually unaffected by an 80-fold excess of either RI alpha or RII alpha. These results demonstrate that mutations at His-87 and Trp-196 alter R interactions with C at a site that is not involved in substrate recognition or enzymatic activity. In contrast to these randomly generated mutations, a site-specific alteration of the autophosphorylated Thr-197 to an Ala resulted in an 80% loss of biological activity and partial resistance to R inhibition. The location and proximity of His-87 and Trp-196 in the crystal structure of C suggest a surface domain that may interact with a region of R that is outside of the substrate/pseudosubstrate site.
Orellana
S A
SA
Department of Pharmacology, University of Washington, Seattle 98195.
McKnight
G S
GS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002822:Choriocarcinoma; D003062:Codon / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D003062:Codon; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D011494:Protein Kinases