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 thr 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.