Second-site mutations in cyclic AMP-Sensitive revertants of a Ka mutant of S49 mouse lymphoma cells reduce the affinity of regulatory subunit of cyclic AMP-Dependent protein kinase for catalytic subunit

Ka mutants of S49 mouse lymphoma cells are generally heterozygous for expression of wild‐type and mutant regulatory (R) subunits of type lα cyclic AMP‐(cAMP)‐dependent protein kinase, where the mutant R subunit has a defect in cAMP‐binding to one of two intrachain cAMP‐binding sites. Several cAMP‐sensitive revertants of such a Ka mutant were found previously to harbor second‐site mutations in the mutant allele, and we have now identified three such mutations by sequence analysis of PCR‐amplified cDNAs. The resulting amino acid changes were Ala98 to Thr, Gly179 to Arg, or Gly224 to Asp. The Ka mutation in these strains (Glu201 to Lys) eliminated cAMP‐binding to the more aminoterminal cAMP‐binding site (site A). None of the second‐site mutations restored this activity in bacterially expressed recombinant R subunit. On the other hand, all three second‐site mutations reduced the apparent affinity of the mutant R subunit for catalytic (C) subunit with the effects of the substitutions at Ala98 and Gly179 substantially greater than the effect of the substitution at Gly224. Patterns of phosphorylation and turnover of wild‐type and mutant R subunits in intact revertant cells were consistent with reduced association of the doubly mutant subunits with C subunit, but the free mutant subunits apparently were more stable than free wild‐type subunits. Differences in metabolic turnover of mutant and wild‐type subunits did not correlate with the sensitivities of the isolated proteins to proteolytic cleavage. © 1995 Wiley‐Liss, Inc.