Hotspots for spontaneous and mutagen-induced lesions in regulatory subunit of cyclic AMP-dependent protein kinase in S49 mouse lymphoma cells.

From an S49 mouse lymphoma cell subline that carries an electrophoretic marker mutation in one allele for a regulatory (R) subunit of cyclic AMP-dependent protein kinase, 130 cyclic AMP-resistant mutants were isolated and characterized. Of the 77 independent spontaneous and mutagen-induced isolates identified, 74 had kinases with increased apparent activation constants (KaS) for cyclic AMP-dependent activation. The "Ka" phenotype was invariably correlated with an apparent structural lesion in one R subunit allele. "Charge-shift" lesions in 43 independent isolates were mapped to small regions within the R subunit by two-dimensional gel analysis of partial proteolysis peptides. Nine Ka mutations were distinguished by differences in charge or peptide maps of mutant R subunits, and the mutations were clustered in two regions associated with the cyclic AMP-binding sites of the R subunit. The relative frequencies of different mutations differed among spontaneous, ethyl methanesulfonate-induced, and N-methyl-N'-nitro-N-nitrosoguanidine-induced isolates. Mutation frequencies were also markedly different for the two R subunit alleles; this allele preference was strongest for mutagen-induced lesions in the more carboxy terminal cyclic AMP-binding site.