A series of mutant cell lines (Kin) were previously isolated from Y1 adrenocortical tumor cells based on their ability to resist the growth-inhibitory effects of 8-bromo cAMP. In these Kin clones, cAMP-dependent protein kinase (cAMPdPK) was resistant to activation by cAMP as the consequence of mutations affecting the type I regulatory subunit (RI) of the enzyme. This study shows that the cAMP-resistant phenotypes of mutant clones Kin-2, Kin-7, and Kin-8 were associated with single base changes causing substitutions, respectively, of Glu for Gly200, Trp for Arg334, and Asp for Gly324 in the RI protein. By expressing the mutant Trp334 and Asp324 forms of RI under the control of an inducible promoter in Y1 cells, the causal relationship between these RI mutations and impairment of cAMP-stimulated adrenocortical responses was studied. Expression of the mutant RI forms rendered cAMPdPK resistant to activation by cAMP and decreased cAMP-stimulated cell rounding, steroid production, and growth inhibition. These observations indicate that the cAMP-resistant phenotype of Kin mutant clones resulted specifically from single mutational events in RI and thus establish the importance of cAMPdPK as an essential regulator of adrenocortical function. Unlike the original Kin mutant clones, transformants expressing the mutant forms of RI had adenylyl cyclases that were resistant to activation by ACTH, forskolin, or sodium fluoride. These results indicate that there may be a hitherto unappreciated mechanism of regulation of adenylyl cyclase activity by cAMPdPK.

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Mutants of S49 mouse lymphoma cells resistant to cytolysis by analogs of cyclic AMP (cAMP) generally have missense mutations in the gene encoding the regulatory subunit of cAMP-dependent protein kinase. We have compared the mutations in 95 spontaneous isolates with those in 60 mutagen-induced isolates by sequence analysis of amplified cDNAs. Twenty-nine single basepair substitutions in 19 codons produced selectable phenotypes. The spontaneous mutant spectrum was dominated by a CpG transition hotspot in the codon for Arg334. This and other nearby CpG sites were found to be methylated in genomic S49 cell DNA by restriction enzyme analyses. Most of the remaining spontaneous mutants had either G-C-->C-G or T-A-->G-C transversions, which have been associated with damage caused by oxygen radicals. In contrast, the majority of mutants induced with the alkylating mutagens ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine had G-C-->A-T mutations at non-CpG sites; in addition, EMS induced several A-T-->G-C, A-T-->T-A, and G-C-->T-A substitutions. A single ICR191-induced mutant analyzed had a unique A-T-->G-C lesion. A number of spontaneous and mutagen-induced isolates had closely linked double or triple substitutions, and two isolates had tandem triple substitutions.

are thought to result from hydrolytic deamination of 5-methylcytosine residues in these sites. The gene for regulatory subunit of murine cyclic AMP-dependent protein kinase has two closely linked CpG sites, one of which is a strong hotspot for spontaneous CG-+TA mutations leading to cyclic AMP resistance in S49 mouse lymphoma cells. About 5% of mutants with a spontaneous mutation at this CpG site had also acquired a second CG-oTA mutation at the nearby CpG site. The two mutations were always at first positions of the Arg codons in which they occurred, and they were always together in a single regulatory subunit allele. Their linked appearance could be attributed to neither the selection conditions nor the preexistence of one mutation in the target cells. The high frequency of these double mutants suggests that their lesions result not from hydrolytic deamination but rather from an endogenous enzymatic mechanism.Spontaneous point mutations are thought to arise from errors in replicative or repair synthesis of DNA, the chemical effects of background ionizing radiation, and/or the chemical instability of normal or modified DNA bases (6, 18). Their low frequency at any one allele has suggested that the flux of DNA-damaging events is low and distributed throughout the genome. Transition mutations at CpG dinucleotide sequences account for about 25 to 40% of known point mutations leading to human genetic disorders or cancer and are thought to arise by spontaneous hydrolytic deamination of 5-methylcytosine (5-Me-C) residues, which occur most frequently at CpG sites (3, 7, 13). Although it is impossible to know a priori whether these human mutations were spontaneous or induced by exposure to mutagens, support for the deamination pathway comes from a recent report showing that CpG hotspots for mutations in the low-density lipoprotein receptor and the p53 tumor suppressor genes are indeed methylated in vivo (19).Mutants of the cyclic AMP (cAMP)-sensitive S49 mouse lymphoma cell line selected for resistance to cAMP analogs have provided abundant material for study of missense mutations in a mammalian gene. The most common mutants have lesions in the regulatory (R) or cAMP-binding subunit of cAMP-dependent protein kinase that increase apparent constants (Kas) of the enzyme for cAMP-dependent activation (16,23). In a recent study of sequence changes underlying such lesions in S49 sublines hemizygous for expression of mutant R subunits with altered charge, we found 8 distinct single-base-change mutations clustered, for the most part, in regions identified with the two cAMP-binding sites of R subunit, sites A and B (22); subsequently, we have identified 14 additional point mutations associated with Ka phenotypes in hemizygous or heterozygous mutant cells (10). Among spontaneous isolates, the most frequent mutation was a CG-+TA transition in the site B region causing substitution of Trp for Arg-334. Among mutants heterozygous for expression of mutant R subunits, we found several mutageninduced isolates that had this Trp-334 muta...

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