On the spontaneous mutability of CpG sites in cultured S49 mouse lymphoma cells.

The frequent occurrence of human mutations at CpG dinucleotide sites has been attributed to cytosine methylation and hydrolytic deamination of the resulting 5-methylcytosine residue. Previously, we reported an unusually strong hotspot for spontaneous transitions at a CpG site in the gene for regulatory (R) subunit of protein kinase A in S49 mouse lymphoma cells. Now, using polymerase chain reaction-based methods to screen mutant populations for mutations at particular CpG sites, we show that two methylated CpG sites in the gene for hypoxanthine phosphoribosyltransferase are much less mutable than the R subunit hotspot site, suggesting that different methylated CpG sites are differentially susceptible to spontaneous mutation. We also present data on spontaneous R subunit mutations in cloned populations of 5-azacytidine-treated S49 cells that had been demethylated at the hotspot site in both R subunit alleles. Of 13 independent mutants isolated from populations grown from fully demethylated cells, seven had the hotspot mutation. We conclude that CG-->TA mutations at strong CpG hotspots do not require prior methylation of CpG sites.

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase.

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.

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 I alpha 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.

Arg-242 is necessary for allosteric coupling of cyclic AMP-binding sites A and B of RI subunit of cyclic AMP-dependent protein kinase.

The functional consequences of Arg-242 to Ser or Lys substitutions in type I alpha regulatory (R) subunits of cAMP-dependent protein kinase were analyzed by using recombinant murine R subunits expressed in Escherichia coli. These mutations arose in cAMP-resistant mutants to S49 mouse lymphoma cells and were shown previously to inhibit cAMP binding to site A, the more amino-terminal of two intrachain cAMP-binding sites. Binding of cAMP to site A of the mutant R subunits could be detected by cAMP-dependent quenching of endogenous tryptophan fluorescence, [3H]cAMP binding to mutant R subunits with the Arg-242 mutations without or with an inactivating mutation in site B, or biphasic dissociation of [3H]cAMP from the mutant subunits at low temperature. The mutations reduced site A affinities by about 25-fold, and the reductions were attributable to accelerated rates of cAMP dissociation. While the presence of cAMP in site A retards dissociation of [3H]cAMP from site B of wild-type R subunits, saturation of site A had little or no effect on dissociation of [3H]cAMP from site B of the mutant subunits. The predominant effect of the mutations, therefore, was loss of allosteric coupling between the two cAMP-binding sites. A second allosteric interaction, that coupling occupation of site A with a reduced affinity of R for catalytic subunit, was inhibited only partially by these mutations at Arg-242.

Autoactivation of catalytic (C alpha) subunit of cyclic AMP-dependent protein kinase by phosphorylation of threonine 197.

We recently found, using cultured mouse cell systems, that newly synthesized catalytic (C) subunits of cyclic AMP-dependent protein kinase undergo a posttranslational modification that reduces their electrophoretic mobilities in sodium dodecyl sulfate (SDS)-polyacrylamide gels and activates them for binding to a Sepharose-conjugated inhibitor peptide. Using an Escherichia coli expression system, we now show that recombinant murine C alpha subunit undergoes a similar modification and that the modification results in a large increase in protein kinase activity. Threonine phosphorylation appears to be responsible for both the enzymatic activation and the electrophoretic mobility shift. The phosphothreonine-deficient form of C subunit had reduced affinities for the ATP analogs p-fluorosulfonyl-[14C]benzoyl 5'-adenosine and adenosine 5'-O-(3-thiotriphosphate) as well as for the Sepharose-conjugated inhibitor peptide; it also had markedly elevated Kms for both ATP and peptide substrates. Autophosphorylation of C-subunit preparations enriched for this phosphothreonine-deficient form reproduced the changes in enzyme activity and SDS-gel mobility that occur in intact cells. A mutant form of the recombinant C subunit with Ala substituted for Thr-197 (the only C-subunit threonine residue known to be phosphorylated in mammalian cells) was similar in SDS-polyacrylamide gel electrophoresis mobility and activity to the phosphothreonine-deficient form of wild-type C subunit. In contrast to the wild-type subunit, however, the Ala-197 mutant form could not be shifted or activated by incubation with the phosphothreonine-containing wild-type form. We conclude that posttranslational autophosphorylation of Thr-197 is a critical step in intracellular expression of active C subunit.