Analysis of the mechanism of activation of cAMP-dependent protein kinase through the study of mutants of the yeast regulatory subunit.

Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP-dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA-dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897-11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total catalytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild-type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1-6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit.