Mutant regulatory subunit of 3',5'-cAMP-dependent protein kinase of yeast Saccharomyces cerevisiae

Summary Four mutants with amino acid substitution(s) at or near the putative phosphorylation site (Arg¹⁴² Arg¹⁴³ Thr¹⁴⁴ Ser¹⁴⁵) of the regulatory subunit of cAMP-dependent protein kinase were obtained by site-directed mutagenesis. Three mutants, BCY1^{Ala 145} (Ser¹⁴⁵ to Ala), BCY1^{His 143} (Arg¹⁴³ to His) and BCY1^{Asn 144, Ala 145} (Thr¹⁴⁴ to Asn and Ser¹⁴⁵ to Ala) complemented a bcy1 mutant, whereas BCY1^{Gly 143} (Arg¹⁴³ to Gly) did not. In addition, mutant, BCY1^{Asn 144, Ala 145} exhibited a dominant coldsensitive phenotype, which can be most easily explained by the functional alteration of the regulatory subunit of cAMP-dependent protein kinase by the mutations. Analyses of these mutant genes revealed that phosphorylation of the regulatory subunit is not a prerequisite for the regulation of the cAMP-dependent protein kinase activity in responding to the cAMP level.