Characterization of Phosphorylated and Dephosphorylated Regulatory Subunit of CAMP-Dependent Protein Kinase II

The regulatory subunit (RII) of cAMP-dependent protein kinase II purified from bovine cardiac muscle contains between 1.2 mol and 2.7 mol phosphate/mol RII monomer (range for six different batches). The protein-bound phosphate had the stability characteristics of an alkylphosphate. Treatment of RII containing 2 mol endogenous phosphate/mol for 20 h with alkaline phosphatase hydrolyzed about 0.4 mol phosphate/mol. In contrast, RII was almost completely dephosphorylated by an acid phosphatase within 4 h. Catalytic subunit added in the presence of cAMP at I/100 of the concentration of dephosphorylated RII or RII containing 2 mol endogenous phosphate/mol incorporated 0.75 mol phosphate/mol of each RII. Catalytic subunit added at higher concentrations incorporated up to 2 mol phosphate/mol dephosphorylated RII as shown by 32P incorporation and chemical phosphate analysis. Dephosphorylated RII and RII containing 2 mol endogenous phosphate/mol recombined with similar affinity with catalytic subunit (approx. Ki 0.3 nM). Holoenzymes reconstituted with dephosphorylated RII and Rll containing 1.6 mol or 2.0 mol endogenous phosphate/mol were half-maximally activated at similar concentra- tions of cAMP (approx. Ka 0.1 μM). Isolated dephosphorylated RII or RII containing 2 mol endogenous phosphate/mol bound 2 mol cAMP/mol RII monomer with identical affinity (approx. Kd 12–20 nM) in the absence and presence of 0.48 M NaCl. Addition of stoichiometric concentrations of catalytic subunit (0.14 pM) to each RII induced apparent negative cooperative binding of cAMP to either RII in the absence of salt and no change in cooperativity in the presence of 0.48 M NaCl. The apparent negative cooperativity observed in the absence of salt was not seen when Mg. ATP was added together with catalytic subunit to each RII. Under the latter condition, an about threefold difference was observed in the affinity of cAMP for dephosphorylated RII and RII containing 2 mol endogenous phosphate/mol (apparent Kd 13 nM and 32 nM, respectively). This difference was not abolished by the addition of 0.48 M NaCl.