Effect of Mg²⁺ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca²⁺-independent, autophosphorylation-dependent protein kinase

In a previous report (Yu and Yang, Biochem. Biophys. Res. Commun.207, 140-147 (1995)], phosphorylase b kinase from rabbit skeletal muscle was found to be phosphorylated and activated by a cyclic nucleotide- and Ca²⁺-independent protein kinase previously identified as an autophosphorylation-dependent multifunctional protein kinase (autokinase) from brain and liver (Yang et al, J. Biol. Chem.262, 7034-7040, 9421-9427 (1987)]. In this report, the effect of Mg²⁺ ion concentration on the auto-kinase-catalyzed activation of phosphorylase b kinase is investigated. The levels of phosphorylation and activation of phosphorylase b kinase catalyzed by auto-kinase are found to be dependent on the concentration of Mg²⁺ ion used. Phosphorylation of phosphorylase b kinase at high Mg²⁺ ion (>9 mM) is 2-3 times higher than that observed at low Mg²⁺ ion (1 mM) and this results in a further 2- to 3-fold activation of the enzyme activity at high Mg²⁺ ion. Analysis of the phosphorylation stoichiometry of α and β subunits of phosphorylase b kinase at different Mg²⁺ ion concentrations further reveals that the phosphorylation level of the β subunit remains almost unchanged, whereas the phosphorylation level of the α subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the β subunit, phosphorylations of myelin basic protein and histone 2A by auto-kinase are also unaffected by Mg²⁺ ion. Taken together, the results provide initial evidence that Mg²⁺ ion may specifically render the a subunit a better substrate for auto-kinase to cause further phosphorylation/activation of phosphorylase b kinase, representing a new mode of control mechanism for the regulation of auto-kinase involved in the phosphorylation and concurrent activation of phosphorylase b kinase.