Effect of Mg2+ concentrations on phosphorylation/activation of phosphorylase b kinase by cAMP/Ca(2+)-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 Ca(2+)-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 alpha and beta subunits of phosphorylase b kinase at different Mg2+ ion concentrations further reveals that the phosphorylation level of the beta subunit remains almost unchanged, whereas the phosphorylation level of the alpha subunit increases dramatically and correlates with the increased enzyme activity. In similarity with the beta 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 alpha 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.