SUBUNIT PHOSPHORYLATION AND ACTIVATION OF SKELETAL-MUSCLE PHOSPHORYLASE-KINASE BY THE CAMP-DEPENDENT PROTEIN-KINASE - DIVALENT METAL-ION, ATP, AND PROTEIN-CONCENTRATION DEPENDENCE

  • 1 January 1985
    • journal article
    • research article
    • Vol. 260  (4) , 2046-2056
Abstract
A characterization of the effects of varying the concentrations of Mg2+, ATP, phosphorylase kinase [rabbit] and the cAMP-dependent protein kinase on the activation and phosphorylation of phosphorylase kinase is provided. The Km for MgATP2- for the cAMP-dependent protein kinase-catalyzed phosphorylation is decreased by increasing Mg2+, probably as a consequence of decreasing the free ATP:MgATP2- ratio and increasing free Mg2+. Whereas .beta. subunit phosphorylation of phosphorylase kinase plays a prominent role in determining its activity, .alpha. subunit phosphorylation can also modulate activity. The phosphorylation of the .alpha. subunit, which occurs following the initial cAMP-dependent phosphorylation of the .beta. subunit, is catalyzed by the cAMP-dependent protein kinase and is not a consequence of EGTA[ethylene glycol bis(.beta.-aminoethyl ether)N,N,N''N''-tetraacetic acid]-insensitive (or EGTA-sensitive) autophosphorylation occurring as a result of the enhanced phosphorylase kinase activity. The relationship between subunit phosphorylation and phosphorylase kinase activation is complex and particularly dependent upon concentrations of cAMP-dependent protein kinase and phosphorylase kinase in the activation reaction. The possibilities that the pathway of phospho-intermediates involved in the activation process probably varies with the activation conditions, that the efficacy of a specific site to be covalently modified is dependent upon the phosphorylation status of other sites and that the effect of phosphorylation in regulating activity may also be dependent on the phosphorylation status of other sites were suggeted. Apparently, the activation process for phosphorylase kinase can be very complex; it is possible that this complexity might have significant physiological ramifications.