Phosphocreatine-dependent protein phosphorylation in rat skeletal muscle

Abstract

Phosphocreatine (PCr) was found to alter the phosphorylation state of two proteins of apparent molecular masses 18 and 29 kDa in dialysed cell-free extracts of rat skeletal muscle in the presence of [gamma-32P]ATP. The 29 kDa protein was identified as phosphoglycerate mutase (PGM), phosphorylated at the active-site histidine residue by 2,3-bisphosphoglycerate (2,3-biPG). 2,3-biPG labelling from [gamma-32P]ATP occurred through the concerted action of phosphoglycerate kinase and 2,3-bisphosphoglycerate mutase. PCr-dependent labelling, which required creatine kinase, resulted from a shift in the phosphoglycerate kinase equilibrium towards 1,3-bisphosphoglycerate (1,3-biPG) synthesis, ultimately resulting in an increase in available [2-32P]2,3-biPG. The maximal catalytic activity of PGM was unaffected by PCr. The 18 kDa protein was transiently phosphorylated at a histidine residue, probably by 1,3-biPG. No proteins of this monomeric molecular mass are known to bind 1,3-biPG, suggesting that the 18 kDa protein is an undescribed phosphoenzyme intermediate. Previous observations of 2- and 3-phosphoglycerate-dependent protein phosphorylation in cytosolic extracts [Ueda & Plagens (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1229-1233; Pek, Usami, Bilir, Fischer-Bovenkerk & Ueda (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4294-4298], attributed to the action of novel kinases, are likely to represent phosphoenzyme intermediates labelled by bisphosphorylated metabolites in a similar manner.