Phosphoglycerates and Protein Phosphorylation: Identification of a Protein Substrate as Glucose-1,6-Bisphosphate Synthetase

Abstract

We have previously reported the occurrence of two endogenous protein phosphorylation systems in mammalian brain that are enhanced in the presence of 3-phosphoglycerate (3PG) and ATP. We present here a study of one of these systems, the phosphorylation of the 72-kDa protein (3PG-PP 72 ). This system was separated into the substrate, 3PG-PP 72 , and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1- 32 P]1,3-bisphosphoglycerate ([1- 32 P]1,3BPG) with an apparent K m of 1.1 n M . Nonradioactive 1,3BPG inhibited 32 P incorporation in the presence of [Γ- 32 P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP 72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1- 32 P]1,3BPG. Moreover, [ 32 P]phosphate incorporated into 3PG-PP 72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP 72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P 2 ) synthetase. Based upon these observations, we conclude that 3PG-PP 72 is G1,6P 2 synthetase and that it is phosphorylated directly by 1,3BPG, which is formed from 3PG and ATP by 3PG kinase present in a crude 3PG-PP 72 preparation.