Phosphoglycerates and protein phosphorylation: identification of a protein substrate as glucose-1,6-bisphosphate synthetase.
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-PP72). This system was separated into the substrate, 3PG-PP72, and a kinase by ammonium sulfate fractionation, hydroxyapatite chromatography, and hydrophobic interaction HPLC. The substrate protein was shown to be directly phosphorylated with [1-32P]1,3-bisphosphoglycerate [( 1-32P]1,3BPG) with an apparent Km of 1.1 nM. Nonradioactive 1,3BPG inhibited 32P incorporation in the presence of [gamma-32P]ATP and 3PG. Phosphopeptide mapping and phosphoamino acid analyses indicated that the site of phosphorylation of 3PG-PP72 observed in the presence of 3PG and ATP is a serine residue identical to that observed with [1-32P]1,3BPG. Moreover, [32P]phosphate incorporated into 3PG-PP72 in the presence of 3PG and ATP was removed by subsequent incubation with glucose-1-phosphate or glucose-6-phosphate. Finally, 3PG-PP72 showed chromatographic behaviors identical to those of glucose-1,6-bisphosphate (G1,6P2) synthetase. Based upon these observations, we conclude that 3PG-PP72 is G1,6P2 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-PP72 preparation.
Morino
H
H
Mental Health Research Institute, University of Michigan, Ann Arbor 48109.
Fischer-Bovenkerk
C
C
Kish
P E
PE
Ueda
T
T
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004163:Diphosphoglyceric Acids / Q000378:metabolism; D005988:Glyceric Acids / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008433:Mathematics; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010449:Peptide Mapping; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism
D000596:Amino Acids; D004163:Diphosphoglyceric Acids; D005988:Glyceric Acids; D009419:Nerve Tissue Proteins; D010761:Phosphorus Radioisotopes; C005156:3-phosphoglycerate; D010770:Phosphotransferases; D011494:Protein Kinases; C020583:glucose 1,6-bisphosphate synthase