Effect of 2,3-diphosphoglycerate on the phosphorylation of protein 4.1 by protein kinase C.
We have previously shown that 2,3-diphosphoglycerate (2,3-DPG) inhibits the phosphorylation of erythrocyte membrane cytoskeletal proteins by endogenous casein kinases. Here, we report that 2,3-DPG stimulates the phosphorylation of protein 4.1 by protein kinase C. Studies with red cell membrane preparations showed that while the phosphorylation of most of the membrane proteins by endogenous membrane-bound kinases and purified kinase C was inhibited by 2,3-DPG, the phosphorylation of protein 4.1 was slightly enhanced by the metabolite. The effect of 2,3-DPG was further examined using purified protein 4.1 preparations. Our results indicate that 2,3-DPG stimulates both the rate and the extent of phosphorylation of purified protein 4.1 by kinase C. The amount of phosphate incorporated was found to double to 2 mol of phosphate per mole of protein 4.1 in the presence of 10 mM 2,3-DPG. The increase in phosphorylation was distributed over all phosphorylation sites as revealed by an analysis of the labeling patterns of phosphopeptides resolved by high performance liquid chromatography, but a significantly higher incorporation was detected in two of the phosphopeptides. The stimulatory effect of 2,3-DPG on the phosphorylation of protein 4.1 was observed only with kinase C. Phosphorylation by the cytosolic erythrocyte casein kinase and the cyclic AMP-dependent protein kinase was inhibited by 2,3-DPG. Moreover, the stimulatory effect of 2,3-DPG seemed to be unique to the phosphorylation of protein 4.1 since a similar effect had not been observed with other protein kinase C substrates. Our results suggest that 2,3-DPG may play an important role in the regulation of cytoskeletal interactions.
Chao
T S
TS
Department of Biochemistry, University of Illinois, College of Medicine, Chicago 60612.
Tao
M
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D019794:2,3-Diphosphoglycerate; D047388:Casein Kinases; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000494:pharmacology; D003598:Cytoskeletal Proteins; D004163:Diphosphoglyceric Acids / Q000494:pharmacology*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009479:Neuropeptides; D010446:Peptide Fragments / Q000302:isolation & purification; D010750:Phosphoproteins / Q000097:blood*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D014357:Trypsin
D003598:Cytoskeletal Proteins; D004163:Diphosphoglyceric Acids; D008565:Membrane Proteins; D009479:Neuropeptides; D010446:Peptide Fragments; D010750:Phosphoproteins; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; D019794:2,3-Diphosphoglycerate; D000242:Cyclic AMP; D011494:Protein Kinases; D047388:Casein Kinases; D011493:Protein Kinase C; D014357:Trypsin