The erythropoietin-sensitive membrane phosphoprotein, pp43, is a protein serine/threonine kinase.
We have shown previously that treatment of isolated erythroid cell plasma membranes with erythropoietin leads to a rapid decrease in pp43, an erythropoietinsensitive membrane phosphoprotein (Choi, H. S., Wojchowski, D. M., and Sytkowski, A. J., J. Biol. Chem. 262, 2933, 1987; Choi, H. S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowski, A. J., J. Biol. Chem. 265, 4143, 1990). We have now demonstrated this effect in intact cells and have obtained further information regarding pp43 function during erythropoietin stimulation. 32P-phosphorylated membranes were subjected to conditions of increasing pH. [32P]pp43 dissociated readily into solution, reaching half-maximal dissociation at pH approximately 9. This dissociation was enhanced markedly by increasing the ionic strength up to a maximum of 0.5 M KCl. These biochemical properties characterize pp43 as a membrane-associated protein. Addition of [gamma-32P]ATP to an aqueous supernatant prepared from unlabeled membranes resulted in the 32P-phosphorylation of pp43 in solution, after dissociation from the plasma membrane. Furthermore, erythropoietin treatment of unlabeled, intact cells followed by fractionation and 32P-phosphorylation resulted in a striking erythropoietin- and time-dependent increase in [32P]pp43 found in the supernatant and a concomitant decrease in [32P]pp43 found in the membrane pellet. This strongly suggests that erythropoietin stimulates the dissociation of pp43 from the plasma membrane and promotes translocation into the supernatant (cytoplasm). Using a renaturation kinase assay, we demonstrated that pp43 is capable of autophosphorylation on serine and threonine, thus identifying it as a new protein serine/threonine kinase. The results suggest a role for pp43 in transmembrane signaling.
Lunn
E D
ED
Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, USA.
Sytkowski
A J
AJ
D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004921:Erythropoietin / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D004915:Leukemia, Erythroblastic, Acute; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009994:Osmolar Concentration; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins; D014407:Tumor Cells, Cultured
D008565:Membrane Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D004921:Erythropoietin; D010769:Phosphothreonine; D010768:Phosphoserine; D011189:Potassium Chloride; D017346:Protein Serine-Threonine Kinases