THE ERYTHROPOIETIN-SENSITIVE MEMBRANE PHOSPHOPROTEIN, PP43, IS A PROTEIN SERINE THREONINE KINASE/

We have shown previously that treatment of isolated erythroid cell pla sma membranes with erythropoietin leads to a rapid decrease in pp43, a n erythropoietin-sensitive membrane phosphoprotein (Choi, H. S., Wojch owski, D. M., and Sytkowski, A. J., J. Biol. Chem. 262, 2933, 1987; Ch oi, H. S., Bailey, S. C., Donahue, K. A., Vanasse, G. J., and Sytkowsk i, A. J., J. Biol. Chem. 265, 4143, 1990). We have now demonstrated th is effect in intact cells and have obtained further information regard ing pp43 function during erythropoietin stimulation. P-32-phosphorylat ed membranes were subjected to conditions of increasing pH. [P-32]pp43 dissociated readily into solution, reaching half-maximal dissociation at pH similar to 9. This dissociation was enhanced markedly by increa sing the ionic strength up to a maximum of 0.5 M KCl. These biochemica l properties characterize pp43 as a membrane-associated protein. Addit ion of [gamma-P-32]ATP to an aqueous supernatant prepared from unlabel ed membranes resulted in the P-32-phosphorylation of pp43 in solution, after dissociation from the plasma membrane. Furthermore, erythropoie tin treatment of unlabeled, intact cells followed by fractionation and P-32-phosphorylation resulted in a striking erythropoietin- and time- dependent increase in [P-32]pp43 found in the supernatant and a concom itant decrease in [P-32]pp43 found in the membrane pellet. This strong ly suggests that erythropoietin stimulates the dissociation of pp43 fr om 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 i dentifying it as a new protein serine/threonine kinase. The results su ggest a le for pp43 in transmembrane signaling. Academic Press.