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 erythropoietin-sensitive 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 ∼9. This dissociation was enhanced markedly by increasing the ionic strength up to a maximum of 0.5mKCl. These biochemical properties characterize pp43 as a membrane-associated protein. Addition of [γ-32P]ATP to an aqueous supernatant prepared from unlabeled membranes resulted in the32P-phosphorylation of pp43 in solution, after dissociation from the plasma membrane. Furthermore, erythropoietin treatment of unlabeled, intact cells followed by fractionation and32P-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.