phosphothreonine and Leukemia--Erythroblastic--Acute

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.

Topics: Animals; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Erythropoietin; Hydrogen-Ion Concentration; Leukemia, Erythroblastic, Acute; Membrane Proteins; Mice; Osmolar Concentration; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphothreonine; Potassium Chloride; Protein Serine-Threonine Kinases; Recombinant Proteins; Tumor Cells, Cultured

Protein phosphorylation is an early event that follows the interaction of erythropoietin (Epo) with its receptor, even though this receptor lacks a kinase domain. To further define the role of protein kinases in Epo-mediated signal transduction, the effect of Epo on serine-threonine kinase activity was examined in the Epo-dependent cell line, HCD-57, using a kinase renaturation assay. In HCD-57 cells synchronized in G0 phase by centrifugal elutriation, multiple serine-threonine kinases were constitutively active, and exposure to Epo was associated with an increase in the activity of kinases with apparent molecular masses of 170, 120, and 90-95 kD. Phosphoamino acid analysis established the covalent incorporation of 32P into serine and threonine for constitutively active kinases and into serine alone for the 90-95 kD kinase. Reelectrophoresis experiments established that 32P incorporation represented kinase autophosphorylation as opposed to protein substrate phosphorylation. Epo-associated serine kinase autophosphorylation was both hormone concentration and time dependent as well as restricted to the G0, G1, and S phases of the cell cycle. Cell fractionation studies localized the activity of the 90-95 kD serine kinase to the plasma membrane.

Topics: Animals; Cell Cycle; Electrophoresis, Polyacrylamide Gel; Erythropoietin; Leukemia, Erythroblastic, Acute; Mice; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; Phosphoserine; Phosphothreonine; Protein Serine-Threonine Kinases; Signal Transduction; Tumor Cells, Cultured