ERYTHROPOIETIN STIMULATES SERINE KINASE-ACTIVITY IN ERYTHROPOIETIN-DEPENDENT CELLS

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 i n Epo-mediated signal transduction, the effect of Epo on serine-threon ine kinase activity was examined in the Epo-dependent cell line, HCD-5 7, using a kinase renaturation assay. In HCD-57 cells synchronized in Go phase by centrifugal elutriation, multiple serine-threonine kinases were constitutively active, and exposure to Epo was associated with a n increase in the activity of kinases with apparent molecular masses o f 170, 120, and 90-95 kD. Phosphoamino acid analysis established the c ovalent incorporation of P-32 into serine and threonine for constituti vely active kinases and into serine alone for the 90-95 kD kinase. Ree lectrophoresis experiments established that P-32 incorporation represe nted kinase autophosphorylation as opposed to protein substrate phosph orylation. Epo-associated serine kinase autophosphorylation was both h ormone concentration and time dependent as well as restricted to the G (0), G(1), and S phases of the cell cycle. Cell fractionation studies localized the activity of the 90-95 kD serine kinase to the plasma mem brane.