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 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.
Spivak
J L
JL
Division of Hematology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Connor
E
E
Isaacs
M
M
D000818:Animals; D002453:Cell Cycle; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000494:pharmacology*; D004915:Leukemia, Erythroblastic, Acute / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism; D010769:Phosphothreonine / Q000032:analysis / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D010761:Phosphorus Radioisotopes; D004921:Erythropoietin; D010769:Phosphothreonine; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases