Erythropoietin stimulates serine kinase activity in erythropoietin-dependent cells

J. L. Spivak, E. Connor, M. Isaacs

Research output: Contribution to journalArticlepeer-review

Abstract

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 reconstitutively 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.

Original languageEnglish (US)
Pages (from-to)1141-1146
Number of pages6
JournalExperimental Hematology
Volume22
Issue number12
StatePublished - 1994

Keywords

  • Autophosphorylation
  • Cell cycle
  • Erythroleukemia cells
  • Erythropoietin
  • Serine threonine kinases

ASJC Scopus subject areas

  • Molecular Biology
  • Hematology
  • Genetics
  • Cell Biology
  • Cancer Research

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