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 language | English (US) |
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Pages (from-to) | 1141-1146 |
Number of pages | 6 |
Journal | Experimental Hematology |
Volume | 22 |
Issue number | 12 |
State | Published - 1994 |
Keywords
- Autophosphorylation
- Cell cycle
- Erythroleukemia cells
- Erythropoietin
- Serine threonine kinases
ASJC Scopus subject areas
- Molecular Biology
- Hematology
- Genetics
- Cell Biology
- Cancer Research