Erythropoietin (EPO) is the major hormone regulating the proliferation of erythroid precursors and their differentiation into erythrocytes. Ligand binding to the erythropoietin receptor (EPO-R), a member of the cytokine receptor family, triggers Tyr phosphorylation of the surface form of the receptor, presumably mediated by the Janus kinase (JAK) 2. To study whether non-surface EPO-R can be phosphorylated, Ba/F3 cells stably transfected with EPO-R were treated with pervanadate (PV), which is widely used as a potent tool to inhibit cellular protein Tyr phosphatases, thus resulting in enhanced Tyr phosphorylation of cellular proteins. PV treatment caused the EPO-R to undergo Tyr phosphorylation in a time-dependent and dose-dependent manner. PV-mediated Tyr phosphorylation of EPO-R occurred at several intracellular sites including the endoplasmic reticulum (ER), because both endoglycosidase H (endo H)-resistant EPO-R and the ER-retained EPO-R mutant (ΔWS1 EPO-R) were Tyr phosphorylated in response to PV. Moreover, in metabolic labelling experiments, endo H-sensitive EPO-R was also phosphorylated. The phosphorylated fraction accounted for only 30-50% of the newly synthesized EPO-R, the fraction that normally exits from the ER. Tyr phosphorylation could not be detected on proteolytic fragments of the EPO-R, suggesting that this is a highly regulated process. Unlike the wild-type (wt) EPO-R, which was phosphorylated both on EPO binding and after inhibition of Tyr phosphatases by PV treatment, an EPO-R mutant (W282R EPO-R) that does not activate JAK2 was phosphorylated after PV treatment but not by EPO binding. Both EPO-R and JAK2 were phosphorylated with similar kinetics by PV treatment, suggesting that JAK2, as well as protein Tyr kinases different from JAK2, might mediate PV-dependent EPO-R phosphorylation. Furthermore the Tyr-phosphorylated ER-retained EPO-R mutant ΔWS1 co-immunoprecipitated with JAK2 kinase, indicating that the EPO-R might interact with JAK2 while in the ER.
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Research Article|
October 15 1997
Phosphorylation of erythropoietin receptors in the endoplasmic reticulum by pervanadate-mediated inhibition of tyrosine phosphatases
Jacob COHEN;
Jacob COHEN
*Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel
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Hanna ALTARATZ;
Hanna ALTARATZ
*Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel
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Yehiel ZICK;
Yehiel ZICK
†Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
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Ursula KLINGMÜLLER;
Ursula KLINGMÜLLER
1
‡Whitehead Institute for Biomedical Research, Cambridge, MA, U.S.A.
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Drorit NEUMANN
Drorit NEUMANN
2
*Department of Cell Biology and Histology, Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel
2To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
December 19 1996
Revision Received:
May 28 1997
Accepted:
June 18 1997
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1997
1997
Biochem J (1997) 327 (2): 391–397.
Article history
Received:
December 19 1996
Revision Received:
May 28 1997
Accepted:
June 18 1997
Citation
Jacob COHEN, Hanna ALTARATZ, Yehiel ZICK, Ursula KLINGMÜLLER, Drorit NEUMANN; Phosphorylation of erythropoietin receptors in the endoplasmic reticulum by pervanadate-mediated inhibition of tyrosine phosphatases. Biochem J 15 October 1997; 327 (2): 391–397. doi: https://doi.org/10.1042/bj3270391
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