Proteasomes Regulate Erythropoietin Receptor and Signal Transducer and Activator of Transcription 5 (STAT5) Activation
POSSIBLE INVOLVEMENT OF THE UBIQUITINATED CIS PROTEIN*
- Frédérique Verdier‡§,
- Stany Chrétien¶,
- Odile Muller‡,
- Paule Varlet‡,
- Akihiko Yoshimura‖,
- Sylvie Gisselbrecht‡,
- Catherine Lacombe‡ and
- Patrick Mayeux‡**
- From the ‡Institut Cochin de Génétique Moléculaire, INSERM U363, Université René Descartes, 27 rue du Faubourg Saint Jacques, F75014 Paris, France,¶Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, F75015 Paris, France, and ‖Institute of Life Science, Kurume University, 2432-3 Aikawa-machi, Kurume 839, Japan
Abstract
Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the erythropoietin receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitorsN-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In erythropoietin-stimulated UT-7 cells, the activation of the erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30–60 min of erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the erythropoietin receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-erythropoietin receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the erythropoietin receptor.
Footnotes
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↵* This work was supported by grants from the Association pour la Recherche sur le Cancer (Contract 1373), the Ligue Nationale Contre le Cancer, and the Comité de la Manche of the Ligue Nationale Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported by Glaxo Wellcome Laboratories.
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↵** To whom correspondence should be addressed: Institut Cochin de Génétique Moléculaire, INSERM U363, Hôpital Cochin, 27 rue du Faubourg Saint Jacques, F75014 Paris, France. Tel.: 33-1-46-33-14-09; Fax: 33-1-46-33-92-97; E-Mail:mayeux{at}cochin.inserm.fr.
- Abbreviations:
- SCF
-
stem cell factor
- CFU-E
-
colony-forming unit-erythroid
- Epo
-
erythropoietin
- GM-CSF
-
granulocyte-macrophage colony-stimulating factor
- GST
-
glutathione S-transferase
- IL
-
interleukin
- LLnL
-
N-acetyl-leucyl-leucyl-nor-leucinal
- STAT
-
signal transducer and activator of transcription
- Jak
-
Janus kinase.
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- Received May 28, 1998.
- Revision received August 3, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
