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 inhibitors N-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.
Highlights
Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation
Cellular extracts were immunoprecipitated with anti-Epo receptor antibodies, with anti-GST control antibodies, or with anti-Cis antibodies, and immunoprecipitated radioactivity was measured by ␥ counting
SOCS1, SOCS2, and SOCS3 are composed of 210 –260 amino acids, and they exhibit a similar structure with an Src homology 2 domain located in the middle of the protein and a conserved motif of ϳ40 amino acids located at the C-terminal part of the molecules
Summary
Frederique Verdier‡§, Stany Chretien¶, Odile Muller‡, Paule Varlet‡, Akihiko Yoshimuraʈ, Sylvie Gisselbrecht‡, Catherine Lacombe‡, and Patrick Mayeux‡**. 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 These proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. Except for SCF, macrophage colony-stimulating factor, and FLT3 ligand recep- Another negative regulatory pathway of Epo receptor signaling involves the protein Cis. Cis (cytokine-inducible Src homology 2-containing protein) gene expression is rapidly induced in hematopoietic cells by IL-2, IL-3, GM-CSF, and Epo [23]. Cis (cytokine-inducible Src homology 2-containing protein) gene expression is rapidly induced in hematopoietic cells by IL-2, IL-3, GM-CSF, and Epo [23] All of these cytokines activate STAT5, and it has been shown that Cis is a target gene of STAT5 [24]. We observed that Cis is a ubiquitinated protein and that proteasome inhibitors induced the accumulation of the ubiquitinated form of Cis and the accumulation of the Cis-Epo receptor complexes
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