Abstract
We examined the biological functions of the gene Cbl in erythropoietin (EPO) signaling using Cbl-deficient F-36P human erythroleukemia cells by the introduction of the Cbl siRNA expression vector. Knockdown of Cbl promoted EPO-dependent proliferation and survival of F-36P cells, especially at a low concentration of EPO (0.01U/mL), similar to serum concentrations of EPO in healthy volunteers (0.005-0.04U/mL). We found that Src was degraded mainly by the proteasomal pathway because the proteasome inhibitor MG-132 but not the lysosome inhibitor NH4Cl suppressed the EPO-induced degradation of Src in F-36P cells and that knockdown of Cbl inhibited EPO-induced ubiquitination and degradation of Src in F-36P cells. The experiments using the Src inhibitor PP1 and co-expression experiments further confirmed that Cbl and the kinase activity of Src are required for the EPO-induced ubiquitination of Src. In addition, the co-expression experiments and in vitro kinase assay demonstrated that the EPO-induced tyrosine phosphorylation and ubiquitination of Cbl were dependent on the kinase activity of Src but not Jak2. Thus, Cbl negatively regulates EPO signaling mainly through the proteasome-dependent degradation of Src, and the E3 ligase activity of Cbl and its tyrosine phosphorylation are regulated by Src but not Jak2.
Keywords:
Cbl; Erythropoietin; Proteasome; Src; Ubiquitination.
Copyright © 2014 Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Ammonium Chloride / pharmacology
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Antineoplastic Agents / pharmacology
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Cell Line, Tumor
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Cell Proliferation / drug effects
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Cell Survival / drug effects
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Erythropoietin / metabolism
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Erythropoietin / pharmacology*
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Gene Expression Regulation
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Humans
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Janus Kinase 2 / genetics
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Janus Kinase 2 / metabolism
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Leupeptins / pharmacology
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Lymphocytes / cytology
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Lymphocytes / drug effects*
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Lymphocytes / metabolism
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Phosphorylation / drug effects
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Proteasome Endopeptidase Complex / drug effects
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Proteasome Endopeptidase Complex / metabolism
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Proteolysis
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Proto-Oncogene Proteins c-cbl / antagonists & inhibitors
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Proto-Oncogene Proteins c-cbl / genetics*
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Proto-Oncogene Proteins c-cbl / metabolism
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Proto-Oncogene Proteins pp60(c-src) / antagonists & inhibitors
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Proto-Oncogene Proteins pp60(c-src) / genetics*
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Proto-Oncogene Proteins pp60(c-src) / metabolism
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Pyrazoles / pharmacology
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Pyrimidines / pharmacology
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RNA, Small Interfering / genetics
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RNA, Small Interfering / metabolism
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Signal Transduction
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Ubiquitination
Substances
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4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine
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Antineoplastic Agents
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EPO protein, human
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Leupeptins
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Pyrazoles
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Pyrimidines
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RNA, Small Interfering
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Ammonium Chloride
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Erythropoietin
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Proto-Oncogene Proteins c-cbl
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JAK2 protein, human
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Janus Kinase 2
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Proto-Oncogene Proteins pp60(c-src)
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Proteasome Endopeptidase Complex
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CBL protein, human
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde