title: Cbl negatively regulates erythropoietin-induced growth and survival signaling through the proteasomal degradation of Src kinase.
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.
authors.affiliation: Department of Community Medicine, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Takamichi
authors.initials: T
authors.lastname: Shintani
authors.affiliation: Department of Internal Medicine, Takamatsu Red Cross Hospital, Kagawa 760-0017, Japan.
authors.forename: Fusako
authors.initials: F
authors.lastname: Ohara-Waki
authors.affiliation: Department of Gastroenterology and Hematology, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kitanaka
authors.affiliation: Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan.
authors.forename: Terukazu
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Community Medicine, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan. Electronic address: yokubota@kms.ac.jp.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Kubota
mesh_terms: D000643:Ammonium Chloride / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004921:Erythropoietin / Q000378:metabolism / Q000494:pharmacology*; D005786:Gene Expression Regulation; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D059748:Proteolysis; D050721:Proto-Oncogene Proteins c-cbl / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D054875:Ubiquitination
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D000970:Antineoplastic Agents; C554270:EPO protein, human; D007976:Leupeptins; D011720:Pyrazoles; D011743:Pyrimidines; D034741:RNA, Small Interfering; D000643:Ammonium Chloride; D004921:Erythropoietin; D050721:Proto-Oncogene Proteins c-cbl; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D016392:Proto-Oncogene Proteins pp60(c-src); D046988:Proteasome Endopeptidase Complex; C067419:CBL protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: Cbl; Erythropoietin; Proteasome; Src; Ubiquitination