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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 (0.01 U/mL), similar to serum concentrations of EPO in healthy volunteers (0.005–0.04 U/mL). We found that Src was degraded mainly by the proteasomal pathway because the proteasome inhibitor MG-132 but not the lysosome inhibitor NH 4 Cl 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. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we examined the role of Src in gemcitabine-induced cell growth suppression in human pancreatic cancer cell lines. In two human pancreatic cancer cell lines, PK-9 and MIA PaCa-2, we found that a selective Src protein tyrosine kinase inhibitor, PP2, inhibited gemcitabine-induced cell growth suppression. When dominant negative src cDNA was constitutively expressed in PK-9 cells (PK-9-Src-DN), the degree of gemcitabine-induced cell growth suppression was decreased compared with that of mock-transfected PK-9 cells. The mechanism of the inhibitory effect of gemcitabine-induced cytotoxicity was found to be the suppression of apoptosis, which was downregulated in PK-9-Src-DN cells. These results indicate that Src mediates signals that culminate in suppressing cell growth and survival in the presence of gemcitabine, at least in particular cell lines. [Copyright &y& Elsevier]

Abstract: Most human leukemia cells are shown to express growth hormone receptor (GHR) and some of them proliferate in response to GH. We demonstrate that Src contributes to GHR-mediated signal transduction via STAT5 activation in F-36P human leukemia cells stimulated with GH. The tyrosine phosphorylation levels of GHR and STAT5 induced by GH decreased in the presence of PP2 Src kinase inhibitor. When GHR and wild-type Src were co-expressed in COS7 cells, GHR was markedly tyrosine phosphorylated as well as when Jak2 was co-expressed with GHR, but not when kinase-inactive Src co-expressed. The treatment of F-36P cells with the antisense src oligonucleotides, which selectively decreased the Src expression, reduced the rhGH-induced tyrosine phosphorylation of the STAT5 activation sites. [Copyright &y& Elsevier]