| Budget Amount *help |
¥1,970,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥270,000)
Fiscal Year 2007: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
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| Research Abstract |
Cbl functions as the ubiquitine ligase and adaptor molecule in a variety of receptor-mediated signaling. However, the function of Cbl in erythropoietin receptor (EPOR)-mediated signaling is still unidentified. In the present study, to examine a physiological function of Cbl in EPOR-mediated signaling, we developed Cbl-deficient F-36P human erythroleukemia (F-36P-Cbl-siRNA) cells by introducing the expression vector for Cbl siRNA. Knockdown of Cbl induced enhancement of proliferation and survival of F-36P cells at a physiological concentration of EPO compared to that of mock-transfected F-36P (F-36P-mock) cells. Furthermore, apoptosis of F-36P-Cbl-siRNA was decreased in the absence of EPO compared to that of F-36P-mock cells. We next examined the mechanism by which Cbl inhibits proliferation of F-36P cells and promotes survival of those cells. Akt was constitutively activated in F-36P-Cbl-siRNA but not in F-36P-mock cells. Knockdown of Cbl did not affect EPO-induced activation of Erk1/2
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in F-36P cells. After EPO treatment, Cbl was phosphorylated on tyrosine residues including Tyr731, a binding site for the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) and associated with p85. EPO-induced phosphorylation of Cbl on Tyr731 in F-36P cells was inhibited by PP1, a selective Src kinase inhibitor, but not by AG490, a selective Jak kinase inhibitor. In F-36P cells expressing the dominant-negative Src, EPO failed to induce the tyrosine phosphorylation of Cbl. Co-expression experiments, In vitro kinase and GST pull-down assays further demonstrated that recombinant Src directly phosphorylated Cbl on Tyr731. Taken together with these findings, Cbl negatively regulates EPO-induced proliferation and survival of F-36P cells through the PI3-kinase/Akt pathway. In addition, Src is likely to be involved in Cbl-mediated regulation of PI3-kinase through inducing association of Cbl with p85, because Src phosphorylates Cbl on Tyr731, thereby leading to providing a binding site for p85. Less
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