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The c-Met receptor tyrosine kinase (MetR) is frequently overexpressed and constitutively

The c-Met receptor tyrosine kinase (MetR) is frequently overexpressed and constitutively phosphorylated in a number of human malignancies. increase in HGF-mediated cell migration with no discernible effect on cell proliferation. By contrast, knockdown of hTid-1 markedly impairs both the onset and amplitude of MetR phosphorylation in response to HGF without altering receptor protein levels. hTid-1-depleted cells display defective migratory properties, coincident with inhibition of ERK/MAP kinase and STAT3 pathways. Taken together, our findings denote hTid-1S as an essential regulatory component of MetR signaling. We propose that the binding of hTid-1S to MetR may stabilize the receptor in a ligand-competent state and this stabilizing function may influence conformational changes that take place during the catalytic cycle that promote kinase activation. Given the prevalence of HGF/MetR pathway activation in human cancers, targeted inhibition of hTid-1 may be a useful therapeutic in the management of MetR-dependent malignancies. gene (cause defects IWP-2 manufacturer in differentiation and morphogenesis of larval imaginal discs leading to neoplastic growth of these cells into lethal tumors (Kurzik-Dumke gene gives rise to two major alternative splice isoforms: a 43-kDa long form hTid-1L and a shorter 40?kDa hTid-1S variant, both of which are widely expressed (Trentin and and growth of ErbB2-dependent mammary carcinomas (Kim reported the binding of the oncogenic fusion protein, tpr-met, to the HDAC-A precursor form of hTid-1, the functional significance of this interaction was not addressed (Schaaf (Nakaigawa value of 0.05 was considered significant. Cell proliferation assay Cells were infected with Ad-GFP, Ad-hTid-1S or Ad-hTid-1L. The following day, 2500 cells were seeded/well of 24-well plates. Cells were serum-starved overnight, IWP-2 manufacturer and left untreated or stimulated with the indicated concentration of HGF. The media was changed every 48?h to fresh starvation media with HGF. Cells were IWP-2 manufacturer counted manually at the indicated time points after contamination using trypan blue exclusion. Growth assays were performed at least three impartial times. Densitometric analysis Densitometric analysis of western blot results was performed using Scion Image software (Scion Corp., Frederick, MD, USA). The pixel densities for both the phosphorylated protein and total protein as indicated were determined and were expressed as a ratio of phosphorylated protein:total protein. Statistical analysis A student’s em t /em -test was used to compare each experimental condition with IWP-2 manufacturer the control condition. A em P /em -value of 0.05 was considered significant. Acknowledgments This work was supported by a grant from the National Cancer Institure of Canada ( to MRA). ER was supported by an Ontario Graduate Scholarship and CML by a Canadian Institutes of Health Research Master’s Award. We thank Kevin Conway for assistance with immunofluorescence microscopy. Notes The authors declare no conflict of interest..