| Summary: | 碩士 === 國立中興大學 === 生物醫學研究所 === 100 === Tumor-associated NADH oxidase (tNOX; ENOX2) expresses on the cell surface of transformed cells. Previous reports have shown that tNOX expression associates with proliferation in cancer cells. In addition, knockdown tNOX lead to reduced cell proliferation and migration. Phosphorylation-dephosphorylation of protein is an important post translational modification that plays a key role in regulation of protein function involving in many cellular signal transductions. We have demonstrated that PKCδ phosphorylate tNOX by in vitro kinase assay. Thus, we investigate phosphorylation status of tNOX on serine-504 though protein kinase Cδ (PKCδ) and whether phosphorylation status of tNOX is involved in regulating tNOX function.
First, we overexpressed wt-tNOX, and serine-504 mutatant alaine (A, to mimic dephosphorylation) in NIH3T3, and observed that PKCδ directly interacted with tNOX by GST pull down assays and immunoprecipitation. Using protein stability assay, we demonstrated that phosphorylation of tNOX on serine-504 enhanced stability. Subsequently we confirmed that phosphorylation of tNOX on serine-504 increased cell proliferation and migration. In addition, used to comfirmed tNOX serine-504 aspartic acid mutatant (D, to mimic phosphorylation) was constructed and used to confirm tNOX serine-504 incrased cell proliferation and migration. Finally, we discovered that tNOX interacted with β-catenin and tNOX serine-504 phosphorylation levels relatived affinity of interaction with β-catenin. Taken together, these results suggest that phosphorylation of tNOX serine-504 through PKCδ modulates the biological function of tNOX such as enhancing proliferation and migration, possibly by β-catenin-mediated signaling pathways.
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