Functional analysis of DDA3, a p53 downstream target

• Main Authors: Pei-Chen Hsieh, 謝佩真
• Other Authors: Fung-Fang Wang
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/70556467797681446541

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 95 === We have previously identified mouse DDA3 as a p53-inducible gene. To explore the functional role of DDA3, we screened a mouse brain cDNA library by the yeast two-hybrid assay, and identified the microtubule plus-end binding protein EB3 as a DDA3 interacting protein. Binding of DDA3 to EB3 was verified by GST pull-down assay and subcellular co-localization; co-immunoprecipitation further indicated that interaction of these two proteins within cells required intact microtubules. Domains of DDA3-EB3 interaction were mapped by GST pull-down assay to amino acids (a.a.) 118-241 and 242-329 of DDA3 and the N- and C-termini of EB3. Immunofluorescence analysis revealed co-localization of DDA3 with microtubules in various cell phases, and regions encompassing a.a. 118-241 and 242-329 contained microtubule-interacting and bundling activities. In vitro microtubule binding assay showed that DDA3 and EB3 associated directly with microtubules, and cooperated with each other for microtubule binding. In addition, DDA3 bound to the EB3 interacting partner APC2, a homologue of the tumor suppressor adenomatous polyposis coli (APC) which is a component of the β-catenin destruction complex. Ectopic expression of DDA3 and EB3 enhanced β-catenin-dependent transactivation and cyclin D1 production, while knockdown of endogenous DDA3 or EB3 inhibited β-catenin-mediated transactivation and the ability of cells to form colonies, implicating DDA3 and EB3 in the β-catenin-mediated growth signaling. Furthermore, overexpression of DDA3 inhibited neurite formation during NGF-induced PC12 differentiation, whereas knockdown of DDA3 expression by small interference RNA resulted in neurite outgrowth in N2a cells. Consistent with these findings, DDA3 expression was downregulated during retinoic acid induced differentiation of N2a neuroblastoma cells. Together, our results uncover novel roles of DDA3 in microtubule dynamics, cell growth and neuronal differentiation.