Identification and Characterization of Associated Proteins for DDA3, a Novel p53 Downstream Target

• Main Authors: Pei-Chen Hsieh, 謝佩真
• Other Authors: Fung-Fang Wang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/45407709141898942543

碩士 === 國立陽明大學 === 生物化學研究所 === 89 === Abstract The p53 tumor suppressor is the most frequently mutated gene in human cancers. p53 functions as a guardian of genome and is normally present at low levels in cells. Upon DNA damage, p53 accumulates in the nucleus, where it transcriptionally activates the effector genes whose products are involved in cell growth arrest, apoptosis and DNA repair. Through the method of mRNA differential display, we have previously identified a novel p53 transcriptional target gene DDA3. DDA3 encodes a protein of 329 amino acids containing one coiled-coil region and 6 PXXP motifs known to interact with SH3-domain containing proteins. A LexA based yeast two-hybrid screening was established to identify DDA3 partners. Among the interacting clones are Ku80, a subunit of the DNA-dependent protein kinase, and EB3. EB3 is a microtubule-associated protein shown to interact with APCL, a homologue of the tumor suppressor APC. The interaction of DDA3 with EB3 and Ku80 was confirmed by GST-pull down assay. Futhermore, association of DDA3 and EB3 in vivo was demonstrated by co-immunoprecipitation with antibodies against each protein. Immunofluorescence analysis using confocal microscopy revealed that DDA3 and EB3 are colocalized in cells; they are present mainly in the cytoplasm that manifested as network patterns. Treatment of cells with nocodazole disrupted the interaction of DDA3 and EB3, suggesting that intact microtubule network is required for interaction. Similarly, no DDA3-EB3 complex formation was detectable in the presence of UV radiation. Because EB3 is a microtubule-associated protein, we examined whether DDA3 is also localized to the microtubules. Immunofluorescence analysis using DDA3 and tubulin-specific antibodies demonstrated colocalization of DDA3 with tubulin; the association was detected in mitotic cells at prophase, and metaphase, as well as in cells undergoing cytokinesis. Our result that the p53 direct target DDA3 is a microtubule-associated protein which interacts with EB3, supports the notion that DDA3 may regulate cell division or cell growth by affecting the microtubule network.