The Functional Study of p53 Downstream Gene DDA3 on ASPP2, and a Further Study on Novel ASPP2 Functions

• Main Authors: Wei-Tzu Sun, 孫瑋孜
• Other Authors: Fung-Fang Chen
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/k4d4wt

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 97 === ASPP2 (53BP2L) and its alternatively spliced form 53BP2S are known as tumor suppressors, which stimulates the p53 transcriptional activity under genotoxic stress and thereby enhance the transcription of p53 downstream target genes, especially those mediating cell apoptosis. In this study, we discovered that DDA3, a p53 downstream target protein, binds ASPP2 as shown in the GST pull-down assay and in vivo immunofluorescence assay. Using luciferase reporter gene assay, we found DDA3 also inhibits ASPP2’s stimulation on p53-mediated Bax transactivation. Through studying the cellular localization of endogenous ASPP2, we have discovered for the first time that ASPP2 is an actin-associated protein. The endogenous ASPP2 localizes with actin-rich cellular structures such as adherens junctions, lamellapodia and filopodia. Using immunofluorescence and FRET techniques, we have confirmed that ASPP2 associates with actin filaments, and the association is mediated by the N-terminal region of ASPP2. Overexpression of ASPP2 induces extensive membrane protrusions resembling lamellapodia in several cell types, and induces the disruption of adherens junction integrity in MDCK cells. In Vero and COS-1 cells, the overexpression of ASPP2 significantly enhances cell migration ability, and the migration-promoting function is not mediated through the ASPP2-p53 mechanism. The overexpression of ASPP2 activates Rac1 by translocating it to the plasma membrane as shown by immunofluorescence assay. Using siRNA knock-down technique, we found that lowered 53BP2L expression is accompanied with reduced Akt activity under insulin stimulation, and the overexpression of ASPP2 itself induces Akt phosphorylation in the absence of insulin. These data suggest ASPP2 play a role in the insulin/Akt stimulated cell motility. We have previously identified DDA3 as a microtubule-associated protein. When both ASPP2 and DDA3 were overexpressed in COS-1 cells, we found that the cellular localization of both proteins were altered, with ASPP2 re-localized to the microtubule structure in some cells, and DDA3 in actin-rich areas in some other cells. This phenomenon might be the consequence of their binding and attracting each other to their original cellular location. Taken together, this study proved that DDA3 binds to ASPP2, and inhibits its stimulation on p53-mediated Bax transactivation. We also discovered that ASPP2 plays a novel role in actin reorganization, probably through affecting the Akt/Rac1 pathways. With these evidences, along with the finding that overexpressed DDA3 “recruits” ASPP2 to the microtubule, we presume the interaction between ASPP2 and DDA3 modulates multiple aspects of biological processes, and the underlying mechanisms, although remain largely unknown, are well worth to explore further.