Structural and functional studies of the apoptosis stimulating proteins of p53

• Main Authors: Yu-Tzu Chan, 詹玉資
• Other Authors: Fung-Fang Wang
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/13887700650402779987

碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === p53 tumor suppressor is the most frequently mutated gene in human cancers. p53 is a stress-induced transcription factor that activates genes whose products are involved in cell cycle arrest and apoptosis, thus preventing the damaged cells from replication. Recent evidence showed that the apoptotic function of p53 is stimulated by a family of p53 binding proteins known as ASPP. The ASPP family members include ASPP1, ASPP2 and its alternatively spliced form 53BP2. Previous finding from this lab indicated that ASPPs distributed to the adherens junction (AJ) and colocalized with β-catenin upon cell confluence. In this study, a series of plasmids expressing various regions of ASPP were constructed for mapping the AJ localization domain. Analysis by confocal microscopy showed that region containing amino acids 97-227 of the ASPP was necessary and sufficient for its AJ localization. Coimmunoprecipitation assay did not reveal the interaction of ASPP with the known AJ components, including E-cadherin, α, β and γ-catenin as well as actin. Mass spectroscopy analysis of the proteins precipitated with the N-terminus of ASPP2 identified nucleolin (C23) as a candidate ASPP interacting protein, which was verified by co-immunoprecipitation and immunofluorescence. Transfection reporter assay using the Bax-promoter driven luciferase construct indicated that the ASPP2- and 53BP2-stimulated luciferase activity was abolished by the presence of JNK inhibitor SP600125. Unexpectedly, we found that the N-terminal AJ localization domain of ASPP2 was necessary and sufficient for stimulating the p53-mediated transcription; the C-terminal p53 binding domain, on the other hand, inhibited the p53-dependent luciferase activation. Flow cytometry assay further supported that the AJ interacting domain stimulated, while the C-terminal p53 binding domain inhibited, cell death induced by p53. Phosphorylation of p53 at serine 6, 9, 15, 20, 46 and 392 was not altered by ectopic expression of ASPP, as revealed by immunoblotting. Together we have mapped the AJ localization domain to amino acids 97-227 of ASPP, and shown that the N-terminal 1-450 amino acids of the AJ localization domain is necessary and sufficient to promote the p53-mediated Bax promoter activation and apoptosis induction.