Identification Of PIAS1 As SUMOylation E3 Ligase Of AIB1 And Relative Functional Research

As a transcriptional coactivator for nuclear receptors and other transcription factors, steroid receptor coactivator-3 (SRC-3/AIB1) is required for animal growth and reproductive development and implicated in breast carcinogenesis. Through covalent interaction with target protein, small molecular SUMO affects target proteins'stability, cellular localization and interactions with other proteins to regulate a series of important processes including transcriptional regulation. AIB1 has been proved covalently modified by SUMO-1, which cooperates with other posttranslational modifications like phosphorylation and ubiquitination to regulate its stability and transcriptional activity, but the potential SUMO E3 ligase taking part in its SUMOylation process was still unknown. PIAS (protein inhibitor of activated STAT) family has been a research highlight concerning in the recent years. PIAS proteins were found to promote target proteins'SUMOylation and final functions and take part in gene transcriptional regulation as SUMO E3 ligases. This research is mean to find the E3 ligase in AIB1 SUMOylation from PIAS family and to explore the effection to AIB1 function by PIAS family members.In this research, Luciferase reporter assays were primarily conducted to find the PIAS member that could inhibit the transcriptional activity of AIB1 and make it as the E3 ligase candidate; then Immunoprecipitation-immunoblotting experiments and Immunofluorescence assays were conducted to prove PIAS1 could interact with AIB1 in vivo and in vitro, which establish a basis for the following functional research on PIAS1; then Luciferase reporter assays and Immunoprecipitation-immunoblotting experiments were conducted to prove AIB1's SUMO-1 modification was strongly enhanced when PIAS1 was overexpressed. And through contracted with the results from the condition of E3 ligase activity losing mutent PIAS1 (C350S) was overexpressed, we found the enchancement of SUMOylation of AIB1 and the following decrease of AIB1's transcriptional activity were both dependent on PIAS1's SUMO E3 ligase activity, but the possibility of PIAS1 affecting AIB1's transcriptional activity through other ways could not be excluded; finally, through Immunoprecipitation and Immunoblotting experiments, we tried to find other effects to AIB1 by PIAS1. And we found overexpression of PIAS 1 promoted the stability of AIB1, which shouled be realized by the enhancement of SUMO-1 modification of AIB1. Moreover, we found overexpression of PIAS1 attenuate the interaction between AIB1 and ERa, which makes a foundation for the future research of PIAS1 affecting other relationships between AIB1 and ERa.In conclusion, for the fist time we found PIAS1 as E3 ligase of AIB1 SUMO-1 modification, and PIAS family especially PIAS1 play roles in the regulation of sever processes including AIB1's transcripitional activity, stability, and interactions with nuclear receptors like ERa. Our research provided important theoretic evidences for the further understanding of AIB1's cotranscripitonal activating machnism and the search of new drug targets for the breast cancer therapy.