Effect Of Negative Regulation Of RLIM On P53 Signaling Pathway By MDMX

This study focuses on the critical tumor suppressor p53. The thesis contains two parts:The first part is about how MDMX negatively regualtes p53 transcription activity enhanced by RLIM. P53 is a crucial tumor suppressor. More than half of all human tumors contain p53 mutations, and in cells which retain wild-type p53, other defects in p53 pathway play important roles in tumorigenesis. MDMX is a homologue of MDM2, and both of them are critical negative regulator of p53. Similarly to MDM2, MDMX was reported to overexpress or amplify in various types of human tumors. MDMX amplification and/or overexpression in tumors were considered as an alternative way to inactivate p53 signal pathway. However, it is still unknow whether mdmx amplifies or overexpresses in tumors of Chinese hepatocellular carcinoma patients. Here, we reported that the mRNA level of MDMX was significantly elevated in the tumor tissues of 90 Chinese IICC patients when comparing to theis adjacent nontumorous tissues (Wilcoxon signed rank test, p<0.00001), suggesting that MDMX might play a role during the occurrence and development of IICC.RLIM (RING finger LIM domain binding protein) is a RING H2 type of E3 ubiquitin ligase. Previous research in our lab indicated that RLIM could interact with both p53 and MDM2, and enhance p53 acetylation and p53 transcriptional activity. However, it is unclear whether MDMX has any effect on the regulation of p53 transcriptional activity enhanced by RLIM. Here, we reported that knocking down MDMX in cells synergistically enhanced the ability of RLIM to activate p53 transcriptional activity. MDMX and RLIM could interact with each other both in vivo and in vitro. We then identified that the C-terminal of MDMX (aa304-490) interacted with two specific domains of RLIM (aa209-226 and aa313-410). MDMX could not regulate RLIM protein levels. IIowever, MDMX protein levels could be elevated by RLIM, and this was dependent on endogenous MDM2. Moreover, overexpression of RLIM does not affect MDMX mRNA levels. We found that knocking down RLIM expression decreased MDMX protein levels in MCF-7 cells. Moreover, MDMX could be ubiquitinated by RLIM.RLIM could inhibit MDMX degradation by MDM2 and increase the steady-state level of MDMX, suggesting that endogenous MDMX inhibited the activation of p53 pathway by RLIM. Furthermore, competitive co-immunoprecipitation experiments confirmed that RLIM was unable to interfere with the interaction between MDM2 and MDMX. We also found that MDMX, MDM2 and RLIM could form heterotrimeric complex using co-immunoprecipitation assay. The formation of this heterotrimeric complex might inhibit the degradation of ubiquitinated MDMX and interfered with p53 transcriptional activity enhanced by RLIM.The second part of this thesis is about the interplay between the four negative regulators of p53:MDM2, MDMX, Pirh2 and COP1. MDM2, Pirh2 and COP1 are important E3 ubiquitin ligases, which target p53 for proteasome-mediated degradation. The MDM2 homologous protein MDMX inhibits p53-mediated transcription activity. Here, we report that there were interactions between these four proteins independently of p53. The protein levels of MDM2, MDMX, Pirh2 and COP1 changed when any two of them were co-transfected. The integrity of MDM2 RING finger domain was crucial for its ability to elevate the protein levels of COP1 and Pirh2. Any two of these four proteins could inhibit p53-mediated transcriptional activity synergistically. Furthermore, COP1 inhibited MDM2 self-ubiquitination and MDMX ubiquitination by MDM2.In summary, in the first part of this thesis, we found that MDMX mRNA level elevated significantly in HCC in tumors of Chinese HCC patients. We uncovered that MDMX interfered with p53 transcriptional activity enhanced by RLIM. The ternary complex formed by MDMX, MDM2 and RLIM might inhibit the degradation of ubiquitinated MDMX and negatively regulate p53 transcriptional activity enhanced by RLIM. We identified a new regulator of MDMX protein, and our results also confirmed that MDMX could not only inhibit p53 activity directly, but also inhibit p53 transcriptional activity enhanced by RLIM. In the second part, we uncovered the interplay between the four p53 negative regulators: MDM2, MDMX, Pirh2 and COP1. Our results suggest that these four proteins might inhibit p53 activity synergistically in vivo.