Specific domains of nucleolin interact with Hdm2 and antagonize Hdm2‐mediated p53 ubiquitination

Abstract

Nucleolin is an abundant multifunctional nucleolar protein with defined roles in ribosomal RNA processing, RNA polymerase I-catalyzed transcription, and the regulation of apoptosis. Earlier we reported that human nucleolin binds to the p53-antagonist Hdm2 as determined by reciprocal co-immunoprecipitation assays using cell lysates. We also demonstrated that nucleolin antagonizes Hdm2-mediated degradation of p53. Here, we identify specific domains of nucleolin and Hdm2 proteins that support mutual interaction and investigated the implications of complex formation on p53-ubiquitination and protein levels. Our data indicate that the nucleolin N-terminus as well as the central RNA-binding domain (RBD) are predominantly involved in binding to Hdm2. The nucleolin RBD robustly bound to the NLS/NES (nuclear localization and export signals) domain of Hdm2 in vitro, while N-terminus of nucleolin preferentially associated with the Hdm2 RING domain expressed in cells. We further demonstrate that the C-terminal GAR (Glycine-Arginine Rich) domain of nucleolin serves as the predominant binding domain for direct interaction with p53. While over-expression of nucleolin or its various domains had no significant effect on Hdm2 auto-ubiquitination, the nucleolin RBD antagonized the Hdm2 E3 ligase activity against p53, leading to p53 stabilization. Conversely, the adjacent GAR domain of nucleolin interacted with p53 causing a modest stimulatory effect on p53 ubiquitination. These data suggest that changes in nucleolin conformation can alter the availabilities of such domains in vivo to modulate the overall impact of nucleolin on Hdm2 activity and hence on p53 stability.

Effects of cadmium on nucleolus in root tips of Hordeum vulgare

“…The nucleolus contains several kinds of NPs such as nucleolin and fibrillarin, which are known as the two major and multifunctional NPs, participating in rRNA processing and cell activities. They take key roles in rDNA transcripts and ribosome assembly (Sobol et al 2006, Ugrinova et al 2007, Tajrishi et al 2011, Bhatt et al 2012. As for what kind of NPs influenced by Cd, there are few reports concerning with them.…”

Nucleolin and nucleophosmin: nucleolar proteins with multiple functions in DNA repair

“…However, domain-mapping experiments suggest that NCL binds p53 and HDM2 via different regionsthe C-terminal RGG domain and N-terminal RBD domains, respectively -and may form a ternary complex with both proteins simultaneously (Bhatt et al 2012). While NCL's interaction with HDM2 has been shown to inhibit HDM2-mediated p53 destabilization, both through driving p53-HDM2 dissociation and by promoting HDM2's own ubiquitination and degradation (Bhatt et al 2012;Saxena et al 2006), D r a f t 15 the formation of an NCL-p53-HDM2 ternary complex was also shown to result in recruitment of a ubiquitin protease, HAUSP, which supports deubiquitination and stabilization of p53, HDM2 and NCL (Lim et al 2015).…”

“…While NCL's interaction with HDM2 has been shown to inhibit HDM2-mediated p53 destabilization, both through driving p53-HDM2 dissociation and by promoting HDM2's own ubiquitination and degradation (Bhatt et al 2012;Saxena et al 2006), D r a f t 15 the formation of an NCL-p53-HDM2 ternary complex was also shown to result in recruitment of a ubiquitin protease, HAUSP, which supports deubiquitination and stabilization of p53, HDM2 and NCL (Lim et al 2015). How these apparently contradictory pathways may be coordinated and/or interact to mediate NCL-dependent p53 stabilization remains to be fully dissected.…”

“…such domains have been reported; dotted brackets for NCL indicate that a region which is not absolutely required for the interaction but enhances the affinity of NCL for the target. Interaction surfaces were determined by truncation mutants and interaction screening in all cases as follows: NCL-NPM1 (Li et al 1996); NCL-RAD50 (Goldstein et al 2013); NCL-HDM2 and NCL-p53 (Bhatt et al 2012); NCL-WRN (Indig et al 2012); NCL-RPA (Kim et al 2005); NCL-ssDNA (Hanakahi et al 2000); NCL-G-quadruplex (González and Hurley 2010;Hanakahi et al 1999); NPM1-p53 (Colombo et al 2002) translationally modified residues discussed in this review. Brackets beneath each protein indicate the reported minimal regions for interactions of each protein with its respective protein partners, where such domains have been reported; dotted brackets for NCL indicate that a region which is not absolutely required for the interaction but enhances the affinity of NCL for the target.…”

“…Brackets beneath each protein indicate the reported minimal regions for interactions of each protein with its respective protein partners, where such domains have been reported; dotted brackets for NCL indicate that a region which is not absolutely required for the interaction but enhances the affinity of NCL for the target. Interaction surfaces were determined by truncation mutants and interaction screening in all cases as follows: NCL-NPM1 (Li et al 1996); NCL-RAD50 (Goldstein et al 2013); NCL-HDM2 and NCL-p53 (Bhatt et al 2012); NCL-WRN (Indig et al 2012); NCL-RPA (Kim et al 2005); NCL-ssDNA (Hanakahi et al 2000); NCL-G-quadruplex (González and Hurley 2010;Hanakahi et al 1999); NPM1-p53 (Colombo et al 2002); NPM1-H3/H4 (Okuwaki et al 2001);NPM1-polyUb (Koike et al 2010); and NPM1-APE1 (Vascotto et al 2009). 182x264mm (300 x 300 DPI)…”