title: 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 human double minute 2 (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 investigate 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 the N-terminus of nucleolin preferentially associated with the Hdm2 RING (really interesting new gene) domain expressed in cells. We further demonstrate that the C-terminal glycine-arginine rich domain of nucleolin serves as the predominant binding domain for direct interaction with p53. While overexpression 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 glycine-arginine rich 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.
authors: New York University School of Medicine, NY, USA.
authors: Purvi
authors: P
authors: Bhatt
authors: Claire
authors: C
authors: d'Avout
authors: Naomi S
authors: NS
authors: Kane
authors: James A
authors: JA
authors: Borowiec
authors: Anjana
authors: A
authors: Saxena
mesh_terms: D001665:Binding Sites; D006801:Humans; D019913:Nuclear Localization Signals; D010750:Phosphoproteins / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism* / Q000502:physiology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors; D054875:Ubiquitination; D000097796:Nucleolin
chemical_list: D019913:Nuclear Localization Signals; D010750:Phosphoproteins; D051736:Proto-Oncogene Proteins c-mdm2; D016601:RNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D044767:Ubiquitin-Protein Ligases; D000097796:Nucleolin; C495901:TP53 protein, human; C497206:MDM2 protein, human