C23 Interacts with B23, A Putative Nucleolar‐Localization‐Signal‐Binding Protein

Abstract

The human protein C23 (nucleolin) is a major nucleolar protein. Its interactions with other proteins were studied with the two-hybrid system which identified nucleolar protein B23 (nucleoph'osmin) as being associated with C23. Both proteins were co-immunoprecipitated from HeLa cell nuclear extract by either monoclonal antLC23 or monoclonal anti-B23. Binding studies utilizing deletion mutants indicated that the binding of C23 and B23 involves specific motifs. In addition to an approximately 46-amino-acidbinding domain in B23 (amino acids 194-239), amino acids 540-628 of C23 were required for binding; this region of C23 is required for the nucleolar localization. In addition, nucleolar protein p120 was also found to be co-immunoprecipitated with B23. A fragment of p120 containing a functional nucleolar localization signal bound to the truncated binding domain of B23, as did C23. These results suggest that the interaction of C23 and B23 may represent a nucleolar-targeting mechanism in which B23 acts as a nucleolar-localization signal-binding protein.Keywords: C23 ; nucleolin; B23 ; nucleophosmin ; protein-protein interaction.The nucleolus is the site where preribosomes are formed. Recent studies showed that the nucleolus may also be important for poly(A)-rich RNA transport and processing [l]. Over the years, a number of nucleolar proteins have been discovered and characterized in our laboratory, including protein C23 (nucleolin) [2], protein B23 (nucleophosmin) [2], and protein p120 [3].Although a great deal of knowledge on the chemistry of these proteins has been accumulated, the role of these proteins in nucleolar structure and function is still unknown. In this regard, we consider that understanding the interactions between certain nucleolar proteins would be useful. Thus, the interaction of nucleolar protein C23 with other proteins was studied.C23 is a highly conserved major nucleolar protein found in multiple species including human, rodent, insect, frog and chicken cells Abbreviations. NOR, nucleolar organizer regions ; RRM, RNA recognition motif; GST, glutathione S-transferase; NoLS, nucleolar-localization signal.College of Medicine, Houston, TX 77030 has also been confirmed by co-immunoprecipitation of the two proteins from HeLa cell nuclear extracts. Further studies utilizing deletion mutants of C23 or B23 revealed that the binding of the two proteins requires specific amino acid motifs and such binding may be important for the nucleolar localization of C23.

NSC348884, a nucleophosmin inhibitor disrupts oligomer formation and induces apoptosis in human cancer cells

“…The structural features of NPM consist of an oligomerization domain, a metal-binding motif, a bipartite nuclear localization signal, phosphorylation sites and a nucleolar localization signal (Wang et al, 1993;Wang et al, 2005). Mounting evidence supports that NPM interacts with a variety of proteins including nucleolin (Li et al, 1996), cell cycle-related protein p120 (Valdez et al, 1994), human immunodeficiency virus (HIV)-1 Rev protein (Fankhauser et al, 1991), Human MDM2 (HDM2) (Kurki et al, 2004), tumor suppressor ARF (Itahana et al, 2003;Bertwistle et al, 2004;Korgaonkar et al, 2005), p53 (Colombo et al, 2002;Li et al, 2004;Maiguel et al, 2004) and pRb (Takemura et al, 1999).…”

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

“…The two proteins represent major constituents of mammalian nucleoli, with NCL in particular comprising up to 10% of all protein content within the nucleolus (Durut and Saez-Vasquez 2015). Subsequent work has shown that these two proteins share numerous physiological features including their ability to undergo nucleocytoplasmic shuttling (Borer et al 1989) and to act as RNA and protein chaperones (Angelov et al 2006 Hanakahi et al 2000;Szebeni and Olson 1999;Thyagarajan et al 1998), their active relocalization from the nucleolus to the nucleoplasm in response cellular stresses (Colombo et al 2011;Tajrishi et al 2011), and their ability to interact with each other, among others (Li et al 1996). Several decades of detailed functional study have revealed that both proteins are remarkably multifunctional, playing overlapping but non-identical roles in a range of cellular survival and proliferation pathways including ribosome biogenesis, cell cycle and apoptosis, transcriptional regulation, and DNA replication and repair (Colombo et al 2011;Tajrishi et al 2011).…”

“…As a histone chaperone, NPM1 binds most efficiently to acetylated H3/H4 heterodimers and, as mentioned above, is able to promote the activities of nucleosome remodeling complexes and to support transcription and chromatin packaging activities (Okuwaki et al 2001;Swaminathan et al 2005). The ability of NPM1 to interact with highly positive sequences, along with its oligomerization activity have D r a f t 9 led several groups to propose that NPM1 may be the scaffolding protein responsible for retention of proteins containing arginine/lysine-rich nucleolar localization signals (NoLSs) within the nucleolus (Emmott and Hiscox 2009;Li et al 1996;Mitrea et al 2016). Through its C-terminal regions, NPM1 is able to bind short ssDNA sequences and promote dsDNA strand annealing (Borggrefe et al 1998) and, independently of those functions, to also interact with structured RNAs in response to genotoxic stress (Yang et al 2002).…”

“…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.…”

Centrosomal nucleolin is required for microtubule network organization

“…Nucleolin and B23 have numerous functions in common. Both proteins interact 56 and are involved in ribosome biogenesis within the nucleoli. In the same manner as nucleolin, B23 is localized to the centrosome during interphase 57 and its absence leads to microtubule polymerization defects.…”