Mapping the Functional Domains of Nucleolar Protein B23

Abstract

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N-and Cterminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N-and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acidbinding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.Ribosome assembly is a multistep process that utilizes numerous proteins and small nucleolar RNAs (1, 2). One candidate for a ribosome assembly factor is an abundant protein called B23 (also known as nucleophosmin/NPM (3), NO38 (4), or numatrin (5)) whose location, abundance, and multiple activities suggest that it plays a major role in ribosome biogenesis. This is supported by the ability of protein B23 to bind nucleic acids (6, 7) and by its association with maturing preribosomal ribonucleoprotein particles (4,8,9). Treatment of cells with drugs that inhibit preribosomal RNA processing or synthesis (10, 11) causes translocation of B23 to the nucleoplasm, which further suggests its presence in nascent preribosomal particles. Finally, protein B23 possesses intrinsic ribonuclease activity that has been implicated in the processing of preribosomal RNA in the internal transcribed spacer region 2 region (12, 13).Protein B23 interacts with other nucleolar proteins, including nucleolin (14), protein p120 (15), and the HIV-1 Rev protein (16). Its ability to shuttle between the nucleus and cytoplasm (17), bind nuclear localization signal containing peptides (18), and stimulate import of proteins into the nucleus (18) suggested a role in nuclear import. The latter activity might be explained by its ability to act as a molecular chaperone (19). In normal cells, this activity may aid in the transport of ribosomal or other nucleolar proteins from their site of synthesis into the nucleus or nucleolus. Alternatively, protein B23 could serve as a chaperone in preventing aggregation of proteins in the very crowded environment of the nucleolus during ribosome assemb...

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

“…Structure-based discovery of small molecular inhibitors of NPM Nucleophosmin forms dimers and higher oligomers via its N-terminal globular domain (residues, 1-110) ( Figure 1a, Hingorani et al, 2000). We hypothesized that an SMI, targeting protein-protein interactions of the dimer interface would inhibit NPM function.…”

“…This is due to the discovery of several specific small molecular agents targeting anti-apoptotic targets such as Bcl-2-Bax (Oltersdorf et al, 2005), MDM2-p53 (Vassilev et al, 2004) and XIAP-SMAC (Oost et al, 2004). Under native condition, NPM exists as oligomers via its Nterminal molecular chaperone domain (Herrera et al, 1996;Hingorani et al, 2000;Namboodiri et al, 2004). We hypothesized that a specific SMI that disrupts the formation of oligomers would inhibit NPM function(s) in cancer cells.…”

B23 acts as a nucleolar stress sensor and promotes cell survival through its dynamic interaction with hnRNPU and hnRNPA1

“…hnRNPU-B23 interaction is mediated by histone-binding domain of B23 and RNA-binding domain of hnRNPU Both B23 and hnRNPU were known to contain functional domains, which account for their distinctive biochemical functions (Hingorani et al, 2000;Kukalev Figure 1 B23 interacts with hnRNPA1 and hnRNPU in the cytosol on ActD treatment. (a) HeLa cells stably expressing GFP were treated with the 0.1% DMSO vehicle, CHX (25 mg/ml) and ActD (1 mg/ml), respectively, followed by immunostaining with anti-B23 antibody (red).…”

“…The multiple functions of B23 make it both a potential oncogene and a potential tumor suppressor, depending on expression levels and environmental stimuli (Grisendi et al, 2006). B23 possesses a nuclear-localization signal and a nuclearexport signal, which allows it to shuttle between nucleus and cytoplasm (Hingorani et al, 2000;Wang et al, 2005). The shuttling property of B23 contributes to its activity in various cellular processes including the transport of pre-ribosomal particles, the response to stress stimuli such as UV irradiation and hypoxia, as well as cell cycle regulation (Yung et al, 1985;Wu et al, 2002;Brady et al, 2004).…”

Increased expression of nucleophosmin/B23 in hepatocellular carcinoma and correlation with clinicopathological parameters

“…Overexpression and tumorigenic activation of the Smoothened (SMO) protooncogene mediates c-myc overexpression, suggesting that SMO may also be a prognostic factor in HCC tumorigenesis (Sicklick et al, 2006). Nucleophosmin (NPM) is a major nucleolar phosphoprotein implicated in multiple cellular functions, including ribosomal protein assembly and transport (Verheggen et al, 2000;Huang et al, 2005), centrosome duplication (Okuda et al, 2000;Okuda, 2002;Grisendi et al, 2005), molecular chaperone activity in preventing protein aggregation (Hingorani et al, 2000;Szebeni et al, 2003), and regulating the activity of the tumour suppressors p53 (Colombo et al, 2002;Li et al, 2004;Maiguel et al, 2004) and p14 ARF (Itahana et al, 2003;Bertwistle et al, 2004;Brady et al, 2004). Earlier studies have shown that the level of NPM is markedly and promptly increased in association with cell commitment to mitogenesis (Feuerstein and Mond, 1987;Feuerstein et al, 1988).…”