Oncogene (2011) 30, 2595–2609 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc REVIEW Nucleophosmin and its complex network: a possible therapeutic target in hematological diseases E Colombo1,2, M Alcalay1,2 and PG Pelicci1,2 1Department of Experimental Oncology, European Institute of Oncology, Milan, Italy and 2Dipartimento di Medicina, Chirurgia e Odontoiatria, University of Milano, Milan, Italy Nucleophosmin (NPM, also known as B23, numatrin or alternative splicing of the NPM1 transcript, have been NO38) is a ubiquitously expressed phosphoprotein belong- reported (Wang et al., 1993) (Figure 1). In the long ing to the nucleoplasmin family of chaperones. NPM is isoform, known as B23.1, exon 9 is spliced to exon 11 mainly localized in the nucleolus where it exerts many of and the coding sequence stops at exon 12, resulting in a its functions, but a proportion of the protein continuously protein of 294 residues. In the short isoform, B23.2, shuttles between the nucleus and the cytoplasm. A growing exon 9 is spliced to exon 10 that immediately contains a number of cellular proteins have been described as stop codon, and as a result, B23.2 is a truncated protein physical interactors of NPM, and consequently, NPM is of 259 residues. The prevalent form expressed in all thought to have a relevant role in diverse cellular tissues is B23.1, although the biological significance and functions, including ribosome biogenesis, centrosome physiological function of B23.2 remain largely unclear. In duplication, DNA repair and response to stress. NPM the human EST database a further transcript variant that has been implicated in the pathogenesis of several human lacks exon 8 has been identified (GenBank accession malignancies and intriguingly, it has been described both number: NM_199185). Its functional role as well as its as an activating oncogene and a tumor suppressor, tissue distribution are currently totally unknown. depending on cell type and protein levels. In fact, NPM is biochemically defined as a histone chaperone increased NPM expression is associated with different (Frehlick et al., 2007). It belongs to the nucleoplasmin/ types of solid tumors whereas an impairment of NPM nucleophosmin family of nuclear chaperones, sharing function is characteristic of a subgroup of hematolologic the core-domain at the N-terminus of the protein. This malignancies. A large body of experimental evidence links portion of the NPM protein includes an acidic tract and the deregulation of specific NPM functions to cellular is responsible for oligomerization and chaperone activ- transformation, yet the molecular mechanisms through ity (Hingorani et al., 2000). Histone chaperones are which NPM contributes to tumorigenesis remain elusive. required for the correct assembly/disassembly of nucleo- In this review, we have summarized current knowledge somes, and therefore are necessary for DNA-dependent concerning NPM functions, and attempted to interpret its activities like transcription, replication and repair multifaceted and sometimes apparently contradictory (Eitoku et al., 2008). Accordingly, NPM has been activities in the context of both normal cellular homeo- isolated as a putative histone chaperone using a series stasis and neoplastic transformation. of in vitro assays that reveal chromatin related functions. Oncogene (2011) 30, 2595–2609; doi:10.1038/onc.2010.646; Okuwaki et al., (2001a) showed that NPM favors published online 31 January 2011 replication of adenovirus DNA in complex with viral basic core proteins (viral nucleosome). They also Keywords: nucleophosmin; leukemia; cancer demonstrated that NPM interacts with human histones, binds preferentially to histone (H3-H4)2 tetramers and is able to transfer nucleosomes to naked DNA favoring chromatin assembly. On the other hand, NPM, like Nucleophosmin (NPM) is a multi-functional cellular other histone chaperones, decondenses sperm chromatin chaperone (Okuwaki et al., 2001b). Therefore, NPM behaves in vitro as a classical histone chaperone favoring NPM is the product of the NPM1 gene, which in assembly and disassembly of chromatin templates. humans contains 12 exons and maps to chromosome Whether this is a major role of NPM also in vivo is 5q35; two isoforms of the NPM protein, derived from less clear. NPM is part of the centromere protein A protein complex, which represents the nucleosome Correspondence: Dr E Colombo or Professor PG Pelicci, Department variant that replaces H3 in centromeric nucleosomes. of Experimental Oncology, Istituto Europeo di Oncologia, Via (Foltz et al., 2006, 2009) However, if NPM is required Adamello 16, Milano 20139, Italy. for centromere-specic assembly of centromere protein A E-mail: [email protected] or [email protected] nucleosomes has not been fully investigated. Received 23 November 2010; revised 24 December 2010; accepted 31 A number of reports implicate NPM in the control December 2010; published online 31 January 2011 of DNA transcription, in most cases through the Nucleophosmin and its complex network E Colombo et al 2596 S4 S10 S70 S125 T199 T219 T234/237 S254 B231.1 NPM1 isoform1 R230 R263 B231.2 NPM1 isoform2 257 259 NPMmut 287 298 NES oligomerization domain (1-119) Nuclear Localization Signal (141-157) Nuclear Export Signals (42-49; 94-102) Moderately basic region (189-257) Acidic domains (120-132; 161-188) Aromatic region (258-294; unique to B23.1 isoform) Central region Nucleolar localization region (WW 288, 290) NPM1 isoform 2 unique region (257-259) Mutated C-terminal domain (287-298; unique NPMmut) Figure 1 Schematic representation of NPM isoforms and AML-associated NPM mutant. Relevant functional domains are highlighted together with the aminoacidic position in parentheses. Modified aminoacids (phosphorylated and sumoylated) are also indicated. S4: serine4, phosphorylated by polo-like kinase 1 (Plk1) in mitosis and by Plk2 in S-phase. S10: serine10, phosphorylated by unknown CDK, involved in G2-M transition. S70: serine70, phosphorylated by unknown CDK, involved in G2-M transition. S125: serine125, phosphorylated by casein kinase II and ATR; involved in DNA damage response, chaperone activity and NPM1 mobility. T199: threonine199, phosphorylated by CDK2 in G1 and CDK1 in mitosis; involved in centrosome duplication and DNA damage response. T219: threonine219, phopshorylated by CDK1 in mitosis; involved in RNA binding. R230: arginine230, sumoylated; involved in stability, localization, proteins interaction, apoptosis. T234/37: threonine234/37, phosphorylated by CDK1 in mitosis; involved in RNA binding. S254: serine 254, phosphorylated by unknown kinase in mitosis. R263: arginine263, sumoylated; involved in stability, localization, proteins interaction and apoptosis. interaction with a variety of different transcription in vivo. In vitro functional studies suggest that NPM factors. NPM can either contribute to transcriptional displays chaperone-like behaviors on a wide variety of activation, as in the case of Myc-interacting zinc finger denatured substrates: (i) NPM inhibits temperature- protein 1 (Miz1) (Wanzel et al., 2008), androgen dependent and independent protein aggregation, due to receptor (Leotoing et al., 2008), ribosomal genes thermal denaturation, protecting enzymes from loss of (Murano et al., 2008), c-Myc (Li et al., 2008) and activity; (ii) NPM promotes renaturation of chemically nuclear factor of k light polypeptide gene enhancer in B- denatured proteins and it appears to preferentially bind cells (NF-kB) (Dhar et al., 2004), or to repression, as for denatured substrates through the exposure of hydro- the activating protein transcription factor 2 (AP2a) (Liu phobic regions in both NPM and substrates (Szebeni et al., 2007a). However, the exact mechanism through and Olson, 1999). Although NPM binds ATP, at least which NPM mediates these regulatory activities is not in vitro(Chang et al., 1998), its binding to substrates is completely elucidated, and it is unclear if it functions as governed by association-dissociation thermodynamics, a general co-factor during transcription or if it is and release of the substrate does not require ATP physically associated to specific promoters. In support hydrolysis (Szebeni and Olson, 1999), as seen with other of a more general role for NPM in transcriptional molecular chaperones including Hsp70 and chaperonins regulation, it has been reported that NPM interacts with (Ruddon and Bedows, 1997). Interestingly, it has been and inhibits the activity of hexamethylene bis-acetamide shown that phosphorylation of NPM by casein kinase II inducible 1 (HEXIM1), a negative regulator of Pol II- induces the release of denaturated substrates (Szebeni dependent transcription (Gurumurthy et al., 2008). et al., 2003). In the case of specific interactions between Furthermore, NPM has been involved in different ways NPM and its binding partners, which involve defined and at different levels as a modulator of chromatin protein domains, such as the human immunodeficiency status. In fact, NPM was found to enhance acetylation- virus-Rev protein, NPM phosphorylation does not dependent transcription of an artificial chromatin affect the interaction (Szebeni et al., 2003). All these template, likely by removing acetylated histones (Swa- data come from in vitro studies using recombinant minathan et al., 2005). In a different experimental proteins, therefore, we do not know if NPM shows the setting, Zou and colleagues recently showed that NPM same behavior in