Nucleophosmin (NPM/B23) is a key regulator in the regulation of a number of processes including centrosome duplication, maintenance of genomic integrity, and ribosome biogenesis. While the mechanisms underlying NPM function are largely uncharacterized, NPM loss results in severe dysregulation of developmental and growth-related events. We show that NPM utilizes a conserved CRM1-dependent nuclear export sequence in its amino terminus to enable its shuttling between the nucleolus/nucleus and cytoplasm. In search of NPM trafficking targets, we biochemically purified NPM-bound protein complexes from HeLa cell lysates. Consistent with NPM's proposed role in ribosome biogenesis, we isolated ribosomal protein L5 (rpL5), a known chaperone for the 5S rRNA. Direct interaction of NPM with rpL5 mediated the colocalization of NPM with maturing nuclear 60S ribosomal subunits, as well as newly exported and assembled 80S ribosomes and polysomes. Inhibition of NPM shuttling or loss of NPM blocked the nuclear export of rpL5 and 5S rRNA, resulting in cell cycle arrest and demonstrating that NPM and its nuclear export provide a unique and necessary chaperoning activity to rpL5/5S.As the most prominent of subnuclear structures, the nucleolus has long been recognized as the site of active transcription of rRNA and ribosome assembly (8). Various nucleolar proteins, RNAs, and other factors have been implicated in the complex process of ribosome production and maturation (18). Recently, several groups reported the successful isolation and mapping of the mammalian nucleolar proteome (1, 2, 44). While these studies clearly identified proteins and ribonucleoproteins with purported roles in ribosome biogenesis, a surprising number of proteins within the nucleolar proteome (Ͼ100) have no known function. In previous decades, it was assumed that all nucleolar proteins must somehow contribute to static ribosome biogenesis simply by virtue of their localization. However, more-recent findings have demonstrated that the nucleolus is a dynamic subnuclear organelle which regulates numerous cellular processes, prompting a broadened view of the potential functions of nucleolar proteins (28).Nucleophosmin (NPM/B23) is an abundant phosphoprotein that resides within the granular regions of the nucleolus (46). Proliferating cells express NPM at high levels (9, 13), and NPM has been associated with a variety of cellular events, including ribosomal biogenesis, protein chaperoning, and centrosome duplication (13,23,35,36). Structurally, NPM is present in both monomeric and multimeric states, although NPM multimers appear predominant in the nucleolus and may be crucial for the assembly of maturing ribosomes (33,34,53). Furthermore, NPM, along with other nucleolar proteins, is believed (or has been shown) to actively mobilize into distinct subcellular pools, supporting the notion that NPM trafficking may be essential for its (proper) function (6). Indeed, NPM's transit from the nucleolus/nucleus is an essential event in S phase progression; when NPM export was...

The ARF tumor suppressor is widely regarded as an upstream activator of p53-dependent growth arrest and apoptosis. However, recent findings indicate that ARF can also regulate the cell cycle in the absence of p53. In search of p53-independent ARF targets, we isolated nucleophosmin (NPM/B23), a protein we show is required for proliferation, as a novel ARF binding protein. In response to hyperproliferative signals, ARF is upregulated, resulting in the nucleolar retention of NPM and concomitant cell cycle arrest. The Mdm2 oncogene outcompetes NPM/B23 for ARF binding, and introduction of Mdm2 reverses ARF's p53-independent properties: in vitro, NPM is released from ARF-containing protein complexes, and in vivo S phase progression ensues. ARF induction by oncogenes or replicative senescence does not alter NPM/B23 protein levels but rather prevents its nucleocytoplasmic shuttling without inhibiting rRNA processing. By actively sequestering NPM in the nucleolus, ARF utilizes an additional mechanism of tumor suppression, one that is readily antagonized by Mdm2.The murine INK4a/ARF locus, encoding both the p16 INK4a and p19 ARF (p14 ARF in humans) tumor suppressors, exhibits an unparalleled efficiency of organization within a mammalian genome. Specifically, p16INK4a and p19 ARF contain distinct promoters and first exons yet splice into a shared second exon that is translated in alternative reading frames (ARF) (33). While both proteins clearly contribute to tumor surveillance in mice and humans, they appear to play coordinate, yet independent, roles within the cell cycle. p16INK4a imposes a G 1 /S phase block via direct inhibition of the cdk4 and cdk6 cyclindependent kinases, maintaining the active, hypophosphorylated state of the retinoblastoma (Rb) tumor suppressor (36). ARF, in response to hyperproliferative signals relayed by the expression of oncoproteins, such as Myc, E2F, E1A, and Ras, binds and sequesters Mdm2 in the nucleolus, thereby promoting p53-dependent pathways of growth arrest or apoptosis through stabilization of the nucleoplasmic pool of p53 (3, 9, 48). Additionally, ARF directly inhibits the ubiquitination of p53 by Mdm2, suggesting that nucleolar sequestration might not be a requisite step in ARF's activation of p53 (13,46).Mounting evidence suggests that the ARF-p53-Mdm2 pathway is not strictly linear. Mice engineered to overexpress a Myc transgene under the control of the immunoglobulin heavy chain enhancer (E) develop B-cell lymphomas that exhibit biallelic ARF deletion, mutation of p53, or Mdm2 overexpression (11). Additional molecular analysis revealed that several tumors which lacked functional p53 also displayed Mdm2 overexpression, arguing against a simple epistatic relationship among ARF, p53, and Mdm2 (11). Additionally, Carnero and colleagues showed that diminished ARF expression resulted in bypassing of replicative senescence, whereas induction of ARF restored ARF's tumor-suppressive properties, even in the presence of a dominant-negative p53 mutant (7). In support of these initial finding...

Since its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.