ARF Impedes NPM/B23 Shuttling in an Mdm2-Sensitive Tumor Suppressor Pathway

Abstract

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...

Binding to nucleophosmin determines the localization of human and chicken ARF but not its impact on p53

“…Reports of a direct interaction between mammalian ARF and NPM (Itahana et al, 2003;Bertwistle et al, 2004;Brady et (Kim et al, 2003).…”

“…A notable example is nucleophosmin (also called NPM, B23, numatrin or NO38), which has been consistently identified in screens for ARF binding proteins in different laboratories, including our own (Itahana et al, 2003;Bertwistle et al, 2004;Brady et al, 2004;Korgaonkar et al, 2005). NPM is an abundant, evolutionarily conserved protein that resides predominantly in the nucleolus and also shuttles between the nucleus and cytoplasm (Grisendi et al, 2006).…”

“…The gene is also a target of chromosomal translocations and frameshift mutations in human leukaemia (Grisendi et al, 2006). Although a number of reports have suggested that ARF regulates NPM function (Itahana et al, 2003;Brady et al, 2004;Enomoto et al, 2006), a more logical interpretation for which there is growing support would be that binding to NPM is responsible for the nucleolar accumulation and stability of ARF (Kuo et al, 2004;Korgaonkar et al, 2005).…”

An Extensive Tumor Array Analysis Supports Tumor Suppressive Role for Nucleophosmin in Breast Cancer

“…8,9 NPM also regulates the tumor suppressor proteins p53 10 -12 and p14 ARF . [13][14][15] Moreover, NPM protein is post-translationally modified by acetylation, 16 sumoylation, 17,18 ubiquitinylation, 19 and phosphorylation. 20 -23 NPM has been heavily implicated in cancer pathogenesis, but its actual role in oncogenesis is controversial.…”

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

“…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).…”