The ARF tumor suppressor is a nucleolar protein that activates p53-dependent checkpoints by binding Mdm2, a p53 antagonist. Despite persuasive evidence that ARF can bind and inactivate Mdm2 in the nucleoplasm, the prevailing view is that ARF exerts its growth-inhibitory activities from within the nucleolus. We suggest ARF primarily functions outside the nucleolus and provide evidence that it is sequestered and held inactive in that compartment by a nucleolar phosphoprotein, nucleophosmin (NPM). Most cellular ARF is bound to NPM regardless of whether cells are proliferating or growth arrested, indicating that ARF-NPM association does not correlate with growth suppression. Notably, ARF binds NPM through the same domains that mediate nucleolar localization and Mdm2 binding, suggesting that NPM could control ARF localization and compete with Mdm2 for ARF association. Indeed, NPM knockdown markedly enhanced ARF-Mdm2 association and diminished ARF nucleolar localization. Those events correlated with greater ARF-mediated growth suppression and p53 activation. Conversely, NPM overexpression antagonized ARF function while increasing its nucleolar localization. These data suggest that NPM inhibits ARF's p53-dependent activity by targeting it to nucleoli and impairing ARF-Mdm2 association.

The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.

The NF-B family of transcription factors plays a fundamental role in development, maintenance of the immune system, and cell viability (1-3). NF-B is composed of heterodimers of DNA-binding subunits (p50 and p52) and subunits with transcriptional activity (p65 (RelA), RelB, or c-Rel). In unstimulated cells, binary complexes of these subunits are restricted to the cytoplasm by interaction with members of a family of inhibitory proteins, inhibitors of B (IBs) 1 (4, 5). In response to extracellular stimuli such as cytokines or UV radiation, IB proteins are phosphorylated, polyubiquitinated, and then degraded by the 26 S proteasome (6 -13). Dissociation from IBs unmasks the nuclear localization sequence of NF-B, permitting it to move into the nucleus, bind the promoters of target genes, and alter gene expression and cell function (10,13,14). The demonstration that phosphorylation of IB proteins initiates events necessary for activation of NF-B led to the discovery of IB kinase (IKK) complexes composed of IKK␣, IKK␤, and IKK␥ (NEMO) (15)(16)(17)(18)(19). IKK␣ and IKK␤ are serine-threonine kinases, and IKK␥ is a scaffolding protein essential for the function of IKK␣ and IKK␤. IKK␣ and IKK␤ share a high degree of amino acid homology and domain organization. The kinases are composed of an N-terminal kinase domain, a leucine zipper that facilitates homo-and heterodimerization, and a helix-loop-helix domain (20). IKK␣ and IKK␤ can be activated by diverse kinases, among which are NF-B-inducing kinase (NIK), MEKK1, Cot, NF-Bactivating kinase (NAK/TBK), protein kinases C␤ and C␦, and MEKK3 (21-28). However, interest remains sustained in identifying other kinases that affect IKK complexes and NF-B activity. One of these is the Akt serine-threonine kinase, a downstream target for activated phosphatidylinositol 3-kinase (PI 3-kinase) (29 -32). Akt is activated by mitogens and cytokines that function as survival factors. Akt mediates its functions by phosphorylating substrates that decrease the activity of pro-apoptotic proteins or increase the activity of anti-apoptotic proteins (32)(33)(34)(35)(36)(37)(38)(39)(40)(41).Akt may affect NF-B through multiple mechanisms. We demonstrated previously that TNF activates Akt, which phosphorylates and activates IKK␣, thus promoting NF-B function (42). TNF and interleukin-1 can also increase the transactivation potential of the RelA/p65 subunit of NF-B through a mechanism in which Akt has been implicated (43-45). PI 3-kinase activated by phorbol esters or lipopolysaccharide and PI 3-kinase/Akt signaling induced by signaling through CD40, interleukin-1, or G protein-coupled receptors activates [46][47][48]. However, PI 3-kinase/Akt signaling induced by TNF in human umbilical vein endothelial cells inhibits apoptosis without playing a significant role in activation of NF-B (49). Furthermore, Akt can activate a member of the mitogenactivated protein kinase kinase kinase (MAP3K) family, Cot, and indirectly affect IKK activity and NF-B (50). Thus, PI 3-kinase/Akt signaling is upstream of diver...

It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal 31 RRPR 34 did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.