Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation.

Nucleophosmin (NPM, B23) is an abundant nucleolar phosphoprotein involved in ribosome biogenesis, and interacts with tumor suppressor proteins p53 and Rb. Here we show that NPM is a UV damage response protein that undergoes nucleoplasmic redistribution and regulates p53 and HDM2 levels and their interaction. By utilizing RNAi approaches and analyses of endogenous and ectopically expressed proteins, we demonstrate that NPM binds HDM2 and acts as a negative regulator of p53-HDM2 interaction. Viral stress, enforced by expression of Kaposi's sarcoma virus K cyclin, causes NPM redistribution, K cyclin-NPM association, and p53 stabilization by dissociation of HDM2-p53 complexes. The results demonstrate novel associations of HDM2 and K cyclin with NPM and implicate NPM as a crucial controller of p53 through inhibition of HDM2.

Reactivation of the p53 pathway as a treatment modality for KSHV-induced lymphomas.

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent for primary effusion lymphoma (PEL), a non-Hodgkin type lymphoma manifesting as an effusion malignancy in the affected individual. Although KSHV has been recognized as a tumor virus for over a decade, the pathways for its tumorigenic conversion are incompletely understood, which has greatly hampered the development of efficient therapies for KSHV-induced malignancies like PEL and Kaposi's sarcoma. There are no current therapies effective against the aggressive, KSHV-induced PEL. Here we demonstrate that activation of the p53 pathway using murine double minute 2 (MDM2) inhibitor Nutlin-3a conveyed specific and highly potent activation of PEL cell killing. Our results demonstrated that the KSHV latency-associated nuclear antigen (LANA) bound to both p53 and MDM2 and that the MDM2 inhibitor Nutlin-3a disrupted the p53-MDM2-LANA complex and selectively induced massive apoptosis in PEL cells. Together with our results indicating that KSHV-infection activated DNA damage signaling, these findings contribute to the specificity of the cytotoxic effects of Nutlin-3a in KSHV-infected cells. Moreover, we showed that Nutlin-3a had striking antitumor activity in vivo in a mouse xenograft model. Our results therefore present new options for exploiting reactivation of p53 as what we believe to be a novel and highly selective treatment modality for this virally induced lymphoma.

Redox state of tumor suppressor p53 regulates its sequence-specific DNA binding in DNA-damaged cells by cysteine 277.

Using a bio-oligo pull-down DNA-binding assay we investigated the binding capacity of endogenous, DNA damage-induced p53 in human diploid fibroblasts to several p53-responsive elements (REs) present in p53-regulated genes. During the course of p53 accumulation, we observed a decrease in p53 binding to the GADD45 but not to the p21(WAF1/CIP1) RE. Using mutated GADD45 sequences we show that this change is dependent on the presence of cytosines at position 3 in RE pentamers and on the p53 redox state. Site-directed mutagenesis experiments demonstrated that Cys277 (a residue directly contacting base 3 in a RE pentamer) is critical for differential regulation of GADD45 in DNA-damaged cells. These data represent a novel mechanism for differential affinity of p53 to distinct REs.

p53 and MDM2 are regulated by PI-3-kinases on multiple levels under stress induced by UV radiation and proteasome dysfunction.

p53 is a key stress responsive cellular component. It is negatively regulated by MDM2, which is also its transcriptional target. Here we have studied the involvement of phosphatidylinositol-3-kinases (PI-3-kinase) in the regulation of p53-MDM2 pathway following cellular stress induced by UV damage and proteasomal downregulation. We show that p53 stabilized both by proteasome inhibition and UV damage is transcriptionally active. However, p53 in proteasomally downregulated cells differs from UV-stabilized p53 in its interaction with MDM2, posttranslational modifications and subnuclear localization. It is known that members of PI-3-kinase family are able to directly phosphorylate p53 and MDM2. We show that these kinases regulate p53 accumulation after UV radiation, but accumulation of MDM2 after proteasome inhibition. Both proteins have earlier been shown to translocate into nucleoli after downregulation of the proteasome. We found this effect to be dependent on PI-3-kinase activity but not on any suggested PI-3-kinase phosphorylation site on MDM2. In conclusion, we show here that PI-3-kinases regulate p53-MDM2 pathway on multiple, earlier unknown levels.

The MELAS mutations 3946 and 3949 perturb the critical structure in a conserved loop of the ND1 subunit of mitochondrial complex I.

The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.

Cellular stress and DNA damage invoke temporally distinct Mdm2, p53 and PML complexes and damage-specific nuclear relocalization.

Mdm2 is a nucleoplasmic and nucleolar protein interacting with p53 and alternative reading frame (ARF) tumor suppressor proteins. Here we demonstrate relocalization and novel interactions of Mdm2 with the promyelocytic leukemia (PML) protein following cellular stress and DNA damage. We show that Mdm2 and PML interact directly in vivo and in vitro depending on the Mdm2 RING finger domain and the PML C-terminus, and that Mdm2 is recruited to the PML nuclear bodies by overexpression of PML. Cellular stress and DNA damage caused by UV-radiation, downregulation of the proteasome and arsenic trioxide promoted Mdm2 and PML damage-specific nuclear relocalization and interaction in a p53-independent manner. However, in vitro analyses showed that PML, Mdm2 and p53 form trimeric complexes. UV-radiation caused rapid rearrangements of PML nuclear bodies and promoted PML-p53 and PML-Mdm2 complex formation, coinciding with p53 stabilization and preceding p53-Mdm2 interaction suggesting temporally distinct complexes. The results demonstrate novel associations between Mdm2 and PML and show the capacity of PML to participate in the activation and stabilization of p53 in response to cellular stress through PML interaction with Mdm2.

Nucleophosmin, HDM2 and p53: players in UV damage incited nucleolar stress response.

p53 tumor suppressor protein acts as a critical monitor preventing survival of cells with irreparable genetic damage. Its levels are tightly controlled by its negative regulator HDM2, and are allowed to rise only during cellular stress. In our recent paper (Kurki, et al. Cancer Cell 2004; 5:465-75) we identify a novel mechanism leading to p53 stabilization following UV damage of the cells. This involves UV damage provoked nucleoplasmic relocalization of a nucleolar protein, nucleophosmin (NPM, B23) and its rapid and transient interactions with both p53 and HDM2. We discuss here implications of recent findings that several p53 pathway proteins interact with NPM and find that its participation in cellular damage responses is limited to transcriptional stress but absent in direct ds DNA breaks. These findings suggest divergence in the routes provoking p53 stability and implicate the nucleolus as a central site participating in transcriptional stress responses.