Defining the molecular basis of Arf and Hdm2 interactions.

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Arf) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14(Arf) are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition from disordered conformations to extended structures comprised of beta-strands. The presence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of five amino acid residues using surface plasmon resonance. Surface plasmon resonance and circular dichroism spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) interact with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the limited size of the protein domains involved provide opportunities for the development of anticancer therapeutics.     

Solution structure of the p53 regulatory domain of the p19Arf tumor suppressor protein

Arf is a tumor suppressor that regulates p53 function and is a frequent target for loss in human cancers. Through two novel mechanisms, Arf inhibits the oncoprotein Hdm2, a negative regulator of p53. (1) Arf inhibits the E3 ubiquitin ligase activity of Hdm2 that leads to p53 degradation, and (2) Arf sequesters Hdm2 within nucleoli. These activities of Arf promote p53-mediated cell cycle arrest and apoptosis. Fundamental to these processes are interactions between Arf and Hdm2. Here we show that a peptide containing the 37 N-terminal amino acids of mouse Arf (mArfN37) localizes to nucleoli, sequesters Hdm2 within nucleoli, and causes cell cycle arrest. Circular dichroism and NMR spectroscopy show that mArfN37 is largely unstructured under aqueous conditions; however, the peptide adopts two alpha-helices (helix 1, residues 4-14; and helix 2, residues 20-29) in 2,2,2-trifluoroethanol (TFE). Each helix contains an amino acid motif that is repeated twice in mArfN37, once in each helix. The two helices, however, do not interact but are connected by an apparently flexible linker. The repeated motif contains Arg residues spaced by a hydrophobic segment that may be involved in Hdm2 recognition and binding. The RRPR nucleolar localization signal, contained within residues 31-34, appears to be disordered under all conditions. The identification of two Arf structural modules suggests that short peptides containing the repeated motif may function as Arf mimics and may allow the design of small molecule Arf mimics in the future.     

Arf-induced turnover of the nucleolar nucleophosmin-associated SUMO-2/3 protease Senp3

The stabilization and subcellular localization of the p19Arf tumor suppressor protein and the SUMO-2/3 deconjugating protease Senp3 each depend upon their binding to the abundant nucleolar protein nucleophosmin (Npm/B23). Senp3 and p19Arf antagonize each otherâ€?s functions in regulating the SUMOylation of target proteins including Npm itself. The p19Arf protein triggers the sequential phosphorylation, polyubiquitination, and rapid proteasomal degradation of Senp3, and this ability of p19Arf to accelerate Senp3 turnover also depends on the presence of Npm. In turn, endogenous p19Arf and Senp3 are both destabilized in viable Npm-null mouse embryo fibroblasts (that also lack p53), and reintroduction of the human NPM protein into these cells reverses this phenotype. NPM mutants that retain their acidic and oligomerization domains can re-stabilize both p19Arf and Senp3 in this setting, but the nucleolar localization of NPM is not strictly required for these effects. Knockdown of Senp3 with shRNAs mimics the anti-proliferative functions of p19Arf in cells that lack p53 alone or in triple knock-out cells that lack the Arf, Mdm2 and p53 genes. These findings reinforce the hypothesis that the p53-independent tumor suppressive functions of p19Arf may be mediated by its ability to antagonize Senp3, thereby inducing cell cycle arrest by abnormally elevating the cellular levels of SUMOylated proteins.     

Nitric oxide-mediated inhibition of Hdm2-p53 binding

It has become increasingly evident that nitric oxide exerts its effects, in part, by S-nitrosylation of cysteine residues. We tested in vitro whether nitric oxide may indirectly control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, a critical step in Hdm2 regulation of p53. The presence of excess amounts of cysteine or dithiothreitol blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies to disrupt Hdm2-p53 binding. This cysteine is proximal to the Hdm2-p53 binding interface and is conserved across species from zebrafish to humans. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation.     

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.     

Jab1 induces the cytoplasmic localization and degradation of p53 in coordination with Hdm2

The biological mechanisms for maintaining the basal level of p53 in normal cells require nuclear exclusion and cytoplasmic degradation. Here, we showed that Jab1 facilitates p53 nuclear exclusion and its subsequent degradation in coordination with Hdm2. p53 was excluded from the nucleus in the presence of Jab1; this exclusion was prevented by leptomycin B treatment. Nuclear export of p53 was accompanied by a decrease in the levels of p53, as well as of its target proteins, which include p21 and Bax. Domain analyses of Jab1 showed that the N-terminal domain, 1-110, was capable of inducing cytoplasmic translocation of p53. Furthermore, 110-191 was required to facilitate the degradation of p53. Neither of these mutants incorporated into the CSN complex, indicating that Jab1 could affect the levels of p53 independent of intact CSN complex. Conversely, Jab1 was incapable of translocating and degrading two p53 mutants, W23S and 6KR, neither of which could be modified by Hdm2. Moreover, Jab1 did not affect the cellular localization or protein levels of p53 in p53 and Hdm2 double-null mouse embryo fibroblasts. We further observed that the ablation of endogenous Jab1 by small interfering RNA prevented Hdm2-mediated p53 nuclear exclusion. Under stressed conditions, which could sequester Hdm2 in its inactive state, Jab1 did not affect p53. Our studies implicate that Jab1 is required to remove post-translationally modified p53 and provide a novel target for p53-related cancer therapies.     

Abrogation of Wip1 expression by RITA-activated p53 potentiates apoptosis induction via activation of ATM and inhibition of HdmX

Inactivation of the p53 tumour suppressor, either by mutation or by overexpression of its inhibitors Hdm2 and HdmX is the most frequent event in cancer. Reactivation of p53 by targeting Hdm2 and HdmX is therefore a promising strategy for therapy. However, Hdm2 inhibitors do not prevent inhibition of p53 by HdmX, which impedes p53-mediated apoptosis. Here, we show that p53 reactivation by the small molecule RITA leads to efficient HdmX degradation in tumour cell lines of different origin and in xenograft tumours in vivo. Notably, HdmX degradation occurs selectively in cancer cells, but not in non-transformed cells. We identified the inhibition of the wild-type p53-induced phosphatase 1 (Wip1) as the major mechanism important for full engagement of p53 activity accomplished by restoration of the ataxia telangiectasia mutated (ATM) kinase-signalling cascade, which leads to HdmX degradation. In contrast to previously reported transactivation of Wip1 by p53, we observed p53-dependent repression of Wip1 expression, which disrupts the negative feedback loop conferred by Wip1. Our study reveals that the depletion of both HdmX and Wip1 potentiates cell death due to sustained activation of p53. Thus, RITA is an example of a p53-reactivating drug that not only blocks Hdm2, but also inhibits two important negative regulators of p53 - HdmX and Wip1, leading to efficient elimination of tumour cells.     

p19Arf suppresses growth, progression, and metastasis of Hras-driven carcinomas through p53-dependent and -independent pathways

Ectopic expression of oncogenes such as Ras induces expression of p19(Arf), which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19(Arf), and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, p19(Arf)-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in p19(Arf)-deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both p19 (Arf)- and p53-deficient mice. Thus, p19(Arf) inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19(Arf) also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not p19( Arf)-null mice, and p53 mutations were more frequently seen in wild-type than in p19( Arf)-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19(Arf).     

Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response

The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G(2)/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.     

Nucleolar Arf sequesters Mdm2 and activates p53

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.     

Further Insight into Substrate Recognition by USP7: Structural and Biochemical Analysis of the HdmX and Hdm2 Interactions with USP7

Ubiquitin-specific protease 7 (USP7) catalyzes the deubiquitination of several substrate proteins including p53 and Hdm2. We have previously shown that USP7, and more specifically its amino-terminal domain (USP7-NTD), interacts with distinct regions on p53 and Hdm2 containing P/AxxS motifs. The ability of USP7 to also deubiquitinate and control the turnover of HdmX was recently demonstrated. We utilized a combination of biochemistry and structural biology to identify which domain of USP7 interacts with HdmX as well as to identify regions of HdmX that interact with USP7. We showed that USP7-NTD recognized two of six P/AxxS motifs of HdmX (⁸AQCS¹¹ and ³⁹⁸AHSS⁴⁰¹). The crystal structure of the USP7-NTD:HdmX^AHSS complex was determined providing the molecular basis of complex formation between USP7-NTD and the HdmX^AHSS peptide. The HdmX peptide interacted within the same residues of USP7-NTD as previously demonstrated with p53, Hdm2, and EBNA1 peptides. We also identified an additional site on Hdm2 (³⁹⁷PSTS⁴⁰⁰) that interacts with USP7-NTD and determined the crystal structure of this complex. Finally, analysis of USP7-interacting peptides on filter arrays confirmed the importance of the serine residue at the fourth position for the USP7-NTD interaction and showed that phosphorylation of serines within the binding sequence prevents this interaction. These results lead to a better understanding of the mechanism of substrate recognition by USP7-NTD.     

West Nile virus capsid protein induces p53-mediated apoptosis via the sequestration of HDM2 to the nucleolus

The capsid protein of the West Nile virus (WNV) functions as an apoptotic agonist via the induction of mitochondrial dysfunction and the activation of caspases-9 and -3. Here, we have determined that the WNV capsid (WNVCp) is capable of binding to and sequestering HDM2 into the nucleolus. WNVCp was shown to interfere with the formation of the HDM2 and p53 complex, thereby causing the stabilization of p53 and the subsequent induction of its target apoptotic protein, Bax. Whereas WNVCp was capable of inducing the p53-dependent apoptotic process in wild-type mouse embryonic fibroblasts (MEF) or SH-SY5Y cells, it exerted no significant effects on p53-null MEF or on p53-knockdown SH-SY5Y cells. This suggests that WNVCp-mediated apoptosis requires p53. Furthermore, when WNV was transfected into cells, endogenous Hdm2 and WNVCp were able to interact physically. WNVCp expressed in wild-type MEF proved able to induce the translocation of the endogenous Hdm2 into the nucleolus. Consistently, WNV was highly pathogenic in the presence of p53, and was less so in the absence of p53. The results of these studies suggest that the apoptotic mechanism mediated by WNV might occur in accordance in a fashion similar to that of the tumour-suppressing mechanism mediated by ARF.