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The p53 family of proteins has an important role in determining cell fate in response to different types of stress, such as DNA damage, hypoxia, or oncogenic stress. In recent years, p53 has also been shown to respond to metabolic stress, and to be induced by the AMP-activated protein kinase (AMPK), a central cellular energy sensor. A bioinformatic analysis revealed three putative AMPK phopshorylation sites in p73, a p53 tumor suppressor paralog. In vitro and in vivo assays confirmed that AMPK phosphorylates p73 on a novel residue, S426. Following specific pharmacologic stimulation of AMPK in cells, p73 protein half-life was prolonged leading to p73 accumulation in the nucleus. We show that p73 escaped the E3 ligase Itch resulting in reduced p73 ubiquitination and proteasomal degradation. Furthermore, chronic activation of AMPK led to apoptosis that was p73 dependent, but only in p53-expressing cells. Surprisingly, we found that p73 was required for p53 stabilization and accumulation under AMPK activation, but was dispensable under DNA damage. Our findings couple p73 with p53 in determining cell fate under AMPK-induced metabolic stress.

The members of the tumor suppressor p53 family are under tight regulation by distinct ubiquitin-protein isopeptide (E3) ligases. The level of p73 is regulated by the E3 ligase Itch. Itch levels are sharply reduced in response to DNA damage with concomitant p73 accumulation and activation. The mechanism of controlling Itch level is not known. We show that the Itch promoter is a target of the transcription activator Runx. Yes-associated protein (Yap1) is a shared transcription co-activator of Runx and p73. Under normal conditions, the Runx-Yap1 complex binds the Itch promoter and supports its transcription and p73 degradation. In response to DNA damage, Yap1 is phosphorylated by c-Abl at the position Tyr-357. The modified Yap1 does not co-activate Runx in supporting Itch transcription. The subsequent reduction in the Itch level gives rise to p73 accumulation. These results demonstrate how Yap1 supports degradation of p73 via Runx and how it plays an opposite role in response to DNA damage.

Rsf-1 interacts with human sucrose nonfermenting protein 2 homologue (hSNF2H) to form a chromatin remodeling complex that participates in several biological processes. We have previously shown that Rsf-1 gene amplification was associated with the most aggressive type of ovarian cancer and cancer cells with Rsf-1 overexpression depended on Rsf-1 to survive. In this report, we determine if formation of the Rsf-1/hSNF2H complex could be one of the mechanisms contributing to tumor cell survival and growth in ovarian carcinomas. Based on immunohistochemistry, we found that Rsf-1 and hSNF2H were co-upregulated in ovarian cancer tissues. Ectopic expression of Rsf-1 in SKOV3 ovarian cancer cells with undetectable endogenous Rsf-1 expression enhanced hSNF2H protein levels and promoted SKOV3 tumor growth in a mouse xenograft model. Our studies also indicated that induction of Rsf-1 expression affected the molecular partnership of hSNF2H and translocated hSNF2H into nuclei where it colocalized with Rsf-1. Furthermore, analysis of Rsf-1 deletion mutants showed that the Rsf-D4 fragment contained the hSNF2H binding site based on coimmunoprecipitation and in vitro competition assays. As compared with other truncated mutants, expression of Rsf-D4 resulted in remarkable growth inhibition in ovarian cancer cells with Rsf-1 gene amplification and overexpression, but not in those without detectable Rsf-1 expression. The above findings suggest that interaction between Rsf-1 and hSNF2H may define a survival signal in those tumors overexpressing Rsf-1.

Upon DNA damage signaling, p73, a member of the p53 tumor suppressor family, accumulates to support transcription of downstream apoptotic genes. p73 interacts with Yes-associated protein 1 (Yap1) through its PPPY motif, and increases p73 transactivation of apoptotic genes. The ubiquitin E3 ligase Itch, like Yap1, interacts with p73. Given the fact that both Itch and Yap1 bind p73 via the PPPY motif, we hypothesized that Yap may also function to stabilize p73 by displacing Itch binding to p73. We show that the interaction of Yap1 and p73 was necessary for p73 stabilization. Yap1 competed with Itch for binding to p73, and prevented Itch-mediated ubiquitination of p73. Treatment of cells with cisplatin leads to an increase in p73 accumulation and induction of apoptosis, but both were dramatically reduced in the presence of Yap1 siRNA. Altogether, our findings attribute a central role to Yap1 in regulating p73 accumulation and function under DNA damage signaling.

Protein degradation is an essential and highly regulated process. The proteasomal degradation of the tumor suppressors p53 and p73 is regulated by both polyubiquitination and by an ubiquitin-independent process. Here, we show that this ubiquitin-independent process is mediated by the 20S proteasomes and is regulated by NQO1. NQO1 physically interacts with p53 and p73 in an NADH-dependent manner and protects them from 20S proteasomal degradation. Remarkably, the vast majority of NQO1 in cells is found in physical association with the 20S proteasomes, suggesting that NQO1 functions as a gatekeeper of the 20S proteasomes. We further show that this pathway plays a role in p53 accumulation in response to ionizing radiation. Our findings provide the first evidence for in vivo degradation of p53 and p73 by the 20S proteasomes and its regulation by NQO1 and NADH level.

Proteasomal degradation of p53 is mediated by two alternative pathways that are either dependent or independent of both Mdm2 and ubiquitin. The ubiquitin-independent pathway is regulated by NAD(P)H: quinone oxidoreductase 1 (NQO1) that stabilizes p53. The NQO1 inhibitor dicoumarol induces ubiquitin-independent p53 degradation. We now show that, like dicoumarol, several other coumarin and flavone inhibitors of NQO1 activity, which compete with NAD(P)H for binding to NQO1, induced ubiquitin-independent p53 degradation and inhibited wild-type p53-mediated apoptosis. Although wild-type p53 and several p53 mutants were sensitive to dicoumarol-induced degradation, the most frequent "hot-spot" p53 mutants in human cancer, R175H, R248H, and R273H, were resistant to dicoumarol-induced degradation, but remained sensitive to Mdm2-ubiquitin-mediated degradation. The two alternative pathways for p53 degradation thus have different p53 structural requirements. Further mutational analysis showed that arginines at positions 175 and 248 were essential for dicoumarol-induced p53 degradation. NQO1 bound to wild-type p53 and dicoumarol, which induced a conformational change in NQO1, inhibited this binding. Compared with wild-type p53, the hot-spot p53 mutants showed increased binding to NQO1, which can explain their resistance to dicoumarol-induced degradation. NQO1 thus has an important role in stabilizing hot-spot p53 mutant proteins in human cancer.

p73 is a structural and functional homologue of the p53 tumor-suppressor protein. Like p53, p73 is activated in response to DNA-damaging insults to induce cell cycle arrest or apoptosis. Under these conditions p73 is tyrosine-phosphorylated by c-Abl, a prerequisite modification for p73 to elicit cell death in fibroblasts. In this study we report that in response to ionizing radiation, p73 undergoes nuclear redistribution and becomes associated with the nuclear matrix. This association is c-Abl-dependent because it was not observed in cells that are defective in c-Abl kinase activation. Moreover, STI-571, a specific c-Abl kinase inhibitor, is sufficient to block significantly p73 alpha nuclear matrix association. The observed c-Abl dependence of nuclear matrix association was recapitulated in the heterologous baculovirus system. Under these conditions p73 alpha but not p53 is specifically tyrosine-phosphorylated by c-Abl. Moreover, the phosphorylated p73 alpha is predominantly found in association with the nuclear matrix. Thus, in response to ionizing radiation p73 is modified in a c-Abl-dependent manner and undergoes nuclear redistribution and translocates to associate with the nuclear matrix. Our data describe a novel mechanism of p73 regulation.

The PHD/LAP (plant homology domain/leukemia associated protein) finger motif is characteristically defined by a histidine and seven cysteines that are spatially arranged in a C4HC3 consensus sequence. This unique zinc finger, found primarily in a wide variety of chromatin-associated proteins, is considered to mediate protein-protein interactions. We have isolated a novel human PHD-finger protein, HBXAP (for hepatitis B virus x associated protein). HBXAP has three alternatively spliced isoforms. We also identified the Drosophila melanogaster HBXAP ortholog, gene CG8677. Based on alignment of four different proteins, we found a novel conserved domain in HBXAP that we designated the HBXAP conserved domain (XCD). We show that HBXAP represses transcription when recruited to DNA via the DNA binding of GAL4. Furthermore, the PHD finger alone suffices to repress transcription, thus attributing a functional role to this domain. The gene HBXAP is localized to the long arm of human chromosome 11 between q13.4 and q14.1. This region is amplified and rearranged in many tumors, suggesting a role for HBXAP in tumorigenesis similar to that of other PHD-containing proteins.

p73 is the first identified homolog of p53, but its function has not been established. Our study investigated the expression of p73 in liver tissue of patients with hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC). RT-PCR was performed on RNA extracted from tumorous and nontumorous liver tissue of HCV-associated HCC, and control tissue and the cDNA were sequenced. Anti-p73 polyclonal antibodies were used for protein analysis and immunohistochemistry, and patients' sera were analyzed for anti-p73 antibodies by radioimmunoassay. Analysis of the p53 gene was performed by SSCP and RFLP-PCR. The p73 mRNA and protein were highly expressed and accumulated in HCC tissues. Immunohistochemical studies revealed significant immunoreactivity in the nuclei of HCC cells. No mutations were detected in the p73 gene or in p53, and no loss of heterozygosity of the p53 gene was found. Anti-p73 antibodies were detected in sera of HCC patients, but were not significantly different from that occurring in non-HCV or non-HCC patients. In conclusion, p73 protein is overexpressed and accumulates in the nuclei of HCV-associated HCCs and may play a role in HCC development.

Hepatitis B virus (HBV) gene expression is mainly regulated at the transcription initiation level. The viral X protein (pX) is a transcription coactivator/mediator targeting TFIIB for the recruitment of RNA polymerase II. Here we report a novel pX nuclear target designated HBXAP (hepatitis B virus X-associated protein). HBXAP is a novel cellular nuclear protein containing a PHD (plant homology domain) finger, a domain shared by many proteins that play roles in chromatin remodeling, transcription coactivation, and oncogenesis. pX physically interacts with HBXAP in vitro and in vivo via the HBXAP region containing the PHD finger. At the functional level HBXAP increases HBV transcription in a pX-dependent manner suggesting a role for this interaction in the virus life cycle. Interestingly, HBXAP collaborates with pX in coactivating the transcriptional activator NF-kappaB. Coactivation of NF-kappaB was also observed in tumor necrosis factor alpha-treated cells suggesting that pX-HBXAP functional collaboration localized downstream to the NF-kappaB nuclear import. Collectively our data suggest that pX recruits and potentiates a novel putative transcription coactivator to regulate NF-kappaB. The implication of pX-HBXAP interaction in the development of hepatocellular carcinoma is discussed.

Wild-type p53 is a tumor-suppressor gene that encodes a short-lived protein that, upon accumulation, induces growth arrest or apoptosis. Accumulation of p53 occurs mainly by posttranslational events that inhibit its proteosomal degradation. We have reported previously that inhibition of NAD(P)H: quinone oxidoreductase 1 (NQO1) activity by dicoumarol induces degradation of p53, indicating that NQO1 plays a role in p53 stabilization. We now have found that wild-type NQO1, but not the inactive polymorphic NQO1, can stabilize endogenous as well as transfected wild-type p53. NQO1-mediated p53 stabilization was especially prominent under induction of oxidative stress. NQO1 also partially inhibited p53 degradation mediated by the human papilloma virus E6 protein, but not when mediated by Mdm-2. Inhibitors of heat shock protein 90 (hsp90), radicicol and geldanamycin, induced degradation of p53 and suppressed p53-induced apoptosis in normal thymocytes and myeloid leukemic cells. Differences in the effectiveness of dicoumarol and hsp90 inhibitors to induce p53 degradation and suppress apoptosis in these cell types indicate that NQO1 and hsp90 stabilize p53 through different mechanisms. Our results indicate that NQO1 has a distinct role in the regulation of p53 stability, especially in response to oxidative stress. The present data on the genetic and pharmacologic regulation of the level of p53 have clinical implications for tumor development and therapy.

Specific protein-protein interactions are involved in a large number of cellular processes and are mainly mediated by structurally and functionally defined domains. Here we report that the nuclear phosphoprotein p73 can engage in a physical association with the Yes-associated protein (YAP). This association occurs under physiological conditions as shown by reciprocal co-immunoprecipitation of complexes from lysates of P19 cells. The WW domain of YAP and the PPPPY motif of p73 are directly involved in the association. Furthermore, as required for ligands to group I WW domains, the terminal tyrosine (Y) of the PPPPY motif of p73 was shown to be essential for the association with YAP. Unlike p73alpha, p73beta, and p63alpha, which bind to YAP, the endogenous as well as exogenously expressed wild-type p53 (wt-p53) and the p73gamma isoform do not interact with YAP. Indeed, we documented that YAP interacts only with those members of the p53 family that have a well conserved PPXY motif, a target sequence for WW domains. Overexpression of YAP causes an increase of p73alpha transcriptional activity. Differential interaction of YAP with members of the p53 family may provide a molecular explanation for their functional divergence in signaling.

p53 is the most frequently inactivated tumor suppressor gene in human cancer, whereas its homologue, p73, is rarely mutated. Similarly to p53, p73 can promote growth arrest or apoptosis when overexpressed in certain p53-null tumor cells. It has previously been shown that some human tumor-derived p53 mutants can exert gain of function activity. The molecular mechanism underlying this activity remains to be elucidated. We show here that human tumor-derived p53 mutants (p53His175 and p53Gly281) associate in vitro and in vivo with p73 alpha, beta, gamma, and delta. This association occurs under physiological conditions, as verified in T47D and SKBR3 breast cancer cell lines. The core domain of mutant p53 is sufficient for the association with p73, whereas both the specific DNA binding and the oligomerization domains of p73 are required for the association with mutant p53. Furthermore, p53His175 and p53Gly281 mutants markedly reduce the transcriptional activity of the various isoforms of p73. Thus, human tumor-derived p53 mutants can associate with p73 not only physically but also functionally. These findings define a network involving mutant p53 and the various spliced isoforms of p73 that may confer upon tumor cells a selective survival advantage.

c-Abl, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of c-Abl. p73, a member of the p53 family of tumour-suppressor proteins, can also induce apoptosis. Here we show that the apoptotic activity of p73alpha requires the presence of functional, kinase-competent c-Abl. Furthermore, p73 and c-Abl can associate with each other, andthis binding is mediated by a PxxP motif in p73 and the SH3 domain of c-Abl. We find that p73 is a substrate of the c-Abl kinase and that the ability of c-Abl to phosphorylate p73 is markedly increased by gamma-irradiation. Moreover, p73 is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving p73 and c-Abl.

Hepatitis B virus (HBV) infects humans and causes a wide range of clinical manifestations, from acute hepatitis to hepatocellular carcinoma (HCC). The HBV genome contains multiple promoters with gene expression regulated predominantly by the cellular transcription initiation machinery. Accordingly, the HBV-encoded pX, the only known viral regulator, is a potent transcription coactivator. We investigated the relationship between pX and cellular coactivators. We show that pX restores wild-type activity to inactive TBPAS mutants with poor TAFII250 and activator-binding activity. This pX-mediated recovery, however, is not obtained with inactive TBPAS mutants in binding of other general transcription factors. Remarkably, ts13, a cell line temperature sensitive for TAFII250 function, exhibiting growth arrest and apoptosis at the restrictive temperature, is rescued partially by pX expression, thus generating a pX-dependent cell growth. Collectively, our results suggest that pX suppresses some of the phenotypes of TBP and TAFII250 mutations, implying that pX circumvents the need for a holo-TFIID complex for transcription activation to proceed.

The transcription program of the hepatitis B virus (HBV) genome is regulated by an enhancer element that binds multiple ubiquitous and liver-enriched transcription activators. HBV transcription and replication are repressed in the presence of p53. Here we describe a novel molecular mechanism that is responsible for this repression. The p53 protein binds to a defined region within the HBV enhancer in a sequence-specific manner, and this, surprisingly, results in p53-dependent transcriptional repression in the context of the whole HBV enhancer. This unusual behavior of the HBV enhancer can be reconstituted by replacing its p53-binding region with the p53-binding domain of the mdm2 promoter. Remarkably, mutation of the EP element of the enhancer reversed the effect of p53 from repression to transcriptional stimulation. Furthermore, EP-dependent modulation of p53 activity can be demonstrated in the context of the mdm2 promoter, suggesting that EP is not only required but is also sufficient to convert p53 activity from positive to negative. Our results imply that the transcriptional effect of DNA-bound p53 can be dramatically modulated by the DNA context and by adjacent DNA-protein interactions.

The enhancer of hepatitis B (HBV) virus displays a liver-specific activity that determines the postreceptor virus-host tropism. Despite the detailed study of this enhancer our knowledge of the mechanisms underlying this behavior is very limited. Here we report that the hepatocyte nuclear factor 3 (HNF3) is at least in part responsible for the liver-specific activity of the enhancer. We demonstrate that recombinant HNF3 alpha binds the enhancer at two sites with different affinity. Transfection studies have demonstrated that the enhancer is active only in liver cells and that integrity of the HNF3 binding sites is important for its full activity. In vitro transcription assays revealed that the enhancer is active only in liver extracts but not in extracts prepared from HeLa cells. Furthermore, the latter extract cannot be activated by addition of recombinant HNF3 alpha. A similar behavior is manifested in transfected cells and, here again, the inactive enhancer is not activated by cotransfected HNF3 beta and alpha. Collectively, our study shows that HNF3 activators are required but not sufficient for full activation of the HBV enhancer and there is a need for additional liver-specific activators or coactivators.

We used the enhancer-binding protein C/EBP as a model to study the nature and the complexity of interaction of an enhancer-binding protein with its target DNA. We found that bacterially expressed C/EBP binds the hepatitis B virus enhancer at multiple sites in a hierarchic and cooperative manner. At low concentrations, only the E element is occupied, but at higher concentrations, additional sites are filled including a site that binds EP, a crucial enhancer-activating protein. This pattern of C/EBP binding may explain the concentration-dependent effect of C/EBP on enhancer activity.

The hepatitis B virus enhancer can be dissected into multiple functional elements, one of which is the E element. We show here that the E element binds multiple nuclear proteins that are essential for its enhancer activity. These findings, together with the ability of this element to respond to at least two different viral transactivators, suggest that the E element is an enhancer modulator capable of binding different factors and responding to multiple stimuli.