The ARF/Oncogene Pathway Activates p53 Acetylation within the DNA Binding Domain

Stabilization of the p53 tumor suppressor is a critical event in the response to various forms of cellular stress. Two distinct signaling pathways are thought to lead to this stabilization, depending on the type of cellular stress encountered. Genotoxic stress, such as chromosomal breaks or lesions induced by chemotherapeutic agents, result in the activation of the well-characterized DNA damage response pathway. Conversely, cellular stress that results from the aberrant activation of oncogenes triggers p53 stabilization via the induction of the p19ARF pathway. While activation of the DNA damage pathway ultimately causes a complex array of post-translational modifications on p53, activation few if any modifications have been demonstrated to occur following activation of the p19ARF pathway. We and others have recently identified a novel modification on p53, acetylation of lysine 120 within the DNA binding domain. This acetylation event is eliminated by tumor-derived mutations in p53 and its presence is required for the tumor suppressor apoptotic function of p53. We demonstrate here that both the DNA damage response pathway and the p19ARF/oncogene stress pathway induce the acetylation of p53 at lysine 120.     

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2

The tumor suppressor p53 is activated in response to many types of cellular and environmental insults via mechanisms involving post-translational modification. Here we demonstrate that, unlike phosphorylation, p53 invariably undergoes acetylation in cells exposed to a variety of stress-inducing agents including hypoxia, anti-metabolites, nuclear export inhibitor and actinomycin D treatment. In vivo, p53 acetylation is mediated by the p300 and CBP acetyltransferases. Overexpression of either p300 or CBP, but not an acetyltransferase-deficient mutant, efficiently induces specific p53 acetylation. In contrast, MDM2, a negative regulator of p53, actively suppresses p300/CBP-mediated p53 acetylation in vivo and in vitro. This inhibitory activity of MDM2 on p53 acetylation is in turn abrogated by tumor suppressor p19(ARF), indicating that regulation of acetylation is a central target of the p53-MDM2-p19(ARF) feedback loop. Functionally, inhibition of deacetylation promotes p53 stability, suggesting that acetylation plays a positive role in the accumulation of p53 protein in stress response. Our results provide evidence that p300/CBP-mediated acetylation may be a universal and critical modification for p53 function.     

Opposing effects of Ras on p53: transcriptional activation of mdm2 and induction of p19ARF

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. Here, we show that the mdm2 gene is also regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2.     

Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53.

We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C-terminal deletion mutants, which contain so-called Mdm2-binding sites, is markedly decreased, compared with that of wild-type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53's tertiary structure and/or C-terminal region may also be important for this reaction. DNA-dependent protein kinase is known to phosphorylate p53 on Mdm2-binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage-induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19(ARF) affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19(ARF)-bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19(ARF) promotes the stabilization of p53 by inactivating Mdm2.     

Inactivation of the p19(ARF) tumor suppressor affects intestinal epithelial cell proliferation and integrity

p19(ARF) is a tumor suppressor that is frequently deleted in human cancer. It lies at chromosome 9p21 and shares exons 2 and 3 with p16(ink4a), which is also inactivated by these cancer-associated deletions. The "canonical pathway" by which p19(ARF) is thought to suppress tumorigenesis through activation of the p53 tumor suppressor. In response to hyperproliferative signals, such as expression of oncogenes, p19(ARF) is induced and binds to the MDM2 ubiquitin ligase, sequestering it in the nucleolus to allow the accumulation of p53. However, p19(ARF) also has MDM2 and p53 independent functions. In human colon cancer, p19(ARF) is only rarely deleted, but it is more frequently silenced by DNA promoter methylation. Here we show that inactivation of p19(ARF) in mice increases the number of cycling cells in the crypts of the colonic epithelium. Moreover, inactivation of p19(ARF) exacerbated the ulceration of the colonic epithelium caused by dextran sodium sulfate (DSS). These effects were similar to those observed in mice lacking myeloid translocation gene-related-1 (Mtgr1), and mice lacking both of these genes showed an even greater sensitivity to DSS. Surprisingly, inactivation of p19(ARF) restored the loss of the secretory lineage in mice deficient in Mtgr1, suggesting an additional role for p19(ARF) in the small intestinal epithelium.     

p19ARF-induced p53-independent apoptosis largely occurs through BAX

Combined disruption of the ARF gene and the p53 gene causes mouse predisposition to tumors of a wider variety and at a higher frequency than disruption of the p53 gene, indicating that the ARF gene has p53-independent anti-tumor function in addition to p53-dependent function. Coincidentally with this notion, ectopic expression of the p19(ARF) induces apoptosis for wild-type mouse embryo fibroblasts which have been immortalized by introduction of the SV40 virus genome (SV40-MEFs). The protein expression levels of p53, p21(Cip1), and Bax were not upregulated by ectopic expression of p19(ARF) in SV40-MEFs, indicating that expression of p19(ARF) induced apoptosis through p53-independent pathways in this system. Ectopic expression of p19(ARF) induced prominent apoptosis even in SV40-Bak-/-MEFs. In contrast, expression of p19(ARF) induced only a very low grade of apoptosis in Bax-/- or Bax-/-/Bak-/-SV40-MEFs. Remarkable attenuation of p19(ARF)-induced apoptosis by disruption of the Bax gene thus leads to the conclusion that Bax plays a major role in p53-independent apoptosis induced by p19(ARF).     

Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.     

Contribution of two independent MDM2-binding domains in p14(ARF) to p53 stabilization

The MDM2 protein targets the p53 tumor suppressor for ubiquitin-dependent degradation [1], and can function both as an E3 ubiquitin ligase [2] and as a regulator of the subcellular localization of p53 [3]. Oncogene activation stabilizes p53 through expression of the ARF protein (p14(ARF) in humans, p19(ARF) in the mouse) [4], and loss of ARF allows tumor development without loss of wild-type p53 [5] [6]. ARF binds directly to MDM2, and prevents MDM2 from targeting p53 for degradation [6] [7] [8] [9] by inhibiting the E3 ligase activity of MDM2 [2] and preventing nuclear export of MDM2 and p53 [10] [11]. Interaction between ARF and MDM2 results in the localization of both proteins to the nucleolus [12] [13] [14] through nucleolar localization signals (NoLS) in ARF and MDM2 [11] [12] [13] [14]. Here, we report a new NoLS within the highly conserved amino-terminal 22 amino acids of p14(ARF), a region that we found could interact with MDM2, relocalize MDM2 to the nucleolus and inhibit the ability of MDM2 to degrade p53. In contrast, the carboxy-terminal fragment of p14(ARF), which contains the previously described NoLS [11], did not drive nucleolar localization of MDM2, although this region could bind MDM2 and weakly inhibit its ability to degrade p53. Our results support the importance of nucleolar sequestration for the efficient inactivation of MDM2. The inhibition of MDM2 by a small peptide from the amino terminus of p14(ARF) might be exploited to restore p53 function in tumors.     

The ARF/p53 pathway

The ARF tumor suppressor connects pathways regulated by the retinoblastoma protein and p53. ARF inactivation reduces p53-dependent apoptosis induced by oncogenic signals. Nucleolar relocalization of Mdm2 by ARF connotes a novel mechanism for preventing p53 turnover and provides a framework for understanding how stress signals cooperate to regulate p53 function.     

Bax loss impairs Myc-induced apoptosis and circumvents the selection of p53 mutations during Myc-mediated lymphomagenesis

The ARF and p53 tumor suppressors mediate Myc-induced apoptosis and suppress lymphoma development in E mu-myc transgenic mice. Here we report that the proapoptotic Bcl-2 family member Bax also mediates apoptosis triggered by Myc and inhibits Myc-induced lymphomagenesis. Bax-deficient primary pre-B cells are resistant to the apoptotic effects of Myc, and Bax loss accelerates lymphoma development in E mu-myc transgenics in a dose-dependent fashion. Eighty percent of lymphomas arising in wild-type E mu-myc transgenics have alterations in the ARF-Mdm2-p53 tumor suppressor pathway characterized by deletions in ARF, mutations or deletions of p53, and overexpression of Mdm2. The absence of Bax did not alter the frequency of biallelic deletion of ARF in lymphomas arising in E mu-myc transgenic mice or the rate of tumorigenesis in ARF-null mice. Furthermore, Mdm2 was overexpressed at the same frequency in lymphomas irrespective of Bax status, suggesting that Bax resides in a pathway separate from ARF and Mdm2. Strikingly, lymphomas from Bax-null E mu-myc transgenics lacked p53 alterations, whereas 27% of the tumors in Bax(+/-) E mu-myc transgenic mice contained p53 mutations or deletions. Thus, the loss of Bax eliminates the selection of p53 mutations and deletions, but not ARF deletions or Mdm2 overexpression, during Myc-induced tumorigenesis, formally demonstrating that Myc-induced apoptotic signals through ARF/Mdm2 and p53 must bifurcate: p53 signals through Bax, whereas this is not necessarily the case for ARF and Mdm2.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death

The tumor suppressor functions of p19(ARF) have been attributed to its ability to induce cell cycle arrest or apoptosis by activating p53 and regulating ribosome biogenesis. Here we describe another cellular function of p19(ARF), involving a short isoform (smARF, short mitochondrial ARF) that localizes to a Proteinase K-resistant compartment of the mitochondria. smARF is a product of internal initiation of translation at Met45, which lacks the nucleolar functional domains. The human p14(ARF) mRNA likewise produces a shorter isoform. smARF is maintained at low levels via proteasome-mediated degradation, but it increases in response to viral and cellular oncogenes. Ectopic expression of smARF reduces mitochondrial membrane potential (DeltaPsim) without causing cytochrome c release or caspase activation. The dissipation of DeltaPsim does not depend on p53 or Bcl-2 family members. smARF induces massive autophagy and caspase-independent cell death that can be partially rescued by knocking down ATG5 or Beclin-1, suggesting a different prodeath function for this short isoform.