Nucleolar p19ARF, unlike mitochondrial smARF, is incapable of inducing p53-independent autophagy

ARF mRNA encodes two distinct proteins, the nucleolar p19(ARF), and a shorter mitochondrial isoform, named smARF. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19(ARF) and smARF proteins. The two ARF isoforms differ not only in their localization but also in their functions. Nucleolar p19(ARF) inhibits cell growth mainly by activating p53 or by inhibiting ribosomal biogenesis. In contrast, mitochondrial smARF can induce dissipation of the mitochondrial membrane potential and autophagy in a p53 independent manner. Recently, it was proposed by Abida et al., that similar to smARF, the nucleolar p19(ARF) can also induce p53 independent autophagy, but in contrast to smARF it does so from within the nucleolus. Our current work shown here indicates, however, that if the ectopic expression of p19(ARF) is restricted to the nucleolus it cannot induce autophagic vesicle formation. Only upon extreme overexpression, when p19(ARF) is localized to extra nuclear compartments, can it trigger p53-independent autophagic vesicle formation. Thus, our experiments indicate that the nucleolar p19(ARF) is incapable of inducing autophagy from within the nucleolus.     

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.     

A smARF way to die: a novel short isoform of p19ARF is linked to autophagic cell death

We recently revealed a novel mechanism by which p19ARF can induce cell death. We found that the p19ARF mRNA encodes an additional shorter isoform from the same open reading frame, named smARF. smARF is a short lived protein, which is rapidly degraded by the proteasome, but accumulates after inappropriate proliferative signals generated by oncogenes. Surprisingly, smARF translocates to the mitochondria, impairs the structure of the mitochondria, and dissipates the mitochondrial membrane potential in a p53 and Bcl-2 family independent manner, ultimately inducing type II caspase-independent autophagic cell death.     

Autophagy and caspase-independent cell death: p19ARF enters the game

ARF, often localized in the nucleolus, controls the p53 pathway and ribosomal biogenesis. In a recent issue of Molecular Cell, Kimchi and colleagues describe a short mitochondrial form of ARF (smARF), produced by internal initiation of translation, that dissipates mitochondrial membrane potential independently of p53 and Bcl-2 family members and triggers caspase-independent cell death. The prodeath function of smARF is dependent on the induction of autophagy.     

A smARF Way to Die: A Novel Short Isoform of p19ARF is Linked to Autophagic Cell Death

We recently revealed a novel mechanism by which p19ARF can induce cell death. We found that the p19ARF mRNA encodes an additional shorter isoform from the same open reading frame, named smARF. smARF is a short lived protein, which is rapidly degraded by the proteasome, but accumulates after inappropriate proliferative signals generated by oncogenes. Surprisingly, smARF translocates to the mitochondria, impairs the structure of the mitochondria and dissipates the mitochondrial membrane potential in a p53 and Bcl-2 family independent manner, ultimately inducing type II caspase-independent autophagic cell death.

Addendum to:

A Short Mitochondrial form of p19(ARF) Induces Autophagy and Caspase-Independent Cell Death

S. Reef, E. Zalckvar, O. Shifman, S. Bialik, H. Sabanay, M. Oren and A. Kimchi

Mol Cell 2006; 22:463-75     

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.     

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.     

A conserved domain in exon 2 coding for the human and murine ARF tumor suppressor protein is required for autophagy induction

The ARF tumor suppressor, encoded by the CDKN2A gene, has a well-defined role regulating TP53 stability; this activity maps to exon 1β of CDKN2A. In contrast, little is known about the function(s) of exon 2 of ARF, which contains the majority of mutations in human cancer. In addition to controlling TP53 stability, ARF also has a role in the induction of autophagy. However, whether the principal molecule involved is full-length ARF, or a small molecular weight variant called smARF, has been controversial. Additionally, whether tumor-derived mutations in exon 2 of CDKN2A affect ARF’s autophagy function is unknown. Finally, whereas it is known that silencing or inhibiting TP53 induces autophagy, the contribution of ARF to this induction is unknown. In this report we used multiple autophagy assays to map a region located in the highly conserved 5′ end of exon 2 of CDKN2A that is necessary for autophagy induction by both human and murine ARF. We showed that mutations in exon 2 of CDKN2A that affect the coding potential of ARF, but not p16INK4a, all impair the ability of ARF to induce autophagy. We showed that whereas full-length ARF can induce autophagy, our combined data suggest that smARF instead induces mitophagy (selective autophagy of mitochondria), thus potentially resolving some confusion regarding the role of these variants. Finally, we showed that silencing Tp53 induces autophagy in an ARF-dependent manner. Our data indicated that a conserved domain in ARF mediates autophagy, and for the first time they implicate autophagy in ARF’s tumor suppressor function.     

Regulation of p14ARF Through Subnuclear Compartmentalization

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23(NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Research 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.     

DNA damage, p14ARF, Nucleophosmin (NPM/B23), and cancer

The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.     

Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function

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.     

Amino terminal hydrophobic import signals target the p14ARF tumor suppressor to the mitochondria

The p14ARF tumour suppressor is frequently targeted for inactivation in many human cancers and in individuals predisposed to cutaneous melanoma. The functions of p14ARF are closely linked with its subcellular distribution. Nucleolar p14ARF dampens ribosome biosynthesis and nucleoplasmic forms of p14ARF activate the p53 pathway and induce cell cycle arrest. p14ARF can also be recruited to mitochondria where it interacts with many mitochondrial proteins, including Bcl-xL and p32 to induce cell death. It has been suggested that the movement of p14ARF to mitochondria requires its interaction with p32, but we now show that the ARF-p32 interaction is not necessary for the accumulation of p14ARF in mitochondria. Instead, highly hydrophobic domains within the amino-terminal half of p14ARF act as mitochondrial import sequences. We suggest that once this hydrophobic pocket is exposed, possibly in a stimulus-dependent manner, it accelerates the mitochondrial import of p14ARF. This allows the interaction of p14ARF with mitochondrial proteins, including p32 and enables p53-independent cell death.     

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.     

Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53

Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here, we show that DHA increased both the level of microtubule-associated protein light-chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.     

Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus

The alternative product of the human INK4a/ARF locus, p14ARF, has the potential to act as a tumour suppressor by binding to and inhibiting the p53 antagonist MDM2. Current models propose that ARF function depends on its ability to sequester MDM2 in the nucleolus. Here we describe situations in which stabilization of MDM2 and p53 occur without relocalization of endogenous MDM2 from the nucleoplasm. Conversely, forms of ARF that do not accumulate in the nucleolus retain the capacity to stabilize MDM2 and p53. We therefore propose that nucleolar localization is not essential for ARF function but may enhance the availability of ARF to inhibit MDM2.     

Mdm2 regulates p53 independently of p19(ARF) in homeostatic tissues

Tumor suppressor proteins must be exquisitely regulated since they can induce cell death while preventing cancer. For example, the p19(ARF) tumor suppressor (p14(ARF) in humans) appears to stimulate the apoptotic function of the p53 tumor suppressor to prevent lymphomagenesis and carcinogenesis induced by oncogene overexpression. Here we present a genetic approach to defining the role of p19(ARF) in regulating the apoptotic function of p53 in highly proliferating, homeostatic tissues. In contrast to our expectation, p19(ARF) did not activate the apoptotic function of p53 in lymphocytes or epithelial cells. These results demonstrate that the mechanisms that control p53 function during homeostasis differ from those that are critical for tumor suppression. Moreover, the Mdm2/p53/p19(ARF) pathway appears to exist only under very restricted conditions.     

ARF Induces Autophagy by Virtue of Interaction with Bcl-xl

The ARF tumor suppressor controls a well-described p53/Mdm2-dependent oncogenic stress checkpoint. In addition, ARF has recently been shown to localize to mitochondria, and to induce autophagy; however, this has never before been demonstrated for endogenous ARF, and the molecular basis for this activity of ARF has not been elucidated. Using an unbiased mass spectrometry-based approach, we show that mitochondrial ARF interacts with the Bcl2 family member Bcl-xl, which normally protects cells from autophagy by inhibiting the Beclin-1/Vps34 complex, which is essential for autophagy. We find that increased expression of ARF decreases Beclin-1/Bcl-xl complexes in cells, thereby providing a basis for ARF-induced autophagy. Our data also indicate that silencing p53 leads to high levels of ARF and increased autophagy, thereby providing a possible basis for the finding by others that p53 inhibits autophagy. The combined data support the premise that ARF induces autophagy in a p53-independent manner in part by virtue of its interaction with Bcl-xl.The ARF tumor suppressor, p14^ARF in humans and p19^ARF in mouse, is a critical growth suppressor that is up-regulated by chronic mitogenic signals and localizes predominantly to the nucleolus. At the nucleolus and in the nucleoplasm, ARF can exert both p53-dependent and -independent growth suppressive function, by virtue of interaction with and inhibition of MDM2, nucleophosmin, E2F-1, CtBP, c-Myc, as well as others (see Ref. 1 for review). Recently, a small molecular weight variant of ARF, generated by translation from an internal methionine, has been discovered to localize primarily to mitochondria and to induce autophagy (2). More recently, another group has shown that full-length ARF, in addition to the small molecular weight variant, can likewise induce autophagy (3). However, neither of these studies revealed a mechanism whereby ARF induces autophagy.Autophagy is an evolutionarily conserved homeostatic process whereby cytosolic components are targeted for removal or turnover in membrane-bound compartments (autophagosomes) that fuse with the lysosome (for review see Ref. 4). This process regulates the turnover of damaged organelles and long-lived proteins that are too large to be delivered to the proteasome. Autophagy occurs constitutively at low levels and is greatly induced during period of metabolic stress, where lysosome-mediated digestion of sequestered molecules serves to release free amino acids and ATP to fuel the continued survival of the cell.Several genes are implicated in the control of autophagy. Perhaps most notable of these is Beclin-1, which is an evolutionarily-conserved mediator of autophagy, with structural similarity to the yeast autophagy gene Apg6/Vps30. Beclin-1 is a component of the class III PI3 kinase complex that includes Vps34; this complex regulates the formation and nucleation of autophagosomes, and the regulation of the activity of this complex is tightly regulated. For example, Beclin-1 possesses a BH3 domain that interacts with the BH3 binding groove of certain members of the Bcl-2 family, including Bcl-2, Bcl-xl, Bcl-w, and to a lesser extent, Mcl-1 (5–9). Binding of Bcl-2 family members to Beclin-1 inhibits autophagy, possibly by decreasing the kinase activity of the Beclin/Bcl-2/Vps34 complex (5) or by negatively regulating Beclin-1 oligomerization (10). The interaction between Beclin-1 and Bcl-2 family members is also regulated; for example, BH3-only proteins can bind directly to Bcl-2 family members and disrupt complex formation with Beclin-1 (11). Additionally, phosphorylation of Bcl-2 by Jun-N-terminal kinase (JNK) can interfere with its ability to bind to Beclin-1 (12). In all cases, dissociation of the Beclin-1/Bcl-2 complex is associated with induction of autophagy.In this report we confirm the findings of others that a fraction of ARF protein localizes to mitochondria and can induce autophagy. We show for the first time that endogenous ARF, up-regulated in non-transformed cells by oncogenes, is capable of inducing autophagy, and further that silencing of p53 is sufficient to de-repress ARF and induce autophagy. We report the identification of Bcl-xl as a mitochondrial ARF-binding protein, and show that ARF-mediated autophagy is enhanced in cells with Bcl-xl silenced. Finally, we show that ARF can reduce complex formation between Bcl-xl and Beclin-1. These data offer the first mechanistic insights into ARF-mediated autophagy. They also point to ARF as a novel regulator of Beclin/Bcl-xl complex formation.     

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.