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.

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The mammalian cell death network comprises three distinct functional modules: apoptosis, autophagy and programmed necrosis. Currently, the field lacks systems level approaches to assess the extent to which the intermodular connectivity affects cell death performance. Here, we developed a platform that is based on single and double sets of RNAi-mediated perturbations targeting combinations of apoptotic and autophagic genes. The outcome of perturbations is measured both at the level of the overall cell death responses, using an unbiased quantitative reporter, and by assessing the molecular responses within the different functional modules. Epistatic analyses determine whether seemingly unrelated pairs of proteins are genetically linked. The initial running of this platform in etoposide-treated cells, using a few single and double perturbations, identified several levels of connectivity between apoptosis and autophagy. The knock down of caspase3 turned on a switch toward autophagic cell death, which requires Atg5 or Beclin-1. In addition, a reciprocal connection between these two autophagic genes and apoptosis was identified. By applying computational tools that are based on mining the protein-protein interaction database, a novel biochemical pathway connecting between Atg5 and caspase3 is suggested. Scaling up this platform into hundreds of perturbations potentially has a wide, general scope of applicability, and will provide the basis for future modeling of the cell death network. The process of programmed cell death (PCD) is driven by a network of proteins connected to each other in an intricate manner. Over the past two decades, many of the network's proteins (nodes) and the interactions among them (edges; mostly post-translation modifications) have been identified. The PCD network is turned on by well-defined input signals, such as activation of death receptors or exposure to DNA damaging agents. The efficiency of its performance determines the individual cell's probability to die, which, when assessed over a large population of cells, can be translated into the percent of cell death. Dying cells can display several distinct cell death phenotypes, each driven by a different subset of proteins and molecular pathways. Examples are the caspase-dependent apoptotic cell death, autophagic cell death and programmed necrosis. 1 Cells exposed to the same input signal can switch from one cell death modality to another in response to specific perturbations, 2-4 and in some cases, a mixed type of cell death can also be observed. [5][6][7] This led us to propose here a working model, according to which the proteins that mediate the three different cell death phenotypes should be integrated within a common network, and the corresponding subsets of proteins should be considered as functional modules within this global network. To study the network as a whole, new strategies capable of analyzing the connectivity within and between the functional modules are required.Here, we developed a platform for dissecting the network's a...

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.

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.