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Autophagy by ARF: A Short Story
Molecular Cell 436
Selected Reading
Liu, Y.C., Penninger, J., and Karin, M. (2005). Nat. Rev. Immunol. 5, 941–952.
Chen, Z.J. (2005). Nat. Cell Biol. 7, 758–765.
Weil, R., and Israel, A. (2004). Curr. Opin. Immunol. 16, 374–381.
Devin, A., Lin, Y., Yamaoka, S., Li, Z., Karin, M., and Liu, Z. (2001). Mol. Cell. Biol. 21, 3986–3994.
Wu, C.J., Conze, D.B., Li, T., Srinivasula, S.M., and Ashwell, J.D. (2006a). Nat. Cell Biol. 8, 398–406.
Ea, C.K., Deng, L., Xia, Z.P., Pineda, G., and Chen, Z.J. (2006). Mol. Cell 22, 245–257.
Wu, Z.H., Shi, Y., Tibbetts, R.S., and Miyamoto, S. (2006b). Science 311, 1141–1146.
Ghosh, S., and Karin, M. (2002). Cell Suppl. 109, S81–S96.
Zhang, S.Q., Kovalenko, A., Cantarella, G., and Wallach, D. (2000). Immunity 12, 301–311.
Li, H., Kobayashi, M., Blonska, M., You, Y., and Lin, X. (2006). J. Biol. Chem. Published online March 16, 2006. 10.1074/jbc. M600620200.
Molecular Cell 22, May 19, 2006 ª2006 Elsevier Inc.
Zhou, H., Wertz, I., O’Rourke, K., Ultsch, M., Seshagiri, S., Eby, M., Xiao, W., and Dixit, V.M. (2004). Nature 427, 167–171.
DOI 10.1016/j.molcel.2006.05.005
Autophagy by ARF: A Short Story
In this issue of Molecular Cell, Reef et al. (2006) describe a shortened unstable form of the ARF tumor suppressor protein that localizes within mitochondria, where it reduces membrane potential and triggers autophagy. Could this account for the Mdm2- and p53-independent tumor suppressive effects of ARF? The mouse p19ARF tumor suppressor (p14ARF in humans) initiates p53-dependent cell cycle arrest and enhances apoptosis triggered by collateral cell death-promoting signals. ARF is induced by increased and sustained mitogenic stimuli conveyed by oncogenes, but not directly by DNA damage. The ARF protein accumulates within the nucleolus where it forms stable complexes with nucleophosmin (NPM/B23), but a minor fraction associates separately with Mdm2 to antagonize its functions and provoke a robust p53 transcriptional response (Sherr et al., 2005). Because mice without functional ARF, Mdm2, and p53 genes are much more tumor prone than those lacking both Mdm2 and p53, p19ARF must also have Mdm2- and p53-independent tumor suppressive functions (Weber et al., 2000). Inhibition of ribosomal biogenesis, antagonism of transcriptional activation by Myc, E2F-1, and NFkB, and promotion of protein sumoylation have each been invoked to explain p53-independent effects of p19ARF (Sherr et al., 2005). A provocative report in this issue of Molecular Cell now points to a novel p53-independent activity of a variant ARF protein. Reef et al. (2006) provide evidence that a short form of the ARF protein is translationally initiated from the single internal methionine codon within both mouse and human ARF mRNA (Met45 in p19ARF [Figure 1] and Met48 in p14ARF). Mutation of the canonical p19ARF initiation codon (Met1) or deletion of sequences encoding ARF’s 40 N-terminal amino acids led to increased production of these variants, which were localized by immunofluorescence staining to mitochondria. Cell fractionation revealed that these ‘‘short mitochondrial’’ (sm) ARF species, like cytochrome c, were confined within the organelle and were resistant to attack by added proteinase K, whereas two surface mitochondrial proteins, Bcl-XL and
Bcl-2, were unprotected and proteolyzed. Production of nucleolar and mitochondrial forms of ARF are transcriptionally coregulated in response to oncogenes, but fulllength p19ARF (half-life 6–8 hr) is much more stable than smARF (half-life <1 hr), which comprises only a small fraction (1%–5%) of the total ARF protein normally detected in vivo. Importantly, smARF lacks all p19ARF N-terminal sequences that direct its nucleolar localization, confer stable binding to Mdm2 and NPM, retard rRNA processing, and trigger p53-dependent cell cycle arrest. So what, if any, is its function? After enforced expression of smARF in both human and mouse cells, mitochondria underwent abnormal morphologic alterations, their membrane potential (DJm) was dissipated, and the transfected cells eventually died. These effects were observed in mouse embryo fibroblasts (MEFs) lacking functional p53 or the proapoptotic mitochondrial proteins Bax and Bak and were not circumvented by overexpressing the antiapoptotic factors Bcl-2 and Bcl-XL. Moreover, smARF did not trigger cytochrome c release from mitochondria or cleavage of PARP-1 and caspase-3, all hallmarks of apoptosis. Instead, smARF was found to stimulate autophagy, a process triggered by nutrient starvation in which cells derive energy by digesting their own components, including organelles such as mitochondria (Gozuacik and Kimchi, 2004; Kondo et al., 2005). Reduced DJm was detected within 24 hr of enforced smARF expression, after which autophagic vesicles accumulated. The timing fits with observations that enforced expression of p19ARF can arrest the outgrowth of p53-null MEFs, but only after a significant delay (Carnero et al., 2000; Weber et al., 2000). Plasmids encoding short-hairpin RNAs directed to ATG5 and Beclin-1, two key regulators of autophagosome formation, attenuated smARFinduced cell death. So, although autophagy can protect cells from stress, in this case, it appears to promote their demise. Reef and colleagues speculate that the two ARF isoforms activate different tumor suppressor pathways: the first involving a rapid, p53-induced nuclear response, and the second utilizing a slowly evolving, mitochondrial-based autophagy program that gains primacy when p53 is dysfunctional. However, these interpretations largely rely on smARF overexpression. Numerous questions arise from this work. How does the single internal ATG codon within ARF mRNA enable
Previews 437
Figure 1. Generation and Composition of smARF The top schematic defines the position of ARF exon-1b (E1b), located midway between the Ink4b and Ink4a genes. The latter two genes encode polypeptide inhibitors (p15Ink4b and p16Ink4a) of cyclin D-dependent kinases. Arrows indicate the positions of the ARF and the two Ink4 promoters. Mouse ARF mRNA, comprised of spliced products of three exons (E1b, E2, and E3 as shown), yields a 169 amino acid protein (bottom) containing >20% arginine residues (denoted by white bars). (E2 sequences within an alternatively spliced Ink4a transcript [E1a, E2, and E3] are translated in a different reading frame to yield part of p16Ink4a.) Residues 1–37 of p19ARF are necessary and sufficient for Mdm2 binding and nucleolar import. Internal initiation of translation at methionine (M) 45 yields the smARF product.
initiation of translation? The argument that smARF is not derived via proteolytic processing of full-length p19ARF stems principally from observations that in vitro translation of the two proteins occurs simultaneously, whereas mutation of Met45 cancels smARF accumulation, both in vitro and in vivo. There is no internal Met codon in the rat ARF gene, and yet paradoxically, expression of the rat ARF cDNA also yielded a shortened form of the protein that was detected when proteasomes were inhibited. Hence, in the rat, smARF must be generated in a different manner. Why is smARF unstable? Although Met1 in p19ARF is removed by methionine aminopeptidase and the penultimate glycyl amino group remains unblocked and is subsequently polyubiquitinated, sequence considerations predict that Met45 in smARF should be retained and undergo cotranslational acetylation (Kuo et al., 2004). Although smARF is highly basic (>20% arginine, see Figure 1), it lacks lysine residues, so no free amino groups would be available for ubiquitination. Misfolded proteins that result from errors in translation are rapidly degraded even if not polyubiquitinated, and when cells
Molecular Cell 22, May 19, 2006 ª2006 Elsevier Inc.
are treated with proteasome inhibitors, they accumulate (Goldberg, 2003). Unlike p19ARF, which is stabilized through interactions with NPM (Kuo et al., 2004), basic smARF is degraded quickly by the proteasome, possibly because it remains unstructured. How, then, does smARF find its way to mitochondria, and how can it exert slowly evolving phenotypic effects that lead to autophagy and cell death? How does smARF avoid ATP-dependent degradation by quality control protease complexes in mitochondria (Suzuki et al., 1997)? With what proteins within the mitochondria does smARF interact, and is this intramitochondrial fraction stabilized in situ? The key issue will be to determine through further genetic analyses, ideally by use of mouse knockin models, the extent to which full-length or smARF proteins alone protect against oncogene-induced cancer development. Would a mutant ARF gene incapable of generating smARF retain p53-independent tumor suppressive functions? Would mice lacking p53 and Mdm2 and engineered to produce only smARF be less tumor prone than animals lacking all three genes? In short, ARF continues to surprise. Charles J. Sherr1,2 Howard Hughes Medical Institute 2 Department of Genetics and Tumor Cell Biology St. Jude Children’s Research Hospital 332 N. Lauderdale Memphis, Tennessee 38105 1
Selected Reading Carnero, A., Hudson, J.D., Price, C.M., and Beach, D.H. (2000). Nat. Cell Biol. 2, 148–155. Goldberg, A.L. (2003). Nature 426, 895–899. Gozuacik, D., and Kimchi, A. (2004). Oncogene 23, 2891–2906. Kondo, Y., Kanazawa, T., Sawaya, R., and Kondo, S. (2005). Nat. Rev. Cancer 5, 726–734. Kuo, M.-L., den Besten, W., Bertwistle, D., Roussel, M.F., and Sherr, C.J. (2004). Genes Dev. 18, 1862–1874. Reef, S., Zalckvar, E., Shifman, O., Bialik, S., Sabanay, H., Oren, M., and Kimchi, A. (2006). Mol. Cell 22, this issue, 463–475. Sherr, C.J., Bertwistle, D., den Besten, W., Kuo, M.-L., Sugimoto, M., Tago, K., Williams, R.T., Zindy, F., and Roussel, M.F. (2005). Cold Spring Harb. Symp. Quant. Biol. LXX, 129–137. Suzuki, C.K., Rep, M., van Dijl, J.M., Suda, K., Grivell, L.A., and Schatz, G. (1997). Trends Biochem. Sci. 22, 118–123. Weber, J.D., Jeffers, J.R., Rehg, J.E., Randle, D.H., Lozano, G., Roussel, M.F., Sherr, C.J., and Zambetti, G.P. (2000). Genes Dev. 14, 2358–2365.
DOI 10.1016/j.molcel.2006.05.001
VHL and p53: Tumor Suppressors Team Up to Prevent Cancer
itive regulator of p53, thus providing insight into the mechanisms by which VHL loss of function leads to cancer.
In a recent issue of Molecular Cell, Roe et al. (2006) report that the von Hippel-Lindau (VHL) protein is a pos-
Cancer is a genetic disease in which mutations dysregulate cell growth and survival pathways (Vogelstein and Kinzler, 2004). These mutations target three types of
Selected Reading
Liu, Y.C., Penninger, J., and Karin, M. (2005). Nat. Rev. Immunol. 5, 941–952.
Chen, Z.J. (2005). Nat. Cell Biol. 7, 758–765.
Weil, R., and Israel, A. (2004). Curr. Opin. Immunol. 16, 374–381.
Devin, A., Lin, Y., Yamaoka, S., Li, Z., Karin, M., and Liu, Z. (2001). Mol. Cell. Biol. 21, 3986–3994.
Wu, C.J., Conze, D.B., Li, T., Srinivasula, S.M., and Ashwell, J.D. (2006a). Nat. Cell Biol. 8, 398–406.
Ea, C.K., Deng, L., Xia, Z.P., Pineda, G., and Chen, Z.J. (2006). Mol. Cell 22, 245–257.
Wu, Z.H., Shi, Y., Tibbetts, R.S., and Miyamoto, S. (2006b). Science 311, 1141–1146.
Ghosh, S., and Karin, M. (2002). Cell Suppl. 109, S81–S96.
Zhang, S.Q., Kovalenko, A., Cantarella, G., and Wallach, D. (2000). Immunity 12, 301–311.
Li, H., Kobayashi, M., Blonska, M., You, Y., and Lin, X. (2006). J. Biol. Chem. Published online March 16, 2006. 10.1074/jbc. M600620200.
Molecular Cell 22, May 19, 2006 ª2006 Elsevier Inc.
Zhou, H., Wertz, I., O’Rourke, K., Ultsch, M., Seshagiri, S., Eby, M., Xiao, W., and Dixit, V.M. (2004). Nature 427, 167–171.
DOI 10.1016/j.molcel.2006.05.005
Autophagy by ARF: A Short Story
In this issue of Molecular Cell, Reef et al. (2006) describe a shortened unstable form of the ARF tumor suppressor protein that localizes within mitochondria, where it reduces membrane potential and triggers autophagy. Could this account for the Mdm2- and p53-independent tumor suppressive effects of ARF? The mouse p19ARF tumor suppressor (p14ARF in humans) initiates p53-dependent cell cycle arrest and enhances apoptosis triggered by collateral cell death-promoting signals. ARF is induced by increased and sustained mitogenic stimuli conveyed by oncogenes, but not directly by DNA damage. The ARF protein accumulates within the nucleolus where it forms stable complexes with nucleophosmin (NPM/B23), but a minor fraction associates separately with Mdm2 to antagonize its functions and provoke a robust p53 transcriptional response (Sherr et al., 2005). Because mice without functional ARF, Mdm2, and p53 genes are much more tumor prone than those lacking both Mdm2 and p53, p19ARF must also have Mdm2- and p53-independent tumor suppressive functions (Weber et al., 2000). Inhibition of ribosomal biogenesis, antagonism of transcriptional activation by Myc, E2F-1, and NFkB, and promotion of protein sumoylation have each been invoked to explain p53-independent effects of p19ARF (Sherr et al., 2005). A provocative report in this issue of Molecular Cell now points to a novel p53-independent activity of a variant ARF protein. Reef et al. (2006) provide evidence that a short form of the ARF protein is translationally initiated from the single internal methionine codon within both mouse and human ARF mRNA (Met45 in p19ARF [Figure 1] and Met48 in p14ARF). Mutation of the canonical p19ARF initiation codon (Met1) or deletion of sequences encoding ARF’s 40 N-terminal amino acids led to increased production of these variants, which were localized by immunofluorescence staining to mitochondria. Cell fractionation revealed that these ‘‘short mitochondrial’’ (sm) ARF species, like cytochrome c, were confined within the organelle and were resistant to attack by added proteinase K, whereas two surface mitochondrial proteins, Bcl-XL and
Bcl-2, were unprotected and proteolyzed. Production of nucleolar and mitochondrial forms of ARF are transcriptionally coregulated in response to oncogenes, but fulllength p19ARF (half-life 6–8 hr) is much more stable than smARF (half-life <1 hr), which comprises only a small fraction (1%–5%) of the total ARF protein normally detected in vivo. Importantly, smARF lacks all p19ARF N-terminal sequences that direct its nucleolar localization, confer stable binding to Mdm2 and NPM, retard rRNA processing, and trigger p53-dependent cell cycle arrest. So what, if any, is its function? After enforced expression of smARF in both human and mouse cells, mitochondria underwent abnormal morphologic alterations, their membrane potential (DJm) was dissipated, and the transfected cells eventually died. These effects were observed in mouse embryo fibroblasts (MEFs) lacking functional p53 or the proapoptotic mitochondrial proteins Bax and Bak and were not circumvented by overexpressing the antiapoptotic factors Bcl-2 and Bcl-XL. Moreover, smARF did not trigger cytochrome c release from mitochondria or cleavage of PARP-1 and caspase-3, all hallmarks of apoptosis. Instead, smARF was found to stimulate autophagy, a process triggered by nutrient starvation in which cells derive energy by digesting their own components, including organelles such as mitochondria (Gozuacik and Kimchi, 2004; Kondo et al., 2005). Reduced DJm was detected within 24 hr of enforced smARF expression, after which autophagic vesicles accumulated. The timing fits with observations that enforced expression of p19ARF can arrest the outgrowth of p53-null MEFs, but only after a significant delay (Carnero et al., 2000; Weber et al., 2000). Plasmids encoding short-hairpin RNAs directed to ATG5 and Beclin-1, two key regulators of autophagosome formation, attenuated smARFinduced cell death. So, although autophagy can protect cells from stress, in this case, it appears to promote their demise. Reef and colleagues speculate that the two ARF isoforms activate different tumor suppressor pathways: the first involving a rapid, p53-induced nuclear response, and the second utilizing a slowly evolving, mitochondrial-based autophagy program that gains primacy when p53 is dysfunctional. However, these interpretations largely rely on smARF overexpression. Numerous questions arise from this work. How does the single internal ATG codon within ARF mRNA enable
Previews 437
Figure 1. Generation and Composition of smARF The top schematic defines the position of ARF exon-1b (E1b), located midway between the Ink4b and Ink4a genes. The latter two genes encode polypeptide inhibitors (p15Ink4b and p16Ink4a) of cyclin D-dependent kinases. Arrows indicate the positions of the ARF and the two Ink4 promoters. Mouse ARF mRNA, comprised of spliced products of three exons (E1b, E2, and E3 as shown), yields a 169 amino acid protein (bottom) containing >20% arginine residues (denoted by white bars). (E2 sequences within an alternatively spliced Ink4a transcript [E1a, E2, and E3] are translated in a different reading frame to yield part of p16Ink4a.) Residues 1–37 of p19ARF are necessary and sufficient for Mdm2 binding and nucleolar import. Internal initiation of translation at methionine (M) 45 yields the smARF product.
initiation of translation? The argument that smARF is not derived via proteolytic processing of full-length p19ARF stems principally from observations that in vitro translation of the two proteins occurs simultaneously, whereas mutation of Met45 cancels smARF accumulation, both in vitro and in vivo. There is no internal Met codon in the rat ARF gene, and yet paradoxically, expression of the rat ARF cDNA also yielded a shortened form of the protein that was detected when proteasomes were inhibited. Hence, in the rat, smARF must be generated in a different manner. Why is smARF unstable? Although Met1 in p19ARF is removed by methionine aminopeptidase and the penultimate glycyl amino group remains unblocked and is subsequently polyubiquitinated, sequence considerations predict that Met45 in smARF should be retained and undergo cotranslational acetylation (Kuo et al., 2004). Although smARF is highly basic (>20% arginine, see Figure 1), it lacks lysine residues, so no free amino groups would be available for ubiquitination. Misfolded proteins that result from errors in translation are rapidly degraded even if not polyubiquitinated, and when cells
Molecular Cell 22, May 19, 2006 ª2006 Elsevier Inc.
are treated with proteasome inhibitors, they accumulate (Goldberg, 2003). Unlike p19ARF, which is stabilized through interactions with NPM (Kuo et al., 2004), basic smARF is degraded quickly by the proteasome, possibly because it remains unstructured. How, then, does smARF find its way to mitochondria, and how can it exert slowly evolving phenotypic effects that lead to autophagy and cell death? How does smARF avoid ATP-dependent degradation by quality control protease complexes in mitochondria (Suzuki et al., 1997)? With what proteins within the mitochondria does smARF interact, and is this intramitochondrial fraction stabilized in situ? The key issue will be to determine through further genetic analyses, ideally by use of mouse knockin models, the extent to which full-length or smARF proteins alone protect against oncogene-induced cancer development. Would a mutant ARF gene incapable of generating smARF retain p53-independent tumor suppressive functions? Would mice lacking p53 and Mdm2 and engineered to produce only smARF be less tumor prone than animals lacking all three genes? In short, ARF continues to surprise. Charles J. Sherr1,2 Howard Hughes Medical Institute 2 Department of Genetics and Tumor Cell Biology St. Jude Children’s Research Hospital 332 N. Lauderdale Memphis, Tennessee 38105 1
Selected Reading Carnero, A., Hudson, J.D., Price, C.M., and Beach, D.H. (2000). Nat. Cell Biol. 2, 148–155. Goldberg, A.L. (2003). Nature 426, 895–899. Gozuacik, D., and Kimchi, A. (2004). Oncogene 23, 2891–2906. Kondo, Y., Kanazawa, T., Sawaya, R., and Kondo, S. (2005). Nat. Rev. Cancer 5, 726–734. Kuo, M.-L., den Besten, W., Bertwistle, D., Roussel, M.F., and Sherr, C.J. (2004). Genes Dev. 18, 1862–1874. Reef, S., Zalckvar, E., Shifman, O., Bialik, S., Sabanay, H., Oren, M., and Kimchi, A. (2006). Mol. Cell 22, this issue, 463–475. Sherr, C.J., Bertwistle, D., den Besten, W., Kuo, M.-L., Sugimoto, M., Tago, K., Williams, R.T., Zindy, F., and Roussel, M.F. (2005). Cold Spring Harb. Symp. Quant. Biol. LXX, 129–137. Suzuki, C.K., Rep, M., van Dijl, J.M., Suda, K., Grivell, L.A., and Schatz, G. (1997). Trends Biochem. Sci. 22, 118–123. Weber, J.D., Jeffers, J.R., Rehg, J.E., Randle, D.H., Lozano, G., Roussel, M.F., Sherr, C.J., and Zambetti, G.P. (2000). Genes Dev. 14, 2358–2365.
DOI 10.1016/j.molcel.2006.05.001
VHL and p53: Tumor Suppressors Team Up to Prevent Cancer
itive regulator of p53, thus providing insight into the mechanisms by which VHL loss of function leads to cancer.
In a recent issue of Molecular Cell, Roe et al. (2006) report that the von Hippel-Lindau (VHL) protein is a pos-
Cancer is a genetic disease in which mutations dysregulate cell growth and survival pathways (Vogelstein and Kinzler, 2004). These mutations target three types of