Δ133p53α, a p53 isoform that can inhibit full-length p53, is downregulated at replicative senescence in a manner independent of mRNA regulation and proteasome-mediated degradation. Here we demonstrate that, unlike full-length p53, Δ133p53α is degraded by autophagy during replicative senescence. Pharmacological inhibition of autophagy restores Δ133p53α expression levels in replicatively senescent fibroblasts, without affecting full-length p53. The siRNA-mediated knockdown of pro-autophagic proteins (ATG5, ATG7 and Beclin-1) also restores Δ133p53α expression. The chaperone-associated E3 ubiquitin ligase STUB1, which is known to regulate autophagy, interacts with Δ133p53α and is downregulated at replicative senescence. The siRNA knockdown of STUB1 in proliferating, early-passage fibroblasts induces the autophagic degradation of Δ133p53α and thereby induces senescence. Upon replicative senescence or STUB1 knockdown, Δ133p53α is recruited to autophagosomes, consistent with its autophagic degradation. This study reveals that STUB1 is an endogenous regulator of Δ133p53α degradation and senescence, and identifies a p53 isoform-specific protein turnover mechanism that orchestrates p53-mediated senescence.

] Laboratory of Human Carcinogenesis Center for Cancer Research National Cancer Institute National Institutes of Health 37 Convent Drive Bethesda Maryland 20892 4258 USA [2]

] Laboratory of Human Carcinogenesis Center for Cancer Research National Cancer Institute National Institutes of Health 37 Convent Drive Bethesda Maryland 20892 4258 USA [2] [3]

Institute of Molecular and Cell Biology 61 Biopolis Drive Proteos Singapore 138673 Singapore

Laboratory of Cell Biology Center for Cancer Research National Cancer Institute National Institutes of Health 37 Convent Drive Bethesda Maryland 20892 4258 USA

Laboratory of Human Carcinogenesis Center for Cancer Research National Cancer Institute National Institutes of Health 37 Convent Drive Bethesda Maryland 20892 4258 USA

Regional Centre for Applied and Molecular Oncology Masaryk Memorial Cancer Institute Zluty Kopec 7 Brno 65653 Czech Republic

Zobrazit více v PubMed

Mizushima N Autophagy: process and function. Genes Dev. 21, 2861–2873 (2007). PubMed

White E Deconvoluting the context-dependent role for autophagy in cancer. Nat. Rev. Cancer 12, 401–410 (2012). PubMed PMC

Kirkin V, McEwan DG, Novak I & Dikic I A role for ubiquitin in selective autophagy. Mol. Cell 34, 259–269 (2009). PubMed

Kraft C, Peter M & Hofmann K Selective autophagy: ubiquitin-mediated recognition and beyond. Nat. Cell Biol. 12, 836–841 (2010). PubMed

Itakura E & Mizushima N Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764–776 (2010). PubMed PMC

Liang XH et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672–676 (1999). PubMed

Mizushima N, Yoshimori T & Levine B Methods in mammalian autophagy research. Cell 140, 313–326 (2010). PubMed PMC

Tanida I, Ueno T & Kominami E LC3 and Autophagy. Methods Mol. Biol 445, 77–88 (2008). PubMed

Johansen T & Lamark T Selective autophagy mediated by autophagic adapter proteins. Autophagy 7, 279–296 (2011). PubMed PMC

Matecic M et al. A microarray-based genetic screen for yeast chronological aging factors. PLoS Genet. 6, e1000921 (2010). PubMed PMC

Rubinsztein DC, Marino G & Kroemer G Autophagy and aging. Cell 146, 682–695 (2011). PubMed

Toth ML et al. Longevity pathways converge on autophagy genes to regulate life span in Caenorhabditis elegans. Autophagy 4, 330–338 (2008). PubMed

Bjedov I et al. Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster. Cell Metab. 11, 35–46 (2010). PubMed PMC

Simonsen A et al. Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila. Autophagy 4, 176–184 (2008). PubMed

Choi AM, Ryter SW & Levine B Autophagy in hum an health and disease. New Engl. J. Med 368, 651–662 (2013). PubMed

Kimmelman AC The dynamic nature of autophagy in cancer. Genes Dev. 25, 1999–2010 (2011). PubMed PMC

Mah LY & Ryan KM Autophagy and cancer. Cold Spring Harb. Perspect. Biol 4, a008821 (2012). PubMed PMC

Campisi J Cancer, aging and cellular senescence. In Vivo 14, 183–188 (2000). PubMed

Collado M, Blasco MA & Serrano M Cellular senescence in cancer and aging. Cell 130, 223–233 (2007). PubMed

Bassham DC et al. Autophagy in development and stress responses of plants. Autophagy 2, 2–11 (2006). PubMed

Kang HT, Lee KB, Kim SY, Choi HR & Park SC Autophagy impairment induces premature senescence in primary human fibroblasts. PLoS ONE 6, e23367 (2011). PubMed PMC

Shay JW & Wright WE Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis 26, 867–874 (2005). PubMed

Bartkova J et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 444, 633–637 (2006). PubMed

Serrano M, Lin AW, McCurrach ME, Beach D & Lowe SW Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602 (1997). PubMed

Nakamura AJ et al. Both telomeric and non-telomeric DNA damage are determinants of mammalian cellular senescence. Epigenetics Chromatin 1, 6 (2008). PubMed PMC

Sedelnikova OA et al. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6,168–170 (2004). PubMed

Herbig U, Jobling WA, Chen BP, Chen DJ & Sedivy JM Telomere shortening triggers senescence of hum an cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol. Cell 14, 501–513 (2004). PubMed

Bourdon JC et al. p53 isoforms can regulate p53 transcriptional activity. Genes Dev. 19, 2122–2137 (2005). PubMed PMC

Khoury MP & Bourdon JC p53 isoforms: an intracellular microprocessor? Genes Cancer 2, 453–465 (2011). PubMed PMC

Fujita K et al. p53 isoforms Δ133p53 and p53β are endogenous regulators of replicative cellular senescence. Nat. Cell Biol 11, 1135–1142 (2009). PubMed PMC

Picksley SM, Vojtesek B, Sparks A & Lane DP Immunochemical analysis of the interaction of p53 with MDM2;--fine mapping of the MDM2 binding site on p53 using synthetic peptides. Oncogene 9, 2523–2529 (1994). PubMed

Surget S, Khoury MP & Bourdon JC Uncovering the role of p53 splice variants in human malignancy: a clinical perspective. Onco. Targets Ther 7, 57–68 (2013). PubMed PMC

Lawrence BP & Brown WJ Inhibition of protein synthesis separates autophagic sequestration from the delivery of lysosomal enzymes. J. Cell Sci 105, 473–480 (1993). PubMed

Munafo DB & Colombo MI A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J. Cell Sci 114, 3619–3629 (2001). PubMed

Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelarova H & Meijer AJ The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur. J. Biochem 243, 240–246 (1997). PubMed

Ding J, Vlahos CJ, Liu R, Brown RF & Badwey JA Antagonists of phosphatidylinositol 3-kinase block activation of several novel protein kinases in neutrophils. J. Biol. Chem 270, 11684–11691 (1995). PubMed

Rubinsztein DC et al. In search of an ‘autophagomometer’. Autophagy 5, 585–589 (2009). PubMed

Tavaria M, Gabriele T, Kola I & Anderson RL A hitchhiker’s guide to the human Hsp70 family. Cell Stress Chaperones 1, 23–28 (1996). PubMed PMC

Esser C, Scheffner M & Hohfeld J The chaperone-associated ubiquitin ligase CHIP is able to target p53 for proteasomal degradation. J. Biol. Chem 280, 27443–27448 (2005). PubMed

Arndt V, Daniel C, Nastainczyk W, Alberti S & Hohfeld J BAG-2 acts as an inhibitor of the chaperone-associated ubiquitin ligase CHIP. Mol. Biol. Cell 16, 5891–5900 (2005). PubMed PMC

Kabbage M & Dickman MB The BAG proteins: a ubiquitous family of chaperone regulators. Cell Mol. Life Sci 65, 1390–1402 (2008). PubMed PMC

McDonough H & Patterson C CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperones 8, 303–308 (2003). PubMed PMC

Hainaut P & Milner J Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation. EMBO J. 11, 3513–3520 (1992). PubMed PMC

Meek DW & Anderson CW Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harb. Perspect. Biol 1, a000950 (2009). PubMed PMC

Coppe JP, Desprez PY, Krtolica A & Campisi J The senescence-associated secretory phenotype: the dark side of tum or suppression. Annu. Rev. Pathol 5, 99–118 (2010). PubMed PMC

Camus S et al. The p53 isoforms are differentially modified by Mdm2. Cell Cycle 11, 1646–1655 (2012). PubMed PMC

Shin Y, Klucken J, Patterson C, Hyman BT & McLean PJ The co-chaperone carboxyl terminus of Hsp70-interacting protein (CHIP) mediates alpha-synuclein degradation decisions between proteasomal and lysosomal pathways. J. Biol. Chem 280, 23727–23734 (2005). PubMed

Li W, Yang Q & Mao Z Chaperone-mediated autophagy: machinery, regulation and biological consequences. Cell Mol. Life Sci. 68, 749–763 (2011). PubMed PMC

Vicencio JM et al. Senescence, apoptosis or autophagy? When a damaged cell must decide its path--a mini-review. Gerontology 54, 92–99 (2008). PubMed

Jaeger PA & Wyss-Coray T All-you-can-eat: autophagy in neurodegeneration and neuroprotection. Mol. Neurodegener 4, 16 (2009). PubMed PMC

Finn PF, Mesires NT, Vine M & Dice JF Effects of small molecules on chaperone-mediated autophagy. Autophagy 1, 141–145 (2005). PubMed

Kettern N, Dreiseidler M, Tawo R & Hohfeld J Chaperone-assisted degradation: multiple paths to destruction. Biol. Chem 391, 481–489 (2010). PubMed

Vakifahmetoglu-Norberg H et al. Chaperone-mediated autophagy degrades mutant p53. Genes Dev. 27, 1718–1730 (2013). PubMed PMC

Rodriguez OC et al. Dietary downregulation of mutant p53 levels via glucose restriction: mechanisms and implications for tum or therapy. Cell Cycle 11, 4436–4446 (2012). PubMed PMC

Tsvetkov P, Adamovich Y, Elliott E & Shaul Y E3 ligase STUB1/CHIP regulates NAD(P)H:quinone oxidoreductase 1 (NQO1) accumulation in aged brain, a process impaired in certain Alzheimer disease patients. J. Biol. Chem 286, 8839–8845 (2011). PubMed PMC

Min JN et al. CHIP deficiency decreases longevity, with accelerated aging phenotypes accompanied by altered protein quality control. Mol. Cell Biol. 28, 4018–4025 (2008). PubMed PMC

Johnson JE, Cao K, Ryvkin P, Wang LS & Johnson FB Altered gene expression in the Werner and Bloom syndromes is associated with sequences having G-quadruplex forming potential. Nucleic Acids Res. 38, 1114–1122 (2010). PubMed PMC

Kelemen O et al. Function of alternative splicing. Gene 514, 1–30 (2013). PubMed PMC

Yang Q et al. Functional diversity of human protection of telomeres 1 isoforms in telomere protection and cellular senescence. Cancer Res. 67, 11677–11686 (2007). PubMed

Fujita K et al. Positive feedback between p53 and TRF2 during telomere-damage signalling and cellular senescence. Nat. Cell Biol 12, 1205–1212 (2010). PubMed PMC

Tang Y et al. Downregulation of splicing factor SRSF3 induces p53β, an alternatively spliced isoform of p53 that promotes cellular senescence. Oncogene 32, 2792–2798 (2013). PubMed PMC

Levine B, Mizushima N & Virgin HW Autophagy in immunity and inflammation. Nature 469, 323–335 (2011). PubMed PMC

Mathew R et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075 (2009). PubMed PMC

Mondal AM et al. p53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes. J. Clin. Invest 123, 5247–5257 (2013). PubMed PMC

Jeram SM, Srikumar T, Pedrioli PG & Raught B Using mass spectrometry to identify ubiquitin and ubiquitin-like protein conjugation sites. Proteomics 9, 922–934 (2009). PubMed

p53 isoforms regulate premature aging in human cells

p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration