, Volume 17, Issue 8, pp 810–820 | Cite as

Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

  • Ozlem Oral
  • Devrim Oz-Arslan
  • Zeynep Itah
  • Atabak Naghavi
  • Remziye Deveci
  • Sabire Karacali
  • Devrim Gozuacik
Original Paper


Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.


Apoptosis Autophagy Death receptor TNF-α TRAIL Caspase-8 Cell survival 



Microtubule-associated protein 1 (MAP1) light chain 3


Phosphatidylinositol 3-kinase


Benzyloxycarbonyl-valyl-alanyl-aspartic-acid (O-methyl)-fluoromethylketone




Tumor necrosis factor


Tumor necrosis factor-related apoptosis-inducing ligand


Fas-associated protein with death domain


Tumor necrosis factor receptor type 1-associated death domain protein




Human embryonic kidney cells

Supplementary material

10495_2012_735_MOESM1_ESM.pdf (382 kb)
Supplementary material 1 (PDF 382 kb)
10495_2012_735_MOESM2_ESM.doc (26 kb)
Supplementary material 2 (DOC 25 kb)


  1. 1.
    Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6:463–477PubMedCrossRefGoogle Scholar
  2. 2.
    Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23:2891–2906Google Scholar
  3. 3.
    Moreau K, Luo S, Rubinsztein DC (2010) Cytoprotective roles for autophagy. Curr Opin Cell Biol 22:206–211PubMedCrossRefGoogle Scholar
  4. 4.
    Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Bio 78:217–245CrossRefGoogle Scholar
  5. 5.
    Maiuri MC, Zalckvar E, Kimchi A, Kroemer G (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741–752PubMedCrossRefGoogle Scholar
  6. 6.
    Kourtis N, Tavernarakis N (2009) Autophagy and cell death in model organisms. Cell Death Differ 16:21–30PubMedCrossRefGoogle Scholar
  7. 7.
    Hengartner MO (2000) Biochemistry of apoptosis. Nature 407:770–776PubMedCrossRefGoogle Scholar
  8. 8.
    Lavrik I, Golks A, Krammer PH (2005) Death receptor signaling. J Cell Sci 118:265–267PubMedCrossRefGoogle Scholar
  9. 9.
    Kroemer G, Galluzzi L, Brenner C (2007) Mitochondrial membrane permeabilization in cell death. Physiol Rev 87:99–163PubMedCrossRefGoogle Scholar
  10. 10.
    Yousefi S, Perozzo R, Schmid I et al (2006) Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol 8:1124–1132PubMedCrossRefGoogle Scholar
  11. 11.
    Betin VMS, Lane JD (2009) Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. J Cell Sci 122:2554–2566PubMedCrossRefGoogle Scholar
  12. 12.
    Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD, Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122:927–939PubMedCrossRefGoogle Scholar
  13. 13.
    Maiuri MC, Le Toumelin G, Criollo A et al (2007) Functional and physical interaction between Bcl-X(L) and a BH3-like domain in Beclin-1. EMBO J 26:2527–2539PubMedCrossRefGoogle Scholar
  14. 14.
    Ciechomska IA, Goemans GC, Skepper JN, Tolkovsky AM (2009) Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function. Oncogene 28:2128–2141PubMedCrossRefGoogle Scholar
  15. 15.
    Cho DH, Jo YK, Hwang JJ et al (2009) Caspase-mediated cleavage of ATG6/Beclin-1 links apoptosis to autophagy in HeLa cells. Cancer Lett 274:95–100PubMedCrossRefGoogle Scholar
  16. 16.
    Luo S, Rubinsztein DC (2010) Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL. Cell Death Differ 17:268–277PubMedCrossRefGoogle Scholar
  17. 17.
    Wirawan E, Vande Walle L, Kersse K, Cornelis S, Claerhout S, Vanoverberghe I et al (2010) Caspase-mediated cleavage of Beclin-1 inactivates Beclin-1-induced autophagy and enhances apoptosis by promoting the release of proapoptotic factors from mitochondria. Cell Death Dis 1:18CrossRefGoogle Scholar
  18. 18.
    Djavaheri-Mergny M, Maiuri MC, Kroemer G (2010) Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1. Oncogene 29:1717–1719PubMedCrossRefGoogle Scholar
  19. 19.
    Zhu Y, Zhao L, Liu L, Gao P, Tian W, Wang X, Jin H, Xu H, Chen Q (2010) Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis. Protein Cell 1:468–477PubMedCrossRefGoogle Scholar
  20. 20.
    Dix MM, Simon GM, Cravatt BF (2008) Global mapping of the topography and magnitude of proteolytic events in apoptosis. Cell 134:679–691PubMedCrossRefGoogle Scholar
  21. 21.
    Norman JM, Cohen GM, Bampton ET (2010) The in vitro cleavage of the hAtg proteins by cell death proteases. Autophagy 6:1042–1056PubMedCrossRefGoogle Scholar
  22. 22.
    Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 27:107–132PubMedCrossRefGoogle Scholar
  23. 23.
    Ichimura Y, Kirisako T, Takao T et al (2000) A ubiquitin-like system mediates protein lipidation. Nature 408:488–492PubMedCrossRefGoogle Scholar
  24. 24.
    Hanada T, Noda NN, Satomi Y, Ichimura Y, Fujioka Y, Takao T, Inagaki F, Ohsumi Y (2007) The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. J Biol Chem 282:37298–37302PubMedCrossRefGoogle Scholar
  25. 25.
    Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 152:657–668PubMedCrossRefGoogle Scholar
  26. 26.
    Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I et al (2005) Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169:425–434PubMedCrossRefGoogle Scholar
  27. 27.
    Sou YS, Waguri S, Iwata J, Ueno T, Fujimura T, Hara T et al (2008) The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice. Mol Biol Cell 19:4762–4775PubMedCrossRefGoogle Scholar
  28. 28.
    Inohara N, Koseki T, del Peso L, Hu Y, Yee C, Chen S, Carrio R, Merino J, Liu D, Ni J, Núñez G (1999) Nod1, an Apaf-1-like activator of caspase-9 and nuclear factor-kappaB. J Biol Chem 274:14560–14567PubMedCrossRefGoogle Scholar
  29. 29.
    Kosar A, Sesen M, Oral O, Itah Z, Gozuacik D (2011) Bubbly cavitating flow generation and investigation of its erosional nature for biomedical applications. IEEE Trans Biomed Eng 58:1337–1346PubMedCrossRefGoogle Scholar
  30. 30.
    Korkmaz G, le Sage C, Tekirdag KA, Agami R, Gozuacik D (2012) miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1. Autophagy 8:165–176PubMedCrossRefGoogle Scholar
  31. 31.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212PubMedCrossRefGoogle Scholar
  32. 32.
    McDevitt H, Munson S, Ettinger R, Wu A (2002) Multiple roles for tumor necrosis factor-alpha and lymphotoxin alpha/beta in immunity and autoimmunity. Arthritis Res 4:141–152CrossRefGoogle Scholar
  33. 33.
    Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728PubMedCrossRefGoogle Scholar
  34. 34.
    Klionsky DJ (2008) Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4:151–175PubMedGoogle Scholar
  35. 35.
    Zhang J, Cado D, Chen A, Kabra N, Winoto A (1998) Fas-mediated apoptosis and activationinduced T-cell proliferation are defective in mice lacking FADD/Mort1. Nature 392:296–300PubMedCrossRefGoogle Scholar
  36. 36.
    Salmena L, Lemmers B, Hakem A et al (2003) Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Genes Dev 17:883–895PubMedCrossRefGoogle Scholar
  37. 37.
    Chau H, Wong V, Chen NJ et al (2005) Cellular FLICE-inhibitory protein is required for T cell survival and cycling. J Exp Med 202:405–413PubMedCrossRefGoogle Scholar
  38. 38.
    Zhang N, He YW (2005) An essential role for c-FLIP in the efficient development of mature T lymphocytes. J Exp Med 202:395–404PubMedCrossRefGoogle Scholar
  39. 39.
    Newton K, Harris A, Bath M, Smith K, Strasser A (1998) A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes. EMBO J 17:706–718PubMedCrossRefGoogle Scholar
  40. 40.
    Walsh C, Wen B, Chinnaiyan A et al (1998) A role for FADD in T cell activation and development. Immunity 8:439–449PubMedCrossRefGoogle Scholar
  41. 41.
    Zornig M, Hueber AO, Evan G (1998) p53-dependent impairment of T-cell proliferation in FADD dominant-negative transgenic mice. Curr Biol 8:467–470PubMedCrossRefGoogle Scholar
  42. 42.
    Beisner DR, Chu IH, Arechiga AF, Hedrick SM, Walsh CM (2003) The requirements for fasassociated death domain signaling in mature T cell activation and survival. J Immunol 171:247–256PubMedGoogle Scholar
  43. 43.
    Ch’en IL, Beisner DR, Degterev A et al (2008) Antigen mediated T cell expansion regulated by parallel pathways of death. Proc Natl Acad Sci USA 105:17463–17468PubMedCrossRefGoogle Scholar
  44. 44.
    Bell BD, Leverrier S, Weist BM et al (2008) FADD and caspase-8 control the outcome of autophagic signaling in proliferating T cells. Proc Natl Acad Sci USA 105:16677–16682PubMedCrossRefGoogle Scholar
  45. 45.
    Gozuacik D, Bialik S, Raveh T, Mitou G, Shohat G, Sabanay H, Mizushima N, Yoshimori T, Kimchi A (2008) DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death. Cell Death Differ 15:1875–1886PubMedCrossRefGoogle Scholar
  46. 46.
    Criollo A, Chereau F, Malik SA et al (2012) Autophagy is required for the activation of NFκB. Cell Cycle 11:194–199PubMedCrossRefGoogle Scholar
  47. 47.
    Hou W, Han J, Lu C, Goldstein LA, Rabinowich H (2010) Autophagic degradation of active caspase-8: a crosstalk mechanism between autophagy and apoptosis. Autophagy 6:891–900PubMedCrossRefGoogle Scholar
  48. 48.
    Kovacs JR, Li C, Yang Q, Li G, Garcia IG, Ju S, Roodman DG, Windle JJ, Zhang X, Lu B (2012) Autophagy promotes T-cell survival through degradation of proteins of the cell death machinery. Cell Death Differ 19:144–152PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Ozlem Oral
    • 1
  • Devrim Oz-Arslan
    • 1
    • 2
  • Zeynep Itah
    • 1
  • Atabak Naghavi
    • 3
  • Remziye Deveci
    • 3
  • Sabire Karacali
    • 3
  • Devrim Gozuacik
    • 1
  1. 1.Faculty of Engineering and Natural Sciences, Biological Sciences and Bioengineering ProgramSabanci UniversityIstanbulTurkey
  2. 2.Faculty of Medicine, Department of BiophysicsAcibadem UniversityIstanbulTurkey
  3. 3.Faculty of Science, Department of BiologyEge UniversityIzmirTurkey

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