Defining regulatory and phosphoinositide-binding sites in the human WIPI-1 β-propeller responsible for autophagosomal membrane localization downstream of mTORC1 inhibition

Journal of Molecular Signaling

Tassula Proikas-Cezanne¹  Anneliese Hoffmann¹  Daniela Bakula¹  Anja Gaugel¹ 

[1] From the Autophagy Laboratory, Department of Molecular Biology, Interfaculty Institute of Cell Biology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 15, 72076, Tuebingen, Germany

关键词: YM201636;    WIPI-1;    PtdIns(3,5)P₂;    PtdIns(3)P;    PtdIns3KC3;    Phagophore;    Atg18;    Atg12;    Autophagy;    Autophagosome;   

Others  :  814968
DOI  :  10.1186/1750-2187-7-16

 received in 2012-08-14, accepted in 2012-09-24,  发布年份 2012

【 摘 要 】

Background

Autophagy is a cytoprotective, lysosomal degradation system regulated upon induced phosphatidylinositol 3-phosphate (PtdIns(3)P) generation by phosphatidylinositol 3-kinase class III (PtdIns3KC3) downstream of mTORC1 inhibition. The human PtdIns(3)P-binding β-propeller protein WIPI-1 accumulates at the initiation site for autophagosome formation (phagophore), functions upstream of the Atg12 and LC3 conjugation systems, and localizes at both the inner and outer membrane of generated autophagosomes. In addition, to a minor degree WIPI-1 also binds PtdIns(3,5)P₂. By homology modelling we earlier identified 24 evolutionarily highly conserved amino acids that cluster at two opposite sites of the open Velcro arranged WIPI-1 β-propeller.

Results

By alanine scanning mutagenesis of 24 conserved residues in human WIPI-1 we define the PtdIns-binding site of human WIPI-1 to critically include S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257. These amino acids confer PtdIns(3)P or PtdIns(3,5)P₂ binding. In general, WIPI-1 mutants unable to bind PtdIns(3)P/PtdIns(3,5)P₂ lost their potential to localize at autophagosomal membranes, but WIPI-1 mutants that retained PtdIns(3)P/PtdIns(3,5)P₂ binding localized at Atg12-positive phagophores upon mTORC1 inhibition. Both, downregulation of mTOR by siRNA or cellular PtdIns(3)P elevation upon PIKfyve inhibition by YM201636 significantly increased the localization of WIPI-1 at autophagosomal membranes. Further, we identified regulatory amino acids that influence the membrane recruitment of WIPI-1. Exceptional, WIPI-1 R110A localization at Atg12-positive membranes was independent of autophagy stimulation and insensitive to wortmannin. R112A and H185A mutants were unable to bind PtdIns(3)P/PtdIns(3,5)P₂ but localized at autophagosomal membranes, although in a significant reduced number of cells when compared to wild-type WIPI-1.

Conclusions

We identified amino acids of the WIPI-1 β-propeller that confer PtdIns(3)P or PtdIns(3,5)P₂ binding (S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257), and that regulate the localization at autophagosomal membranes (R110, R112, H185) downstream of mTORC1 inhibition.

【 授权许可】

   
2012 Gaugel et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Mizushima N, Levine B, Cuervo AM, Klionsky DJ: Autophagy fights disease through cellular self-digestion. Nature 2008, 451:1069-1075.
• [2]Yang Z, Klionsky DJ: Eaten alive: a history of macroautophagy. Nat Cell Biol 2010, 12:814-822.
• [3]Moreau K, Luo S, Rubinsztein DC: Cytoprotective roles for autophagy. Curr Opin Cell Biol 2010, 22:206-211.
• [4]Singh R, Cuervo AM: Autophagy in the cellular energetic balance. Cell Metab 2011, 13:495-504.
• [5]Hamasaki M, Yoshimori T: Where do they come from? Insights into autophagosome formation. FEBS Lett 2010, 584:1296-1301.
• [6]Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A: A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 2009, 11:1433-1437.
• [7]Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL: 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 2009, 5:1180-1185.
• [8]Ravikumar B, Moreau K, Jahreiss L, Puri C, Rubinsztein DC: Plasma membrane contributes to the formation of pre-autophagosomal structures. Nat Cell Biol 2010, 12:747-757.
• [9]Mari M, Griffith J, Rieter E, Krishnappa L, Klionsky DJ, Reggiori F: An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis. J Cell Biol 2010, 190:1005-1022.
• [10]Mizushima N, Yoshimori T, Ohsumi Y: The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol 2011, 27:107-132.
• [11]Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, Iemura S, Natsume T, Takehana K, Yamada N, Guan JL, Oshiro N, Mizushima N: Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 2009, 20:1981-1991.
• [12]Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, Kundu M, Kim DH: ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol Biol Cell 2009, 20:1992-2003.
• [13]Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, Vasquez DS, Joshi A, Gwinn DM, Taylor R, Asara JM, Fitzpatrick J, Dillin A, Viollet B, Kundu M, Hansen M, Shaw RJ: Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 2011, 331:456-461.
• [14]Lee JW, Park S, Takahashi Y, Wang HG: The association of AMPK with ULK1 regulates autophagy. PLoS One 2010, 5:e15394.
• [15]Kim J, Kundu M, Viollet B, Guan KL: AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol 2011, 13:132-141.
• [16]Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ: Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem 1995, 270:2320-2326.
• [17]Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P: Distinct classes of phosphatidylinositol 3'-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem 2000, 275:992-998.
• [18]Knaevelsrud H, Simonsen A: Lipids in autophagy: constituents, signaling molecules and cargo with relevance to disease. Biochim Biophys Acta 2012, 1821:1133-1145.
• [19]Obara K, Ohsumi Y: PtdIns 3-Kinase Orchestrates Autophagosome Formation in Yeast. J Lipids 2011, 2011:498768.
• [20]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 1997, 243:240-246.
• [21]Noda T, Matsunaga K, Taguchi-Atarashi N, Yoshimori T: Regulation of membrane biogenesis in autophagy via PI3P dynamics. Semin Cell Dev Biol 2010, 21:671-676.
• [22]Noda T, Matsunaga K, Yoshimori T: Atg14L recruits PtdIns 3-kinase to the ER for autophagosome formation. Autophagy 2011, 7:438-439.
• [23]Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, Ktistakis NT: Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 2008, 182:685-701.
• [24]Polson HE, de Lartigue J, Rigden DJ, Reedijk M, Urbe S, Clague MJ, Tooze SA: Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. Autophagy 2010, 6:506-522.
• [25]Itakura E, Mizushima N: Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 2010, 6:764-776.
• [26]Simonsen A, Birkeland HC, Gillooly DJ, Mizushima N, Kuma A, Yoshimori T, Slagsvold T, Brech A, Stenmark H: Alfy, a novel FYVE-domain-containing protein associated with protein granules and autophagic membranes. J Cell Sci 2004, 117:4239-4251.
• [27]Ridley SH, Ktistakis N, Davidson K, Anderson KE, Manifava M, Ellson CD, Lipp P, Bootman M, Coadwell J, Nazarian A, Erdjument-Bromage H, Tempst P, Cooper MA, Thuring JW, Lim ZY, Holmes AB, Stephens LR, Hawkins PT: FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments. J Cell Sci 2001, 114:3991-4000.
• [28]Isakson P, Holland P, Simonsen A: The role of ALFY in selective autophagy. Cell Death Differ 2012.
• [29]Proikas-Cezanne T, Waddell S, Gaugel A, Frickey T, Lupas A, Nordheim A: WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy. Oncogene 2004, 23:9314-9325.
• [30]Krick R, Busse RA, Scacioc A, Stephan M, Janshoff A, Thumm M, Kühnel K: Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a beta-propeller protein family. Proc Natl Acad Sci USA 2012, 109:E2042-E2049.
• [31]Baskaran S, Ragusa MJ, Boura E, Hurley JH: Two-Site Recognition of Phosphatidylinositol 3-Phosphate by PROPPINs in Autophagy. Mol Cell 2012, 3:339-348.
• [32]Mauthe M, Jacob A, Freiberger S, Hentschel K, Stierhof YD, Codogno C, Proikas-Cezanne T: Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation. Autophagy 2011, 7:1448-1461.
• [33]Proikas-Cezanne T, Robenek H: Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes. J Cell Mol Med 2011, 15:2007-2010.
• [34]Tooze SA, Jefferies HB, Kalie E, Longatti A, McAlpine FE, McKnight NC, Orsi A, Polson HE, Razi M, Robinson DJ, Webber JL: Trafficking and signaling in mammalian autophagy. IUBMB Life 2010, 62:503-508.
• [35]Codogno P, Mehrpour M, Proikas-Cezanne T: Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat Rev Mol Cell Biol 2012, 13:7-12.
• [36]Jeffries TR, Dove SK, Michell RH, Parker PJ: PtdIns-specific MPR pathway association of a novel WD40 repeat protein, WIPI49. Mol Biol Cell 2004, 15:2652-2663.
• [37]Proikas-Cezanne T, Ruckerbauer S, Stierhof YD, Berg C, Nordheim A: Human WIPI-1 puncta-formation: a novel assay to assess mammalian autophagy. FEBS Lett 2007, 581:3396-3404.
• [38]Proikas-Cezanne T, Pfisterer SG: Assessing mammalian autophagy by WIPI-1/Atg18 puncta formation. Methods Enzymol 2009, 452:247-260.
• [39]Mauthe M, Yu W, Krut O, Krönke M, Gotz F, Robenek H, Proikas-Cezanne T: WIPI-1 Positive Autophagosome-Like Vesicles Entrap Pathogenic Staphylococcus aureus for Lysosomal Degradation. Int J Cell Biol 2012, 2012:179207.
• [40]Jefferies HB, Cooke FT, Jat P, Boucheron C, Koizumi T, Hayakawa M, Kaizawa H, Ohishi T, Workman P, Waterfield MD, Parker PJ: A selective PIKfyve inhibitor blocks PtdIns(3,5)P(2) production and disrupts endomembrane transport and retroviral budding. EMBO Rep 2008, 9:164-170.
• [41]Pfisterer SG, Mauthe M, Codogno P, Proikas-Cezanne T: Ca2+/calmodulin-dependent kinase (CaMK) signaling via CaMKI and AMP-activated protein kinase contributes to the regulation of WIPI-1 at the onset of autophagy. Mol Pharmacol 2011, 80:1066-1075.
• [42]Berendzen KW, Bohmer M, Wallmeroth N, Peter S, Vesi M, Zhou Y, Tiesler FK, Schleifenbaum F, Harter K: Screening for in planta protein-protein interactions combining bimolecular fluorescence complementation with flow cytometry. Plant Methods 2012, 8:25. BioMed Central Full Text
• [43]Frickey T, Lupas A: CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics 2004, 20:3702-3704.
• [44]Van de Peer Y, Frickey T, Taylor J, Meyer A: Dealing with saturation at the amino acid level: a case study based on anciently duplicated zebrafish genes. Gene 2002, 295:205-211.