LY294002 is a synthetic quercetin-like compound, which, unlike wortmannin, is more specific inhibitor of phosphatidylinositol 3-kinase (PI3K). It inhibits endocytosis and vacuolar transport. We report here on the proteome-wide effects of LY294002 on Arabidopsis roots focusing on proteins involved in vesicular trafficking and stress response. At the subcellular level, LY294002 caused swelling and clustering of late endosomes leading to inhibition of vacuolar transport. At the proteome level, this compound caused changes in abundances of proteins categorized to 10 functional classes. Among proteins involved in vesicular trafficking, a small GTPase ARFA1f was more abundant, indicating its possible contribution to the aggregation and fusion of late endosomes triggered by LY294002. Our study provides new information on storage proteins and vacuolar hydrolases in vegetative tissues treated by LY294002. Vacuolar hydrolases were downregulated, while storage proteins were more abundant, suggesting that storage proteins were protected from degradation in swollen multivesicular bodies upon LY294002 treatment. Upregulation of 2S albumin was validated by immunoblotting and immunolabeling analyses. Our study also pointed to the control of antioxidant enzyme machinery by PI3K because LY294002 downregulated two isozymes of superoxide dismutase. This most likely occurred via PI3K-mediated downregulation of protein AtDJ1A. Finally, we discuss specificity differences of LY294002 and wortmannin against PI3K, which are reflected at the proteome level. Compared with wortmannin, LY294002 showed more narrow and perhaps also more specific effects on proteins, as suggested by gene ontology functional annotation.

Centre of the Region Haná for Biotechnological and Agricultural Research Department of Cell Biology Faculty of Science Palacký University Šlechtitelů 11 CZ 783 71 Olomouc Czech Republic

Zobrazit více v PubMed

Munnik T, Vermeer JEM. Osmotic stress-induced phosphoinositide and inositol phosphate signalling in plants. Plant Cell Environ. 2010;33:655–669. PubMed

Stenmark H, Gillooly DJ. Intracellular trafficking and turnover of phosphatidylinositol 3-phosphate. Seminars in Cell & Developmental Biology. 2001;12:193–199. PubMed

Matsuoka K, Bassham DC, Raikhel NV, Nakamura K. Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells J. Cell Biol. 1995;130:1307–1318. PubMed PMC

Takáč T, Pechan T, Šamajová O, Ovečka M, Richter H, Eck C, Niehaus K, Šamaj J. Wortmannin treatment induces changes in Arabidopsis root proteome post-Golgi compartments J. Proteome Res. 2012;11:3127–3142. PubMed

Vlahos CJ, Matter WF, Hui KY, Brown RF. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) J. Biol. Chem. 1994;269:5241–5248. PubMed

Mueller-Roeber B, Pical C. Inositol phospholipid metabolism in Arabidopsis Characterized and putative isoforms of inositol phospholipid kinase and phosphoinositide-specific phospholipase C. Plant Physiol. 2002;130:22–46. PubMed PMC

Brunn GJ, Williams J, Sabers C, Wiederrecht G, Lawrence JC, Abraham RT. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996;15:5256–5267. PubMed PMC

Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000;351:95–105. PubMed PMC

Gharbi SI, Zvelebil MJ, Shuttleworth SJ, Hancox T, Saghir N, Timms JF, Waterfield MD. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem. J. 2007;404:15–21. PubMed PMC

Walker EH, Pacold ME, Perisic O, Stephens L, Hawkins PT, Wymann MP, Williams RL. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. Mol. Cell. 2000;6:909–919. PubMed

Etxeberria E, Baroja-Fernandez E, Muñoz FJ, Pozueta-Romero J. Sucrose-inducible endocytosis as a mechanism for nutrient uptake in heterotrophic plant cells. Plant Cell Physiol. 2005;46:474–481. PubMed

Kim DH, Eu YJ, Yoo CM, Kim YW, Pih KT, Jin JB, Kim SJ, Stenmark H, Hwang I. Trafficking of phosphatidylinositol 3-phosphate from the trans-Golgi network to the lumen of the central vacuole in plant cells. Plant Cell. 2001;13:287–301. PubMed PMC

Lee Y, Bak G, Choi Y, Chuang W-I, Cho H-T, Lee Y. Roles of phosphatidylinositol 3-kinase in root hair growth. Plant Physiol. 2008;147:624–635. PubMed PMC

Bar M, Avni A. EHD2 inhibits ligand-induced endocytosis and signaling of the leucine-rich repeat receptor-like protein LeEix2. Plant J. 2009;59:600–611. PubMed

Jung J-Y, Kim Y-W, Kwak JM, Hwang J-U, Young J, Schroeder JI, Hwang I, Lee Y. Phosphatidylinositol 3- and 4-phosphate are required for normal stomatal movements. Plant Cell. 2002;14:2399–2412. PubMed PMC

Joo JH, Yoo HJ, Hwang I, Lee JS, Nam KH, Bae YS. Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase. FEBS Lett. 2005;579:1243–1248. PubMed

Voigt B, Timmers ACJ, Šamaj J, Hlavacka A, Ueda T, Preuss M, Nielsen E, Mathur J, Emans N, Stenmark H, Nakano A, Baluska F, Menzel D. Actin-based motility of endosomes is linked to the polar tip growth of root hairs. Eur. J. Cell Biol. 2005;84:609–621. PubMed

Takáč T, Pechan T, Richter H, Müller J, Eck C, Böhm N, Obert B, Ren H, Niehaus K, Šamaj J. Proteomics on brefeldin A-treated Arabidopsis roots reveals profilin 2 as a new protein involved in the cross-talk between vesicular trafficking and the actin cytoskeleton. J. Proteome Res. 2011;10:488–501. PubMed

Hurkman WJ, Tanaka CK. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 1986;81:802–806. PubMed PMC

Hajduch M, Ganapathy A, Stein JW, Thelen JJ. A systematic proteomic study of seed filling in soybean Establishment of high-resolution two-dimensional reference maps, expression profiles, and an interactive proteome database. Plant Physiol. 2005;137:1397–1419. PubMed PMC

Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 1988;60:2299–2301. PubMed

Donaldson JR, Nanduri B, Burgess SC, Lawrence ML. Comparative proteomic analysis of Listeria monocytogenes strains F2365 and EGD. Appl. Environ. Microbiol. 2009;75:366–373. PubMed PMC

Nanduri B, Lawrence ML, Vanguri S, Pechan T, Burgess SC. Proteomic analysis using an unfinished bacterial genome: the effects of subminimum inhibitory concentrations of antibiotics on Mannheimia haemolytica virulence factor expression. Proteomics. 2005;5:4852–4863. PubMed

Sauer M, Paciorek T, Benková E, Friml J. Immunocytochemical techniques for whole-mount in situ protein localization in plants. Nat Protoc. 2006;1:98–103. PubMed

Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure, some applications. Proc. Natl. Acad Sci U.S.A. 1979;76:4350–4354. PubMed PMC

Shimada T, Yamada K, Kataoka M, Nakaune S, Koumoto Y, Kuroyanagi M, Tabata S, Kato T, Shinozaki K, Seki M, Kobayashi M, Kondo M, Nishimura M, Hara-Nishimura I. Vacuolar processing enzymes are essential for proper processing of seed storage proteins in Arabidopsis thaliana . J. Biol. Chem. 2003;278:32292–32299. PubMed

Lin C-L, Chen H-J, Hou W-C. Activity staining of glutathione peroxidase after electrophoresis on native and sodium dodecyl sulfate polyacrylamide gels. Electrophoresis. 2002;23:513–516. PubMed

Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M. KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res. 2012;40:D109–D114. PubMed PMC

Ovečka M, Berson T, Beck M, Derksen J, Šamaj J, Baluška F, Lichtscheidl IK. Structural sterols are involved in both the initiation and tip growth of root hairs in Arabidopsis thaliana . Plant Cell. 2010;22:2999–3019. PubMed PMC

Vernoud V, Horton AC, Yang Z, Nielsen E. Analysis of the small GTPase gene superfamily of Arabidopsis . Plant Physiol. 2003;131:1191–1208. PubMed PMC

Miao Y, Li KY, Li H-Y, Yao X, Jiang L. The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells. Plant J. 2008;56:824–839. PubMed

Xu XM, Møller SG. ROS removal by DJ-1. Plant Signal Behav. 2010;5:1034–1036. PubMed PMC

Chen Y, Chen T, Shen S, Zheng M, Guo Y, Lin J, Baluška F, Šamaj J. Differential display proteomic analysis of Picea meyeri pollen germination and pollen-tube growth after inhibition of actin polymerization by latrunculin B. Plant J. 2006;47:174–195. PubMed

Wu X, Chen T, Zheng M, Chen Y, Teng N, Šamaj J, Baluška F, Lin J. Integrative proteomic cytological analysis of the effects of extracellular Ca(2+) influx on Pinus bungeana pollen tube development. J Proteome Res. 2008;7:4299–4312. PubMed

Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21:3674–3676. PubMed

Robaglia C, Thomas M, Meyer C. Sensing nutrient energy status by SnRK1 and TOR kinases. Curr. Opin. Plant Biol. 2012;15:301–307. PubMed

Leshem Y, Seri L, Levine A. Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance. Plant J. 2007;51:185–197. PubMed

Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA. The proteolytic processing of seed storage proteins in Arabidopsis embryo cells starts in the multivesicular bodies. Plant Cell. 2006;18:2567–2581. PubMed PMC

Pimpl P, Hanton SL, Taylor JP, Pinto-daSilva LL, Denecke J. The GTPase ARF1p controls the sequence-specific vacuolar sorting route to the lytic vacuole. Plant Cell. 2003;15:1242–1256. PubMed PMC

Xu J, Scheres B. Cell polarity: ROPing the ends together. Curr Opin Plant Biol. 2005;8:613–618. PubMed

Böhlenius H, Mørch SM, Godfrey D, Nielsen ME, Thordal-Christensen H. The Multivesicular Body-Localized GTPase ARFA1b/1c Is Important for Callose Deposition and ROR2 Syntaxin-Dependent Preinvasive Basal Defense in Barley. Plant Cell. 2010;22:3831–3844. PubMed PMC

Asaoka R, Uemura T, Ito J, Fujimoto M, Ito E, Ueda T, Nakano A. Arabidopsis RABA1 GTPases are involved in transport between the trans-Golgi network and the plasma membrane, and are required for salinity stress tolerance. Plant J. 2013;73:240–249. PubMed

Wang J, Cai Y, Miao Y, Lam SK, Jiang L. Wortmannin induces homotypic fusion of plant prevacuolar compartments. J. Exp. Bot. 2009;60:3075–3083. PubMed PMC

Scheuring D, Viotti C, Krüger F, Künzl F, Sturm S, Bubeck J, Hillmer S, Frigerio L, Robinson DG, Pimpl P, Schumacher K. Multivesicular bodies mature from the trans-Golgi network/early endosome in Arabidopsis . Plant Cell. 2011;23:3463–3481. PubMed PMC

Liu J, Zhou J, Xing D. Phosphatidylinositol 3-kinase plays a vital role in regulation of rice seed vigor via altering NADPH oxidase activity. PLoS ONE. 2012;7:e33817. PubMed PMC

Advantages and limitations of shot-gun proteomic analyses on Arabidopsis plants with altered MAPK signaling

Endosomal Interactions during Root Hair Growth