UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes

BMC Cell Biology

Deborah A Brown²  Azad L Gucwa¹ 

[1] Department of Biomedical Sciences, Long Island University at Post, Brookville, NY 11548-1300, USA;Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA

关键词: Receptor down-regulation;    Ubiquitin-interacting motif;    Early endosome;    Endocytosis;   

Others  :  1088722
DOI  :  10.1186/1471-2121-15-34

 received in 2014-01-10, accepted in 2014-09-22,  发布年份 2014

【 摘 要 】

Background

Eps15 is an endocytic adaptor protein that stimulates clathrin-mediated endocytosis. Among other interactions, Eps15 binds ubiquitin via UIM domains, recruiting ubiquitinated cargo into clathrin-coated vesicles. In EGF-treated cells, Eps15 also localizes to endosomes. The basis of this localization is not known.

Results

We show that accumulation of ubiquitinated cargo can recruit Eps15 to endosomes via UIM domain interactions. First, treatment of SK-Br-3 breast cancer cells, which overexpress the EGFR family member ErbB2, with geldanamycin to promote receptor ubiquitination and endosomal transport, recruited FLAG-Eps15 to endosomes. Two in-frame ubiquitin constructs, PM-GFP-Ub (retained in endosomes after endocytosis), and GFP-FYVE-UbΔGG (targeted directly to endosomes) also recruited Eps15 to endosomes, as did slowing endosome maturation with constitutively-active Rab5-Q79L. Endosomal recruitment required the UIM domains, but not the N-terminal EH domains or central coiled-coil domains, of Eps15. Silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes. In fact, Hrs silencing itself modestly recruited Eps15 to endosomes, probably by accumulating endogenous ubiquitinated cargo. Eps15 silencing did not affect lysosomal degradation of ubiquitinated ErbB2; however, GFP-FYVE-UbΔGG overexpression inhibited internalization of EGFR and transferrin receptor.

Conclusions

We show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.

【 授权许可】

   
2014 Gucwa and Brown; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Le Roy C, Wrana JL: Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat Rev Mol Cell Biol 2005, 6:112-126.
• [2]Polo S, Di Fiore PP: Endocytosis conducts the cell signaling orchestra. Cell 2006, 124:897-900.
• [3]Reider A, Wendland B: Endocytic adaptors–social networking at the plasma membrane. J Cell Sci 2011, 124:1613-1622.
• [4]van Bergen En Henegouwen PM: Eps15: a multifunctional adaptor protein regulating intracellular trafficking. Cell Commun Signal 2009, 7:24. BioMed Central Full Text
• [5]Salcini AE, Confalonieri S, Doria M, Santolini E, Tassi E, Minenkova O, Cesareni G, Pelicci PG, Di Fiore PP: Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module. Genes Dev 1997, 11:2239-2249.
• [6]Tebar F, Confalonieri S, Carter RE, Di Fiore PP, Sorkin A: Eps15 is constitutively oligomerized due to homophilic interaction of its coiled-coil region. J Biol Chem 1997, 272:15413-15418.
• [7]Sengar AS, Wang W, Bishay J, Cohen S, Egan SE: The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15. EMBO J 1999, 18:1159-1171.
• [8]Parachoniak CA, Park M: Distinct recruitment of Eps15 via its coiled-coil domain is required for efficient down-regulation of the Met receptor tyrosine kinase. J Biol Chem 2009, 284:8382-8394.
• [9]Benmerah A, Poupon V, Cerf-Bensussan N, Dautry-Varsat A: Mapping of Eps15 domains involved in its targeting to clathrin-coated pits. J Biol Chem 2000, 275:3288-3295.
• [10]Polo S, Sigismund S, Faretta M, Guidi M, Capua MR, Bossi G, Chen H, De Camilli P, Di Fiore PP: A single motif responsible for ubiquitin recognition and monoubiquitination in endocytic proteins. Nature 2002, 416:451-455.
• [11]Tebar F, Sorkina T, Sorkin A, Ericsson M, Kirchhausen T: Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits. J Biol Chem 1996, 271:28727-28730.
• [12]Cupers P, Jadhav AP, Kirchhausen T: Assembly of clathrin coats disrupts the association between Eps15 and AP-2 adaptors. J Biol Chem 1998, 273:1847-1850.
• [13]Edeling MA, Mishra SK, Keyel PA, Steinhauser AL, Collins BM, Roth R, Heuser JE, Owen DJ, Traub LM: Molecular switches involving the AP-2 β2 appendage regulate endocytic cargo selection and clathrin coat assembly. Dev Cell 2006, 10:329-342.
• [14]Benmerah A, Lamaze C, Bègue B, Schmid SL, Dautry-Varsat A, Cerf-Bensussan N: AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J Cell Biol 1998, 140:1055-1062.
• [15]Morgan JR, Prasad K, Jin S, Augustine GJ, Lafer EM: Eps15 homology domain-NPF motif interactions regulate clathrin coat assembly during synaptic vesicle recycling. J Biol Chem 2003, 278:33583-33592.
• [16]Fazioli F, Minichiello L, Matoskova B, Wong WT, Di Fiore PP: eps15, a novel tyrosine kinase substrate, exhibits transforming activity. Mol Cell Biol 1993, 13:5814-5828.
• [17]Confalonieri S, Salcini AE, Puri C, Tacchetti C, Di Fiore PP: Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis. J Cell Biol 2000, 150:905-912.
• [18]Torrisi MR, Lotti LV, Belleudi F, Gradini R, Salcini AE, Confalonieri S, Pelicci PG, Di Fiore PP: Eps15 is recruited to the plasma membrane upon epidermal growth factor receptor activation and localizes to components of the endocytic pathway during receptor internalization. Mol Biol Cell 1999, 10:417-434.
• [19]de Melker AA, van der Horst G, Borst J: c-Cbl directs EGF receptors into an endocytic pathway that involves the ubiquitin-interacting motif of Eps15. J Cell Sci 2004, 117:5001-5012.
• [20]Barriere H, Nemes C, Lechardeur D, Khan-Mohammad M, Fruh K, Lukacs GL: Molecular basis of oligoubiquitin-dependent internalization of membrane proteins in mammalian cells. Traffic 2006, 7:282-297.
• [21]Hawryluk MJ, Keyel PA, Mishra SK, Watkins SC, Heuser JE, Traub LM: Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein. Traffic 2006, 7:262-281.
• [22]Murakami T, Felinski EA, Antonetti DA: Occludin phosphorylation and ubiquitination regulate tight junction trafficking and vascular endothelial growth factor-induced permeability. J Biol Chem 2009, 284:21036-21046.
• [23]Catarino SM, Ramalho JS, Marques C, Pereira PC, Girao H: Ubiquitin-mediated internalization of Connexin43 is independent on the canonical endocytic tyrosine-sorting signal. Biochem J 2011, 15:255-267.
• [24]Dores MR, Schnell JD, Maldonado-Baez L, Wendland B, Hicke L: The function of yeast epsin and Ede1 ubiquitin-binding domains during receptor internalization. Traffic 2010, 11:151-160.
• [25]Sorkina T, Bild A, Tebar F, Sorkin A: Clathrin, adaptors and eps15 in endosomes containing activated epidermal growth factor receptors. J Cell Sci 1999, 112:317-327.
• [26]Kanazawa C, Morita E, Yamada M, Ishii N, Miura S, Asao H, Yoshimori T, Sugamura K: Effects of deficiencies of STAMs and Hrs, mammalian class E Vps proteins, on receptor downregulation. Biochem Biophys Res Commun 2003, 309:848-856.
• [27]Bache KG, Raiborg C, Mehlum A, Stenmark H: STAM and Hrs are subunits of a multivalent ubiquitin-binding complex on early endosomes. J Biol Chem 2003, 278:12513-12521.
• [28]van Delft S, Schumacher C, Hage WVA, van Bergen en Henegouwen PM: Association and colocalization of Eps15 with adaptor protein-2 and clathrin. J Cell Biol 1997, 136:811-821.
• [29]Roxrud I, Camilla Raiborg NMP, Stang E, Stenmark H: An endosomally localized isoform of Eps15 interacts with Hrs to mediate degradation of epidermal growth factor receptor. J Cell Biol 2008, 180:1205-1218.
• [30]Hurley JH: ESCRT complexes and the biogenesis of multivesicular bodies. Curr Opin Cell Biol 2008, 20:4-11.
• [31]Bean AJ, Davanger S, Chou MF, Gerhardt B, Tsujimoto S, Chang Y: Hrs-2 regulates receptor-mediated endocytosis via interactions with Eps15. J Biol Chem 2000, 275:15271-15278.
• [32]Mayers JR, Wang L, Pramanik J, Johnson A, Sarkeshik A, Wang Y, Saengsawang W, Yates JR 3rd, Audhya A: Regulation of ubiquitin-dependent cargo sorting by multiple endocytic adaptors at the plasma membrane. Proc Natl Acad Sci U S A 2013, 110:11857-11862.
• [33]Barr DJ, Ostermeyer-Fay AG, Matundan RA, Brown DA: Clathrin-independent endocytosis of ErbB2 in geldanamycin-treated human breast cancer cells. J Cell Sci 2008, 121:3155-3166.
• [34]Citri A, Kochupurakkal BS, Yarden Y: The Achilles heel of ErbB-2/HER2: regulation by the Hsp90 chaperone machine and potential for pharmacological intervention. Cell Cycle 2004, 3:51-60.
• [35]Wandinger SK, Richter K, Buchner J: The Hsp90 chaperone machinery. J Biol Chem 2008, 283:18473-18477.
• [36]Chen H, De Camilli P: The association of epsin with ubiquitinated cargo along the endocytic pathway is negatively regulated by its interaction with clathrin. Proc Natl Acad Sci U S A 2005, 102:2766-2771.
• [37]Austin CD, De Mazière AM, Pisacane PI, van Dijk SM, Eigenbrot C, Sliwkowski MX, Klumperman J, Scheller RH: Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol Biol Cell 2004, 15:5268-5282.
• [38]Sigismund S, Woelk T, Puri C, Maspero E, Tacchetti C, Transidico P, Di Fiore PP, Polo S: Clathrin-independent endocytosis of ubiquitinated cargos. Proc Natl Acad Sci U S A 2005, 102:2760-2765.
• [39]Xu W, Yuan X, Jung YJ, Yang Y, Basso A, Rosen N, Chung EJ, Trepel J, Neckers L: The heat shock protein 90 inhibitor geldanamycin and the ErbB inhibitor ZD1839 promote rapid PP1 phosphatase-dependent inactivation of AKT in ErbB2 overexpressing breast cancer cells. Cancer Res 2003, 15:7777-7784.
• [40]Su C, Lan K, Li C, Chao Y, Lin L, Lee S, Lee W: Phosphorylation accelerates geldanamycin-induced Akt degradation. Arch Biochem Biophys 2013, 536:6-11.
• [41]Kovarova M, Tolar P, Arudchandran R, Draberova L, Rivera J, Draber P: Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fc-epsilon receptor I aggregation. Mol Cell Biol 2001, 21:8318-8328.
• [42]Klapisz E, Sorokina I, Lemeer S, Pijnenburg M, Verkleij AJ, van Bergen en Henegouwen PM: A ubiquitin-interacting motif (UIM) is essential for Eps15 and Eps15R ubiquitination. J Biol Chem 2002, 277:30746-30753.
• [43]Cupers P, ter Haar E, Boll W, Kirchhausen T: Parallel dimers and anti-parallel tetramers formed by epidermal growth factor receptor pathway substrate clone 15. J Biol Chem 1997, 272:33430-33434.
• [44]Hurley JH, Lee S, Prag G: Ubiquitin-binding domains. Biochem J 2006, 399:361-372.
• [45]Stenmark H, Parton RG, Steele-Mortimer O, Lutcke A, Gruenberg J, Zerial M: Inhibition of Rab5 GTPase activity stimulates membrane fusion in endocytosis. EMBO J 1994, 13:1287-1296.
• [46]Rink J, Ghigo E, Kalaidzidis Y, Zerial M: Rab conversion as a mechanism of progression from early to late endosomes. Cell 2005, 122:735-749.
• [47]Huang F, Khvorova A, Marshall W, Sorkin A: Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference. J Biol Chem 2004, 279:16657-16661.
• [48]Thomas M, Lu JJ, Ge Q, Zhang C, Chen J, Klibanov AM: Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc Natl Acad Sci U S A 2005, 102:5679-5684.
• [49]Liu P, Sudhaharan T, Koh RML, Hwang LC, Ahmed S, Maruyama IN, Wohland T: Investigation of the dimerization of proteins from the epidermal growth factor receptor family by single wavelength fluorescence cross-correlation spectroscopy. Biophys J 2007, 93:684-698.
• [50]Volpicelli LA, Lah JJ, Levey AI: Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies. J Biol Chem 2001, 276:47590-47598.
• [51]Bolte S, Cordelières FP: A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 2006, 224:213-232.
• [52]Schroeder RJ, Ahmed SN, Zhu Y, London E, Brown DA: Cholesterol and sphingolipid enhance the Triton X-100-insolubility of GPI-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains. J Biol Chem 1998, 273:1150-1157.