BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Human Intersectin 2 (ITSN2) binds to Eps8 protein and enhances its degradation

  • Ding, Xiaofeng (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Yang, Zijian (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Zhou, Fangliang (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Hu, Xiang (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Zhou, Chang (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Luo, Chang (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • He, Zhicheng (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Liu, Qian (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Li, Hong (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Yan, Feng (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Wang, Fangmei (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Xiang, Shuanglin (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University) ;
  • Zhang, Jian (Key Laboratory of Protein Chemistry and Development Biology of State Education Ministry of China, College of Life Science, Hunan Normal University)
  • Received : 2011.09.15
  • Accepted : 2011.12.06
  • Published : 2012.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Participates in actin remodeling through Rac and receptor endocytosis via Rab5. Here, we used yeast two-hybrid system with Eps8 as bait to screen a human brain cDNA library. ITSN2 was identified as the novel binding factor of Eps8. The interaction between ITSN2 and Eps8 was demonstrated by the in vivo co-immunoprecipitation and colocalization assays and the in vitro GST pull-down assays. Furthermore, we mapped the interaction domains to the region between amino acids 260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition, protein stability assays and immunofluorescence analysis showed ITSN2 overexpression induced the degradation of Eps8 proteins, which was markedly alleviated with the lysosome inhibitor NH4Cl treatment. Taken together, our results suggested ITSN2 interacts with Eps8 and stimulates the degradation of Eps8 proteins.

Keywords

References

  1. Fazioli, F., Minichiello, L., Matoska, V., Castagnino, P., Miki, T., Wong, W. T. and Di Fiore, P. P. (1993) Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. Embo J. 12, 3799-3808.
  2. Castagnino, P., Biesova, Z., Wong, W. T., Fazioli, F., Gill, G. N. and Di Fiore, P. P. (1995) Direct binding of eps8 to the juxtamembrane domain of EGFR is phosphotyrosine- and SH2-independent. Oncogene 10, 723-729.
  3. Yap, L. F., Jenei, V., Robinson, C. M., Moutasim, K., Benn, T. M., Threadgold, S. P., Lopes, V., Wei, W., Thomas, G. J. and Paterson, I. C. (2009) Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation. Oncogene 28, 2524-2534. https://doi.org/10.1038/onc.2009.105
  4. Wang, H., Patel, V. Miyazaki, H., Gutkind, J. S. and Yeudall, W. A. (2009) Role for EPS8 in squamous carcinogenesis. Carcinogenesis 30, 165-174. https://doi.org/10.1093/carcin/bgn252
  5. Wang, H., Teh, M. T., Ji, Y., Patel, V., Firouzabadian, S., Patel, A. A., Gutkind, J. S. and Yeudall, W. A. (2010) EPS8 upregulates FOXM1 expression, enhancing cell growth and motility. Carcinogenesis 31, 1132-1141. https://doi.org/10.1093/carcin/bgq058
  6. Forman-Kay, J. D. and Pawson, T. (1999) Diversity in protein recognition by PTB domains. Curr. Opin. Struct. Biol. 9, 690-695.
  7. Margolis, B., Borg, J. P., Straight, S. and Meyer, D. (1999) The function of PTB domain proteins. Kidney Int. 56, 1230-1237. https://doi.org/10.1046/j.1523-1755.1999.00700.x
  8. Di Fiore, P. P. and Scita, G. (2002) Eps8 in the midst of GTPases. Int. J. Biochem. Cell Biol. 34, 1178-1183. https://doi.org/10.1016/S1357-2725(02)00064-X
  9. Kavanaugh, W. M., Turck, C. W. and Williams, L. T. (1995) PTB domain binding to signaling proteins through a sequence motif containing phosphotyrosine. Science 268, 1177-1179. https://doi.org/10.1126/science.7539155
  10. Ong, S. H., Guy, G. R., Hadari, Y. R., Laks, S., Gotoh, N., Schlessinger, J. and Lax, I. (2000) FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors. Mol. Cell Biol. 20, 979-989. https://doi.org/10.1128/MCB.20.3.979-989.2000
  11. Funato, Y., Terabayashi, T., Suenaga, N., Seiki, M., Takenawa, T. and Miki, H. (2004) IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness. Cancer Res. 64, 5237-5244. https://doi.org/10.1158/0008-5472.CAN-04-0327
  12. Mayer, B. J. (2001) SH3 domains: complexity in moderation. J. Cell Sci. 114, 1253-1263.
  13. Kishan, K. V., Scita, G., Wong, W. T., Di Fiore, P. P. and Newcomer, M. E. (1997) The SH3 domain of Eps8 exists as a novel intertwined dimer. Nat. Struct. Biol. 4, 739-743. https://doi.org/10.1038/nsb0997-739
  14. Biesova, Z., Piccoli, C. and Wong, W. T. (1997) Isolation and characterization of e3B1, an eps8 binding protein that regulates cell growth. Oncogene 14, 233-241. https://doi.org/10.1038/sj.onc.1200822
  15. Matoskova, B., Wong, W. T., Nomura, N., Robbins, K. C. and Di Fiore, P. P. (1996) RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation. Oncogene 12, 2679-2688.
  16. Lanzetti, L., Rybin, V., Malabarba, M. G., Christoforidis, S., Scita, G., Zerial, M. and Di Fiore, P. P. (2000) The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408, 374-377. https://doi.org/10.1038/35042605
  17. Scita, G., Tenca, P., Areces, L. B., Tocchetti, A., Frittoli, E., Giardina, G., Ponzanelli, I., Sini, P., Innocenti, M. and Di Fiore, P. P. (2001) An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine. J. Cell Biol. 154, 1031-1044. https://doi.org/10.1083/jcb.200103146
  18. Scita, G., Nordstrom, J., Carbone, R., Tenca, P., Giardina, G., Gutkind, S., Bjarnegard, M., Betsholtz, C. and Di Fiore, P. P. (1999) EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401, 290-293. https://doi.org/10.1038/45822
  19. Chen, H., Wu, X., Pan, Z. K., and Huang, S. (2010) Integrity of SOS1/EPS8/ABI1 tri-complex determines ovarian cancer metastasis. Cancer Res. 70, 9979-9990. https://doi.org/10.1158/0008-5472.CAN-10-2394
  20. Matoskova, B., Wong, W. T., Salcini, A. E., Pelicci, P. G. and Di Fiore, P. P. (1995) Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation. Mol. Cell Biol. 15, 3805-3812. https://doi.org/10.1128/MCB.15.7.3805
  21. Chen, Y. J., Shen, M. R., Chen, Y. J., Maa, M. C. and Leu, T. H. (2008) Eps8 decreases chemosensitivity and affects survival of cervical cancer patients. Mol. Cancer Ther. 7, 1376-1385. https://doi.org/10.1158/1535-7163.MCT-07-2388
  22. Maa, M. C., Lee, J. C., Chen, Y. J., Chen, Y. J., Lee, Y. C., Wang, S. T., Huang, C. C., Chow, N. H. and Leu, T. H. (2007) Eps8 facilitates cellular growth and motility of colon cancer cells by increasing the expression and activity of focal adhesion kinase. J. Biol. Chem. 282, 19399-19409. https://doi.org/10.1074/jbc.M610280200
  23. Zhang, W., Wang, L., Liu, Y., Xu, J., Zhu, G., Cang, H., Li, X., Bartlam, M., Hensley, K., Li, G., Rao, Z. and Zhang, X. (2009) Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione. Genes Dev. 23, 1387-1392. https://doi.org/10.1101/gad.1789209
  24. Welsch, T., Endlich, K., Giese, T., Buchler, M. W. and Schmidt, J. (2007) Eps8 is increased in pancreatic cancer and required for dynamic actin-based cell protrusions and intercellular cytoskeletal organization. Cancer Lett. 255, 205-218. https://doi.org/10.1016/j.canlet.2007.04.008
  25. Bashir, M., Kirmani, D., Bhat, H. F., Baba, R. A., Hamza, R., Naqash, S., Wani, N. A., Andrabi, K. I., Zargar, M. A. and Khanday, F. A. (2010) P66shc and its downstream Eps8 and Rac1 proteins are upregulated in esophageal cancers. Cell Commun. Signal 8, 13. https://doi.org/10.1186/1478-811X-8-13
  26. Yang, T. P., Chiou, H. L., Maa, M. C. and Wang, C. J. (2010) Mithramycin inhibits human epithelial carcinoma cell proliferation and migration involving downregulation of Eps8 expression. Chem. Biol. Interact 183, 181-186. https://doi.org/10.1016/j.cbi.2009.09.018
  27. Leu, T. H., Yeh, H. H., Huang, C. C., Chuang, Y. C., Su, S. L. and Maa, M. C. (2004) Participation of p97Eps8 in Src-mediated transformation. J. Biol. Chem. 279, 9875-9881. https://doi.org/10.1074/jbc.M309884200
  28. Pucharcos, C., Estivill, X., and de la Luna, S. (2000) Intersectin 2, a new multimodular protein involved in clathrin-mediated endocytosis. FEBS Lett. 478, 43-51. https://doi.org/10.1016/S0014-5793(00)01793-2
  29. Innocenti, M., Frittoli, E., Ponzanelli, I., Falck, J. R., Brachmann, S. M., Di Fiore, P. P. and Scita, G. (2003) Phosphoinositide 3-kinase activates Rac by entering in a complex with Eps8, Abi1, and Sos-1. J. Cell Biol. 160, 17-23. https://doi.org/10.1083/jcb.200206079
  30. Offenhauser, N., Borgonovo, A., Disanza, A., Romano, P., Ponzanelli, I., Iannolo, G., Di Fiore, P. P. and Scita, G. (2004) The eps8 family of proteins links growth factor stimulation to actin reorganization generating functional redundancy in the Ras/Rac pathway. Mol. Biol. Cell 15, 91-98. https://doi.org/10.1091/mbc.E03-06-0427
  31. Yamabhai, M., Hoffman, N. G., Hardison, N. L., McPherson, P. S., Castagnoli, L., Cesareni, G. and Kay, B. K. (1998) Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains. J. Biol. Chem. 273, 31401-31407. https://doi.org/10.1074/jbc.273.47.31401
  32. Mohney, R. P., Das, M., Bivona, T. G., Hanes, R., Adams, A. G., Philips, M. R. and O'Bryan, J. P. (2003) Intersectin activates Ras but stimulates transcription through an independent pathway involving JNK. J. Biol. Chem. 278, 47038-47045. https://doi.org/10.1074/jbc.M303895200
  33. Hussain, N. K., Jenna, S., Glogauer, M., Quinn, C. C., Wasiak, S., Guipponi, M., Antonarakis, S. E., Kay, B. K., Stossel, T. P., Lamarche-Vane, N. and McPherson, P. S. (2001) Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP. Nat. Cell Biol. 3, 927-932. https://doi.org/10.1038/ncb1001-927
  34. Snyder, J. T., Worthylake, D. K., Rossman, K. L., Betts, L., Pruitt, W. M., Siderovski, D. P., Der, C. J. and Sondek, J. (2002) Structural basis for the selective activation of Rho GTPases by Dbl exchange factors. Nat. Struct. Biol. 9, 468-475. https://doi.org/10.1038/nsb796
  35. Klein, I. K., Predescu, D. N., Sharma, T., Knezevic, I., Malik, A. B. and Predescu, S. (2009) Intersectin-2L regulates caveola endocytosis secondary to Cdc42-mediated actin polymerization. J. Biol. Chem. 284, 25953-25961. https://doi.org/10.1074/jbc.M109.035071
  36. McGavin, M. K., Badour, K., Hardy, L. A., Kubiseski, T. J., Zhang, J. and Siminovitch, K. A. (2001) The intersectin 2 adaptor links Wiskott Aldrich Syndrome protein (WASp)- mediated actin polymerization to T cell antigen receptor endocytosis. J. Exp. Med. 194, 1777-1787. https://doi.org/10.1084/jem.194.12.1777
  37. Burkhard, P., Stetefeld, J. and Strelkov, S. V. (2001) Coiled coils: a highly versatile protein folding motif. Trends Cell Biol. 11, 82-88. https://doi.org/10.1016/S0962-8924(00)01898-5
  38. Schatz, G. and Dobberstein, B. (1996) Common principles of protein translocation across membranes. Science 271, 1519-1526. https://doi.org/10.1126/science.271.5255.1519
  39. Luo, C., Ding, X., Sun, Y. and Han, M. (2008) Subcellular Location of EPS8 by Its Expression and Preparation of Antiserum. J. Natu. Scie. Hunan Norm. Univ. 31, 100-104.
  40. Ding, X., Fan, C., Zhou, J., Zhong, Y., Liu, R., Ren, K., Hu, X., Luo, C., Xiao, S., Wang, Y., Feng, D. and Zhang, J. (2006) GAS41 interacts with transcription factor AP-2b and stimulates AP-2b-mediated transactivation. Nucleic Acids Res. 34, 2570-2578. https://doi.org/10.1093/nar/gkl319

Cited by

  1. A Conserved Mechanism for Binding of p53 DNA-Binding Domain and Anti-Apoptotic Bcl-2 Family Proteins vol.37, pp.3, 2014, https://doi.org/10.14348/molcells.2014.0001
  2. ITSN2L Interacts with and Negatively Regulates RABEP1 vol.16, pp.12, 2015, https://doi.org/10.3390/ijms161226091
  3. Emerging Roles for Intersectin (ITSN) in Regulating Signaling and Disease Pathways vol.14, pp.4, 2013, https://doi.org/10.3390/ijms14047829
  4. Rac1-mediated cytoskeleton rearrangements induced by intersectin-1s deficiency promotes lung cancer cell proliferation, migration and metastasis vol.15, pp.1, 2016, https://doi.org/10.1186/s12943-016-0543-1
  5. Akt attenuates apoptotic death through phosphorylation of H2A under hydrogen peroxide-induced oxidative stress in PC12 cells and hippocampal neurons vol.6, pp.1, 2016, https://doi.org/10.1038/srep21857
  6. Induction of Apoptosis by Fucoidan in Human Leukemia U937 Cells through Activation of p38 MAPK and Modulation of Bcl-2 Family vol.11, pp.7, 2013, https://doi.org/10.3390/md11072347
  7. Intersectin-Cdc42 interaction is required for orderly meiosis in porcine oocytes pp.00219541, 2018, https://doi.org/10.1002/jcp.27510