The polybasic region of cytohesin-2 determines paxillin binding specificity to mediate cell migration
Author(s)
Tomohiro Torii*,
Yuki Miyamoto,
Kohji Nishimura,
Masahiro Maeda,
Akito Tanoue,
Junji Yamauchi
Affiliation(s)
Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan. Department of Molecular and Functional Genomics, Shimane University, Matsue, Japan. Immuno-Biological Laboratories (IBL) Co. Ltd., Fujioka, Japan. The Japan Health Sciences Foundation (JHSF), Tokyo, Japan.
Department of Pharmacology, National Research Institute for Child Health and Development, Tokyo, Japan. Department of Molecular and Functional Genomics, Shimane University, Matsue, Japan. Immuno-Biological Laboratories (IBL) Co. Ltd., Fujioka, Japan. The Japan Health Sciences Foundation (JHSF), Tokyo, Japan.
ABSTRACT
The intracellular signaling pathways through ADP-ribosylation factors (Arfs) of the small GTPase family control cell morphological changes by regulating membrane components and/or cytoskeletal protein dynamics. We previously reported that cytohesin-2 (CYTH2), an Arf-guanine-nucleotide exchange factor (GEF), binds to the cytoskeletal scaffold protein paxillin through C-terminal region of CYTH2 and promotes the migration of mouse 3T3-L1 fibroblasts. In mammals, CYTH family GEFs are composed of four subfamilies. Among them, CYTH2 and CYTH3 are widely expressed in tissues and it remains to be clarified to determine whether they have specific biochemical and cellular functions or are redundant. Here, we show that the C-terminal short polybasic region of CYTH2 is necessary and sufficient for binding to paxillin to mediate cell migration. Although 3T3-L1 cells primarily express CYTH2 and CYTH3 of four CYTH family members, neither knockdown of CYTH3 by the specific siRNA nor expression of its C-terminal region inhibits migration. Importantly, replacing the C-terminal region of CYTH3 with that of CYTH2 adds the ability of paxillinbinding and mediating migration to CYTH3. Conversely, replacing the C- terminal region of CYTH2 with that of CYTH3 leads to loss of these abilities of CYTH2. These results reveal that paxillin is a unique binding partner with CYTH2 in migrating cells, presenting the first CYTH family GEF’s region that is involved in the selectivity of the binding protein.
The intracellular signaling pathways through ADP-ribosylation factors (Arfs) of the small GTPase family control cell morphological changes by regulating membrane components and/or cytoskeletal protein dynamics. We previously reported that cytohesin-2 (CYTH2), an Arf-guanine-nucleotide exchange factor (GEF), binds to the cytoskeletal scaffold protein paxillin through C-terminal region of CYTH2 and promotes the migration of mouse 3T3-L1 fibroblasts. In mammals, CYTH family GEFs are composed of four subfamilies. Among them, CYTH2 and CYTH3 are widely expressed in tissues and it remains to be clarified to determine whether they have specific biochemical and cellular functions or are redundant. Here, we show that the C-terminal short polybasic region of CYTH2 is necessary and sufficient for binding to paxillin to mediate cell migration. Although 3T3-L1 cells primarily express CYTH2 and CYTH3 of four CYTH family members, neither knockdown of CYTH3 by the specific siRNA nor expression of its C-terminal region inhibits migration. Importantly, replacing the C-terminal region of CYTH3 with that of CYTH2 adds the ability of paxillinbinding and mediating migration to CYTH3. Conversely, replacing the C- terminal region of CYTH2 with that of CYTH3 leads to loss of these abilities of CYTH2. These results reveal that paxillin is a unique binding partner with CYTH2 in migrating cells, presenting the first CYTH family GEF’s region that is involved in the selectivity of the binding protein.
Cite this paper
Torii, T. , Miyamoto, Y. , Nishimura, K. , Maeda, M. , Tanoue, A. and Yamauchi, J. (2012) The polybasic region of cytohesin-2 determines paxillin binding specificity to mediate cell migration. Advances in Biological Chemistry, 2, 291-300. doi: 10.4236/abc.2012.23037.
Torii, T. , Miyamoto, Y. , Nishimura, K. , Maeda, M. , Tanoue, A. and Yamauchi, J. (2012) The polybasic region of cytohesin-2 determines paxillin binding specificity to mediate cell migration. Advances in Biological Chemistry, 2, 291-300. doi: 10.4236/abc.2012.23037.
References
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Journal of Biological Chemistry, 275, 16827-16836. doi:10.1074/jbc.275.22.16827 [18] Kitano, J., Kimura, K., Yamazaki, Y., Soda, T., Shigemoto, R., Nakajima, Y. and Nakanishi, S. (2002) Tamalin, a PDZ domain-containing protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins. Journal of Neuroscience, 22, 1280-1289. [19] Mansour, M., Lee, S.Y. and Pohajdak, B. (2002) The N-terminal coiled coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with the scaffolding protein CASP. Journal of Biological Chemistry, 277, 32302-32309. doi:10.1074/jbc.M202898200 [20] Boehm, T., Hofer, S., Winklehner, P., Kellersch, B., Geiger, C., Trockenbacher, A., Neyer, S., Fiegl, H., Ebner, S., Ivarsson, L., Schneider, R., Kremmer, E., Heufler, C. and Kolanus, W. (2003) Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration. EMBO Journal, 22, 1014-1024. doi:10.1093/emboj/cdg101 [21] Tang, P., Cheng, T.P., Agnello, D., Wu, C.Y., Hissong, B.D., Watford, W.T., Ahn, H.J., Galon, J., Moss, J., Vaughan, M., O’Shea, J.J. and Gadina, M. (2002) Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity. Proceedings of the National Academy of Sciences USA, 99, 2625-2629. doi:10.1073/pnas.052712999 [22] Venkateswarlu, K. (2003) Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity. Journal of Biological Chemistry, 278, 43460-43469. doi:10.1074/jbc.M304078200 [23] Ikenouchi, J. and Umeda, M. (2010) FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex. Proceedings of the National Academy of Sciences USA, 107, 748-753. doi:10.1073/pnas.0908423107 [24] Lim, J., Zhou, M., Veenstra, T.D. and Morrison, D.K. (2010) The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling. Genes & Development, 24, 1496-1506. doi:10.1101/gad.1904610 [25] Bill, A., Schmitz, A., Albertoni, B., Song, J.N., Heukamp, L.C., Walrafen, D., Thorwirth, F., Verveer, P.J., Zimmer, S., Meffert, L., Schreiber, A., Chatterjee, S., Thomas, R.K., Ullrich, R.T., Lang, T. and Famulok, M. (2010) Cytohesins are cytoplasmic ErbB receptor activators. Cell, 143, 201-211. doi:10.1016/j.cell.2010.09.011 [26] Hofmann, I., Thompson, A., Sanderson, C.M. and Munro, S. (2007) The Arl4 family of small G proteins can recruit the cytohesin Arf6 exchange factors to the plasma membrane. Current Biology, 17, 711-716. doi:10.1016/j.cub.2007.03.007 [27] Li, C.C., Chiang, T.C., Wu, T.S., Pacheco-Rodriguez, G., Moss, J. and Lee, F.J. (2007) ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling. Molecular Biology of the Cell, 18, 4420-4437. doi:10.1091/mbc.E07-02-0149 [28] Oh, S. J. and Santy, L. C. (2010) Differential effects of cytohesins 2 and 3 on beta1 integrin recycling. Journal of Biological Chemistry, 285, 14610-14616. doi:10.1074/jbc.M109.043935 [29] Hafner, M., Schmitz, A., Grune, I., Srivatsan, S.G., Paul, B., Kolanus, W., Quast, T., Kremmer, E., Bauer, I. and Famulok, M. (2006) Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature, 444, 941-944. doi:10.1038/nature05415 [30] Fuss, B., Becker, T., Zinke, I. and Hoch, M. (2006) The cytohesin Steppke is essential for insulin signalling in Drosophila. Nature, 444, 945-948. doi:10.1038/nature054
[1] Bar-Sagi, D. and Hall, A. (2000) Ras and Rho GTPases: A family reunion. Cell, 103, 227-238. doi:10.1016/S0092-8674(00)00115-X [2] Schmidt, A. and Hall, A. (2002) Guanine nucleotide exchange factors for Rho GTPases: Turning on the switch. Genes & Development, 16, 1587-1609. doi:10.1101/gad.1003302 [3] Rossman, K.L., Der, C.J. and Sondek, J. (2005) GEF- means go: Turning on RHO GTPases with guanine nucleotide-exchange factors. Nature Reviews Molecular Cell Biology, 6, 167-180. doi:10.1038/nrm1587 [4] Miyamoto, Y. and Yamauchi, J. (2010) The cellular signaling of Dock family in neural function. Cellular Signalling, 22, 175-182. doi:10.1016/j.cellsig.2009.09.036 [5] Donaldson, J.G. and Honda, A. (2005) Localization and function of Arf family GTPases. Biochemical Society Transactions, 33, 639-642. [6] Kahn, R.A., Cherfils, J., Elias, M., Lovering, R.C., Munro, S. and Schurmann, A. (2006) Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins. Journal of Cell Biology, 172, 645-650. doi:10.1083/jcb.200512057 [7] D’Souza-Schorey, C. and Chavrier, P. (2006) ARF proteins: Roles in membrane traffic and beyond. Nature Reviews Molecular Cell Biology, 7, 347-358. doi:10.1038/nrm1910 [8] Casanova, J.E. (2007) Regulation of Arf activation: The Sec7 family of guanine nucleotide exchange factors. Traffic, 8, 1476-1485. doi:10.1111/j.1600-0854.2007.00634.x [9] DiNitto, J.P., Delprato, A., Gabe Lee, M.T., Cronin, T.C., Huang, S., Guilherme, A., Czech, M.P. and Lambright, D.G. (2007) Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. Molecular Cell, 28, 569-583. doi:10.1016/j.molcel.2007.09.017 [10] Brown, M.C. and Turner, C.E. (2004) Paxillin: Adapting to change. Physiological Reviews, 84, 1315-1339. doi:10.1152/physrev.00002.2004 [11] Torii, T., Miyamoto, Y., Sanbe, A., Nishimura, K., Yamauchi, J. and Tanoue, A. (2010) Cytohesin-2/ARNO, through its interaction with focal adhesion adaptor protein paxillin, regulates preadipocyte migration via the downstream activation of Arf6. Journal of Biological Chemistry, 285, 24270-24281. doi:10.1074/jbc.M110.125658 [12] Heo, W.D., Inoue, T., Park, W.S., Kim, M.L., Park, B.O., Wandless, T.J. and Meyer, T. (2006) PI(3,4,5)P3 and PI (4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science, 314, 1458-1461. doi:10.1126/science.1134389 [13] Yamauchi, J., Miyamoto, Y., Chan, J.R. and Tanoue, A. (2008) ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration. Journal of Cell Biology, 181, 351-365. doi:10.1083/jcb.200709033 [14] Yamauchi, J., Miyamoto, Y., Torii, T., Mizutani, R., Nakamura, K., Sanbe, A., Koide, H., Kusakawa, S. and Tanoue, A. (2009) Valproic acid-inducible Arl4D and cytohesin-2/ARNO, acting through the downstream Arf6, regulate neurite outgrowth in N1E-115 cells. Experimental Cell Research, 315, 2043-2052. doi:10.1016/j.yexcr.2009.03.012 [15] Mizutani, R. Yamauchi, J., Kusakawa, S., Nakamura, K., Sanbe, A., Torii, T., Miyamoto, Y. and Tanoue, A. (2009) Sorting nexin 3, a protein upregulated by lithium, contains a novel phosphatidylinositol-binding sequence and mediates neurite outgrowth in N1E-115 cells. Cellular Signalling, 21, 1586-1594. doi:10.1016/j.cellsig.2009.06.005 [16] Klarlund, J.K., Holik, J., Chawla, A., Park, J.G., Buxton, J. and Czech, M.P. (2001) Signaling complexes of the FERM domain-containing protein GRSP1 bound to ARF exchange factor GRP1. Journal of Biological Chemistry, 276, 40065-40070. doi:10.1074/jbc.M105260200 [17] Nevrivy, D.J., Peterson, V.J., Avram, D., Ishmael, J.E., Hansen, S.G., Dowell, P., Hruby, D.E., Dawson, M.I. and Leid, M. (2000) Interaction of GRASP, a protein encoded by a novel retinoic acid-induced gene, with members of the cytohesin family of guanine nucleotide exchange factors. Journal of Biological Chemistry, 275, 16827-16836. doi:10.1074/jbc.275.22.16827 [18] Kitano, J., Kimura, K., Yamazaki, Y., Soda, T., Shigemoto, R., Nakajima, Y. and Nakanishi, S. (2002) Tamalin, a PDZ domain-containing protein, links a protein complex formation of group 1 metabotropic glutamate receptors and the guanine nucleotide exchange factor cytohesins. Journal of Neuroscience, 22, 1280-1289. [19] Mansour, M., Lee, S.Y. and Pohajdak, B. (2002) The N-terminal coiled coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with the scaffolding protein CASP. Journal of Biological Chemistry, 277, 32302-32309. doi:10.1074/jbc.M202898200 [20] Boehm, T., Hofer, S., Winklehner, P., Kellersch, B., Geiger, C., Trockenbacher, A., Neyer, S., Fiegl, H., Ebner, S., Ivarsson, L., Schneider, R., Kremmer, E., Heufler, C. and Kolanus, W. (2003) Attenuation of cell adhesion in lymphocytes is regulated by CYTIP, a protein which mediates signal complex sequestration. EMBO Journal, 22, 1014-1024. doi:10.1093/emboj/cdg101 [21] Tang, P., Cheng, T.P., Agnello, D., Wu, C.Y., Hissong, B.D., Watford, W.T., Ahn, H.J., Galon, J., Moss, J., Vaughan, M., O’Shea, J.J. and Gadina, M. (2002) Cybr, a cytokine-inducible protein that binds cytohesin-1 and regulates its activity. Proceedings of the National Academy of Sciences USA, 99, 2625-2629. doi:10.1073/pnas.052712999 [22] Venkateswarlu, K. (2003) Interaction protein for cytohesin exchange factors 1 (IPCEF1) binds cytohesin 2 and modifies its activity. Journal of Biological Chemistry, 278, 43460-43469. doi:10.1074/jbc.M304078200 [23] Ikenouchi, J. and Umeda, M. (2010) FRMD4A regulates epithelial polarity by connecting Arf6 activation with the PAR complex. Proceedings of the National Academy of Sciences USA, 107, 748-753. doi:10.1073/pnas.0908423107 [24] Lim, J., Zhou, M., Veenstra, T.D. and Morrison, D.K. (2010) The CNK1 scaffold binds cytohesins and promotes insulin pathway signaling. Genes & Development, 24, 1496-1506. doi:10.1101/gad.1904610 [25] Bill, A., Schmitz, A., Albertoni, B., Song, J.N., Heukamp, L.C., Walrafen, D., Thorwirth, F., Verveer, P.J., Zimmer, S., Meffert, L., Schreiber, A., Chatterjee, S., Thomas, R.K., Ullrich, R.T., Lang, T. and Famulok, M. (2010) Cytohesins are cytoplasmic ErbB receptor activators. Cell, 143, 201-211. doi:10.1016/j.cell.2010.09.011 [26] Hofmann, I., Thompson, A., Sanderson, C.M. and Munro, S. (2007) The Arl4 family of small G proteins can recruit the cytohesin Arf6 exchange factors to the plasma membrane. Current Biology, 17, 711-716. doi:10.1016/j.cub.2007.03.007 [27] Li, C.C., Chiang, T.C., Wu, T.S., Pacheco-Rodriguez, G., Moss, J. and Lee, F.J. (2007) ARL4D recruits cytohesin-2/ARNO to modulate actin remodeling. Molecular Biology of the Cell, 18, 4420-4437. doi:10.1091/mbc.E07-02-0149 [28] Oh, S. J. and Santy, L. C. (2010) Differential effects of cytohesins 2 and 3 on beta1 integrin recycling. Journal of Biological Chemistry, 285, 14610-14616. doi:10.1074/jbc.M109.043935 [29] Hafner, M., Schmitz, A., Grune, I., Srivatsan, S.G., Paul, B., Kolanus, W., Quast, T., Kremmer, E., Bauer, I. and Famulok, M. (2006) Inhibition of cytohesins by SecinH3 leads to hepatic insulin resistance. Nature, 444, 941-944. doi:10.1038/nature05415 [30] Fuss, B., Becker, T., Zinke, I. and Hoch, M. (2006) The cytohesin Steppke is essential for insulin signalling in Drosophila. Nature, 444, 945-948. doi:10.1038/nature054