v-Crk Induces Rac-dependent Membrane Ruffling and Cell Migration in CAS-deficient Embryonic Fibroblasts

  • Sung, Bong Hwan (Center for Distributed Sensor Network (CDSN), Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Yeo, Myoung Gu (Center for Distributed Sensor Network (CDSN), Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Oh, Hye Jin (Center for Distributed Sensor Network (CDSN), Department of Life Sciences, Gwangju Institute of Science and Technology) ;
  • Song, Woo Keun (Center for Distributed Sensor Network (CDSN), Department of Life Sciences, Gwangju Institute of Science and Technology)
  • Received : 2007.07.19
  • Accepted : 2007.09.10
  • Published : 2008.02.29

Abstract

Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.

Keywords

Acknowledgement

Supported by : Ministry of Science and Technology

References

  1. Abassi, Y.A. and Vuori, K. (2002). Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling. EMBO J. 21, 4571-4582 https://doi.org/10.1093/emboj/cdf446
  2. Allen, W.E., Jones, G.E., Pollard, J.W., and Ridley, A.J. (1997). Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J. Cell Sci. 110, 707-720
  3. Altun-Gultekin, Z.F., Chandriani, S., Bougeret, C., Ishizaki, T., Narumiya, S., de Graaf, P., Van Bergen en Henegouwen, P., Hanafusa, H., Wagner, J.A., and Birge, R.B. (1998). Activation of Rho-dependent cell spreading and focal adhesion biogenesis by the v-Crk adaptor protein. Mol. Cell. Biol. 18, 3044-3058 https://doi.org/10.1128/MCB.18.5.3044
  4. Bear, J.E., Loureiro, J.J., Libova, I., Faesler, R., Wehland, J., and Gertler, F.B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteins. Cell 101, 717-728 https://doi.org/10.1016/S0092-8674(00)80884-3
  5. Bear, J.E., Svitkina, T.M., Krause, M., Schafer, D.A., Loureiro, J.J., Strasser, G.A., Maly, I. V., Chaga, O.Y., Cooper, J.A., and Borisy, G.G. (2002). Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109, 509−521
  6. Benard, V. and Bokoch, G.. (2002). Assay of CDC42, Rac, and Rho GTPase activation by affinity methods. In Methods in Enzymology, R. Iyengar and J.D. Hildebrandt, eds. (California USA: Academic Press), pp. 349-359
  7. Craig, S.W. and Chen, H. (2003). Lamellipodia protrusion: moving interactions of vinculin and Arp2/3. Curr. Biol. 13, 236-238 https://doi.org/10.1016/S0960-9822(03)00010-1
  8. del Pozo, M.A., Price, L.S., Alderson, N.B., Ren, X.D., and Schwartz, M.A. (2000). Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J. 19, 2008-2014 https://doi.org/10.1093/emboj/19.9.2008
  9. Gu, J., Sumida, Y., Sanzen, N., and Sekiguchi, K. (2001). Laminin-10/11 and fibronectin differentially regulate integrin-dependent Rho and Rac activation via p130Cas-CrkII-DOCK180 pathway. J. Biol. Chem. 276, 27090-27097 https://doi.org/10.1074/jbc.M102284200
  10. Hinz, B., Alt, W., Johnen, C., Herzog, V., and Kaiser, H.W. (1999). Quantifying lamella dynamics of cultured cells by SACED, a new computer-assisted motion analysis. Exp. Cell. Res. 251, 234-243 https://doi.org/10.1006/excr.1999.4541
  11. Honda, H., Oda, H., Nakamoto, T., Honda, Z., Sakai, R., Suzuki, T., Saito, T., Nakamura, K., Nakao, K., Ishikawa, T., et al. (1998). Cardiovascular anomaly, impaired actin bundling and resistance to Src-induced transformation in mice lacking p130Cas. Nat. Genet. 19, 361-365 https://doi.org/10.1038/1246
  12. Honda, H., Nakamoto, T., Sakai, R., and Hirai, H. (1999). p130Cas, an assembling molecule of actin filaments, promotes cell movement, cell migration, and cell spreading in fibroblasts. Biochem. Biophys. Res. Commun. 262, 25-30 https://doi.org/10.1006/bbrc.1999.1162
  13. Huttenlocher, A., Sandborg, R.R., and Horwitz, A.F. (1995). Adhesion in cell migration. Curr. Opin. Cell. Biol. 7, 697-706 https://doi.org/10.1016/0955-0674(95)80112-X
  14. Kanner, S.B., Reynolds, A.B., Wang, H. C., Vines, R.R., and Parsons, J.T. (1991). The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 10, 1689-1698
  15. Kiyokawa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H., Kurata, T., and Matsuda, M. (1998). Activation of Rac1 by a Crk SH3-binding protein, DOCK180. Genes Dev. 12, 3331-3336 https://doi.org/10.1101/gad.12.21.3331
  16. Klemke, R.L., Leng, J., Molander, R., Brooks, P.C., Vuori, K., and Cheresh, D.A. (1998). CAS/Crk coupling serves as a "molecular switch" for induction of cell migration. J. Cell Biol. 140, 961-972 https://doi.org/10.1083/jcb.140.4.961
  17. Lauffenburger, D.A. and Horwitz, A.F. (1996). Cell migration: a physically integrated molecular process. Cell 84, 359-369 https://doi.org/10.1016/S0092-8674(00)81280-5
  18. Legg, J.A., Bompard, G., Dawson, J., Morris, H.L., Andrew, N., Cooper, L., Johnston, S.A., Tramountanis, G., and Machesky, L.M. (2007). N-WASP involvement in dorsal ruffle formation in mouse embryonic fibroblasts. Mol. Biol. Cell 18, 678-687 https://doi.org/10.1091/mbc.E06-06-0569
  19. Lili, J., Yuchen, S., and Zhengtao, W. (2006). The Chemokine SDF-1alpha suppresses fibronectin-mediated in vitro lymphocytes adhesion. Mol. Cells 22, 308-313
  20. Matsuda, M., Mayer, B.J., Fukui, Y., and Hanafusa, H. (1990). Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins. Science 248, 1537-1539 https://doi.org/10.1126/science.1694307
  21. Mayer, B.J., Hamaguchi, M., and Hanafusa, H. (1988). A novel viral oncogene with structural similarity to phospholipase C. Nature 332, 272-275 https://doi.org/10.1038/332272a0
  22. Nievers, M.G., Birge, R.B., Greulich, H., Verkleij, A.J., Hanafusa, H., and van Bergen en Henegouwen, P.M. (1997). v-Crk-induced cell transformation: changes in focal adhesion composition and signaling. J. Cell Sci. 110, 389-399
  23. Pinco, K.A., He, W., and Yang, J.T. (2002). Alpha4beta1 integrin regulates lamellipodia protrusion via a focal complex/focal adhesion-independent mechanism. Mol. Biol. Cell 13, 3203-3217 https://doi.org/10.1091/mbc.02-05-0086
  24. Price, L.S., Leng, J., Schwartz, M.A., and Bokoch, G.M. (1998). Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell 9, 1863-1871 https://doi.org/10.1091/mbc.9.7.1863
  25. Ridley, A.J. (2001). Rho GTPases and cell migration. J. Cell Sci. 114, 2713-2722
  26. Ridley, A.J., Paterson, H.F., Johnston, C.L., Diekmann, D., and Hall, A. (1992). The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70, 401-410 https://doi.org/10.1016/0092-8674(92)90164-8
  27. Rottner, K., Behrendt, B., Small, J.V., and Wehland, J. (1999). VASP dynamics during lamellipodia protrusion. Nat. Cell Biol. 1, 321-322 https://doi.org/10.1038/13040
  28. Stam, J.C., Geerts, W. J.C., Versteeg, H.H., Verkleij, A.J., and en Henegouwen, P. (2001). The v-Crk oncogene enhances cell survival and induces activation of protein kinase B/Akt. J. Biol. Chem. 276, 25176-25183 https://doi.org/10.1074/jbc.M009825200
  29. Suetsugu, S., Yamazaki, D., Kurisu, S., and Takenawa, T. (2003). Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. Dev. Cell 5, 595-609 https://doi.org/10.1016/S1534-5807(03)00297-1
  30. Sugihara, K., Nakatsuji, N., Nakamura, K., Nakao, K., Hashimoto, R., Otani, H., Sakagami, H., Kondo, H., Nozawa, S., and Aiba, A. (1999). Rac 1 is required for the formation of three germ layers during gastrulation. Oncogene 17, 3427-3433 https://doi.org/10.1038/sj.onc.1202595
  31. Tsuda, M., Tanaka, S., Sawa, H., Hanafusa, H., and Nagashima, K. (2002). Signaling adaptor protein v-Crk activates Rho and regulates cell motility in 3Y1 rat fibroblast cell line. Cell Growth Differ. 13, 131-139
  32. Tsuda, M., Makino, Y., Iwahara, T., Nishihara, H., Sawa, H., Nagashima, K., Hanafusa, H., and Tanaka, S. (2004). Crk associates with ERM proteins and promotes cell motility toward hyaluronic acid. J. Biol. Chem. 279, 46843-46850 https://doi.org/10.1074/jbc.M401476200
  33. Wu, C.-C., Su, H.-W., Lee, C.-C., Tang, M.-J., and Su, F.-C. (2005). Quantitative measurement of changes in adhesion force involving focal adhesion kinase during cell attachment, spread, and migration. Biochem. Biophys. Res. Commun. 329, 256-265 https://doi.org/10.1016/j.bbrc.2005.01.126
  34. Yamada, K. M. and Geiger, B. (1997). Molecular interactions in cell adhesion complexes. Curr. Opin. Cell Biol. 9, 76-85 https://doi.org/10.1016/S0955-0674(97)80155-X
  35. Yeo, M.G., Sung, B.H., Oh, H.J., Park, J.Y., Marcantonio, E.E., and Song, W.K. (2008). Focal adhesion targeting of v-Crk is essential for FAK phosphorylation and cell migration in mouse embryo fibroblasts deficient Src family kinases or p130CAS. J. Cell. Physiol. 214, 604-613 https://doi.org/10.1002/jcp.21247