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Identification of New Interacting Partners for Atypical Rho GTPases: A SILAC-Based Approach

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Rho GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 827))

Abstract

In contrast to typical Rho GTPases the regulation of atypical Rho GTPases, such as the members of the RhoBTB subfamily, rarely depends on GEFs and/or GAPs. Instead, they are regulated at the level of their expression, by post-translational modifications, by their rate of degradation as well as through binding of diverse cell-specific interactors. Stable Isotope Labeling by Amino acids in Cell culture (SILAC) is a powerful cutting-edge mass-spectrometry-based technology allowing for protein-interaction studies in vitro with removal of false-positive identifications. In this chapter, we describe how the SILAC technology can be applied to the identification of new interacting partners for atypical – constitutively active – Rho GTPases, i.e. RhoBTB3.

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References

  1. Aspenstrom, P., Ruusala, A., and Pacholsky, D. (2007) Taking Rho GTPases to the next level: the cellular functions of atypical Rho GTPases. Exp Cell Res 313, 3673–3679.

    Article  PubMed  Google Scholar 

  2. Hall, A., and Lalli, G. (2010) Rho and Ras GTPases in axon growth, guidance, and branching. Cold Spring Harb Perspect Biol 2, a001818.

    Article  PubMed  Google Scholar 

  3. Heasman, S.J., and Ridley, A.J. (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9, 690–701.

    Article  PubMed  CAS  Google Scholar 

  4. Ridley, A.J. (2006) Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 16, 522–529.

    Article  PubMed  CAS  Google Scholar 

  5. Tybulewicz, V.L., and Henderson, R.B. (2009) Rho family GTPases and their regulators in lymphocytes. Nat Rev Immunol 9, 630–644.

    Article  PubMed  CAS  Google Scholar 

  6. Vega, F.M., and Ridley, A.J. (2008) Rho GTPases in cancer cell biology. FEBS Lett 582, 2093–2101.

    Article  PubMed  CAS  Google Scholar 

  7. Saras, J., Wollberg, P., and Aspenstrom, P. (2004) Wrch1 is a GTPase-deficient Cdc42-like protein with unusual binding characteristics and cellular effects. Exp Cell Res 299, 356–369.

    Article  PubMed  CAS  Google Scholar 

  8. Aspenström, P., Fransson, A., and Saras, J. (2004) Rho GTPases have diverse effects on the organization of the actin filament system. Biochem J 377, 327–337.

    Article  PubMed  Google Scholar 

  9. Wennerberg, K., and Der, C.J. (2004) Rho-family GTPases: it’s not only Rac and Rho (and I like it). J Cell Sci 117, 1301–1312.

    Article  PubMed  CAS  Google Scholar 

  10. Chardin, P. (2006) Function and regulation of Rnd proteins. Nat Rev Mol Cell Biol 7, 54–62.

    Article  PubMed  CAS  Google Scholar 

  11. Chenette, E.J., Abo, A., and Der, C.J. (2005) Critical and distinct roles of amino- and ­carboxyl-terminal sequences in regulation of the biological activity of the Chp atypical Rho GTPase. J Biol Chem 280, 13784–13792.

    Article  PubMed  CAS  Google Scholar 

  12. Berthold, J., Schenkova, K., Ramos, S., Miura, Y., Furukawa, M., Aspenström, P., and Rivero, F. (2008) Characterization of RhoBTB-dependent Cul3 ubiquitin ligase complexes--evidence for an autoregulatory mechanism. Exp Cell Res 314, 3453–3465.

    Article  PubMed  CAS  Google Scholar 

  13. Katoh, H., Harada, A., Mori, K., and Negishi, M. (2002) Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers. Mol Cell Biol 22, 2952–2964.

    Article  PubMed  CAS  Google Scholar 

  14. Madigan, J.P., Bodemann, B.O., Brady, D.C., Dewar, B.J., Keller, P.J., Leitges, M., Philips, M.R., Ridley, A.J., Der, C.J., and Cox, A.D. (2009) Regulation of Rnd3 localization and function by protein kinase C alpha-mediated phosphorylation. Biochem J 424, 153–161.

    Article  PubMed  CAS  Google Scholar 

  15. Riento, K., Totty, N., Villalonga, P., Garg, R., Guasch, R., and Ridley, A.J. (2005) RhoE function is regulated by ROCK I-mediated phosphorylation. EMBO J 24, 1170–1180.

    Article  PubMed  CAS  Google Scholar 

  16. Cusick, M.E., Klitgord, N., Vidal, M., and Hill, D.E. (2005) Interactome: gateway into systems biology. Hum Mol Genet 14 Spec No. 2, R171–181.

    Google Scholar 

  17. Ong, S.E., Foster, L.J., and Mann, M. (2003) Mass spectrometric-based approaches in quantitative proteomics. Methods 29, 124–130.

    Article  PubMed  CAS  Google Scholar 

  18. Ong, S.E., and Mann, M. (2006) A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC). Nat Protoc 1, 2650–2660.

    Article  PubMed  CAS  Google Scholar 

  19. Ong, S. E., and Mann, M. (2007) Stable isotope labeling by amino acids in cell culture for quantitative proteomics. Methods Mol Biol 359, 37–52.

    Article  PubMed  CAS  Google Scholar 

  20. Foster, L.J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L.M., Bilan, P.J., Mann, M., and Klip, A. (2006) Insulin-dependent interactions of proteins with GLUT4 revealed through stable isotope labeling by amino acids in cell culture (SILAC). J Proteome Res 5, 64–75.

    Article  PubMed  CAS  Google Scholar 

  21. Mann, M. (2006) Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol 7, 952–958.

    Article  PubMed  CAS  Google Scholar 

  22. Selbach, M., and Mann, M. (2006) Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods 3, 981–983.

    Article  PubMed  CAS  Google Scholar 

  23. Dobreva, I., Fielding, A., Foster, L.J., and Dedhar, S. (2008) Mapping the integrin-linked kinase interactome using SILAC. J Proteome Res 7, 1740–1749.

    Article  PubMed  CAS  Google Scholar 

  24. Trinkle-Mulcahy, L., Andersen, J., Lam, Y.W., Moorhead, G., Mann, M., and Lamond, A.I. (2006) Repo-Man recruits PP1 gamma to chromatin and is essential for cell viability. J Cell Biol 172, 679–692.

    Article  PubMed  CAS  Google Scholar 

  25. Mortensen, P., Gouw, J.W., Olsen, J.V., Ong, S.E., Rigbolt, K.T., Bunkenborg, J., Cox, J., Foster, L.J., Heck, A..J., Blagoev, B., Andersen, J.S., and Mann, M. (2010) MSQuant, an open source platform for mass spectrometry-based quantitative proteomics. J Proteome Res 9, 393–403.

    Article  PubMed  CAS  Google Scholar 

  26. van Breukelen, B., van den Toorn, H.W., Drugan, M.M., and Heck, A.J. (2009) StatQuant: a post-quantification analysis toolbox for improving quantitative mass spectrometry. Bioinformatics 25, 1472–1473.

    Article  PubMed  Google Scholar 

  27. Fransson, S., Ruusala, A., and Aspenström, P. (2006) The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking. Biochem Biophys Res Commun 344, 500–510.

    Article  PubMed  CAS  Google Scholar 

  28. Weihofen, A., Thomas, K.J., Ostaszewski, B.L., Cookson, M.R., and Selkoe, D.J. (2009) Pink1 forms a multiprotein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking. Biochemistry 48, 2045–2052.

    Article  PubMed  CAS  Google Scholar 

  29. Wilkins, A., Ping, Q., and Carpenter, C.L. (2004) RhoBTB2 is a substrate of the mammalian Cul3 ubiquitin ligase complex. Genes Dev 18, 856–861.

    Article  PubMed  CAS  Google Scholar 

  30. Espinosa, E.J., Calero, M., Sridevi, K., and Pfeffer, S.R. (2009) RhoBTB3: a Rho GTPase-family ATPase required for endosome to Golgi transport. Cell 137, 938–948.

    Article  PubMed  CAS  Google Scholar 

  31. Gasman, S., Kalaidzidis, Y., and Zerial, M. (2003) RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase. Nat Cell Biol 5, 195–204.

    Article  PubMed  CAS  Google Scholar 

  32. Zanata, S.M., Hovatta, I., Rohm, B., and Puschel, A.W. (2002) Antagonistic effects of Rnd1 and RhoD GTPases regulate receptor activity in Semaphorin 3A-induced cytoskeletal collapse. J Neurosci 22, 471–477.

    PubMed  CAS  Google Scholar 

  33. Wang, H., Zeng, X., Fan, Z., and Lim, B. (2011) RhoH modulates pre-TCR and TCR signalling by regulating LCK. Cell Signal 23, 249–258.

    Article  PubMed  CAS  Google Scholar 

  34. Oda, H., Fujimoto, M., Patrick, M.S., Chida, D., Sato, Y., Azuma, Y., Aoki, H., Abe, T., Suzuki, H., and Shirai, M. (2009) RhoH plays critical roles in Fc epsilon RI-dependent signal transduction in mast cells. J Immunol 182, 957–962.

    PubMed  CAS  Google Scholar 

  35. Gu, Y., Chae, H.D., Siefring, J.E., Jasti, A.C., Hildeman, D.A., and Williams, D.A. (2006) RhoH GTPase recruits and activates Zap70 required for T cell receptor signaling and thymocyte development. Nat Immunol 7, 1182–1190.

    Article  PubMed  CAS  Google Scholar 

  36. Alan, J.K., Berzat, A.C., Dewar, B.J., Graves, L.M., and Cox, A.D.(2010) Regulation of the Rho family small GTPase Wrch-1/RhoU by C-terminal tyrosine phosphorylation requires Src. Mol Cell Biol 30, 4324–4338.

    Article  PubMed  CAS  Google Scholar 

  37. Misko, A., Jiang, S., Wegorzewska, I., Milbrandt, J., and Baloh, R.H. (2010) Mitofusin 2 is necessary for transport of axonal mitochondria and interacts with the Miro/Milton complex. J Neurosci 30, 4232–4240.

    Article  PubMed  CAS  Google Scholar 

  38. Brady, D.C., Alan, J.K., Madigan, J.P., Fanning, A.S., and Cox, A.D. (2009) The transforming Rho family GTPase Wrch-1 disrupts epithelial cell tight junctions and epithelial morphogenesis. Mol Cell Biol 29, 1035–1049.

    Article  PubMed  CAS  Google Scholar 

  39. Ruusala, A., and Aspenström, P. (2008) The atypical Rho GTPase Wrch1 collaborates with the nonreceptor tyrosine kinases Pyk2 and Src in regulating cytoskeletal dynamics. Mol Cell Biol 28, 1802–1814.

    Article  PubMed  CAS  Google Scholar 

  40. Harada, A., Katoh, H., and Negishi, M. (2005) Direct interaction of Rnd1 with FRS2 beta regulates Rnd1-induced down-regulation of RhoA activity and is involved in fibroblast growth factor-induced neurite outgrowth in PC12 cells. J Biol Chem 280, 18418–18424.

    Article  PubMed  CAS  Google Scholar 

  41. Vayssiere, B., Zalcman, G., Mahe, Y., Mirey, G., Ligensa, T., Weidner, K.M., Chardin, P., and Camonis, J. (2000) Interaction of the Grb7 adapter protein with Rnd1, a new member of the Rho family. FEBS Lett 467, 91–96.

    Article  PubMed  CAS  Google Scholar 

  42. Toyofuku, T., Yoshida, J., Sugimoto, T., Zhang, H., Kumanogoh, A., Hori, M., and Kikutani, H. (2005) FARP2 triggers signals for Sema3A-mediated axonal repulsion. Nat Neurosci 8, 1712–1719.

    Article  PubMed  CAS  Google Scholar 

  43. Oinuma, I., Katoh, H., Harada, A., and Negishi, M. (2003) Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells. J Biol Chem 278, 25671–25677.

    Article  PubMed  CAS  Google Scholar 

  44. Tong, Y., Chugha, P., Hota, P.K., Alviani, R.S., Li, M., Tempel, W., Shen, L., Park, H.W., and Buck, M. (2007) Binding of Rac1, Rnd1, and RhoD to a novel Rho GTPase interaction motif destabilizes dimerization of the plexin-B1 effector domain. J Biol Chem 282, 37215–37224.

    Article  PubMed  CAS  Google Scholar 

  45. Li, Y.H., Ghavampur, S., Bondallaz, P., Will, L., Grenningloh, G., and Puschel, A.W. (2009) Rnd1 regulates axon extension by enhancing the microtubule destabilizing activity of SCG10. J Biol Chem 284, 363–371.

    Article  PubMed  CAS  Google Scholar 

  46. Karaulanov, E., Bottcher, R.T., Stannek, P., Wu, W., Rau, M., Ogata, S., Cho, K.W., and Niehrs, C. (2009) Unc5B interacts with FLRT3 and Rnd1 to modulate cell adhesion in Xenopus embryos. PLoS One 4, e5742.

    Article  PubMed  Google Scholar 

  47. Wennerberg, K., Forget, M.A., Ellerbroek, S.M., Arthur, W. T., Burridge, K., Settleman, J., Der, C.J., and Hansen, S.H. (2003) Rnd proteins function as RhoA antagonists by activating p190 RhoGAP. Curr Biol 13, 1106–1115.

    Article  PubMed  CAS  Google Scholar 

  48. Naud, N., Toure, A., Liu, J., Pineau, C., Morin, L., Dorseuil, O., Escalier, D., Chardin, P., and Gacon, G. (2003) Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells. Biochem J 372, 105–112.

    Article  PubMed  CAS  Google Scholar 

  49. Uesugi, K., Oinuma, I., Katoh, H., and Negishi, M. (2009) Different requirement for Rnd GTPases of R-Ras GAP activity of Plexin-C1 and Plexin-D1. J Biol Chem 284, 6743–6751.

    Article  PubMed  CAS  Google Scholar 

  50. Tanaka, H., Katoh, H., and Negishi, M. (2006) Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity. J Biol Chem 281, 10355–10364.

    Article  PubMed  CAS  Google Scholar 

  51. Fujita, H., Katoh, H., Ishikawa, Y., Mori, K., and Negishi, M. (2002) Rapostlin is a novel effector of Rnd2 GTPase inducing neurite branching. J Biol Chem 277, 45428–45434.

    Article  PubMed  CAS  Google Scholar 

  52. Tanaka, H., Fujita, H., Katoh, H., Mori, K., and Negishi, M. (2002) Vps4-A (vacuolar protein sorting 4-A) is a binding partner for a novel Rho family GTPase, Rnd2. Biochem J 365, 349–353.

    Article  PubMed  CAS  Google Scholar 

  53. Riento, K., Guasch, R.M., Garg, R., Jin, B., and Ridley, A.J. (2003) RhoE binds to ROCK I and inhibits downstream signaling. Mol Cell Biol 23, 4219–4229.

    Article  PubMed  CAS  Google Scholar 

  54. Goh, L. L., and Manser, E. (2010) The RhoA GEF Syx is a target of Rnd3 and regulated via a Raf1-like ubiquitin-related domain. PLoS One 5, e12409.

    Article  PubMed  Google Scholar 

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Acknowledgments

We thank Dr. Francisco Rivero for kindly providing us a RhoBTB3 expressing vector. This work was supported by the FP7 program (FP7-PEOPLE-IEF-2008/236777/AXOGLIA), COMPETE (FCOMP-01-0124-FEDER-011182), and the Fundação para a Ciência e a Tecnologia (PTDC/SAU-NEU/69831/2006; PTDC/SAU-NEU/099007/2008).

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Authors and Affiliations

  1. Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal

    Laura Montani, Ana Filipa Domingues & João Bettencourt Relvas

  2. Institute of Molecular Systems Biology, NCCR Neuro Center for Proteomics, ETH Zürich, Zürich, Switzerland

    Damaris Bausch-Fluck & Bernd Wollscheid

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  1. Laura Montani
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  2. Damaris Bausch-Fluck
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  3. Ana Filipa Domingues
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  4. Bernd Wollscheid
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  5. João Bettencourt Relvas
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Correspondence to Damaris Bausch-Fluck .

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Editors and Affiliations

  1. Hull York Medical School, Centre for Biomedical Research, University of Hull, Cottingham Road, Hull, HU6 7RX, United Kingdom

    Francisco Rivero

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Montani, L., Bausch-Fluck, D., Domingues, A.F., Wollscheid, B., Relvas, J.B. (2012). Identification of New Interacting Partners for Atypical Rho GTPases: A SILAC-Based Approach. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 827. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-442-1_20

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  • DOI: https://doi.org/10.1007/978-1-61779-442-1_20

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