Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Resource
  • Published:

Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms

Nature Cell Biology volume 22, pages 120–134 (2020)Cite this article

Subjects

An Author Correction to this article was published on 10 February 2020

This article has been updated

Abstract

Guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs) coordinate the activation state of the Rho family of GTPases for binding to effectors. Here, we exploited proximity-dependent biotinylation to systematically define the Rho family proximity interaction network from 28 baits to produce 9,939 high-confidence proximity interactions in two cell lines. Exploiting the nucleotide states of Rho GTPases, we revealed the landscape of interactions with RhoGEFs and RhoGAPs. We systematically defined effectors of Rho proteins to reveal candidates for classical and atypical Rho proteins. We used optogenetics to demonstrate that KIAA0355 (termed GARRE here) is a RAC1 interactor. A functional screen of RHOG candidate effectors identified PLEKHG3 as a promoter of Rac-mediated membrane ruffling downstream of RHOG. We identified that active RHOA binds the kinase SLK in Drosophila and mammalian cells to promote Ezrin–Radixin–Moesin phosphorylation. Our proximity interactions data pave the way for dissecting additional Rho signalling pathways, and the approaches described here are applicable to the Ras family.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: The large-scale Rho GTPases proximity interaction network.
Fig. 2: Mappings of RhoGEF and RhoGAP specificities.
Fig. 3: Mappings and identifications of Rho-family-specific candidate effectors.
Fig. 4: GARRE is a direct interactor of RAC1.
Fig. 5: The GEF PLEKHG3 is an effector of RHOG.
Fig. 6: SLK binds RHOA in an evolutionarily conserved manner.
Fig. 7: SLK induces RHOA-dependent phosphorylation of ERM proteins.
Fig. 8: RHOAG14V directly binds the C-terminal CCD of SLK.

Similar content being viewed by others

Data availability

The raw proteomics data have been deposited into ProteomeXchange (http://www.proteomexchange.org) with accession number PXD015918. The BioID data in this manuscript can also be explored in the supplementary tables and on a dedicated website (http://prohits-web.lunenfeld.ca/GIPR/Datasets.php?projectID=25&m_num=m3). Source data for Figs. 2–8 and Extended Data Figs. 1 and 4–6 are available online. All data that support the findings of this study are available from the corresponding author upon reasonable request.

Change history

References

  1. Rojas, A. M., Fuentes, G., Rausell, A. & Valencia, A. The Ras protein superfamily: evolutionary tree and role of conserved amino acids. J. Cell Biol. 196, 189–201 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hodge, R. G. & Ridley, A. J. Regulating Rho GTPases and their regulators. Nat. Rev. Mol. Cell Biol. 17, 496–510 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Cherfils, J. & Zeghouf, M. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiol. Rev. 93, 269–309 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Laurin, M. & Cote, J. F. Insights into the biological functions of Dock family guanine nucleotide exchange factors. Genes Dev. 28, 533–547 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rossman, K. L., Der, C. J. & Sondek, J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat. Rev. Mol. Cell Biol. 6, 167–180 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Tcherkezian, J. & Lamarche-Vane, N. Current knowledge of the large RhoGAP family of proteins. Biol. Cell 99, 67–86 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Mott, H. R. & Owen, D. Structures of Ras superfamily effector complexes: what have we learnt in two decades? Crit. Rev. Biochem. Mol. Biol. 50, 85–133 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Olson, M. F. Rho GTPases, their post-translational modifications, disease-associated mutations and pharmacological inhibitors. Small GTPases 9, 203–215 (2018).

    Article  CAS  PubMed  Google Scholar 

  9. Krauthammer, M. et al. Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. Nat. Genet. 44, 1006–1014 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Yoo, H. Y. et al. A recurrent inactivating mutation in RHOA GTPase in angioimmunoblastic T cell lymphoma. Nat. Genet. 46, 371–375 (2014).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Mueller, P. M. et al. Spatial Organization of Rho GTPase signaling by RhoGEF/RhoGAP proteins. Preprint at bioRxiv https://www.biorxiv.org/content/10.1101/354316v3 (2018).

  13. Gingras, A. C., Abe, K. T. & Raught, B. Getting to know the neighborhood: using proximity-dependent biotinylation to characterize protein complexes and map organelles. Curr. Opin. Chem. Biol. 48, 44–54 (2019).

    Article  CAS  PubMed  Google Scholar 

  14. Roux, K. J., Kim, D. I., Raida, M. & Burke, B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J. Cell Biol. 196, 801–810 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lai, C. C., Boguski, M., Broek, D. & Powers, S. Influence of guanine nucleotides on complex formation between Ras and CDC25 proteins. Mol. Cell. Biol. 13, 1345–1352 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Garrett, M. D., Self, A. J., van Oers, C. & Hall, A. Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins. J. Biol. Chem. 264, 10–13 (1989).

    Article  CAS  PubMed  Google Scholar 

  17. Youn, J. Y. et al. High-density proximity mapping reveals the subcellular organization of mRNA-associated granules and bodies. Mol. Cell 69, 517–532.e11 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Gupta, G. D. et al. A dynamic protein interaction landscape of the human centrosome–cilium interface. Cell 163, 1484–1499 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Couzens, A. L. et al. Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions. Sci. Signal. 6, rs15 (2013).

    Article  PubMed  CAS  Google Scholar 

  20. St-Denis, N. et al. Phenotypic and interaction profiling of the human phosphatases identifies diverse mitotic regulators. Cell Rep. 17, 2488–2501 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. & Kirschner, M. W. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418, 790–793 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Tyler, J. J., Allwood, E. G. & Ayscough, K. R. WASP family proteins, more than Arp2/3 activators. Biochem. Soc. Trans. 44, 1339–1345 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ishizaki, T. et al. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase. EMBO J. 15, 1885–1893 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Rose, R. et al. Structural and mechanistic insights into the interaction between Rho and mammalian Dia. Nature 435, 513–518 (2005).

    Article  CAS  PubMed  Google Scholar 

  25. Glaven, J. A., Whitehead, I. P., Nomanbhoy, T., Kay, R. & Cerione, R. A. Lfc and Lsc oncoproteins represent two new guanine nucleotide exchange factors for the Rho GTP-binding protein. J. Biol. Chem. 271, 27374–27381 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Ren, Y., Li, R., Zheng, Y. & Busch, H. Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases. J. Biol. Chem. 273, 34954–34960 (1998).

    Article  CAS  PubMed  Google Scholar 

  27. Xie, X., Chang, S. W., Tatsumoto, T., Chan, A. M. & Miki, T. TIM, a Dbl-related protein, regulates cell shape and cytoskeletal organization in a Rho-dependent manner. Cell Signal. 17, 461–471 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Mitin, N., Rossman, K. L. & Der, C. J. Identification of a novel actin-binding domain within the Rho guanine nucleotide exchange factor TEM4. PLoS ONE 7, e41876 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rodrigues, N. R. et al. Characterization of Ngef, a novel member of the Dbl family of genes expressed predominantly in the caudate nucleus. Genomics 65, 53–61 (2000).

    Article  CAS  PubMed  Google Scholar 

  30. Miki, T., Smith, C. L., Long, J. E., Eva, A. & Fleming, T. P. Oncogene ect2 is related to regulators of small GTP-binding proteins. Nature 362, 462–465 (1993).

    Article  CAS  PubMed  Google Scholar 

  31. Zheng, Y., Olson, M. F., Hall, A., Cerione, R. A. & Toksoz, D. Direct involvement of the small GTP-binding protein Rho in lbc oncogene function. J. Biol. Chem. 270, 9031–9034 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Shinohara, M. et al. SWAP-70 is a guanine-nucleotide-exchange factor that mediates signalling of membrane ruffling. Nature 416, 759–763 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Brugnera, E. et al. Unconventional Rac-GEF activity is mediated through the Dock180–ELMO complex. Nat. Cell Biol. 4, 574–582 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Cote, J. F. & Vuori, K. Identification of an evolutionarily conserved superfamily of DOCK180-related proteins with guanine nucleotide exchange activity. J. Cell Sci. 115, 4901–4913 (2002).

    Article  CAS  PubMed  Google Scholar 

  35. Yajnik, V. et al. DOCK4, a GTPase activator, is disrupted during tumorigenesis. Cell 112, 673–684 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Vives, V. et al. The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. J. Bone Miner. Res. 26, 1099–1110 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Miyamoto, Y., Yamauchi, J., Sanbe, A. & Tanoue, A. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth. Exp. Cell Res. 313, 791–804 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Salazar, M. A. et al. Tuba, a novel protein containing Bin/amphiphysin/Rvs and Dbl homology domains, links dynamin to regulation of the actin cytoskeleton. J. Biol. Chem. 278, 49031–49043 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Abiko, H. et al. Rho guanine nucleotide exchange factors involved in cyclic-stretch-induced reorientation of vascular endothelial cells. J. Cell Sci. 128, 1683–1695 (2015).

    CAS  PubMed  Google Scholar 

  40. Harada, Y. et al. DOCK8 is a Cdc42 activator critical for interstitial dendritic cell migration during immune responses. Blood 119, 4451–4461 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gadea, G., Sanz-Moreno, V., Self, A., Godi, A. & Marshall, C. J. DOCK10-mediated Cdc42 activation is necessary for amoeboid invasion of melanoma cells. Curr. Biol. 18, 1456–1465 (2008).

    Article  CAS  PubMed  Google Scholar 

  42. Abe, K. et al. Vav2 is an activator of Cdc42, Rac1, and RhoA. J. Biol. Chem. 275, 10141–10149 (2000).

    Article  CAS  PubMed  Google Scholar 

  43. Bellanger, J. M. et al. The two guanine nucleotide exchange factor domains of Trio link the Rac1 and the RhoA pathways in vivo. Oncogene 16, 147–152 (1998).

    Article  CAS  PubMed  Google Scholar 

  44. Fukuhara, S., Murga, C., Zohar, M., Igishi, T. & Gutkind, J. S. A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J. Biol. Chem. 274, 5868–5879 (1999).

    Article  CAS  PubMed  Google Scholar 

  45. Yamauchi, J., Miyamoto, Y., Chan, J. R. & Tanoue, A. ErbB2 directly activates the exchange factor Dock7 to promote Schwann cell migration. J. Cell Biol. 181, 351–365 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Manser, E. et al. PAK kinases are directly coupled to the PIX family of nucleotide exchange factors. Mol. Cell 1, 183–192 (1998).

    Article  CAS  PubMed  Google Scholar 

  47. Zhuang, B., Su, Y. S. & Sockanathan, S. FARP1 promotes the dendritic growth of spinal motor neuron subtypes through transmembrane Semaphorin6A and PlexinA4 signaling. Neuron 61, 359–372 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Chuang, T. H. et al. Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. Proc. Natl Acad. Sci. USA 92, 10282–10286 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Ellerbroek, S. M. et al. SGEF, a RhoG guanine nucleotide exchange factor that stimulates macropinocytosis. Mol. Biol. Cell 15, 3309–3319 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Reuther, G. W. et al. Leukemia-associated Rho guanine nucleotide exchange factor, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA. J. Biol. Chem. 276, 27145–27151 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Nguyen, T. T. et al. PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front. Proc. Natl Acad. Sci. USA 113, 10091–10096 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Amin, E. et al. Deciphering the molecular and functional basis of RHOGAP family proteins: a systematic approach toward selective inactivation of Rho family proteins. J. Biol. Chem. 291, 20353–20371 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Lamarche-Vane, N. & Hall, A. CdGAP, a novel proline-rich GTPase-activating protein for Cdc42 and Rac. J. Biol. Chem. 273, 29172–29177 (1998).

    Article  CAS  PubMed  Google Scholar 

  54. Chen, P. W., Jian, X., Yoon, H. Y. & Randazzo, P. A. ARAP2 signals through Arf6 and Rac1 to control focal adhesion morphology. J. Biol. Chem. 288, 5849–5860 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Toure, A. et al. MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells. J. Biol. Chem. 273, 6019–6023 (1998).

    Article  CAS  PubMed  Google Scholar 

  56. Wells, C. D. et al. A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells. Cell 125, 535–548 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Omelchenko, T. & Hall, A. Myosin-IXA regulates collective epithelial cell migration by targeting RhoGAP activity to cell–cell junctions. Curr. Biol. 22, 278–288 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Myagmar, B. E. et al. PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector. Biochem. Biophys. Res. Commun. 329, 1046–1052 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. Wennerberg, K. et al. Rnd proteins function as RhoA antagonists by activating p190 RhoGAP. Curr. Biol. 13, 1106–1115 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Lo, H. F. et al. Association of dysfunctional synapse defective 1 (SYDE1) with restricted fetal growth—SYDE1 regulates placental cell migration and invasion. J. Pathol. 241, 324–336 (2017).

    Article  CAS  PubMed  Google Scholar 

  61. Katoh, H. & Negishi, M. RhoG activates Rac1 by direct interaction with the Dock180-binding protein Elmo. Nature 424, 461–464 (2003).

    Article  CAS  PubMed  Google Scholar 

  62. Chenette, E. J., Abo, A. & Der, C. J. 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 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Spektor, A., Tsang, W. Y., Khoo, D. & Dynlacht, B. D. Cep97 and CP110 suppress a cilia assembly program. Cell 130, 678–690 (2007).

    Article  CAS  PubMed  Google Scholar 

  64. Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Bashaw, G. J., Hu, H., Nobes, C. D. & Goodman, C. S. A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion. J. Cell Biol. 155, 1117–1122 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Hipfner, D. R., Keller, N. & Cohen, S. M. Slik sterile-20 kinase regulates moesin activity to promote epithelial integrity during tissue growth. Genes Dev. 18, 2243–2248 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Viswanatha, R., Ohouo, P. Y., Smolka, M. B. & Bretscher, A. Local phosphocycling mediated by LOK/SLK restricts ezrin function to the apical aspect of epithelial cells. J. Cell Biol. 199, 969–984 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Matsui, T., Yonemura, S., Tsukita, S. & Tsukita, S. Activation of ERM proteins in vivo by Rho involves phosphatidyl-inositol 4-phosphate 5-kinase and not ROCK kinases. Curr. Biol. 9, 1259–1262 (1999).

    Article  CAS  PubMed  Google Scholar 

  69. Hebert, M. et al. Rho-ROCK-dependent ezrin–radixin–moesin phosphorylation regulates Fas-mediated apoptosis in Jurkat cells. J. Immunol. 181, 5963–5973 (2008).

    Article  CAS  PubMed  Google Scholar 

  70. Panneton, V. et al. Regulation of catalytic and non-catalytic functions of the Drosophila Ste20 kinase Slik by activation segment phosphorylation. J. Biol. Chem. 290, 20960–20971 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Kovalski, J. R. et al. The functional proximal proteome of oncogenic Ras includes mTORC2. Mol. Cell 73, 830–844.e12 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Paul, F. et al. Quantitative GTPase affinity purification identifies Rho family protein interaction partners. Mol. Cell. Proteomics 16, 73–85 (2017).

    Article  CAS  PubMed  Google Scholar 

  73. Patel, M., Chiang, T. C., Tran, V., Lee, F. J. & Cote, J. F. The Arf family GTPase Arl4A complexes with ELMO proteins to promote actin cytoskeleton remodeling and reveals a versatile Ras-binding domain in the ELMO proteins family. J. Biol. Chem. 286, 38969–38979 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Methot, S. P. et al. A licensing step links AID to transcription elongation for mutagenesis in B cells. Nat. Commun. 9, 1248 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  75. Liu, G. et al. Using ProHits to store, annotate, and analyze affinity purification-mass spectrometry (AP-MS) data. Curr. Protoc. Bioinformatics 39, 8.16.1–8.16.32 (2012).

    Article  Google Scholar 

  76. Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).

    Article  CAS  PubMed  Google Scholar 

  77. Knight, J. D. R. et al. ProHits-viz: a suite of web tools for visualizing interaction proteomics data. Nat. Methods 14, 645–646 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Findlay, S. et al. SHLD2/FAM35A co-operates with REV7 to coordinate DNA double-strand break repair pathway choice. EMBO J. 37, e100158 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Meller, J., Vidali, L. & Schwartz, M. A. Endogenous RhoG is dispensable for integrin-mediated cell spreading but contributes to Rac-independent migration. J. Cell Sci. 121, 1981–1989 (2008).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank A. Echard (Institut Pasteur, France), O. Rocks (Max-Delbrueck-Center for Molecular Medicine, Germany), L. Sabourin (OHRI, Canada) for providing plasmids and antibodies. They also acknowledge the gift of Flp-In T-REx HeLa cells from S. Taylor (University of Manchester, UK). They also thank A. Pelletier, M. Tucholska and K. Oh for excellent technical assistance and J.-P. Lambert for helpful suggestions on data analyses. They thank the IRCM Proteomics facility for the processing of MS samples, C. Poitras for installing ProHits, and D. Filion, É. Lécuyer and X. Wang for microscopy assistance. This work was supported by operating grants from the National Science and Engineering Research Council of Canada (RGPIN-2017-05819 to D.R.H.; RGPIN-2016-04808 to J.-F.C) and the Canadian Institutes of Health Research (FDN144301 to A.-C.G.; H.B., N.S., I.E.E. and V.T. were recipients of FRQS Doctoral studentships). I.E.E. was also supported by an IRCM Foundation-TD scholarship. J.-F.C. holds the Transat Chair in Breast Cancer Research.

Author information

Author notes

  1. These authors contributed equally: David R. Hipfner, Anne-Claude Gingras.

Authors and Affiliations

  1. Montreal Clinical Research Institute (IRCM), Montréal, Québec, Canada

    Halil Bagci, Neera Sriskandarajah, Amélie Robert, Jonathan Boulais, Islam E. Elkholi, Viviane Tran, Marie-Pier Thibault, Nadia Dubé, Denis Faubert, David R. Hipfner & Jean-François Côté

  2. Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada

    Halil Bagci & Jean-François Côté

  3. Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, Québec, Canada

    Neera Sriskandarajah & David R. Hipfner

  4. Molecular Biology Programs, Université de Montréal, Montréal, Québec, Canada

    Islam E. Elkholi, David R. Hipfner & Jean-François Côté

  5. Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada

    Viviane Tran

  6. Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada

    Zhen-Yuan Lin & Anne-Claude Gingras

  7. Department of Medicine, Université de Montréal, Montréal, Québec, Canada

    David R. Hipfner & Jean-François Côté

  8. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Anne-Claude Gingras

Authors
  1. Halil Bagci
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Neera Sriskandarajah
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Amélie Robert
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jonathan Boulais
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Islam E. Elkholi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Viviane Tran
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Zhen-Yuan Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Marie-Pier Thibault
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Nadia Dubé
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Denis Faubert
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. David R. Hipfner
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Anne-Claude Gingras
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jean-François Côté
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.-F.C., A.-C.G., D.R.H., N.D., H.B., A.R, J.B. and I.E.E. designed the research. H.B., N.S., A.R., J.B., I.E.E., V.T., Z.-Y.L., M.-P.T., N.D. and D.R.H. performed the research. H.B., N.S., A.R., J.B., I.E.E., N.D., D.F., D.R.H., A.-C.G. and J.-F.C. analysed the data. J.-F.C., A.R., J.B., H.B., D.R.H. and A.-C.G. wrote the paper with input from all other authors.

Corresponding author

Correspondence to Jean-François Côté.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Validation of BirA*-Flag-RHO GTPases expression constructs.

(a) Immunoblots of lysates from Flp-In T-REx HEK293 cells expressing the indicated constructs after induction with tetracycline (Tet). (b) Immunoblots of lysates from Flp-In T-REx HeLa cells expressing the indicated constructs before (-) and after ( + ) induction with tetracycline. (c) Western blots show the expression of the indicated BirA*-Flag-RHO GTPases as compared to their endogenous counterpart in Flp-In T-REx HeLa cells treated with tetracycline. GADPH was used as loading control. All the data presented in Extended Data Fig 1 are representative of three independent experiments. See unmodified scans in Unprocessed Blots Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Functionality of the BirA*-Flag-RHO GTPases constructs.

BirA*-Flag-RHO GTPases induce cytoskeletal changes. Flp-In T-REx HeLa cell lines were treated with tetracycline together with biotin to induce the expression of the indicated BirA*-Flag-RHO GTPases and the biotinylation of their proximal interactors. Confocal microscopy images of F-actin (Alexa Fluor 488-phalloidin) and biotin (Alexa Fluor 647-streptavidin) are shown. In comparison to control cells, expression of constitutively active RHO-subfamily led to the formation of thick stress fibres while expression of RAC-subfamily proteins promoted the development of large lamellipodia. Expression of the CDC42-subfamily proteins revealed different phenotypes with CDC42G12V enhancing growth of filopodia while RHOJG40V and RHOQG18V promoting both membrane ruffles and filopodia formation. Expression of constitutively active versions of RHOD/F-subfamily proteins led to the formation of long filopodia. The constitutively active versions of the fast cycling atypical RHOU/V-subfamily, as well as RHOHWT, induced the formation of both membrane ruffles and filopodia. The RND proteins did not mediate strong phenotypes with the exception that RND3 expressing cells that showed less actin fibres. Finally, members from the RHOBTB-subfamily did not alter the cytoskeleton. Data are representative of two independent experiments. Bars, 10 µm.

Extended Data Fig. 3 The constitutively active forms of the RHO GTPases baits are more efficient than the corresponding wild type forms to identify GAPs and effectors.

The constitutively active forms of the RHO GTPases enrich more interactions than the corresponding WT forms to identify GAPs and effectors. SAINT express analyses were performed on the following sample sizes (number of interactions): n = 5381 for NF in HEK, n = 7462 for NF in HeLa, n = 17548 for Active in HEK and n = 22042 for Active in HeLa. Only proximity interactions displaying an AvgP ≥ 0.95 (below the Bayesian 1% FDR estimate) were kept and deemed of high confidence. (a) Enrichment of BioID interactions of well-known downstream effectors and complexes with the indicated RHO GTPases. (b) Enrichment of BioID interactions of RHOGAPs with the indicated RHO GTPases.

Extended Data Fig. 4 Basic characterization of GARRE (KIAA0355).

(a) The Phylogenetic tree of GARRE orthologues shows its recent evolutionary origin. (b) PHYRE2 threading software identifies a BAR domain in GARRE similar to the BAR domain of Amphiphysin. Conserved amino acids are in red. (c) Schematic of GARRE displaying the location of the DUF4745 superfamily domain as revealed by amino acid Blast alignment of the GARRE sequence. Note that the DUF domain overlaps with the position of the BAR domain (in yellow). (d) GARRE has no GAP activity on RAC1. Active RAC1 was pulled-down using purified GST-PAK-PDB in cell lysates from HeLa cells expressing Flag-DOCK180 and/or Flag-GARRE. Data are representative of four independent experiments. See unmodified scans in Unprocessed Blots Extended Data Fig. 4.

Source data

Extended Data Fig. 5 A functional siRNA screen for the top RHOGG12V BioID interactors.

A functional siRNA screen for the top RHOGG12V BioID interactors reveals PLEKHG3 as an effector (a) Schematic illustration of the functional siRNA screening approach for RHOG. Flp-In T-REx HeLa cells expressing Flag-RHOGG12V in a tetracycline-inducible manner were transfected with a set of ON-target SmartPool siRNAs targeting the top 22 RHOGG12V BioID effectors prior to tetracycline induction of RHOGG12V and were next subjected to anti-Flag and F-actin staining. Samples were analyzed using low-resolution high throughput microscopy and percentage of cells presenting RHOGG12V and F-actin-enriched membrane ruffles was quantified. (b) High-resolution confocal microscopy images show that RHOGWT induces multiple discrete ruffles while RHOGG12V causes the formation of a uniform membrane ruffle, which is inhibited by RAC1 siRNA. Data are representative of three independent experiments. Bar, 10 µm. (c, d) Computational analyses of the cell shape reveal that the increase in circularity induced by RHOGG12V expression is inhibited by RAC1, ELMO2 or PLEKHG3 siRNA. A circularity value of 1.0 indicates a perfect circle. As the value approaches 0.0, it indicates an increasingly elongated polygon. (c) The graph shows the frequency distribution of circularity for control cells (expressing GFP or GFP−RHOGWT) in grey versus GFP-RHOGG12V expressing cells in black. (n = 377 control cells or 220 RHOGG12V-expressing cells from 3 independent experiments). (d) The graph compares the frequency distribution of circularity of GFP-RHOGG12V-expressing cells 72 h after treatment with the indicated siRNAs. (n = 287 cells for siControl, 363 cells for siRAC1, 382 for siELMO2 and 325 for siPLEHG3 from 3 independent experiments). See Statistical Source Data_Extended Data Fig. 5.

Source data

Extended Data Fig. 6 Activation of ROCK1/2 downstream of RHOA contributes to ERM proteins phosphorylation.

(a) Western blot showing SLK, LOK and ROCK1 depletion after 72 h treatment with indicated siRNAs. Tubulin was used as a loading control. Data are representative of 2 independent experiments. (b) Confocal images of p-ERM staining in Flag-RHOA Flp-In T-REx HeLa cells before (control) or after induction with tetracycline for 16 h. Note that the treatment of RHOAG14V-expressing cells with the ROCK inhibitor Y-27632 (10 µM for 30 min) was sufficient to reduce p-ERM. Data are representative of three independent experiments. Bar = 10 µm. (c) Cells were treated as in b and p-ERM mean fluorescence intensity (MFI) was measured. The graph shows the mean + /- SD of the relative p-ERM MFI as compared to the control (Flag-RHOA Flp-In T-REx HeLa cells without tetracycline induction). n = 22 images of control, n = 19 images of RHOAWT, n = 22 images of RHOAG14V and n = 25 images of RHOAG14V + Y27632 from two independent experiments (See Statistical Source Data Extended Data Fig. 5). P-value was calculated using the Mann-Whitney non-parametric two-tailed test ****, p < 0.0001.

Source data

Supplementary information

Reporting Summary

Supplementary Video 1

Lamellipodia formation following local PA-RAC1 photoactivation in TagRFP-expressing cells. Hela cells were transfected with PA-RAC1 together with TagRFP. The red arrow points to the membrane protrusion induced by PA-RAC1 activation by repeated local illumination with blue light (cyan circle). This video is associated with Fig. 4i (upper panel). Data are representative of two independent experiments.

Supplementary Video 2

Recruitment of GARRE-TagRFP following the local photoactivation of PA-RAC1. Hela cells were transfected with PA-RAC1 together with GARRE-TagRFP. The yellow arrow points to the recruitment of GARRE-TagRFP into structures reminiscent of tubular membranes after PA-RAC1 activation by repeated local illumination with blue light (cyan circle). This video is associated with Fig. 4i (lower panel). Data are representative of two independent experiments.

Supplementary Tables

Supplementary Tables 1–16.

Supplementary Data 1

Cytoscape file (Cytoscape.org) containing the networks of every RHO GTPases in active or nucleotide free (NF) forms.

Source data

Source Data Fig. 2

Unprocessed Western Blots and/or gels

Source Data Fig. 3

Unprocessed Western Blots and/or gels

Source Data Fig. 4

Statistical Source Data

Source Data Fig. 4

Unprocessed Western Blots and/or gels

Source Data Fig. 5

Statistical Source Data

Source Data Fig. 5

Unprocessed Western Blots and/or gels

Source Data Fig. 6

Statistical Source Data

Source Data Fig. 6

Unprocessed Western Blots and/or gels

Source Data Fig. 7

Statistical Source Data

Source Data Fig. 7

Unprocessed Western Blots and/or gels

Source Data Fig. 8

Statistical Source Data

Source Data Fig. 8

Unprocessed Western Blots and/or gels

Source Data Extended Data Fig. 1

Unprocessed Western Blots and/or gels

Source Data Extended Data Fig. 4

Unprocessed Western Blots and/or gels

Source Data Extended Data Fig. 5

Statistical Source Data

Source Data Extended Data Fig. 6

Statistical Source Data

Source Data Extended Data Fig. 6

Unprocessed Western Blots and/or gels

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bagci, H., Sriskandarajah, N., Robert, A. et al. Mapping the proximity interaction network of the Rho-family GTPases reveals signalling pathways and regulatory mechanisms. Nat Cell Biol 22, 120–134 (2020). https://doi.org/10.1038/s41556-019-0438-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-019-0438-7

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing