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


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.

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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.

Data availability

The raw proteomics data have been deposited into ProteomeXchange ( with accession number PXD015918. The BioID data in this manuscript can also be explored in the supplementary tables and on a dedicated website ( Source data for Figs. 28 and Extended Data Figs. 1 and 46 are available online. All data that support the findings of this study are available from the corresponding author upon reasonable request.

Change history

  • 10 February 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.


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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

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.

Correspondence to Jean-François Côté.

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

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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).

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