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

Nat Cell Biol. 2020 Jan;22(1):120-134. doi: 10.1038/s41556-019-0438-7.

Halil Bagci ¹ ², Neera Sriskandarajah ¹ ³, Amélie Robert ¹, Jonathan Boulais ¹, Islam E Elkholi ¹ ⁴, Viviane Tran ¹ ⁵, Zhen-Yuan Lin ⁶, Marie-Pier Thibault ¹, Nadia Dubé ¹, Denis Faubert ¹, David R Hipfner ^# ¹ ³ ⁴ ⁷, Anne-Claude Gingras ^# ⁶ ⁸, Jean-François Côté ⁹ ¹⁰ ¹¹ ¹²

Affiliations

1 Montreal Clinical Research Institute (IRCM), Montréal, Québec, Canada.
2 Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada.
3 Division of Experimental Medicine, Department of Medicine, McGill University, Montréal, Québec, Canada.
4 Molecular Biology Programs, Université de Montréal, Montréal, Québec, Canada.
5 Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Québec, Canada.
6 Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Ontario, Canada.
7 Department of Medicine, Université de Montréal, Montréal, Québec, Canada.
8 Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
9 Montreal Clinical Research Institute (IRCM), Montréal, Québec, Canada. jean-francois.cote@ircm.qc.ca.
10 Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada. jean-francois.cote@ircm.qc.ca.
11 Molecular Biology Programs, Université de Montréal, Montréal, Québec, Canada. jean-francois.cote@ircm.qc.ca.
12 Department of Medicine, Université de Montréal, Montréal, Québec, Canada. jean-francois.cote@ircm.qc.ca.
# Contributed equally.

PMID: 31871319 DOI: 10.1038/s41556-019-0438-7

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.

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