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Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non‐redundant Functions in Vivo

46 Pages Posted: 7 Jun 2018 Sneak Peek Status: Review Complete

See all articles by Matthias Schaks

Matthias Schaks

Helmholtz Centre for Infection Research - Department of Cell Biology

Shashi Prakash Singh

Beatson Institute for Cancer Research

Frieda Kage

Helmholtz Centre for Infection Research - Department of Cell Biology

Peter Thomason

Beatson Institute for Cancer Research

Thomas Klünemann

Helmholtz Centre for Infection Research - Department of Structure and Function of Proteins

Anika Steffen

Helmholtz Centre for Infection Research - Department of Cell Biology

Wulf Blankenfeldt

Helmholtz Centre for Infection Research - Department of Structure and Function of Proteins

Theresia E. Stradal

Helmholtz Centre for Infection Research - Department of Cell Biology

Robert Insall

Beatson Institute for Cancer Research

Klemens Rottner

Helmholtz Centre for Infection Research - Department of Cell Biology; Technology University of Braunschweig - Division of Molecular Cell Biology; Helmholtz Centre for Infection Research - Molecular Cell Biology Group

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Abstract

Cell migration often involves the formation of sheet‐like lamellipodia, generated by actin filament branching through Arp2/3 complex. In these structures, the latter is activated by WAVE regulatory complex (WRC) downstream of small GTPases of the Rac family. Recent structural studies defined two independent Rac1 binding sites on WRC within the Sra‐1/PIR121 subunit of the pentameric WRC, but the functions of these sites in vivo have remained unknown. Here we used CRISPR/Cas9‐mediated gene disruption of Sra‐1 and its paralogue PIR121 in murine B16‐F1 cells combined with Sra‐1 mutant rescue to dissect the mechanism of WRC activation and the in vivo relevance of distinct Rac binding sites on Sra‐1. We found that the A site, positioned adjacent to the binding region of WAVE‐WCA mediating actin‐ and Arp2/3 complex binding, is the main site for allosteric activation of WRC. In contrast, the D site towards the C‐terminus is dispensable for WRC activation but required for optimal lamellipodium morphology and function. These results were confirmed in evolutionarily distant Dictyostelium amoebas. Moreover, the phenotype seen in D site mutants was recapitulated in Rac1 E31 and F37 mutants , we conclude these residues are important for Rac/D site interaction. Finally, lamellipodia formation was observable even after disruption of both Rac interaction sites in constitutively active WRC, showing Rac interaction is not essential for membrane recruitment. Our data establish that physical interaction with Rac is required for WRC activation, in particular through the A site, but not mandatory for WRC accumulation in the lamellipodium.

Suggested Citation

Schaks, Matthias and Singh, Shashi Prakash and Kage, Frieda and Thomason, Peter and Klünemann, Thomas and Steffen, Anika and Blankenfeldt, Wulf and Stradal, Theresia E. and Insall, Robert and Rottner, Klemens, Distinct Interaction Sites of Rac GTPase with WAVE Regulatory Complex Have Non‐redundant Functions in Vivo (2018). Available at SSRN: https://ssrn.com/abstract=3188452 or http://dx.doi.org/10.2139/ssrn.3188452
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Matthias Schaks

Helmholtz Centre for Infection Research - Department of Cell Biology

Inhoffenstraße 7
Braunschweig, 38124
Germany

Shashi Prakash Singh

Beatson Institute for Cancer Research

Switchback Rd
Bearsden, Glasgow G61 1BD
United Kingdom

Frieda Kage

Helmholtz Centre for Infection Research - Department of Cell Biology

Inhoffenstraße 7
Braunschweig, 38124
Germany

Peter Thomason

Beatson Institute for Cancer Research

Switchback Rd
Bearsden, Glasgow G61 1BD
United Kingdom

Thomas Klünemann

Helmholtz Centre for Infection Research - Department of Structure and Function of Proteins

Inhoffenstraße 7
Braunschweig, 38124
Germany

Anika Steffen

Helmholtz Centre for Infection Research - Department of Cell Biology

Inhoffenstraße 7
Braunschweig, 38124
Germany

Wulf Blankenfeldt

Helmholtz Centre for Infection Research - Department of Structure and Function of Proteins

Inhoffenstraße 7
Braunschweig, 38124
Germany

Theresia E. Stradal

Helmholtz Centre for Infection Research - Department of Cell Biology

Inhoffenstraße 7
Braunschweig, 38124
Germany

Robert Insall

Beatson Institute for Cancer Research ( email )

Switchback Rd
Bearsden, Glasgow G61 1BD
United Kingdom

Klemens Rottner (Contact Author)

Helmholtz Centre for Infection Research - Department of Cell Biology ( email )

Inhoffenstraße 7
Braunschweig, 38124
Germany

Technology University of Braunschweig - Division of Molecular Cell Biology ( email )

Abt-Jerusalem-Str. 7
Braunschweig, D-38106
Germany

Helmholtz Centre for Infection Research - Molecular Cell Biology Group ( email )

Inhoffenstraße 7
Braunschweig, 38124
Germany

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