IQGAP1 Stimulates Actin Assembly through the N-Wasp-Arp2/3 Pathway

Alexey Veligodskiy,Dominik Schlaepfer,Christophe Le Clainche,Diep Le,Mirko Klingauf,Katarina Grohmanova,Coumaran Egile,Aldo Ferrari,Marie-France Carlier,Ruth Kroschewski,Dominique Didry

doi:10.1074/jbc.m607711200

Abstract

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.

Highlights

Directional cell migration results from the coordination of protrusion formation and cell adhesion
Extension of lamellipodia is driven by stimulus-responsive WASP family proteins (N-WASP, WASP, and Scar/WAVE) Cortactin and CARMIL, which activate the Arp2/3 complex to catalyze the formation of a branched actin filament array at the leading edge (28 –34)
Lamellipodium Extension Is Promoted by N-WASP upon Activation by IQGAP1—Extension of lamellipodia and filopodia is mediated by the maintenance of dynamic meshworks of actin filaments

Summary

Introduction

Directional cell migration results from the coordination of protrusion formation and cell adhesion. IQGAP1 Activates N-WASP-mediated Actin Polymerization between the CRIB domain and the VCA domain. 16 h later cells were stimulated with minimal essential medium containing 10% fetal calf serum for 10 min, fixed at 37 °C, and stained with primary antibodies against Arp3, IQGAP1, and N-WASP followed by secondary antibodies conjugated with Coumarin Phalloidin, Alexa488, Alexa594, or Alexa647 (Molecular Probes, Invitrogen).

Results

Conclusion