Characterization of a Novel GTPase-activating Protein Associated with Focal Adhesions and the Actin Cytoskeleton

Benjamin Geiger,Irena Lavelin

doi:10.1074/jbc.m411990200

Abstract

In the present study we characterize a novel RhoGAP protein (RC-GAP72) that interacts with actin stress fibers, focal adhesions, and cell-cell adherens junctions via its 185-amino acid C-terminal region. Overexpression of RC-GAP72 in fibroblasts induces cell rounding with partial or complete disruption of actin stress fibers and formation of membrane ruffles, lamellipodia, and filopodia. RC-GAP72 mutant truncated downstream of the GTPase-activating protein (GAP) domain retains the ability to stimulate membrane protrusions but fails to affect stress fiber integrity or induce cell retraction. A mutant protein consisting of the C terminus of RC-GAP72 and lacking the GAP domain does not exert any visible effect on cellular morphology. Inactivation of the GAP domain by a point mutation does not abolish the effect of RC-GAP72 on actin stress fibers but moderates its capability to induce membrane protrusions. Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GAP domain irrespective of binding to the actin cytoskeleton. We propose that RC-GAP72 affects cellular morphology by targeting activated Cdc42 and Rac1 GTPases to specific subcellular sites, triggering local morphological changes. The overall physiological functions of RC-GAP72 are presently unknown, yet our data suggest that RC-GAP72 plays a role in regulating cell morphology and cytoskeletal organization.

Highlights

Rho-family GTPases are regulators of diverse biological activities including the organization of the actin cytoskeleton
Our data imply that the cytoskeletal localization of RC-GAP72 and its interaction with GTPases are essential for its effect on the integrity of actin stress fibers, whereas the induction of lamellipodia and filopodia depends on the activity of the GTPase-activating protein (GAP) domain irrespective of binding to the actin cytoskeleton
In this paper we describe a novel, uncharacterized member of the RhoGAP family that was isolated in a microscopy-based screen due to its association with focal contacts and stress fibers

Summary

Introduction

Rho-family GTPases are regulators of diverse biological activities including the organization of the actin cytoskeleton. Activation of RhoA in fibroblasts induces the formation of stress fibers and focal adhesion, whereas active Rac and Cdc induce the extension of lamellipodia and filopodia, respectively [1,2,3,4] Each of these proteins exerts its particular cytoskeletal effect via a variety of target proteins that affect actin polymerization or actomyosin contractility. A conserved arginine residue present in most RasGAP and RhoGAP proteins functions to stabilize the conformation needed for GTPase acceleration, and its mutation results in loss of GAP activity (8 –12) Outside their GAP domain RhoGAPs exhibit high sequence diversity and have various signaling motifs that can target them to specific subcellular sites, including SH2, SH3, pleckstrin homology or phorbol ester binding domains [13,14,15,16,17,18]. Various RhoGAP proteins can display diverse tissue-specific expression patterns, some ubiquitous and others displaying narrow tissue specificity [22, 25, 34, 35], contributing to the differential regulation of Rho GTPase activity in different cell types

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