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FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling

Abstract

FilGAP is a newly recognized filamin A (FLNa)-binding RhoGTPase-activating protein. The GTPase-activating protein (GAP) activity of FilGAP is specific for Rac and FLNa binding targets FilGAP to sites of membrane protrusion, where it antagonizes Rac in vivo. Dominant-negative FilGAP constructs lacking GAP activity or knockdown of endogenous FilGAP by small interference RNA (siRNA) induce spontaneous lamellae formation and stimulate cell spreading on fibronectin. Knockdown of endogenous FilGAP abrogates ROCK-dependent suppression of lamellae. Conversely, forced expression of FilGAP induces numerous blebs around the cell periphery and a ROCK-specific inhibitor suppresses bleb formation. ROCK phosphorylates FilGAP, and this phosphorylation stimulates its RacGAP activity and is a requirement for FilGAP-mediated bleb formation. FilGAP is, therefore, a mediator of the well-established antagonism of Rac by RhoA that suppresses leading edge protrusion and promotes cell retraction to achieve cellular polarity.

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Figure 1: Sequence of FilGAP and its interaction with FLNa.
Figure 2: GTPase-stimulating activity of FilGAP and its regulation by FLNa.
Figure 3: Localization of FLNa and FilGAP.
Figure 4: FilGAP suppresses lamellae formation.
Figure 5: FilGAP suppresses cell spreading on fibronectin.
Figure 6: Requirement of FilGAP activity for ROCK-dependent suppression of lamellae.
Figure 7: ROCK regulates FilGAP.
Figure 8: Proposed roles of FilGAP and other FLNa-binding partners in determining cell polarity.

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Acknowledgements

We thank: S. Hattori (University of Tokyo, Japan) for helpful advice and assistance with the GAP assays and two-hybrid screening; Y. Imai (National Institute of Neuroscience, Japan) for a human spleen cDNA library and GST–PAK–CRIB construct; F. Nakamura for purified Sf9 FLNa and FilGAP proteins; R. Vadlamudi and R. Kumar (MD Anderson Cancer Center, Houston, TX) for the FLNaD23 construct; S Narumiya (Kyoto University, Japan) for ROCK constructs; and M. Daya and M. Tukey for technical assistance. Supported by U.S. Public Health Service grant HL19429.

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Correspondence to Yasutaka Ohta.

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Supplementary Figures S1, S2, S3, S4, S5,Supplementary Tables S1 and S2 (PDF 404 kb)

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Ohta, Y., Hartwig, J. & Stossel, T. FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling. Nat Cell Biol 8, 803–814 (2006). https://doi.org/10.1038/ncb1437

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