The small GTPase RalA targets filamin to induce filopodia.
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Journal: 1999/April - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 10051605
Abstract:
The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA's effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.
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Proc Natl Acad Sci U S A 96(5): 2122-2128
PMID: 10051605

The small GTPase RalA targets filamin to induce filopodia

Division of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187, Japan; and Division of Hematology, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, 221 Longwood Avenue, Boston, MA 02115
To whom reprint requests should be addressed. e-mail: pj.og.pncn.pxancn@atho.
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 1997.
Contributed by Thomas P. Stossel
This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 29, 1997.
Contributed by Thomas P. Stossel
Accepted 1998 Dec 8.
Copyright © 1999, The National Academy of Sciences

Abstract

The Ras-related small GTPases Rac, Rho, Cdc42, and RalA bind filamin, an actin filament-crosslinking protein that also links membrane and other intracellular proteins to actin. Of these GTPases only RalA binds filamin in a GTP-specific manner, and GTP-RalA elicits actin-rich filopods on surfaces of Swiss 3T3 cells and recruits filamin into the filopodial cytoskeleton. Either a dominant negative RalA construct or the RalA-binding domain of filamin 1 specifically block Cdc42-induced filopod formation, but a Cdc42 inhibitor does not impair RalA’s effects, which, unlike Cdc42, are Rac independent. RalA does not generate filopodia in filamin-deficient human melanoma cells, whereas transfection of filamin 1 restores the functional response. RalA therefore is a downstream intermediate in Cdc42-mediated filopod production and uses filamin in this pathway.

Abstract

Activation of the Rho family of Ras-related small GTPases causes the construction of particular actin-rich surface structures in cells. For example, forced expression of constitutively active forms of RhoA, Rac1, and Cdc42 in serum-starved Swiss 3T3 cells induces actin stress fibers, membrane ruffles, and filopodia, respectively (1). These GTPases do not appear to affect actin dynamics directly, but rather engage downstream intermediates. Such targets include the Rho-activated phosphorylation of myosin, which leads to cellular contractile activity (2), and the phosphorylation of proteins of the ezrin-moesin-radixin group to promote their ability to link actin filaments to cell membranes (3). Rac1-induced polyphosphoinositide synthesis causes cellular actin assembly by uncapping actin filament ends (4), and the actin filament severing and barbed end capping protein gelsolin participates in this process (5). N-WASP, a profilin-binding protein, is an intermediate in filopodial extension, possibly by acquiring actin filament depolymerizing activity when ligated by Cdc42 (6, 7).

We report two related connections in the pathway between Cdc42 and filopodial formation. One part of this connection involves the Ras-related small GTPase RalA (8). Expression of dominant negative RalA delays border cell migration in Drosophila oogenesis (9), and transfection of the guanine nucleotide releasing stimulator of RalA affects cell morphology (10), suggesting a linkage between RalA and the actin cytoskeleton. The second part of this connection is the actin filament crosslinking protein filamin. Three distinct filamin cDNAs have been reported, which originate from three different chromosomes, with additional variants ascribable to alternative mRNA splicing (11–14). Of these protein isoforms, filamin 1 (also called ABP-280) is the founding and most widely distributed (11, 15). Filamin efficiently crosslinks actin filaments and is a docking site for various cell surface receptors and certain intracellular proteins involved in signal transduction or endocytosis (16–22). Genetic evidence shows that filamin 1 in particular is an essential component for stabilization of the cell surface and for efficient translocational motility of melanocytic, neuronal, and other cells (23, 24). Although phosphorylation of filamin occurs in vitro and in cells and may affect its actin-binding properties (25), little information is available about filamin’s cellular regulation. Filamin was found previously to associate with unidentified GTP-binding proteins in human platelets (26, 27). We report here that filamin binds Rac, Rho, and Cdc42. We present evidence for the functional importance of the RalA-filamin interaction in vivo.

The percentages of filopod- or lamellipod-positive cells were calculated and data are expressed as the mean ± SE. The GST-filamin 1 fragment did not affect the actions of 0.8 mg/ml Rac or 0.8 mg/ml RhoA, which produced respectively lamellipodia or stress fibers in 60–70% of injected cells.

Experimental conditions are described and the data are expressed as in Table ​Table1.1. The concentration of microinjected RhoA and Racl are RhoA (1.0 mg/ml) and Racl (1.8 mg/ml), respectively. F, filopodia; L, lamellipodia, SF, stress fibers; N.D., not determined.

Acknowledgments

We thank Seisuke Hattori for helpful discussions, Yuko Niino for subcloning cDNAs, Akemi Takayama for technical assistance, Jim McGrath for video microscopy, and Ikuroh Ohsawa for help with confocal microscopy. N.S. is on leave from the Third Department of Medicine, Nippon Medical School, Tokyo 113 and thanks Ichiji Wakabayashi for support. Y.O. is supported by the Science and Technology Agency of Japan. J.H.H. is supported by National Institutes of Health Grants HL54145, HL56252, HL56949, and HL56993. T.P.S. is supported by National Institutes of Health Grant HL19429 and a Clinical Research Professorship of the American Cancer Society.

Acknowledgments

ABBREVIATIONS

GSTglutathione S-transferase
GTPγSguanosine 5′-γ-thiotriphosphate
FITCfluorescein isothiocyanate
ABBREVIATIONS

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