GRB2 Links Signaling to Actin Assembly by Enhancing Interaction of Neural Wiskott-Aldrich Syndrome Protein (N-WASp) with Actin-related Protein (ARP2/3) Complex

doi:10.1074/jbc.M000687200

GRB2 Links Signaling to Actin Assembly by Enhancing Interaction of Neural Wiskott-Aldrich Syndrome Protein (N-WASp) with Actin-related Protein (ARP2/3) Complex*

From the^‡Dynamique du Cytosquelette,^¶Cristallographie et RMN Biologiques, Laboratoire d'Enzymologie et Biochimie Structurale, CNRS 91198 Gif-sur-Yvette,^**Pathogénicité Microbienne Moléculaire, Institut Pasteur, 28 Rue du Dr Roux, 75 724 Paris and ^‡INSERM U 266/UMR CNRS 8600, Facultéde Pharmacie, 4 Avenue de l'Observatoire, 75248 Paris, France

Abstract

Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5′-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. Here we show that the SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp that stimulates actin polymerization by increasing the amount of N-WASp·Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to its active monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway.

Footnotes

↵* This work was supported in part by the Association pour la Recherche Contre le Cancer, the Association Française Contre les Myopathies, and by Human Frontiers in Science Grant RG 227/98.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ To whom correspondence should be addressed. Tel.: 33 01 69 82 34 65; Fax: 33 01 69 82 31 29; E-mail: carlier@lebs.cnrs-gif.fr.
↵‖ Supported by the Ligue Nationale Française contre le Cancer.
Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M000687200
Abbreviations:

WASp

Wiskott-Aldrich Syndrome protein

N-WASp

neural Wiskott-Aldrich Syndrome protein

PH

pleckstrin homology

WH1

WASp homology 1

PIP₂

phosphatidylinositol bisphosphate

EGF

epidermal growth factor

EDC

1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide

NHS

N-hydroxysuccinimide

GTPγS

guanosine 5′-3-O-(thio)triphosphate

PAGE

polyacrylamide gel electrophoresis

WA

WH₂-Acidic

Aha

aminohexanoic acid
- Received January 24, 2000.
- Revision received March 30, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.