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J. Biol. Chem., Vol. 275, Issue 29, 21946-21952, July 21, 2000

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

Marie-France Carlier^§, Pierre Nioche^¶, Isabelle Broutin-L'Hermite^¶, Rajaa Boujemaa, Christophe Le Clainche, Coumaran Egile^**, Christiane Garbay, Arnaud Ducruix^¶, Philippe Sansonetti^**, and Dominique Pantaloni

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, Faculté de Pharmacie, 4 Avenue de l'Observatoire, 75248 Paris, France

Received for publication, January 24, 2000, and in revised form, March 30, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Actin polymerization is the primary process that elicits the motile response of living cells to extracellular stimuli such as hormones or growth factors. Recent works have shown that the Arp2/3 complex, which contains 7 polypeptides including the actin-related proteins Arp2 and Arp3, is the universal downstream cytoskeletal target of a variety of signaling cascades (see Refs. 1 and 2 for reviews). The Arp2/3 complex stimulates the formation of new actin filaments that actively grow from their barbed ends at specified locations beneath the plasma membrane. Barbed end growth develops the protrusive force responsible for lamellipodia extension and ruffling. The recent reconstitution of actin-based motility from pure proteins (3) has demonstrated that in addition to the site-directed nucleating activity of activated Arp2/3, the maintenance of a highly dynamic steady state of actin assembly in the medium by actin depolymerizing factor/cofilin and capping proteins was crucial in eliciting actin-based movement.

The actin nucleating activity of Arp2/3 is revealed upon interaction with proteins of the WASp¹/Scar family (4-9, see Ref. 10 for review). These multidomain proteins are thought to connect the signaling pathways to Arp2/3 and actin and are involved in the formation of filopodia (11), lamellipodia (12), and podosomes (13). However, the nature of the connections is not completely understood. All proteins of the WASp/Scar family (14-17) contain an N-terminal pleckstrin homology/WASp homology 1 (PH/WH1) domain, a proline-rich region, and a C-terminal domain WH₂-Acidic (WA) which consists of a verprolin/WASp homology 2 (WH2) domain, a short so-called cofilin homology sequence, and an acidic extreme C-terminal region. The PH/WH1 domain contains binding sites for WASp-interacting protein (18), a homolog of the actin-binding protein End5p/verprolin in yeast (19, 20), and for F-actin (9) and may also be responsible for membrane attachment and PIP₂ binding (6). The proline-rich region has been shown to bind receptor tyrosine kinases Fgr and Fyn and the SH2/SH3 domain-containing adaptors Grb2 and Nck, via their SH3 domains (21-25). The WA domain of all members of the WASp family is responsible for the stimulation of actin nucleation by Arp2/3 complex and does so by interacting both with Arp2/3 complex via the A region and with G-actin via the WH2 region (4, 26). The interaction with G-actin via the WH2 region confers a profilin-like function to the WA domain, shuttling actin subunits to the growing barbed ends (9). In addition, the WASp and N-WASp members of the WASp/Scar family contain a CRIB domain that binds Cdc42 in the GTP-bound form (27-30). The isolated full-length N-WASp molecule activates Arp2/3 to a lesser extent than the WA domain; however, upon binding Cdc42 or PIP₂, N-WASp activates Arp2/3 in an enhanced fashion (6), leading to a dramatic enhancement of actin polymerization in vitro. This result suggested that the WA domain may be buried in the N-WASp protein due to some internal interaction, and N-WASp would switch to an active state, in which the WA domain is exposed, upon binding Cdc42 or PIP₂. Very recent NMR studies (31) demonstrate that the so-called "cofilin-homology region" in the WA domain of WASp interacts with the CRIB domain, in an autoinhibited complex. Activation of N-WASp/Arp2/3 by the Shigella flexneri bacterial pathogen has also been described (9). S. flexneri undergoes actin-based propulsion in host cells and is one of the best tools for a molecular approach of actin-based motility. Initiation of actin assembly at the surface of Shigella is mediated by a single protein, IcsA (32), that harnesses N-WASp (33). IcsA mimics Cdc42 in inducing a structural change in N-WASp, exposing WA and stimulating Arp2/3-induced actin assembly and bacterial motility (9). The structural basis for activation of N-WASp by IcsA is not yet known.

The above results suggest that the function of WASp proteins in a physiological context is to link Cdc42 activation to de novo actin assembly. However, the activation of N-WASp may be driven by other ligands binding to the proline-rich region of N-WASp via SH3 domains, such as the adaptors Grb2 and Nck (22, 23, 34, 35). The SH2/SH3 adaptor Grb2 (36, 37) is conventionally considered to link the tyrosine-phosphorylated EGF receptor (via its SH2 domain) and the Ras guanine nucleotide exchange factor Sos (via its SH3 domains), without affecting the nucleotide exchange function (38-42). On the other hand, several studies suggest that Grb2 binds to a large number of other targets (see Ref. 43 for a recent review), including proteins involved in the organization of the actin cytoskeleton. Therefore, Grb2 could act not only as an adaptor but also as an effector of actin-dependent processes in response to signaling. Microinjection of anti-Grb2 antibodies abolishes actin-based membrane ruffling in response to EGF (44), binding of Grb2 to dynamin (45) activates its GTPase activity (46), and inhibitors of the SH2 domain of Grb2 block cell motility in hepatocyte growth factor-stimulated Madin-Darby canine kidney cells (47). The role of these interactions in the regulation of cytoskeletal reorganization has not been clearly established; however, addition of Grb2 to cell lysates increases the amount of N-WASp that coimmunoprecipitates with the activated EGF receptor (23). Here we use in vitro actin polymerization assays with pure Arp2/3 and N-WASp and motility assays of Shigella in a medium reconstituted from pure proteins (3) to show that Grb2 directly activates N-WASp and mediates Arp2/3 complex-dependent actin nucleation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Proteins-- Actin was purified from rabbit muscle and was isolated as monomeric CaATP-G-actin by gel filtration in G buffer (5 mM Tris-Cl, pH 7.8, 1 mM dithiothreitol, 0.2 mM ATP, 0.1 mM CaCl₂, 0.01% NaN₃) and pyrenyl-labeled as described (48). Arp2/3 complex was purified from bovine brain (9). Recombinant histidine-tagged human N-WASp was expressed in Sf21 cells using the baculovirus system (9). Profilin was purified from bovine spleen (48). Recombinant Grb2 wild type was bacterially expressed in Escherichia coli and purified (49). The P49L, P206L, and G203R mutated forms of Grb2 were purified using a protocol derived from Guilloteau et al. (49). Isolated N-terminal and C-terminal SH3 domains of Grb2 were obtained by solid phase peptide synthesis as described (50, 51). The Sos-derived peptide dimer (VPPPVPPRRR-Aha-K-Aha-RRRPPVPPPV) was synthesized as described (52). The SH2 domain binding phosphotyrosine m-arylphospho-Tyr--methylphospho-Tyr-Asn-NH₂ tripeptide (pYpYN) was prepared as described (53). Recombinant Cdc42 was expressed, purified, and loaded with GTPS as described (9).

Polymerization Assays-- Polymerization of actin was monitored by the increase in fluorescence of pyrenyl-labeled actin. The assays were carried out at 20 °C in a Spex fluorolog2 or a Safas flx spectrofluorimeter. Samples (80 µl) contained typically 2.5 µM MgATP-G-actin (10% pyrenyl-labeled), 10-20 nM Arp2/3 complex, and N-WASp, Grb2, and other reagents as indicated in F buffer (G buffer supplemented with 0.1 M KCl and 1 mM MgCl₂). The maximum slope of the polymerization curves was taken as a measure of the activation of Arp2/3 complex.

Modeling of the Kinetic Data in Terms of N-WASp Monomer-Dimer Equilibrium-- N-WASp (W) was assumed to bind Arp2/3 (Y) and Grb2 (X) and to associate into a dimer W₂ with an equilibrium dissociation constant K_D. The different equilibria to be considered (Fig. 6) were described by the following Equations 1-6.

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

(Eq. 6)

The modeling of data shown in Fig. 2a was carried out calculating the concentrations of WX, WY, WXY, W₂, and W₂Y₂ and all other complexes as a function of incremented W, using the above equations, which express that ligands (X and Y) bind independently to N-WASp. The total concentration of W was the sum of the concentrations of all the W containing species. It was considered that [X]_total = [X], i.e. Grb2 is in large excess over all complexes of N-WASp and Arp2/3. The value of Y was adjusted by iteration until the total concentration of Y was exactly equal to the concentration of Arp2/3 in the experiment. The value of [WY] + [WXY], which is the concentration of activated N-WASp-Arp2/3 complex, carrying or not Grb2, was taken as representing the activity measured in the polymerization assays.

Motility Assays of E. coli (IcsA)-- The effect of Grb2 on actin-based movement of E. coli (IcsA), a good substitute for Shigella (54), was tested by examining the motile behavior of bacteria in the motility medium reconstituted from pure proteins (3). Bacteria (6×10⁹ bacteria/ml) were precoated with N-WASp by preincubation in X buffer (20 mM HEPES, pH 7.5, 0.1 M KCl, 1 mM MgCl₂, 0.2 mM CaCl₂, 50 mM sucrose) containing 0.2 µM N-WASp and Grb2 as indicated, at 20 °C for 10 min. Bacteria were then sedimented at 13,000 × g for 4 min, resuspended in the original volume of X buffer, and diluted 30-fold in the motility medium. The motility medium consisted of 8 µM F-actin, 5 µM actin depolymerizing factor, 50 nM Capping protein, 2.5 µM profilin, and 0.1 µM Arp2/3 complex and was supplemented or not with Grb2 at the same concentration as in the preincubation step. Motility was observed in phase contrast optical microscopy as described (3). The time course of generation of actin tails and establishment of the steady rate of propulsion was monitored for each sample, and the average rates were measured over at least 10 bacteria.

EDC/NHS Cross-linking of N-WASp-- N-WASp was equilibrated in 20 mM HEPES, 60 mM KCl buffer, pH 7.5, and incubated at 1 µM with 5 mM EDC and 5 mM NHS for 45 min at 0 °C. Samples were immediately denatured and submitted to SDS-PAGE (9% acrylamide), electrotransferred onto nitrocellulose, and Western blotted using a polyclonal anti-N-WASp at a 1:500 dilution (9) as primary antibody and peroxidase-coupled anti-rabbit IgG as secondary antibody, followed by ECL chemiluminescent detection (Amersham Pharmacia Biotech).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Grb2 Enhances the Interaction of N-WASp with Arp2/3, Leading to the Stimulation of Actin Polymerization-- The binding of Cdc42-GTPS or of the Shigella protein IcsA to N-WASp leads to the activation of actin polymerization by Arp2/3. The molecular mechanism by which activated Arp2/3 complex induces the seemingly autocatalytic polymerization of actin is not fully understood. The current interpretation of the activation of Arp2/3 by N-WASp is that when N-WASp binds effectors like Cdc42 or IcsA, the C-terminal WA domain of N-WASp is exposed and is able to interact with Arp2/3 and G-actin to stimulate actin filament nucleation and barbed end growth (6, 9). The pyrene fluorescence assay for actin polymerization provides a rapid, accurate, and quantitative test of the function of putative effectors leading to the structural change and activation of N-WASp (or of antagonists of this reaction).

The effect of Grb2, an SH3-SH2-SH3 multidomain adaptor, on the function of N-WASp was addressed. Grb2 stimulated actin polymerization in the presence of Arp2/3 complex and N-WASp (Fig. 1a). The effect of Grb2 was maximum at a concentration of 1 µM, and half-effect was observed at 0.1-0.2 µM (data not shown). Under conditions of maximum activation, in the presence of 1 µM Grb2, the N-WASp concentration dependence of the rate of actin polymerization was consistent with the binding of Grb2·N-WASp complex to Arp2/3 with an equilibrium dissociation constant of 5 nM (Fig. 1b). In the absence of Grb2, however, the activity measurements did not reflect normal saturation of Arp2/3 by N-WASp. First, the half-maximum was obtained at a concentration of N-WASp representing 25% of the Arp2/3 concentration. Second, activation of polymerization first increased with N-WASp and then decreased at high concentration (Fig. 2a). Third, the percent activation provided by Grb2 increased with the concentration of N-WASp (Fig. 2a, inset). The decrease in activation of polymerization upon increasing N-WASp was observed in a range of N-WASp concentration (0.1-0.8 µM) in which no appreciable sequestration of G-actin by WA can occur. The two opposite effects of N-WASp therefore suggest that N-WASp exists in two states that can interact with Arp2/3. At low concentration (10⁹-10⁸ M), N-WASp binds Arp2/3 in a complex that stimulates polymerization, whereas at high concentration (10⁷-10⁶ M) it also binds Arp2/3, but in a state that does not stimulate polymerization. The stronger stimulating effect of Grb2 at high N-WASp concentration suggests that Grb2 shifts N-WASp to its active state. To get more insight into the possible association state of N-WASp and its control by Grb2, sedimentation velocity and cross-linking experiments were carried out. At 4 µM N-WASp had a sedimentation coefficient s_20,w of 5.45 S. This value is much higher than the one expected (3.98 S) for a monomeric 56-kDa globular protein and is suggestive of a dimeric structure of N-WASp. Cross-linking of N-WASp by EDC/NHS showed evidence for a triplet of covalently cross-linked polypeptides migrating with apparent molecular masses of 150-190 kDa in SDS-PAGE (Fig. 2b), supporting the view that N-WASp is in a self-association equilibrium. The different cross-linked species may correspond to different locations of the cross-links in the amino acid sequence. The amount of cross-linked polypeptides decreased in the presence of Grb2, suggesting that Grb2 stabilizes the monomeric form of N-WASp. No covalently cross-linked Grb2-N-WASp polypeptide was detected. As will be discussed under "Discussion," the polymerization data shown in Fig. 2a are satisfactorily modeled (continuous line through the data points) assuming that N-WASp undergoes self-association upon increasing concentration and that the monomeric form activates actin polymerization and is favored by Grb2 binding.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Grb2 activates N-WASp to stimulate actin polymerization by Arp2/3 complex. a, activation of actin assembly by Grb2 in the presence of Arp2/3 and N-WASp. MgATP-actin (2.5 µM, 10% pyrenyl-labeled) was polymerized in the presence of 15 nM Arp2/3 alone (a) and with the following additions: 42 nM N-WASp without (b) or with (c) 0.75 µM Grb2. The polymerization process is monitored by the increase in pyrenyl-actin fluorescence. b, N-WASp interacts with Arp2/3 in a 1:1 complex in the presence of Grb2. MgATP-actin (2.5 µM) was polymerized in the presence of 18.5 nM Arp2/3 complex, 1.05 µM Grb2 and N-WASp at the indicated concentrations. Inset, the maximum rate of polymerization was measured on each curve and plotted versus the concentration of N-WASp. A value of 5 nM was derived for the equilibrium dissociation constant for binding of N-WASp to Arp2/3 in the presence of a saturating amount of Grb2.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Structure-function relationship of N-WASp and effect of Grb2. a, N-WASp concentration dependence of the stimulation of actin polymerization. MgATP-G-actin (2.5 µM) was polymerized in the presence of 9 nM Arp2/3 complex and N-WASp at the indicated concentrations, in the absence (closed circles) or in the presence (open circles) of 1 µM Grb2. The maximum rate of polymerization is plotted versus the concentration of N-WASp. Thin lines are theoretical curves obtained assuming that N-WASp is in a reversible monomer-dimer equilibrium (K = 90 nM); Arp2/3 binds equally well monomeric and dimeric N-WASp, but only the monomeric species is active in stimulating polymerization and binds Grb2 preferentially. Inset, percent activation by Grb2 at different concentrations of N-WASp (the line is the theoretical curve). b, evidence for N-WASp self-association by covalent cross-linking. N-WASp (1 µM) was incubated in the presence of Grb2 at the indicated concentrations and submitted to EDC/NHS cross-linking, followed by SDS-PAGE and Western blotting (see "Materials and Methods").

Interaction of the C-terminal SH3 Domain of Grb2 with N-WASp Is Responsible for the Stimulation of Actin Assembly by Arp2/3 Complex-- The above results suggest that one or both of the two SH3 domains of Grb2 activate N-WASp by binding to the proline-rich region of the protein. This possibility was tested using mutated forms of Grb2 and specific targets of the SH3 domains as follows (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Effects of mutated forms of Grb2 and inhibitors of Grb2 SH2 and SH3 domains on the activation of N-WASp. A, MgATP-actin was polymerized at 2.5 µM without additions or with 1.4 µM Grb2 (a), in the presence of 11.5 nM Arp2/3 only (b), and in the presence of Arp2/3 and the following additions: 1.4 µM Grb2 WT (c); 1.4 µM P49L-Grb2 (d); 42 nM N-WASp (e); 42 nM N-WASp and either 1.4 µM Grb2 wild type (f) or 1.4 µM P49L-Grb2 (g); 42 nM N-WASp, 1.4 µM Grb2 wild type or P49L-Grb2 and 2.5 µM V33V peptidimer (h); 42 nM N-WASp and 2.5 µM V33V (i); 42 nM N-WASp and 2.5 µM pYpYN (j); 42 nM N-WASp, 1.4 µM Grb2, 2.5 µM pYpYN (k); 42 nM N-WASp and 1 µM G203R-Grb2 (l); 42 nM N-WASp and 1 µM P206L-Grb2 (m). B, schematic representation of Grb2 with one SH2 domain between two SH3 domains. Binding locations of pYpYN and V33V peptides are indicated. The position of the three Grb2 mutations shows that P49L and P206L prevent binding of the proline-rich peptides to the N-terminal and C-terminal SH3 domains, respectively, whereas G203R is a structural interface mutant. C, SDS-PAGE of the different proteins used in this work. 1 µg each of the following proteins was electrophoresed on SDS-15% acrylamide gels. Lane a, Grb2; lane b, P49L-Grb2; lane c, P206L-Grb2; lane d, G203R-Grb2; lane e, N-terminal SH3 domain of Grb2; lane f, C-terminal SH3 domain of Grb2; lane g, SH2 domain of Grb2; lane h, Cdc42.

First, the activating effect of Grb2 wild type (curve f in Fig. 3A) was totally inhibited by 2.5 µM of the Sos-derived peptide dimer VPPPVPPRRR-Aha-K-Aha-RRRPPVPPPV (curve h in Fig. 3A), which binds both SH3 domains (Fig. 3B) and blocks the Ras signaling pathway (52). The pYpYN phosphorylated tripeptide, which mimics the targets of the SH2 domain of Grb2, caused a weak inhibition in the absence as well as in the presence of Grb2. However, activation of N-WASp by Grb2 in itself was not affected by pYpYN tripeptide (compare curves j and k). In conclusion, the SH2 domain of Grb2 is not responsible for the activation of N-WASp, and binding of the SH2 domain of Grb2 to a phosphotyrosine peptide does not affect the affinity of its SH3 domains for proline-rich targets, in agreement with Cussac et al. (55).

The G203R point mutation in the C-terminal SH3 domain of Grb2 is located at the interface of the two SH3 domains (Fig. 3B). It has been shown to impair sex myoblast migration (56). The G203R Grb2 mutant was totally unable to activate N-WASp (curve l in Fig. 3A). This effect may be due to the arginine bulging off the surface of the SH3 domain or to a local structural perturbation.

The P49L point mutation in the N-terminal SH3 domain of Grb2 is known to greatly weaken the interaction between Grb2 and Sos (42). This mutation is lethal in Caenorhabditis elegans (56). However, the P49L Grb2 mutant activated N-WASp (curve g) with an efficiency that varied, among different preparations, between 50 and 100% of the one observed with wild type Grb2. The effect was abolished by the peptide dimer inhibitor. This result suggests that the C-terminal domain of Grb2 is mainly responsible for its interaction with and activation of N-WASp. In agreement with this conclusion, the P206L point mutation in the C-terminal SH3 domain of Grb2 yielded a protein that failed to activate N-WASp (curve m).

Control assays showed that Grb2 does not affect the polymerization of actin alone and that in the absence of N-WASp, Grb2 and P49L Grb2 activate Arp2/3 to a small extent (curves c and d), indicating that in the overall complex comprising N-WASp, Grb2, and Arp2/3, contacts exist between Arp2/3 and N-WASp, Grb2 and N-WASp, and Grb2 and Arp2/3. A similar conclusion was reached concerning the activation of N-WASp by the Shigella protein IcsA (9).

The activation of N-WASp by the isolated N-terminal and C-terminal SH3 domains of Grb2 was tested next. Fig. 4 shows that in the presence of 40 nM N-WASp, half-maximal activation was observed with 0.25 and 2.5 µM C-terminal and N-terminal SH3 domains, respectively. In conclusion, the C-terminal domain of Grb2 activated N-WASp with an apparent affinity comparable to that of Grb2 and about 10-fold higher than the N-terminal domain. This result corroborates the conclusions derived from the studies with the P49L and P206L Grb2 mutants. In contrast, in the association of Grb2 with Sos and with dynamin, the N-terminal SH3 domain of Grb2 was found to be the most efficient (57).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   The C-terminal SH3 domain of Grb2 is more efficient than the N-terminal SH3 domain to activate N-WASp. MgATP-actin was polymerized at 2.5 µM in the presence of 18 nM Arp2/3 complex in the absence of N-WASp (control) and in the presence of 31 nM N-WASp and either Grb2 C-terminal SH3 domain (a) or Grb2 N-terminal domain (b) at the indicated concentrations.

Finally, the isolated SH2 domain of Grb2 did not activate actin polymerization in the presence of Arp2/3 and N-WASp. In contrast, a concentration-dependent decrease in the rate of polymerization was observed (data not shown). The inhibitory effect of the SH2 domain was not abolished by the pYpYN peptide, which rules out the possibility that the inhibition be mediated by binding of SH2 to a phospho-tyrosine motif in N-WASp.

Cdc42 and Grb2 Bind to N-WASp Simultaneously, Causing Superactivation of Actin Polymerization-- Cdc42 binds to the CRIB domain of N-WASp, and Grb2 binds the proline-rich region. Hence it is theoretically possible that these two effectors of N-WASp, which both independently activate the protein, bind simultaneously to the same N-WASp molecule. This possibility was tested in the experiment shown in Fig. 5. Actin was polymerized in the presence of 20 nM Arp2/3, 30 nM N-WASp, and saturating amounts (1 µM) of either Cdc42-GTPS or Grb2 then in the presence of both effectors together. The same level of Arp2/3 activation was reached with either Grb2 or Cdc42-GTPS. A higher activation was obtained when both effectors were added together, indicating that Grb2 and Cdc42 can be bound simultaneously to N-WASp and that the structure/activity of the N-WASp molecule is different when the two effectors are bound, allowing synergistic behavior.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Cdc42-GTPS and Grb2 bind simultaneously to N-WASp and activate actin polymerization synergistically. MgATP-actin was polymerized at 2.5 µM in the presence of 18.5 nM Arp2/3 and the following additions: none (a); 0.12 µM N-WASp (b); 0.12 µM N-WASp and either 1 µM Grb2 or 2 µM Cdc42-GTPS, or 1 µM Grb2 and 2 µM Cdc42-GTPS as indicated by the arrows.

Grb2 Promotes the Onset of Actin-based Motility of E. coli (IcsA)-- The actin-based propulsion of E. coli (IcsA) is acknowledged as a model for lamellipodium extension, which is suitable for biochemical analysis, and has recently been reconstituted from a set of pure proteins (3). The effect of Grb2 on actin-based motility of E. coli (IcsA) was tested as described under "Materials and Methods." Briefly, bacteria were first preincubated in the presence of N-WASp to allow tight association of N-WASp with IcsA. N-WASp-coated bacteria were then placed in the motility medium. The binding of N-WASp to IcsA at the surface of E. coli was not inhibited by the presence of Grb2 in the preincubation step. Both the rate of propulsion and actin tail length were unchanged upon addition of Grb2 to the motility medium. On the other hand, the time required for bacteria to start moving, i.e. the pre-steady-state step, was shortened from 30 to 5 min when Grb2 was present at concentrations 1-4 µM in the N-WASp-precoating step. These observations, summarized in Table I, indicate first that Grb2 and IcsA do not compete for binding to the same site on N-WASp. Second, the effect of Grb2 on the delay preceding the onset of movement appears linked to the enhanced interaction between N-WASp and IcsA during the precoating of E. coli (IcsA) by N-WASp, suggesting that, in stabilizing the active monomeric form of N-WASp, Grb2 facilitates the interaction of N-WASp with IcsA at the bacterium surface.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present results show that in vitro the SH2/SH3 adaptor Grb2 acts as an effector of N-WASp, leading to the stimulation of actin assembly via N-WASp·Arp2/3 complex. The present data, involving sedimentation velocity, EDC cross-linking, together with the concentration dependence of N-WASp activity in actin polymerization and the effect of Grb2 on N-WASp·Arp2/3-mediated actin polymerization and actin-based motility of E. coli (IcsA) suggest that the structural basis for N-WASp activity involves at least a monomer-dimer equilibrium, shifted toward the active monomer form by Grb2, according to the minimum scheme displayed in Fig. 6. The data shown in Fig. 2a could be satisfactorily accounted for by this scheme (see "Materials and Methods" for detailed equations), assuming that 1) N-WASp dimerizes with an equilibrium dissociation constant K_D of 90 nM; 2) Grb2 binds preferentially monomeric N-WASp; 3) both monomeric and dimeric forms of N-WASp bind Arp2/3 with the same affinity (K_Y = 5 nM), independently of Grb2 binding (Fig. 1b, inset), but only the monomeric forms (N-WASp-Arp2/3 and Grb2-N-WASp-Arp2/3) are active in filament branching. This model explains why in the absence of Grb2 full activation of Arp2/3 is not reached upon increasing N-WASp concentration; the formation of the inactive Arp2/3-N-WASp dimer (W₂Y₂) is thought to overlap the saturation of Arp2/3 by monomeric N-WASp. Consistently, Grb2 is more effective at higher N-WASp concentration (Fig. 2a, inset) because it is thought to shift the inactive dimer toward the active monomer form. In the reports published so far, it has been puzzling to observe quantitatively different effects of IcsA and Cdc42 (9) on N-WASp-mediated activation of Arp2/3, as well as additive effects of different ligands (Cdc42 and PIP₂ (6); Cdc42 and Grb2 in the present work). Such data are well accommodated by a monomer-dimer equilibrium of N-WASp that could be shifted to different extents to the active monomer form by ligand binding. The proposal of regulation of N-WASp by self-association is not incompatible with the evidence for an interaction between peptides of the C-terminal domain of N-WASp and peptides of the CRIB domain (26, 31). Such an interaction may take place via internal folding of a monomer as well as via monomer-dimer interaction (or a higher degree of self-association), as observed in Ezrin, another linker protein of the cytoskeleton (58). However, only the self-association hypothesis can account for the lower activity of N-WASp at high concentration reported here (Fig. 2a). The present functional study is understood as preliminary and calls for more extensive structural studies to understand fully the regulation of N-WASp.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Thermodynamic scheme describing the structural basis for N-WASp-Arp2/3 activation and its regulation by Grb2. The model proposes that N-WASp (W) undergoes monomer-dimer equilibrium 2W  232 W2, with KD = 90 nM. Both monomer and dimer forms bind Arp2/3 (Y) with the same affinity KY = 3 nM, and KYY = (KY)2. Only the complex of monomeric N-WASp with Arp2/3 (WY) is active to stimulate actin polymerization by filament branching. Grb2 (X) binds preferentially the monomeric form of N-WASp with KX = 5 nM, and the dimeric form of N-WASp with a 32-fold lower affinity (KXX = 5000 (nM)2). The WX complex associates with Arp2/3 to form a WXY active complex. The net effect of Grb2 on the stimulation of actin polymerization is to increase the proportion of N-WASp in the active monomer state (WXY). The active complexes of monomeric N-WASp and Arp2/3 (WY and WXY) are boxed.

Overall, our results give full biochemical/molecular support to the view (44, 23, 47) that Grb2 may play a role in actin-based motility, and they suggest that it could act also in vivo by modulating the interaction between N-WASp and Arp2/3 complex. As indicated by She et al. (23), Grb2 could mediate the motile response of cells to EGF by directly linking the tyrosine-phosphorylated EGF receptor to the N-WASp·Arp2/3 complex. In addition to the PH and the CRIB domains, the proline-rich region of N-WASp, which contains five consensus PPPPPXR motifs, now appears to be another signal-responsive region of this fascinating connector in actin-based motility. It is possible that the other SH2/SH3 domain adaptor Nck, which has recently been demonstrated to be involved in the recruitment of Arp2/3 complex in actin-based motility of vaccinia virus (35), activates N-WASp like Grb2 to elicit actin tail formation. Whether the interaction of N-WASp with the SH3 domains of the tyrosine kinases Fgr or Fyn regulates N-WASp function is an open issue.

In the absence of N-WASp, Grb2 elicited a weak activation of Arp2/3. It is possible that the ridge of negatively charged amino acids created by the relative positions of the two SH3 domains at the surface of Grb2 participates in electrostatic interactions with Arp2/3 complex, like the acidic C terminus of N-WASp, resulting in stimulation of the nucleating activity. A similar observation has been made with IcsA (9).

Use of mutated forms of Grb2, of specific inhibitors of SH2 and SH3 domains, and of the isolated N-terminal and C-terminal SH3 domains of Grb2 demonstrates that the C-terminal SH3 domain of Grb2 is the main active target of the proline-rich region on N-WASp. This result is surprising when compared with binding studies of Grb2 to the homologous WASp (23), which indicated that the N-terminal SH3 domain of Grb2 bound slightly more strongly than the C-terminal SH3 domain. This discrepancy may be due either to differences in the proline-rich regions of WASp and N-WASp or to the difference in the methods used. Binding studies relied on pull down assays of WASp from whole cell lysates of hemagglutinin-WASp-transfected kidney cells using glutathione S-transferase fusion proteins coupled to glutathione-agarose beads, and here the biological activity of pure proteins or isolated domains was measured.

The physiological significance of multiple SH3 domains in adaptors like Grb2 (two SH3 domains) or Nck (three SH3 domains) has been discussed (43, 57). It is generally thought that the presence of several SH3 domains strengthens the binding of the whole protein, even though individual SH3 domains may display differential affinity. The N-terminal SH3 domain of Grb2 is the only one that is essential in mediating high affinity binding to dynamin (54), and it displays a predominant contribution in binding to Sos (37, 39), whereas here it is the C-terminal SH3 domain of Grb2 that plays a predominant role in activation of N-WASp. The two SH3 domains of Grb2 may interact differentially in different signaling pathways, making Grb2 a highly versatile adaptor. Our results suggest that transfection of cells with the P49L Grb2 mutant, which blocks the Ras signaling pathway, should not inhibit the function of Grb2 in actin-based motility.

The N-WASp molecule can be activated by Cdc42 and Grb2 in a synergistic fashion. Similarly, PIP₂ and Cdc42 have been shown to cooperate in activating N-WASp (6). These results support the view that the multidomain organization of N-WASp has functional significance. PIP₂ and Grb2 also cooperate in activation of dynamin GTPase (43).

The possible regulation of the activity of N-WASp in vivo by Grb2 is an open issue. Since both Grb2 and N-WASp are soluble proteins, the simple in vitro approach suggests that Grb2 could activate N-WASp and stimulate actin polymerization in the cytosol, whereas in principle in the cell N-WASp is activated at the plasma membrane by signaling. It is possible that in the in vivo context, the cytosolic N-WASp is maintained inactive, perhaps in a self-associated form, and is unable to bind Grb2, and it binds Grb2 when it is recruited to the membrane by other effectors. The possible regulation of N-WASp by phosphorylation of a tyrosine in the N-terminal domain has not been considered yet. Further mutagenesis and biochemical and structural studies of N-WASp are required to understand fully the complex behavior of this important connector between signaling molecules and actin.

	ACKNOWLEDGEMENTS

We are very grateful to Dominique Didry for assistance in actin, N-WASp, and Arp2/3 preparation and discussions and W. Q. Liu, F. Cornille, and C. Lenoir from Professor Roques's laboratory for the chemical synthesis of the N- and C-terminal SH3 domains of Grb2.

	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

The abbreviations used are: 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; GTPS, guanosine 5'-3-O-(thio)triphosphate; PAGE, polyacrylamide gel electrophoresis; WA, WH₂-Acidic; Aha, aminohexanoic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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