Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation

Abstract

WAVE2 is a member of the Wiskott–Aldrich syndrome protein family of cytoskeletal regulatory proteins shown to link Rac activation to actin remodeling via induction of Arp 2/3 activity. WAVE2 is thought to be regulated by its positioning in a macromolecular complex also containing the Abelson-(Abl) interactor-1 (Abi-1) adaptor, but the molecular basis and biologic relevance of WAVE2 inclusion in this complex are ill defined. Here we show that Abi-1 binding to WAVE2 is mediated by discrete motifs in the Abi-1 coiled-coil and WAVE2 WAVE-homology domains and increases markedly in conjunction with Abi-1-WAVE2 translocation and colocalization at the leading edge in B16F1 cells after fibronectin stimulation. Abi-1 also couples WAVE2 to Abl after cell stimulation, an interaction that triggers Abl membrane translocation with WAVE2, Abi-1, and activated Rac, as well as Abl-mediated tyrosine phosphorylation and WAVE2 activation. By contrast, mutation of tyrosine residue Y150, identified here as the major site of Abl-mediated WAVE2 tyrosine phosphorylation, as well as disruption of WAVE2-Abi-1 binding, impairs induction of WAVE2-driven actin polymerization and its membrane translocation in association with activated Rac. Similarly, WAVE2 tyrosine phosphorylation and induction of membrane actin rearrangement are abrogated in fibroblasts lacking the Abl family kinase. Together, these data reveal that Abi-1-mediated coupling of Abl to WAVE2 promotes Abl-evoked WAVE2 tyrosine phosphorylation required to link WAVE2 with activated Rac and with actin polymerization and remodeling at the cell periphery.

The actin cytoskeleton is a dynamic structure integrally involved in the coupling of extracellular stimuli to cell activation and concomitant changes in morphology, motility, and many other fundamental cellular processes. Among the many effectors involved in poststimulatory actin-based cellular responses, members of the Wiskott–Aldrich syndrome protein (WASp) family are now recognized as particularly important players in connecting stimulatory signals to actin cytoskeletal reorganization. In addition to WASp, this family includes the WASp ortholog, N-WASp, and, in mammalian cells, three WAVE/SCAR isoforms, WAVEs 1, 2, and 3 (1–3). All of these cytosolic proteins contain a C-terminal verprolin homology/connecting region/acidic region (VCA) domain, which mediates binding to actin monomers and the Arp 2/3 complex and consequent induction of Arp 2/3 actin nucleating activity (4). Immediately upstream of the VCA domain, the WASp family members all contain a proline-rich region that mediates interaction with profilin and SH3 domain-containing proteins, the latter of which modulate subcellular location and possibly activation of WASp/N-WASp (5–7). WASp and N-WASp also contain an N-terminal EVH1 and cdc42/Rac interactive binding domain, which respectively mediate interactions with the WASp-interacting protein and the activated form of the Rho family GTPase, cdc42 (8, 9). WASp/N-WASp tyrosine phosphorylation and cdc42 binding after cell stimulation appear to release autoinhibitory structural constraints to enable VCA domain-mediated triggering of Arp 2/3 actin polymerizing activity (10, 11). Thus, the capacity of WASp and N-WASp to couple extracellular stimuli to actin polymerization at selected sites within the cell reflects their inducible interactions with multiple signaling effectors that evoke WASp/N-WASp activation and cellular redistribution.

WAVE proteins also play key roles in the induction of actin-based processes, promoting, for example, membrane ruffling, lamellipodia formation, and many other outcomes of actin remodeling processes (12). In contrast to WASp/N-WASp, WAVEs contain an N-terminal WAVE homology domain (WHD) instead of cdc42/Rac interactive binding and EVH1 domains, and their mechanisms of activation and biologic roles are not well defined. WAVEs are thought to act downstream of the Rac GTPase, connecting Rac activation to induction of Arp 2/3-mediated actin polymerization (7). Coupling of Rac to WAVE effector activity was initially ascribed to the mutual interaction of these effectors with the insulin receptor tyrosine kinase substrate (IRSp53), but this interaction appears restricted to WAVE2 and recent data identify cdc42, rather than Rac, as the Rho GTPase that binds IRSp53 (12, 13). WAVE1 and -2 have also been shown to associate with the Abelson-(Abl) interactor adaptors (Abis), Abi-1 and -2, respectively, which appear to position the WAVE proteins in a macromolecular complex containing the Nck-associated protein 125, p53-inducible PIR121 and, in the WAVE1 complex, an actin-stimulating peptide, HSPC300 (14–16). Although results of one study suggest that WAVEs are kept constitutively inactive by maintenance within these complexes and activated by dissociation from such complexes after Rac activation (14), other data suggest that the Abi-1-mediated positioning of WAVE2 within these complexes is sustained after Rac activation and is required for induction of WAVE2 effects on actin nucleation (15). Thus the contribution of the complex and its individual components to the regulation of WAVE function remains unclear.

A major role for Abis in regulating WAVE function is consistent with data implicating both Abis and their binding partner, the Abl protein tyrosine kinase, in the regulation of Rac activation and Rac-dependent actin remodeling (17, 18). Thus resolution of the mechanisms governing WAVE activation requires understanding of the molecular basis and biologic significance of WAVE interaction with Abi-1. To address this issue, our group has followed up on our MS-based identification of Abi-1 as the predominant binding partner of WAVE2 in activated T cells by investigating the mechanisms regulating WAVE2 and Abi-1 association and the relevance of Abl activity to this interaction. Results of these analyses reveal that Abi-1 not only binds WAVE2 directly but also mediates WAVE2 translocation to the membrane after cell stimulation and connects WAVE2 to Abl to permit Abl-mediated WAVE2 tyrosine phosphorylation required for linking WAVE2 to activated Rac and inducing WAVE2 effects on actin nucleation and remodeling.

Materials and Methods

Reagents. Reagents used for these studies included: polyclonal antibodies specific for Abi-1 (raised against a synthetic peptide representing Abi-1 residues 166–177), WAVE2, and Abl (Santa Cruz Biotechnology); murine monoclonal antibodies for GFP (AbCam), DsRed and Flag (Sigma–Aldrich), GST (Santa Cruz Biotechnology), phosphotyrosine (pTyr) (Upstate Biotechnology), Rac (Upstate Biotechnology), and Abl (Oncogene Science); horseradish peroxidase-conjugated goat anti-rabbit, donkey anti-goat, and goat anti-mouse IgG (Bio-Rad); rhodamine, FITC, and Alexa 350-phalloidin (Molecular Probes); and FITC-anti-goat, Cy3 and Cy5-anti-mouse and rhodamine, FITC, and Cy5-anti-rabbit antibodies (Jackson ImmunoResearch). Expression constructs were derived by subcloning cDNAs encoding Abi-1 and Abi-1ΔCoil (Δaa1–106), ΔPro (Δaa336–362), and ΔSH3 (Δaa389–446) into the pEGFP-C3 vector (Clontech), Flag-tagged WAVE2 and WAVE2ΔWHD (Δaa45–170), ΔPro (Δaa247–400) and ΔVCA (Δaa436–495), and other WAVE2 deletion mutants into the pDsRed vector (Clontech), and Rac, Abl, Abl P131L, and Abl K290R (from A. M. Pendergast, Duke University Medical Center, Durham, NC) into pcDNA3. WAVE2 point mutation (tyrosine to phenylalanine) Flag-tagged expression constructs were generated by PCR mutagenesis of each WAVE2 tyrosine residue and subcloned into pDsRed. Fusion proteins containing Abi-1, Abl, the Rac/cdc42-binding region (PAK-CD; from J. Collard, Netherlands Cancer Institute, Amsterdam) or the WAVE2 WHD region (Δaa1–170) were constructed in the pGEX2T vector and, for Abi-1, also in the pQE-30 vector (Qiagen, Valencia, CA) and were expressed in Escherichia coli and purified from glutathione-linked Sepharose 4B beads or Ni-NTA agarose beads (Qiagen). SiRNA vectors for Abi-1 knockdown were derived by subcloning into pSilencer 2.1 (pSi; Ambion, Austin, TX) 50-residue oligomers encoding short-hairpin RNAs representing a scrambled sequence or targeting Abi-1 amino acids 500–518.

MS. Cell lysates prepared from anti-CD3 antibody (10 μg/ml)-stimulated Jurkat E6 cells were immunoprecipitated with anti-WAVE2 antibody (see below), resolved by SDS/PAGE and Coomassie-stained protein bands, then excised and digested with trypsin. Digested samples were analyzed by MALDI-MS in MS and MS/MS mode (QSTAR XL, MDS Sciex, Concord, ON, Canada) and the data searched against the National Center for Biotechnology Information nonredundant protein sequence database by using the MASCOT search algorithm (Matrix Science, Boston). Tolerance set for establishing protein identities was 50 ppm for the peptide masses and 0.1 Da for the MS/MS fragments.

Transfection and Immunofluorescence Assays. B16F1, Cos-7 cells, and mouse embryo fibroblasts (MEFs) doubly null for Abl and Arg or Abl reconstituted (ref. 19; from A. J. Koleske, Yale University, New Haven, CT) were cultured in 10% FBS-supplemented DMEM, lipofectamine (Invitrogen)-transfected with selected plasmids, and then stimulated with 10 ng/ml platelet-derived growth factor (PDGF; Invitrogen) or seeded onto uncoated or fibronectin (FN) (Roche Diagnostics)-coated (100 μg/cm²) plates or glass coverslips for 1–18 h. For RNA interference, B16F1 cells were transfected with the Abi-1 siRNA vectors and cultured in 1.2 μg/ml G418. For immunofluorescence assays, fixed cells were treated with 2% BSA/PBS or, for intracellular staining, first permeabilized with 0.1%Triton X-100 PBS and the cells then incubated with primary antibody and fluorophore-conjugated secondary antibodies. Images were analyzed by using an Olympus (Melville, NY) 1×-70 inverted microscope and deltavision deconvolution software (Applied Precision, Bratislava, Slovakia).

Immunoprecipitation and Immunoblotting. Cells were lysed in Nonidet P-40 lysis buffer as described (11), the lysate proteins (1 mg) incubated for 2 h with the appropriate antibody or IgG preimmune serum, and the immune complexes collected over Protein A Sepharose beads (Amersham Pharmacia). Alternatively, lysate proteins were incubated for 1 h at 4°C with 20 μl of glutathione–Sepharose beads coupled to the GST fusion protein containing the p21 activated kinase cdc42/Rac interactive binding domain (GST-PAK CD). Complexes were eluted from the beads, electrophoresed through 10% polyacrylamide gels, and transferred to PVDF membranes (Millipore). After blocking, filters were incubated sequentially with primary and diluted horseradish peroxidase-secondary antibodies and complexes detected by ECL (Amersham Pharmacia).

In Vitro Protein-Binding Assays. For in vitro protein-binding studies, 5 μg of GST-WAVE2 WHD protein immobilized on glutathione Sepharose 4B beads were incubated for 1 h at 4°C with 2 μg of 6×His-tagged Abi-1. After washing, protein complexes were eluted from the beads, electrophoresed through 10% polyacrylamide gels, and visualized by immunoblotting with anti-His antibody. To derive an Abi-1 peptide array, 20-mer peptides (with two amino acid overlaps) spanning the Abi-1 coiled-coil region were synthesized [Abimed 431 (Analysentechnik, Langenfeld, Germany)], spotted on cellulose, and the filter then treated with 2% acetic acid anhydride in dimethyl formamide. After peptide deprotection and blocking, the filter was incubated overnight with 1 mg GST-WAVE2 WHD protein and bound fusion protein detected by immunoblotting with anti-GST antibody.

Actin Polymerization Assay. For these assays, DsRed Flag WAVE2 and WAVE2 Y150F were expressed in and then immunoprecipitated from Cos-7 cells (protein purity >98% by SDS/PAGE and Coomassie staining), incubated for 30 min with 100 units recombinant Abl in Abl reaction buffer and, after heat inactivation, incubated at 100 nM concentration alone or with 200 nM purified GST-Abi-1 protein in actin polymerization buffer (Cytoskeleton, Denver) containing 50 nM Arp 2/3 complex and 100 μl of monomer pyrene actin stock in 0.2 mM ATP-containing G buffer (final G actin concentration, 2.8 μM). Fluorescence changes were monitored by using a Photo Technology International (Lawrenceville, NJ) fluorometer with filters for excitation at 365 nm and emission at 407 nm.

Cell Spreading and Migration Assays. B16F1 cells were trypsinized 48 h posttransfection with pSi vectors, incubated for 18 h at 37°C in 96-well plates (500 cells/well), coated with FN alone or with FN and 2 μm fluorescent latex beads, and cells then fixed and labeled with TRITC-phalloidin alone or with Hoechst 33342 to gauge cell mobility and spreading, respectively. Plates were analyzed with Arrayscan II Reader at ×5 objective by using cellomics cell migration software (Cellomics, Pittsburgh), migration gauged by measuring of phagokinetic tracks detected as negative images in the fluorescent bead background, and spreading gauged by measuring cell area.

Results and Discussion

Characterization of WAVE2 Interaction with Abi-1. To delineate binding partners for WAVE2 and thereby improve understanding of its cellular functions, WAVE2 was immunoprecipitated from anti-CD3 antibody-stimulated Jurkat T cells and the bound peptides then resolved by SDS/PAGE and identified by mass spectrometric analysis. Among the proteins identified, the most prominent species were the Abi-1 protein and the Arp 3 subunit of the Arp 2/3 complex (data not shown). Evaluation of the WAVE2-Abi-1 association by anti-Abi-1 immunoblotting analysis of WAVE2 immunoprecipitates from Jurkat cells revealed Abi-1 to be coimmunoprecipitated with WAVE2 from these cells and indicated the association of these proteins to be increased after T cell antigen receptor engagement (Fig. 1A). Abi-1 binding to WAVE2 was also increased after FN stimulation of B16F1 cells, a melanoma cell line lacking endogenous WAVES 1 and -3 (20), and immunofluorescence analysis revealed WAVE2 and Abi-1 to be colocalized with one another and with actin in these cells (Fig. 1B). These findings are therefore consistent with recent data identifying Abi-1 as a component of a WAVE2 signaling complex (14, 15) and suggest that complex assembly is enhanced after cell stimulation. The molecular basis for the WAVE2–Abi-1 interaction was then evaluated by using Cos-7 cells expressing Flag-tagged WAVE2 together with GFP-tagged full length Abi-1 or Abi-1 species lacking the SH3 (ΔSH3), proline-rich (ΔPro), or coiled-coil (ΔCoil) domains. Although equivalently expressed in these cells (data not shown), GFP-Abi-1, Abi-1ΔSH3 and ΔPro, but not Abi-1ΔCoil, were coimmunoprecipitated with WAVE2 (Fig. 1C). Conversely, when coexpressed in Cos-7 cells with Flag-tagged WAVE2 or WAVE2 mutants lacking either the WHD (ΔWHD), proline-rich region (ΔPro) or VCA (ΔVCA) domains, Abi-1 coimmunoprecipitated with each WAVE2 species except WAVE2ΔWHD (Fig. 1D). These data indicate WAVE2 interaction with Abi-1 to be mediated by binding of the WAVE2 WHD to the Abi-1 coiled-coil domain, an interaction that appears to be direct because polyhistidine-tagged Abi-1 was precipitated by glutathione Sepharose-bound GST WAVE2 WHD but not by GST protein (Fig. 1E). As is consistent with a critical role for the WHD in the Abi-1–WAVE2 interaction, when coexpressed in Cos-7 cells with WAVE2, WASp, or N-WASp, Abi-1 coimmunoprecipitated with WAVE2 but with neither WASp (Fig. 1F) nor N-WASp (data not shown), both of which lack a WHD.

Fig. 1.

WAVE2 inducibly binds and colocalizes with Abi-1. (A) Lysate proteins prepared from anti-CD3 antibody-stimulated Jurkat (Left) or FN-stimulated B16F1 cells (Right) at the indicated times after stimulation were immunoprecipitated with anti-WAVE2 antibody or lgG and subjected to immunoblotting analysis with anti-Abi-1 and anti-WAVE2 antibodies. (B) B16F1 cells were stained for actin, WAVE2, and Abi-1 and the images visualized by immunofluorescent microscopy. (C) Lysate proteins prepared from Cos-7 cells cotransfected with WAVE2, and indicated pEGFP-Abi-1 cDNAs were immunoprecipitated with anti-WAVE2 antibody and the precipitated proteins subjected to anti-GFP immunoblotting analysis. (D) Lysate proteins prepared from Cos-7 cells cotransfected with the Abi-1 and indicated Flag-tagged WAVE2 cDNAs were immunoprecipitated with anti-Abi-1 antibody and the precipitated proteins immunoblotted with anti-Flag antibody. (E) Purified His-tagged Abi-1 fusion protein was incubated with glutathione-Sepharose-bound GST-WAVE2 WHD or GST fusion proteins and the complexes and aliquots of His-Abi-1 (Input) subjected to sequential immunoblotting with anti-His and -GST antibodies. (F) Lysate proteins prepared from Cos-7 cells cotransfected with Abi-1 and WAVE2 or WASp cDNAs were immunoprecipitated with anti-Abi-1 antibody or lgG and the precipitates or lysate proteins immunoblotted with the indicated antibodies.

To further define the regions mediating WAVE2 binding to Abi-1, DsRed-tagged WAVE2 or WAVE2 WHD deletion mutants were coexpressed with Abi-1 in Cos-7 cells and the effects of each deletion on WAVE2–Abi-1 interaction examined. As shown in Fig. 2 A and B, WAVE2 as well as WAVE2 species truncated at the most N-terminal (Δ1–32) or more C-terminal (Δ66–110, Δ110–170; not shown) regions of the WHD were coimmunoprecipitated with Abi-1 from these cells. By contrast, WAVE2 mutants (Δ1–110, Δ1–66, and Δ32–66) lacking amino acid residues 32–66 were not detected in the Abi-1 immunoprecipitates, suggesting that residues across or within this 34-aa segment are critical for WHD-mediated WAVE2 binding to Abi-1. The structural basis for Abi-1 interaction with WAVE2 was also explored by evaluating the binding of GST-WAVE WHD protein to a solid-phase array of overlapping 20-aa (two amino acid overlaps) peptides spanning the Abi-1 coiled-coil domain. As shown in Fig. 2B, results of anti-GST antibody immunoblotting analysis revealed specific binding of the WHD fusion protein to peptides containing amino acids 48–65, thus delineating an 18-aa segment within the Abi-1 coiled-coil domain as a region necessary and sufficient to bind the WAVE2 WHD in vitro. Neither this segment nor its cognate binding region in the WAVE2 WHD corresponds to known protein-binding motifs and thus needs to be further studied for potential involvement in other signaling protein interactions.

Fig. 2.

Refinement of regions mediating WAVE2 binding to Abi-1. (A) Lysate proteins prepared from Cos-7 cells cotransfected with the Abi-1 cDNA and DsRed-WAVE2 full-length cDNA or DsRed-WAVE2 cDNAs with the indicated amino acid deletions (▵) were immunoprecipitated with anti-Abi-1 antibody and the precipitated proteins subjected to immunoblotting analysis with anti-DsRed and then anti-Abi-1 antibody (Abi-1 precipitates) or anti-DsRed antibody alone (lysate proteins). (B) Peptides (20 mers) from across the Abi-1 coiled-coil domain (amino acids 45–107) were synthesized and spotted on a cellulose filter, the filter then incubated with GST-WAVE2 WHD, and bound fusion detected by immunoblotting with anti-GST antibody. Sequences of the two WAVE2 WHD-binding peptides (1 and 2) are shown.

Abi-1 Directs WAVE2 Translocation to the Membrane After Cell Stimulation. Both Abi-1 and WAVE2 are known to localize to actin-based membrane protrusions, such as membrane ruffles and lamellipodia, after growth factor or integrin-mediated cell stimulation (12, 21). To investigate whether its association with Abi-1 is required for inducible WAVE2 membrane translocation, DsRed WAVE2 protein was coexpressed in B16F1 cells with GFP-tagged wild-type Abi-1 or an Abi-1ΔCoil protein lacking the capacity to bind WAVE2 and the subcellular locations of these proteins after cell plating over FN examined. As shown in Fig. 3 A and B, this analysis revealed Abi-1 and WAVE2 to be colocalized within the cytosol in unstimulated cells but to translocate after FN exposure to the cell membrane, where they colocalize with polymerized actin at the leading edge of F-actin-rich lamellipodia protrusions triggered by cell stimulation. By contrast, coexpression of Abi-1ΔCoil with WAVE2 abrogated FN-induced WAVE2 translocation to the membrane, WAVE2 instead relocating primarily to the nucleus (Fig. 3C). Cell polarization and lamellipodia formation were also substantively reduced in these cells. Coexpression of GFP-Abi-1 with DsRed-WAVE2ΔWHD also evoked constitutive WAVE2 nuclear relocalization and disrupted induction of Abi-1/WAVE2 membrane translocation as well as cell polarization and other actin-based morphological changes normally engendered by B16F1 plating on FN (Fig. 3 D and E). Together, these data suggest a critical role for Abi-1 and, by extension, for the WAVE2 WHD, in mediating WAVE2 membrane translocation after cell stimulation and in the consequent coupling of WAVE2 activity to actin remodeling at the cell surface. These data also suggest a role for the Abi-1–WAVE2 interaction in minimizing WAVE2 shuttling to the nucleus to maintain a cytosolic pool of WAVE2 molecules before cell stimulation.

Fig. 3.

Abi-1 is required for WAVE2 membrane translocation. B16F1 cells were transfected with pEGFP-Abi-1 and either DsRed WAVE2 (a and b) or DsRed WAVE2ΔWHD (d and e) or with pEGFP-Abi-1ΔCoil and DsRed WAVE2 (c) and the subcellular distribution of these proteins as well as Alexa 350-phalloidin-stained F-actin examined in resting (–) or 6-h FN-stimulated (+) cells by immunofluorescent microscopy. Images are representative of six independent experiments.

Abi-1 Promotes Abl-Mediated WAVE2 Tyrosine Phosphorylation. Structural analyses of WAVE2 complexes have suggested that Abi-1 interactions with other complex components allow for Rac recruitment and coupling to WAVE2 (15). As a binding partner and target for the Abl protein tyrosine kinase (22), Abi-1 may also connect WAVE2 with Abl. To address this possibility, Abi-1 immunoprecipitates from PDGF-stimulated B16F1 cells were evaluated for the presence of Abl. Although Abl–Abi-1 association was detectable in resting cells, this interaction was markedly increased after cell stimulation (Fig. 4A). To determine whether Abi-1 might recruit Abl into WAVE2 complexes, the association of these effectors with activated Rac was next investigated in FN-treated B16F1 cells by using a GST fusion protein containing GST-PAK CD, which binds GTP but not GDP-bound Rac (23). As shown in Fig. 4B, Rac GTP, Abl, Abi-1, and WAVE2 were all coprecipitated from these cells by GST-PAK CD, and the amounts of each protein precipitated substantively increased after cell stimulation. Abl also colocalized with Rac, WAVE2, and Abi-1 at the actin-rich lamellipodial leading edge of FN-stimulated B16F1 cells (Fig. 4C). Thus induction of Abl binding to Abi-1 appears to position Abl in WAVE2 and activated Rac-containing complexes that are translocated to the cell membrane, wherein actin remodeling ensues.

Fig. 4.

Abl associates and colocalizes with the WAVE2–Abi-1 complex. (A) Lysate proteins from PDGF-stimulated B16F1 cells were immunoprecipitated with anti-Abi-1 antibody and the precipitated proteins subjected to sequential immunoblotting analysis with anti-Abl and -Abi-1 antibodies. (B) Lysate proteins prepared from FN-stimulated B16F1 cells were precipitated with glutathione-Sepharose-bound GST-PAK CD or GST fusion proteins and complexes or input lysate protein (bottom blot) immunoblotted with the indicated antibodies. (C) B16F1 cells plated for 6 h over FN were stained for actin and various combinations of Abi-1, WAVE2, Rac, and Abl and the images visualized by immunofluorescent microscopy. Images are representative of eight independent experiments.

The capacity of Abi-1 to juxtapose Abl with WAVE2 suggests that WAVE2, like Abi-1, may represent an Abl substrate. This possibility was investigated by anti-pTyr immunoblotting analysis of WAVE2 immunoprecipitates from resting and integrin (data not shown) or PDGF-stimulated B16F1 cells. As shown in Fig. 5A, both WAVE2 and the coimmunoprecipitated Abi-1 species were inducibly tyrosine phosphorylated in these cells. Interestingly, amounts of Abi-1 associated with WAVE2 also increased in conjunction with WAVE2 tyrosine phosphorylation, suggesting that Abi-1–WAVE2 interaction is enhanced by the tyrosine phosphorylation of one or both of these proteins. To further evaluate the relevance of both Abi-1 and Abl to WAVE2 tyrosine phosphorylation, WAVE2 and Abi-1 were coexpressed in Cos-7 cells together with constitutively active (P131L) and catalytically inert (K290R) forms of Abl. As shown by anti-pTyr immunoblotting analysis of WAVE2 immunoprecipitates from these cells, both WAVE2 and Abi-1 became tyrosine-phosphorylated in the presence of Abl but showed no tyrosine phosphorylation in the absence of either functional Abl or, interestingly, Abi-1 (Fig. 5B). Again, amounts of Abi-1 coimmunoprecipitated with WAVE2 were increased in the presence of activated Abl. Thus Abl tyrosine phosphorylates WAVE2, but appears to do so only in the presence of Abi-1 and to thereby induce increased Abi-1 association with WAVE2.

Fig. 5.

Abl tyrosine phosphorylates WAVE2 Y150 upon cell stimulation. (A) Lysate proteins prepared from PDGF-stimulated B16F1 cells were immunoprecipitated with anti-WAVE2 antibody and subjected to sequential immunoblotting analysis with the indicated antibodies. (B) Lysate proteins from Cos-7 cells cotransfected with WAVE2 and either the Abi-1, Abl P131L, or Abl K290R cDNAs were immunoprecipitated with anti-WAVE2 antibody and the precipitated proteins immunoblotted sequentially with the indicated antibodies. (C) Lysate proteins prepared from PDGF-stimulated Abl-reconstituted Abl/Arg-deficient (Abl⁺/Arg^–) and Abl/Arg-deficient (Abl^–/Arg^–) MEFs were immunoprecipitated with anti-WAVE2 antibody and sequentially immunoblotted with the indicated antibodies. (D) Lysate proteins from Cos-7 cells cotransfected with a Flag-tagged WAVE2 cDNA and Abi-1 plus Abl or Abl K290R cDNAs or with Flag-tagged WAVE2 cDNAs carrying the indicated Y→F substitutions and the Abi-1 plus Abl cDNAs were immunoprecipitated with anti-Flag antibody and the precipitates immunoblotted sequentially with anti-pTyr and anti-Flag antibodies.

To determine whether Abl not only targets WAVE2 but is also required for WAVE2 tyrosine phosphorylation, PDGF-induced WAVE2 tyrosine phosphorylation was compared between MEFs deficient for both Abl and the Abl-related Arg protein tyrosine kinase (Abl/Arg-deficient) and Arg-deficient MEFs derived by Abl reconstitution of Abl/Arg-deficient cells. As revealed by anti-pTyr immunoblotting analysis of WAVE2 immunoprecipitates from these cells, Abi-1 and WAVE2 were both inducibly tyrosinephosphorylated after PDGF stimulation of Arg-deficient MEFs but failed to undergo tyrosine phosphorylation in Abl/Arg-deficient MEFs lacking any Abl kinase family activity (Fig. 5C). To identify the WAVE2 tyrosine residues phosphorylated by Abl, cDNAs in which each of the 11 tyrosine residues within WAVE2 were individually replaced with phenylalanine (Y→F) were expressed as Flag-tagged proteins in Cos-7 cells together with Abi-1 and Abl and phosphorylation status of the mutant proteins examined. As shown by anti-pTyr immunoblotting analysis of anti-Flag immunoprecipitates from these cells (Fig. 5D), tyrosine phosphorylation of both WAVE2 and Abi-1 was abrogated by Y150F expression, although unaffected by all other mutations (Y124F and Y230 not shown). These data suggest that both Abl and Abi-1 are required for WAVE2 tyrosine phosphorylation, at least in the cells under study, and identify Y150 as the major site on WAVE2 targeted for Abl-mediated phosphorylation. Importantly, Y150 maps downstream of the Abi-1-binding segment within the WAVE2 WHD and is therefore positioned such that its phosphorylation may induce conformational changes enabling the increases in Abi-1–WAVE2 interaction observed after cell stimulation.

Abl Promotes WAVE2–Abi-1 Complex Assembly and WAVE2-Evoked Actin Nucleation. The capacity of Abl to induce WAVE2 tyrosine phosphorylation and thereby enhance WAVE2 interaction with Abi-1 implies that Abl may promote assembly of the signaling complexes within which WAVE2 is linked to activated Rac. To address this possibility, WAVE2, Abi-1, or Abi-ΔCoil were expressed alone or in combination with the activated Abl or kinasedead Abl proteins in Cos-7 cells and Abl effects on the association of activated Rac with WAVE2 and Abi-1 examined again by using GST-PAK CD fusion protein to precipitate activated Rac. As shown by anti-Rac immunoblotting analysis (Fig. 6A), amounts of activated Rac precipitated from cells expressing WAVE2 alone were negligible but were increased slightly by coexpression of Abi-1 and markedly by coexpressing both Abi-1 and Abl. Amounts of Abi-1 and WAVE2 coprecipitated with Rac GTP were also increased by Abl coexpression. By contrast, amounts of Abi-1, WAVE2, and Rac GTP precipitated in conjunction with Abl or WAVE2 Y150F expression were not increased above levels detected in cells expressing WAVE2 alone. Similarly, the disruption of WAVE2–Abi-1 interaction by Abi-1ΔCoil expression was associated with negligible Rac GTP and WAVE2 and no Abi-1 in the GST-PAK CD precipitates. These findings suggest Abl involvement in promoting the assembly of WAVE2–Abi-1–Rac GTP complexes and imply that Abi-1 contribution to the formation of such complexes is augmented by WAVE2 tyrosine phosphorylation and requires Abi-1 association with WAVE2.

Fig. 6.

Abl augments WAVE2 complex formation and effects on actin polymerization. (A) Lysates were prepared from Cos-7 cells transfected with the WAVE2 cDNA alone or with the WAVE2 or WAVE2 Y150F cDNAs plus the Abi-1 or Abi-1ΔCoil cDNAs and the Abl P131L or Abl K290R cDNAs and the lysate proteins precipitated with glutathione-Sepharose-bound GST-PAK CD protein. Complexes or input lysate protein were then immunoblotted with the indicated antibodies. (B) Abl-reconstituted Abl/Arg-deficient (Abl⁺/Arg^–) and Abl/Arg-deficient (Abl^–/Arg^–) MEFs were stained for F-actin, WAVE2, and Abi-1 and the individual or merged (Lower) images examined by immunofluorescent microscopy. (C) Polymerization of 2.8 μM pyrene-labeled actin-monomer was assayed in the presence of 50 nM Arp 2/3 and varying combinations of WAVE2 or WAVE2 Y150F and GST-Abi-1 and Abl fusion proteins.

The potential role for Abl in promoting the assembly of WAVE2/Abi-1-based signaling complexes suggests that Abl may modulate WAVE2 effects in Arp 2/3-mediated actin nucleation. To address this possibility, the effects of Abl deficiency on the induction of WAVE2 membrane translocation and actin nucleating effects were also examined in the Abl/Arg-deficient and control (Arg-deficient) MEFs. As shown in Fig. 6B, plating of the Arg-deficient MEFs over FN was associated with intense formation of F-actin rich membrane microspikes within which WAVE2, Abi-1, and Abl colocalized. By contrast, as is consistent with a role for Abl in modulating WAVE2 effects on actin polymerization, the induction of these actin-rich structures at the cell membrane was essentially abrogated in the Abl/Arg-deficient cells. Based on these findings, the possibility that Abl modulates the ability of WAVE2 to activate the Arp 2/3 complex was tested by using a pyrene fluorescence in vitro assay of actin polymerization. As shown in Fig. 6C and observed by others (15), WAVE2 alone stimulates some polymerization of pyrene-labeled actin in this assay. However, the effect of WAVE2 on Arp 2/3 actin nucleating activity was markedly increased in the presence of Abi-1 and Abl, although negligibly increased by addition of WAVE2 Y150F even in the presence of Abl and Abi-1. These observations strongly suggest that WAVE2 effects on actin polymerization are modulated by Abl-mediated phosphorylation of Y150. Importantly, when combined with WAVE2 in the absence of Abl, Abi-1 appears to modestly impede WAVE2 actin polymerizing function. This finding is consistent with suggestions of Abi-1 involvement in repressing WAVE2 activity (14) but also reinforces the critical role for Abi-1-mediated Abl recruitment in WAVE2 activation, a role that may reflect WHD conformational changes consequent to Y150 phosphorylation.

Abi-1 Plays an Essential Role in Modulating WAVE2 Effector Function. The key roles of Abi-1 in coupling Abl to WAVE2 tyrosine phosphorylation and subcellular location are consistent with recent data showing Abi-1 to be required for Rac-dependent actin remodeling at the cell membrane. To confirm the essential role for Abi-1 in modulating WAVE2 subcellular location and the actin-cytoskeletal changes mediated via Rac-WAVE2 activation, the effects of Abi-1 deficiency on actin dynamics were examined in B16F1 cells stably expressing an Abi-1 siRNA vector enabling >90% reduction of Abi-1 expression (Fig. 7A). As is consistent with Abi-1 involvement in actin remodeling, Abi-1 deficiency was associated with marked changes in cell spreading on FN, the Abi-1-deficient cells showing negligible polarization, lamellipodia formation, and spreading compared with wild-type cells at both 1 and 6 h after plating.

Fig. 7.

Abi-1 deficiency impairs WAVE2 membrane translocation and cell spreading and migration. (A) Lysate proteins from B16F1 cells expressing scrambled (pSiSc) or Abi-1 silencing (pSi-Abi-1) short-hairpin RNAs were immunoblotted with the indicated antibodies (Left) or plated on FN, stained for actin, and examined by immunofluorescent microscopy (Right). (B and C) pSiSc or pSi-Abi-1 B16F1 transfectants were plated on FN (C) or on FN in the presence of fluorescent latex beads (D), the cells fixed and stained after 18 h, and average cell areas and tracking distances evaluated by using an Arrayscan II Reader (Cellomics, Pittsburgh). Values represent means (±SEM) of two independent experiments. (D) B16F1 cells transfected with the pSiSc and pSi-Abi-1 vectors were plated on FN for 6 h, stained for actin, and Rac and WAVE2 and images examined by immunofluorescent microscopy.

Quantitation of serum-free spreading and migration of these cells over FN was also carried out by using an image analysis algorithm to measure the size and motility of TRITC-phalloidin and Hoechst 33342-stained cells. Results of these analyses again revealed both spreading and migration over FN to be abrogated by Abi-1 deficiency (Fig. 7 B and C). As is consistent with these findings and with a central role for Abi-1 in WAVE2 translocation and activity, immunofluorescence analyses of the Abi-1-deficient cells also revealed the dramatic reduction of F-actin accumulation at the cell membrane after FN exposure as well as impaired membrane translocation of both WAVE2 and Rac (Fig. 7D). These data therefore confirm Abi-1 involvement in modulating cellular actin dynamics and suggest that Abi-1 subserves this role via its effects on the Rac–WAVE2 complex and signaling axis.

Conclusion

In the current study, the mechanisms regulating WAVE2 function were investigated and were shown to involve induction of WAVE2 association with the Abi-1 adaptor after cell stimulation and Abi-1-mediated recruitment of the Abl protein tyrosine kinase into a macromolecular complex containing WAVE2, Abi-1, and activated Rac. Although other proteins within this complex have been shown to couple Abi-1-bound WAVE2 to activated Rac, the current data reveal a critical role for Abi-1 in regulating both WAVE2 activation and membrane translocation and reveal this role to be subserved at least in part by Abi-1-mediated coupling of Abl and WAVE2. Although the data are also consistent with an inhibitory role for Abi-1 in constitutive suppression of WAVE2 activity, the findings indicate a major positive effect of Abi-1 on WAVE2 function after cell stimulation because Abi-1 binding to WAVE2 is required for Abl to tyrosine phosphorylate and thereby activates WAVE2. Thus, although the structural basis for Abl effects on the WAVE2–Abi-1 interaction requires further investigation, these data provide compelling evidence that WAVEs, like the related WASp/N-WASp proteins, are functionally as well as positionally regulated by tyrosine phosphorylation and identify the Abl/Abi-1/WAVE2 axis as a critical pathway for coupling stimulatory signals to actin cytoskeletal remodeling.