SPIN90-Irsp53 Complex Participates in Rac-Induced Membrane Ruffling

EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419

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Research Article SPIN90-IRSp53 complex participates in Rac-induced membrane ruffling

Carmen Teodorof a, Jeom Il Baea, Seon-Myung Kima, Hye Jin Oha, Yong Seok Kanga, Jeonghoon Choib, Jang-Soo Chuna, Woo Keun Songa,⁎ aBio Imaging Research Center, Cell Dynamics Research Center, and Department of Life Science, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, South Korea bDepartment of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, South Korea

ARTICLE INFORMATION ABSTRACT

Article Chronology: SPIN90 is a key regulator of actin cytoskeletal organization. Using the BioGRIDbeta database Received 10 December 2008 (General Repository for Interaction Datasets), we identified IRSp53 as a binding partner of SPIN90, Revised version received and confirmed the in vivo formation of a SPIN90–IRSp53 complex mediated through direct 11 May 2009 association of the proline-rich domain (PRD) of SPIN90 with the SH3 domain of IRSp53. SPIN90 Accepted 14 May 2009 and IRSp53 positively cooperated to mediate Rac activation, and co-expression of SPIN90 and Available online 19 May 2009 IRSp53 in COS-7 cells led to the complex formation of SPIN90-IRSp53 in the leading edge of cells. PDGF treatment induced strong colocalization of SPIN90 and IRSp53 at membrane protrusions. Keywords: Within such PDGF-induced protrusions, knockdown of SPIN90 protein using siRNA significantly SPIN90 reduced lamellipodia-like protrusions as well as localization of IRSp53 at those sites. Finally, IRSp53 competitive inhibition of SPIN90-IRSp53 binding by SPIN90 PRD dramatically reduced ruffle Rac1 formation, further suggesting that SPIN90 plays a key role in the formation of the membrane Cytoskeleton protrusions associated with cell motility. Membrane ruffling © 2009 Elsevier Inc. All rights reserved. Actin

Introduction filopodia [4]. Their activation can be stimulated by growth factors or integrin receptors via a number of pathways [5,6]. For example, Cell motility is a complex biological process initiated by the phosphoinositide 3-kinase (PI3K) is an important upstream formation of actin-based membrane protrusions, including lamel- regulator of Rac GEFs (guanine-nucleotide exchange factors); lipodia, filopodia or membrane ruffles, which rely on the regulated other Rac activators include Vav, Sos, PIX, Tiam, and SWAP-70, of recruitment of molecular scaffolding and the coordinated organi- which at least SWAP-70 participates in membrane ruffle formation zation of actin filaments [1]. These membrane protrusions are [7,8]. In addition, p21-activated kinase (PAK) is a downstream regulated by small GTPases of the Rho family, which act as effector of Rac and Cdc42 coupled to lamellipodia protrusion, while molecular switches by cycling between GTP-bound (active) and Scar/WAVE proteins are known to transduce Rac-mediated GDP-bound (inactive) states to regulate actin cytoskeletal lamellipodium formation via the Arp2/3 complex [9–12]. dynamics [2,3]. Two members of the Rho family, Rac and Cdc42, Insulin receptor tyrosine kinase substrate p53 (IRSp53), also respectively contribute to the formation of lamellipodia and referred to as a brain-specific angiogenesis inhibitor 1-associated

⁎ Corresponding author. Department of Life Science, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500- 712, South Korea. Fax: +82 62 970 2484. E-mail address: [email protected] (W.K. Song).

0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.05.010 EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419 2411 protein 2 (BAIAP2), is known to regulate both Rac and Cdc42 (I403P) was subcloned into pcDNA3.0-6Myc vector and Myc- [13–16]. IRSp53 is a multifunctional adaptor protein that contains a IRSp53 WT plasmid was a gift from Dr. Hiroaki Miki (from unique N-terminal Rac1-binding domain (RCB) also termed as IMD University of Tokyo, Japan). GFP-IRSp53 WT was gift from Dr. (IRSp53/MIM-Missing in metastasis homology domain), a half Kwanghee Kim (from Mount Sinai School of Medicine, USA) and Cdc-42/Rac interactive binding (CRIB) motif, a proline-rich human GST-Rac1Q61L was a gift from Dr. Theresia Stradal (from domain, a Src homology 3 (SH3) domain, a potential WW domain GBF, Germany). HA-Rac1 G12V plasmid was from cDNA Resource binding motif (WWB) and a PDZ domain binding site in some Center (Missouri University of Science and Technology, USA). The isoforms [17–24]. The CRIB motif of IRSp53 is required for Cdc42 antibodies used were: anti-SPIN90 monoclonal and polyclonal binding and provides a molecular link between GTPases and antibody, generated in our laboratory from both rabbits and mice, regulators of the actin cytoskeleton. The SH3 domain of IRSp53 has immunized with purified GST-SPIN90-N-term (amino acids 1– been shown to bind the proline-rich sequences of Wiskott–Aldrich 279) and affinity-purified using the same GST fusion proteins that Syndrome Protein (WASP)-family verprolin-homologous protein served as the immunogen (27), anti-IRSp53 polyclonal rabbit (WAVE)2, vasodilator-stimulated phosphoprotein (Mena/VASP), antibody was kindly provided by Dr. Jeonghoon Choi (from KAIST, Eps8, mDIA, espin, dentatorubral-pallidoluysian atrophy protein South Korea), anti-HA tagged epitope monoclonal antibody 16B12 (DRLPA), Shank-1, synaptopodin. These SH3-mediated interactions (Covance, Berkeley, CA, USA), anti-HA high affinity rat monoclonal are crucial for recruiting F-actin regulators to control cortical actin antibody (3F10, Roche, Germany), anti-GFP polyclonal antibody dynamics, and the various binding partners lend strong support to (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Myc (9E10, the signaling, scaffolding and adaptor properties of IRSp53 [16].In Cell Signaling Technology, MA, USA) and anti-Rac1 clone 23A8 addition, it was recently suggested that IRSp53 stimulates filopodia (Upstate, NY, USA) were purchased. formation by coupling membrane protrusion with actin dynamics via a new SH3 domain binding partner, N-WASP [25]. However, the Co-immunoprecipitation molecular mechanism by which IRSp53 mediates lamellipodia extension or filopodia formation, and even in the signaling Cells were washed with cold PBS (phosphate-buffered saline) and network involved, remains unclear. extracted for 1 h at 4 °C in extraction buffer (50 mM Tris-HCl, We previously demonstrated that SPIN90 is a widely expressed pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 10 mM NaF, and 1 mM

NCK-binding protein that contains an SH3 domain, SH3-binding Na3VO4) supplemented with protease inhibitors. The cell extracts proline-rich region (PRD) and a hydrophobic C-terminal domain, were then clarified by centrifugation for 10 min at 12,000x g and and that it binds proteins involved in regulating actin cytoskeletal subjected to immunoprecipitation overnight. The immunopreci- networks, such as those involving Nck, βPix, WASP and dynamin pitates were incubated for 4 h at 4 °C with protein A/G-Sepharose [26]. SPIN90 is also known to be involved in the activation of the beads (Amersham Pharmacia Biotech, CA, USA), extensively Arp2/3 complex, and treating COS-7 cells with PDGF causes washed with the same extraction buffer and immunoblotted with redistribution of SPIN90 to the cell cortex and formation of respective antibodies. lamellipodia (or membrane ruffles) [27]. Moreover, DIP (mDia interacting protein), which is SPIN90 under a different name, COS-7 cell culture and transfection reportedly regulates Rho GTPase activity (Rho and Rac) in a Src- dependent manner [28,29]. WISH, an N-WASP-binding protein, is a COS-7 cells were grown in Dulbecco's modified Eagle's medium mouse ortholog of SPIN90 that enhances N-WASP-dependent and (DMEM) supplemented with 10% FBS (fetal bovine serum) and -independent activation of the Arp2/3 complex, resulting in rapid 100 U/ml antibiotic/antimycotic and maintained at 37 °C in 5% actin polymerization [30]. CO2. The plasmids constructs were transfected into the cells using Their multiple binding domains enable both SPIN90 and IRSp53 Lipofectamine LTX according to the manufacturer's protocol to mediate multimolecular interactions and to contribute to actin- (Invitrogen, CA, USA). based membrane protrusion. Thus an understanding of SPIN90- IRSp53 is necessary if one is to understand the regulation of Immunofluorescence membrane dynamics underlying cell motility. In the present study, we found that SPIN90 binds IRSp53 via a PRD-SH3 interaction and COS-7 cells were washed three times with phosphate-buffered that it participates in Rac-induced membrane ruffling. saline (PBS) containing 1 mM CaCl2 and 1 mM MgCl2, fixed for 10 min in 3.5% paraformaldehyde, and then permeabilized for 10 min in 0.2% Triton X-100. Once permeabilized, the cells were incubated at Materials and methods room temperature with primary antibody for 1 h and then with Alexa Fluor-conjugated secondary antibodies (Invitrogen, CA, USA) Plasmid construction and antibodies for an additional 40 min. To assess the cytoskeleton, filamentous actin was labeled with Texas Red-phalloidin (Molecular Probes, cDNAs encoding full length SPIN90 (amino acids 1–722) and Eugene, OR, USA), and visualized using Leica DMRBE microscope variant deletion mutant (PRD, Proline rich region, amino acids equipped with a 63x (1.4NA) oil objective and fluoresceine 146–279) were subcloned into hemagglutinin (HA) pcDNA 3.0 isothiocyanate- or Texas Red-optimized filter sets (OmegaR Optical (Invitrogen, CA, USA), pEGFP C1 vector (Clontech, USA) or Inc, Brattleboro, VT, USA). Images were acquired using a CoolSNAP ™ pGEX4T-1 (Amersham Pharmacia Biotech, CA, USA). Full length fx CCD camera driven by a MetaMorph imaging software (Universal SPIN90 was subcloned into pCS2-MT-6Myc vector. HA-IRSp53 WT Imaging Co, Downingtown, PA, USA). In some cases, cells were and HA-IRSp53 SH3 mutant (I403P) plasmids were a gift from Dr. serum-starved for 18 h in serum-free DMEM and then exposed to Jeonghoon Choi (from KAIST, South Korea). IRSp53 SH3 mutant 50 ng/ml PDGF (Sigma, USA) for indicated times. For RNA 2412 EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419 interference experiments, the SPIN90-targeting si-824 psiRNA- the cells starved for 24 h. To test the endogeneous active Rac1 level, hH1GFPzeo G2 vector was used to knockdown SPIN90 and the a pull-down assay with GST-PAK-PBD was used because the PBD empty vector was used as control. Plasmids were transfected in COS- motif of PAK selectively binds activated Rac. Cell lysates expressing 7 cells for 72 h using lipofectamine with Plus reagent (Invitrogen, CA, various plasmid constructs were mixed with 60 μg of GST-PAK-PBD USA). SPIN90 siRNAs were previously described [27]. immobilized on Glutathione beads in Rac assay buffer (25 mM

Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1% Nonidet P-40, 1 mM Pull-down assays DDT, 5% glycerol) supplemented with protease inhibitors. After 45 min rotation at 4 °C, the beads were washed and bound proteins GST pull-down assays were performed by mixing the GST fusion were analyzed. proteins bounded on Glutathione Agarose 4B (Peptron, South Korea) beads with cell lysates expressing different type of plasmid constructs. After 4 h incubation with rotation at 4 °C in binding Results buffer (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.2% Nonidet P-40) supplemented with protease inhibitors, the beads Identification of proteins associating with SPIN90 were washed four times with the same buffer and the bounded proteins were subjected to SDS-PAGE. The separated proteins were Using BioGRIDbeta (General Repository for Interaction Datasets, stained with Coomassie Brilliant Blue or transferred to polyviny- http://www.thebiogrid.org), we were able to identify several lidene difluoride membranes (Bio-Rad, USA) for western blotting proteins (GRB2, NCK2, DMRTB1, NCK1, BAIAP2) that associate with analysis. SPIN90 [NCKIPSD (NCK-interacting protein with SH3 domain)] [32]. Among these proteins, we selected BAIAP2 (IRSp53) for Rac activation assay further study because of its association with lamellipodia formation (Fig. 1A). To test whether SPIN90 binds IRSp53, we initially Rac assay was performed as Funato et al. with minor modifications carried out an immunoprecipitation analysis using COS-7 cells (31). COS-7 cells were transfected with 10 μg of plasmid in a transiently cotransfected with Myc-SPIN90 WT (wild type) and/or 150 mm dish. 18 h after transfection, the medium was replaced and HA-IRSp53 WT (wild type). We found that Myc-SPIN90 WT co-

Fig. 1 – SPIN90 interacts with IRSp53 in vivo. (A) BioGRIDbeta (a General Repository for Interaction Datasets, http://www.thebiogrid. org) was used to identify several SPIN90 binding proteins, including BAIAP2 (IRSp53). (B) Schematic diagram of the domain structures of IRSp53 and SPIN90. (C) COS-7 cells were transfected with Myc-SPIN90 WT and/or HA-IRSp53 WT. The lysates (500 μg) were immunoprecipitated with anti-Myc antibody and blotted with anti-HA or anti-Myc antibodies. (D) COS-7 cell lysates (2000 μg) were precipitated with anti-IRSp53 antibody or preimmune rabbit immunoglobulin (Rb IgG) and blotted with anti-IRSp53 and SPIN90 antibodies. RCB; Rac1 binding domain, SH3; Src homology 3 domain, CRIB; a half Cdc-42/Rac interactive binding motif, PRD; proline-rich domain. EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419 2413 precipitated with HA-ISRp53 WT but not with control vector (Fig. SPIN90 colocalizes with IRSp53 at membrane ruffles after 1C). Moreover, subsequent analysis confirmed that endogenous PDGF treatment SPIN90 interacts with IRSp53, suggesting that SPIN90 participates in IRSp53-mediated signaling via protein–protein interactions (Fig. To investigate the cellular localization of SPIN90, we next 1D), most likely between the PRD of SPIN90 and the SH3 domain of characterized the distribution of SPIN90 and IRSp53 within COS- IRSp53 (Fig. 1B). 7 cells. We found that in unstimulated cells endogenous SPIN90 as

Fig. 2 – SPIN90 colocalizes with IRSp53 at membrane ruffles after PDGF treatment. (A) COS-7 cells were serum-starved for 16 h, treated with PDGF (50 ng/ml) for 5 min and then immunostained with mouse anti-SPIN90 or rabbit anti-IRSp53 antibodies. Cell images were obtained using widefield fluorescence microscopy. (B) COS-7 cells transfected with GFP-SPIN90 or HA-IRSp53 were serum-starved for 16 h and then treated with PDGF for 5 min. SPIN90 was detected with anti-GFP antibody and IRSp53 with anti-HA antibody and processed for examination under fluorescence microscope. Arrows (A and B) indicate membrane ruffles. (C) COS-7 cells with or without PDGF treatment were double-stained with mouse anti-SPIN90 and rabbit anti-IRSp53 antibodies. (D) Cells cotransfected with GFP-SPIN90 and HA-IRSp53 were treated with or without PDGF and processed as in (B). Arrows (C and D) indicate colocalization of SPIN90 and IRSp53 at membrane ruffles. (E) PDGF-stimulated COS-7 cells were cotransfected with GFP-SPIN90 siRNA and HA-IRSp53. Cells transfected with GFP-SPIN90 siRNA were stained with anti-GFP antibody, HA-IRSp53 proteins were seen with anti-HA antibody and actin filaments were stained with labeled phalloidin. Arrows indicate the reduced membrane ruffle formation. Scale bars represent 5 μm. 2414 EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419 well GFP-SPIN90 WT was widely distributed throughout the exhibited moderate lamellipodia formation or membrane ruffling, cytoplasm, and that treating the cells with PDGF caused the which was also described previously (Fig. 2B) [33]. When SPIN90 redistribution of SPIN90 to the membrane ruffles, as we reported and IRSp53 were co-expressed in COS-7 cells, a moderate amount previously (Figs. 2A and B) [27]. IRSp53 was detected in the of SPIN90 was translocated to the membrane, where it colocalized cytoplasm and a significant portion of IRSp53 was also found at with IRSp53, even in the absence of PDGF treatment. PDGF- lamellipodia in untreated COS-7 cells with an increased localiza- induced a much stronger redistribution of SPIN90 to the tion in this area after PDGF treatment (Fig. 2A). After PDGF membrane, where it again colocalized with IRSp53 (Fig. 2D). treatment, SPIN90 and IRSp53 colocalized at membrane ruffles of Conversely, transfection of COS-7 cells with siRNAs targeting COS-7 cells (Fig. 2C). COS-7 cells expressing HA-IRSp53 WT alone SPIN90 inhibited lamellipodia formation, even with PDGF EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419 2415 treatment and localization of IRSp53 in the membrane ruffles was Rac) bound to GST beads with lysates from COS-7 cells expressing dramatically diminished (Fig. 2E). In these cells, strong stress Myc-SPIN90 WT and various amounts of Myc-IRSp53 WT, and then fibers were induced, which might be a consequence of a carried out GST pull-down assays. We found that SPIN90 co- subsequent Rac and Rho activation by growth factors like PDGF precipitated with the active Rac1, and that the amount of SPIN90 as previously reported [4,34,35]. precipitated increased with increasing expression of IRSp53 protein. Thus IRSp53 appears to act as an intermediary link SPIN90 plays a key role in Rac-induced membrane ruffling between SPIN90 and active Rac1 (Fig. 3D). We did not confirm the direct interaction between SPIN90 and active Rac. In fact, SPIN90 Activated Rac is known to strongly induce membrane ruffling in does not have Rac1 binding domain. fibroblasts [34]. Similarly, we observed that transfecting COS-7 cells with Rac1G12V, a dominant active Rac mutant, led to The role of the SPIN90–IRSp53 complex in Rac-induced prominent membrane ruffling and the accumulation of membrane ruffling Rac1G12V in the ruffled areas (Fig. 3A). In parallel experiments, we co-expressed GFP-IRSp53 WT or GFP-SPIN90 WT with HA- To assess the importance to membrane ruffling of the relation Rac1G12V in COS-7 cells, and found that the ectopically expressed between SPIN90 and IRSp53, we initially confirmed the direct IRSp53 or SPIN90 colocalizes with active Rac at membrane ruffles interaction between SPIN90 and IRSp53. SPIN90 PRD readily (Figs. 3A and B). The introduction to SPIN90 siRNA into COS-7 cells bound to IRSp53 WT, but the binding was strongly impaired by expressing active Rac (Rac1G12V) dramatically inhibited Rac- an I403P substitution within the SH3 domain of IRSp53 (Fig. 4A). In induced membrane ruffles but reintroduction of SPIN90 WT addition, a pull-down assay showed that the interaction of SPIN90 rescued the phenotype (Figs. 3B and C). To assess complex and IRSp53 was apparent in the absence of SPIN90 PRD but became formation of these three proteins, we mixed Rac1Q61L (active interrupted when SPIN90 PRD was expressed (Fig. 4B). Apparently,

Fig. 3 – SPIN90 plays a key role in Rac-induced membrane ruffling. (A) COS-7 cells transfected with HA-Rac1G12V were stained with anti-HA antibodies to detect active Rac and with labeled phalloidin to visualize the actin filaments. COS-7 cells co-expressing HA-Rac1G12V and GFP-IRSp53 WT were stained with anti-HA antibody for the active Rac and with anti-GFP antibody to detect IRSp53. Arrows indicate Rac-induced membrane ruffles (top row) and colocalization of active Rac and IRSp53 at membrane ruffles (bottom row). (B) COS-7 cells co-expressing HA-Rac1G12V and GFP-SPIN90 WT or GFP-SPIN90 siRNA were stained as in (A). Ectopically expressed SPIN90 colocalized with active Rac at membrane ruffles (arrows; top row), but introduction of SPIN90 siRNA into COS-7 cells expressing active Rac dramatically inhibited Rac-induced membrane ruffles (arrows; bottom row). (C) Cells were triple transfected with HA-Rac1G12V, GFP-SPIN90 siRNA and Myc-SPIN90 WT and stained with anti-HA antibody to detect active Rac, anti-GFP antibody to detect cells transfected with GFP-SPIN90 siRNA construct and anti-Myc antibody to detect SPIN90 WT. In parallel staining experiments, cells were labeled with phalloidin to visualize actin filaments. Arrows indicate membrane ruffles. Scale bar represents 5 μm. (D) GST beads only or beads coupled with GST-Rac1Q61L were incubated with lysates from COS-7 cells expressing Myc-SPIN90 WT and various levels of Myc-IRSp53 WT. The bound proteins were subjected to immunoblot analysis with anti-Myc antibody. Histograms represent the intensity ratio of Myc-SPIN90 bound to active Rac and intensity ratio of Myc-IRSp53 expression level in the cell lysates. 2416 EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419

SPIN90 binds to the SH3 domain of IRSp53 via its PRD. We then that in mind, we focused on the role played by the SPIN90–IRSp53 used SPIN90 PRD as a competitive inhibitor to assess the complex in the regulation of Rac. To assess endogenous Rac activa- importance of SPIN90–IRSp53 binding to Rac-induced membrane tion in the presence of SPIN90 and IRSp53, we carried out pull-down ruffling. The expression of Rac1G12V in COS-7 cells induced robust assays using GST-PAK-PBD, as the PBD motif of PAK is known to bind ruffle formation, but co-expression with SPIN90 PRD significantly activated Rac. We detected no significant Rac activation in lysates inhibited membrane ruffling, suggesting the SPIN90–IRSp53 from COS-7 cells expressing Myc-vector only, Myc-SPIN90 WT or complex plays a key role in that process (Fig. 4C). Myc-IRSp53 WT. Notably, however, when the two proteins were co-expressed, the amount of precipitated Rac was dramatically SPIN90 and IRSp53 cooperates to activate Rac increased (Fig. 5A). Conversely, the co-expression of Myc-SPIN90 WT with Myc-IRSp53 SH3 mutant (I403P) defective in SPIN90 binding, DIP (SPIN90) mediates Src kinase-dependent activation of Rac in the failed to induce Rac activation (Fig. 5B). These results suggest that membrane fraction following growth factor stimulation [29].With Rac is positively regulated by the SPIN90–IRSp53 complex.

Fig. 4 – The SPIN90–IRSp53 interaction is necessary for Rac-induced membrane ruffling. (A) COS-7 cells transfected with HA-IRSp53 WT or HA-IRSp53 SH3 mutant with substitution of isoleucine with proline (I403P) were pulled down using GST-SPIN90 PRD and immunoblotted with anti-HA antibody. (B) GST pull-down assays using GST beads only or beads coupled with GST-IRSp53 WT were performed with lysates from COS-7 cells expressing HA-SPIN90 WT with or without HA-SPIN90 PRD in dose dependent manner. The bound proteins were visualized with appropriate antibodies. (C) Activated Rac (HA-Rac1G12V) was expressed alone or together with GFP-SPIN90 PRD in COS-7 cells. Images shown are the distributions of SPIN90 PRD detected with anti-GFP antibody, active Rac detected with anti-HA antibody and actin filaments visualized with labeled phalloidin. Arrows marked membrane ruffles induced by active Rac expression (top row) and inhibition of Rac-induced membrane ruffles by co-expression of SPIN90 PRD (bottom row). Scale bar represents 5 μm. EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419 2417

effector proteins capable of signaling to the actin cytoskeleton Discussion [1,34]. We demonstrated in the present study that the SPIN90– IRSp53 complex likely coordinates the activity of cytoskeletal SPIN90 has been shown to play a key role in actin dynamics and scaffolding proteins to promote the actin remodeling underlying PDGF-induced lamellipodia formation in COS-7 cells [27]. This led membrane ruffle formation. It has been suggested, for example, us to hypothesize that SPIN90 regulates actin rearrangement and that Wave/Scars, a member of the WASP family, is involved in might act in concert with Rac to mediate membrane ruffling. To membrane ruffle formation, acting downstream of Rac, and that test that idea, we examined the actions of SPIN90 in mediating IRSp53 binds both Rac and WAVE2, thereby inducing formation of cellular protrusions such as membrane ruffling. In unstimulated a Rac/IRSp53/WAVE2 complex which is important in membrane cells SPIN90 was widely distributed throughout the cytoplasm and ruffling [11,17]. It has also been demonstrated that the IRSp53- the induction of membrane ruffling by PDGF led to the colocaliza- WAVE2 complex has an enhanced affinity for Rac, which appears to tion of SPIN90 and IRSp53 at the membrane ruffles, but molecular reflect a conformational change in IRSp53 induced by WAVE2- genetic inhibition of SPIN90 using siRNA significantly reduced binding [20]. There is, so far, no evidence of a direct interaction membrane ruffling and diminished localization of IRSp53 at those between Rac and WAVE2. sites. Moreover, SPIN90 and IRSp53 colocalized with active Rac at On the other hand, another group has shown that IRSp53 acts membrane ruffles and the downregulation of SPIN90 by siRNA downstream of Cdc42, not Rac1, and that GTP-loaded Cdc42 binds inhibited Rac-induced membrane ruffling. Thus, SPIN90/IRSp53 to IRSp53's CRIB motif to mediate filopodia formation in fibroblasts complex apparently participates in Rac-induced membrane ruf- and neuronal cells [18,19]. IRSp53 would thus seem to link fling, and SPIN90 and IRSp53 likely act together to regulate Rac activated Rac1 or Cdc42 to downstream effectors through its SH3 activity at the leading edge of migrating cells. domain. Alternatively, by acting both upstream and downstream of Membrane protrusions, such as ruffling formation, are caused Rac, IRSp53 may be a crucial molecule involved in generating a by actin remodeling mediated by Rac and a number of other positive feedback loop that underlies the sustained activation

Fig. 5 – Cooperative activation of Rac by SPIN90 and IRSp53. (A) Active Rac was isolated using GST-PAK-PBD immobilized on GST beads from starved COS-7 cell lysates that express Myc-vector, Myc-SPIN90 WT and/or Myc-IRSp53 WT. Precipitated active Rac (Rac-GTP) and total Rac were immunoblotted with anti-Rac antibody. (B) The active Rac was investigated in COS-7 cell expressing Myc-vector, Myc-SPIN90 WT and/or Myc-IRSp53 SH3 mutant (I403P). The immunoprecipitates and immunoblots were performed as in (A). 2418 EXPERIMENTAL CELL RESEARCH 315 (2009) 2410– 2419

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