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Vol. 18, Issue 1, 253-264, January 2007
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PIX
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Departments of *Anesthesiology and Critical Care Medicine, Biomedical Engineering, Cell Biology, and ||Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287; and School of Biological Sciences, Seoul National University, Seoul 151-742, South Korea
Submitted March 17, 2006;
Revised October 25, 2006;
Accepted October 27, 2006
Monitoring Editor: Carole Parent
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PIX and thereby increased its binding to Rac1. In addition, PIX facilitated the targeting of activated Rac1 to focal adhesions and the efficiency of cell spreading. These data indicate that FAK has a role in the activation and focal adhesion translocation of Rac1 through the tyrosine phosphorylation of PIX. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Schaller et al., 1992; Romer et al., 2006). Autophosphorylation of FAK at Tyr-397 creates a binding site for Src. After binding to FAK, Src phosphorylates FAK on several other tyrosine residues, including Tyr-925 and Tyr-576/577 to achieve full FAK activation and scaffolding potential (Schlaepfer et al., 1999; Schaller 2001). Tyr-576 and -577 are in the FAK kinase activation loop, and phosphorylation on these sites enhances catalytic activity (Ruest et al., 2000). Phosphorylation on Tyr-925 induces the recruitment of Grb2 and promotes the activation of the Ras/Raf/MEK/ERK pathway (Schlaepfer et al., 1997). In addition to Src, phosphorylation on FAK Tyr-397 also induces the recruitment of Shc and p130CAS to focal adhesions (Schlaepfer et al., 1997; Takahashi et al., 1999). Src and FAK also directly mediate the tyrosine phosphorylation of p130CAS and paxillin, leading in turn to the recruitment of Crk and Nck and the assembly of multiphosphocomponent signaling complexes at focal adhesions (Schaller and Parsons 1995; Schlaepfer et al., 1999; Turner, 2000; Romer et al., 2006).
FAK's role in cell spreading has been investigated since reports that FAK null fibroblasts from knockout mice exhibited similar plating efficiency but poor spreading when compared with normal controls (Ilic et al., 1995). Reexpression of FAK in the FAK null cells restores their ability to spread on fibronectin (Owen et al., 1999; Sieg et al., 1999), whereas overexpression of either the dominant negative FAK protein FRNK, or the FAK-inactivating phosphatases PTEN or Shp-2, results in delayed or impaired cell spreading (Richardson and Parsons 1996; Gu et al., 1998; Yu et al., 1998). FAK was once thought to be a direct catalyst for the formation of focal adhesions—anchor points for cell motility (Burridge et al., 1992; Richardson et al., 1997). Later it was discovered that FAK suppresses Rho activity and promotes focal adhesion turnover (Ren et al., 2000; Schaller, 2001; Ezratty et al., 2005).
Cell spreading is regulated by coordinated changes in integrin-mediated adhesions to ECM and reorganization of the actin cytoskeleton by Rho family GTP-binding proteins (Machesky and Hall 1997; Nobes and Hall, 1999; Clark et al., 1998; Ren et al., 1999; Hall, 2005). Rac1 and Cdc42 promote actin polymerization, inducing the formation of lamellipodia and filopodia respectively to drive extension of the leading cell edge. On the other hand, Rho-regulated myosin-dependent contractile force is transiently reduced by mechanisms involving Src, FAK, and the Rho inactivating protein p190RhoGAP (Ren et al., 1999, 2000; Arthur et al., 2000). Arthur et al. (2000) demonstrated that p190RhoGAP is tyrosine-phosphorylated in a Src-dependent manner, and tyrosine phosphorylation of p190RhoGAP by FAK has been shown in vitro by Holinstat et al. (2006). Thus, Src and FAK inactivate RhoA activity via p190RhoGAP after integrin-mediated adhesion. This "relaxes" cytoskeletal tension, allowing the formation of membrane extensions.
The Rho family GTPases cycle between inactive GDP-bound and active GTP-bound forms and their activation is mediated by guanine nucleotide exchange factors (GEFs; Hall, 2005). FAK interactions with Rac-activation mechanisms that may positively mobilize cell spreading are still incompletely understood and may proceed along multiple pathways. Thus, Hsia and coworkers showed that viral Src transformation does not fully restore Rac-dependent invasive behavior in FAK null cells. In fact, the transient accumulation of FAK at the lamellipodia of FAK-expressing fibroblasts is associated with the formation of a signaling complex with Src, p130CAS, and Dock180 and elevation of both Rac and JNK activity (Hsia et al., 2003). Rac1 function is positively regulated by PIX and is modulated by PAK. PIX (PAK-interacting exchange factor)/Cool (cloned out of library) protein was identified as a PAK-binding protein (Manser et al., 1998). Overexpression of PIX drives formation of membrane ruffles via activation of Rac1. Targets for PAK on PIX are S525 and T526. After PAK-mediated phosphorylation, PIX specifically localizes to lamellipodia at neuronal growth cones in response to bFGF, and mutation of both S525 and T526 to alanines causes defective lamellipodial targeting (Shin et al., 2002). Shin et al. (2004) also showed that Rac activation induced by PIX was increased after phosphorylation on S525 and T526 by PAK. In addition to PAK, PIX has been shown to bind to the ArfGAP family protein paxillin kinase linker (PKL; Turner et al., 1999) and to the G protein–coupled receptor kinase-interacting protein GIT-1 (Zhao et al., 2000), which both interact with paxillin (Turner et al., 1999; Zhao et al., 2000; West et al., 2001). Mechanisms of PIX targeting and turnover at focal adhesions and the implications for Rac function are foci of intense investigation and debate (Brown et al., 2002; ten Klooster et al., 2006).
Our results delineate a signaling pathway directly linking FAK to Rac1 activation and Rac1-mediated cell spreading via PIX. We quantitate the positive impact of FAK expression on cell spreading and Rac1 activation and targeting. We show that FAK can tyrosine-phosphorylate PIX and increase its binding to Rac1. FAK-facilitated activation of Rac1 via PIX may be an important mechanism for the promotion of cell spreading.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and were maintained in Dulbecco's modified medium (DMEM; Sigma, St. Louis, MO) that was supplemented with 10% fetal bovine serum (Atlanta Biological, Atlanta, GA), 2 mM L-glutamine (Invitrogen-BRL, Grand Island, NY), penicillin G, streptomycin, and amphotericin B (Invitrogen-BRL). The same medium was used for mouse embryo fibroblasts (MEFs) that were obtained from ATCC (Rockville, MD). Mouse fibroblasts homozygous for the deletion of genes for Src, Yes, and Fyn (SYF) were a generous gift from Phillippe Soriano (Fred Hutchinson Cancer Center, Seattle, WA) and were maintained in the same medium. GFP chimeras of L61Rac1 and N17Rac1 were gifts from Dr. Klaus Hahn (University of North Carolina at Chapel Hill). Flag-tagged PIX constructs, including the wild-type and a doubly mutated PIX that neither functions as a GEF nor binds to PAK (PIX-SH3/DH: W43J, L238R, L239S) were made as described previously (Shin et al., 2004), as was PIX-GST (ten Klooster et al., 2006). EGFP-FAK was made in the Romer lab as described elsewhere (Cooley et al., 2000). The avian FAK kinase domain (amino acids 411-686) and a kinase dead (K454R) mutant were generated in baculovirus-transfected Hifive insect cells, and were generously provided by Michael Eck, Dana-Farber Cancer Institute (Harvard University, Boston, MA). Myristylated FAK was a gift from Dr. Silvio Gutkind (National Institutes of Health, Bethesda, MD). The dominant negative FAK construct Dter is an 18-kDa truncation mutant of the FAK carboxy-terminus and was a gift from Dr. Mike Schaller (UNC, Chapel Hill, NC; Thomas et al., 1999; Prutzman et al., 2004). Transient transfections were accomplished using Lipofectamine Plus (Invitrogen-BRL) and standard product protocols. Briefly, cells were plated 24 h before transfection. Cells were washed and then incubated for 3 h in serum-free DMEM medium containing plasmid DNA mixed with Lipofectamine and Plus reagent. The medium was then replaced with DMEM containing 10% FBS and incubated for 48 h before cells were prepared for immunofluorescence or immune replica analysis.
Cell-spreading Assay
Phase-contrast movies were made by plating cells onto fibronectin-coated 35-mm glass bottom microwell dishes (Plastek Cultureware, Ashland, MA). Phase-contrast images were acquired at 1-min intervals up to 50 min after initial plating using a Nikon TE200 microscope (Melville, NY) a Coolsnap HQ CCD camera (Roper, Duluth, GA), and Openlab software (Improvision, Lexington, MA), and converted to QuickTime movie format. Cell spreading rates were determined by measuring cell area as a function of time. Coverslips (no. 1, Warner Instruments, Hamden, CT) were incubated with 25 µg/ml fibronectin in PBS (Invitrogen-BRL) at 4°C overnight and then assembled into flow chambers (Warner). Cells were plated onto coverslips in standard culture medium and were observed until initial attachment was achieved. Cells were then perfused with standard culture media at a flow rate of 
2 ml/min. For all work with live cells, temperature was maintained at 37°C in the chamber with both an inline fluid heater (Ismatec, Boston, MA) and an objective heater (Bioptechs, Eugene, OR). An original MATLAB code (Mathworks, Natick, MA) was used to determine cell area from each phase-contrast image. A Gaussian smoothing filter was applied to each image to remove any sudden intensity changes due to noise. A Laplacian transformation was then applied to the smoothed image to determine points of sudden intensity change, which correlate with the cell edge. The result was a binary image of the cell edge. The area contained within this edge was calculated and converted to units of area (µm2).
Antibodies
Primary antibodies used in this study include the following: Anti-FAK polyclonal antisera 5158 (made by the Romer lab); anti-Flag polyclonal clone F7425 (Sigma, St. Louis, MO); anti-talin polyclonal rabbit antisera TnC22 (a gift from Dr. Susan Craig, Johns Hopkins University, Baltimore, MD); monoclonal anti-Rac1 clones 23A8 (Upstate, Charlottesville, VA) and 102 (BD Biosciences, San Diego CA); polyclonal AB3829 against PIX (Chemicon, Temecula, CA); anti-integrin 1 rat monoclonal clone 9EG7 (BD Biosciences); monoclonal anti-vinculin clone 7F9 (a gift from Dr. Alexey Belkin, Holland Labs, Rockville, MD); polyclonal ab6556 rabbit antisera against GFP (Abcam, Cambridge, MA); rabbit anti-GST polyclonal antibody (Invitrogen), and monoclonal anti-phosphotyrosine clone py20 (BD Biosciences). Affinity cross- adsorbed secondary antibodies including rhodamine-conjugated donkey anti-Rat IgG, Cy3-conjugated goat anti-mouse and goat anti-rabbit IgG, Cy5-conjugated goat anti-mouse and goat anti-rabbit IgG, and FITC-conjugated goat anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). FITC-conjugated donkey anti-mouse IgG was purchased from Chemicon (Temecula, CA). Rhodamine-conjugated phalloidin for actin staining was purchased from Molecular Probes (Eugene, OR). Horseradish peroxidase–conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from ICN Biochemicals (Costa Mesa, CA).
Immunofluorescence Staining and Epifluorescence Microscopy
Cells were plated on FN-coated glass coverslips for 2 h. Cells were then permeabilized for 2 min with 0.5% Triton X-100 (Fisher Scientific, Hampton, NH) in 3% paraformaldehyde (Sigma) followed by fixation with 3% paraformaldehyde for 20 min. Antibody incubations were done for 30 min. Cells were observed on an epifluorescence Nikon TE-200 microscope. Images were captured with a Coolsnap HQ camera (Roper) with Openlab software (Improvision).
Analysis of Rac1 Localization at Focal Adhesions
Images of endogenous or ectopically expressed Rac1 or the focal adhesion proteins (vinculin, talin, or 1 integrin) were acquired with Openlab software as described above. The percentage of focal adhesion areas occupied by Rac1 was determined using an original automation. First, a binary mask corresponding to total focal adhesion area was made from the vinculin or talin or 1 integrin images, and the total focal adhesion area was calculated. Then, a mask corresponding to regions of Rac1 localization at focal adhesions was made. The ratio of the two binary masks yielded the percentage of focal adhesion area showing Rac1 colocalization. Optimal spectral separation of Rac1 and other focal adhesion protein detection was achieved when possible by using Cy5-conjugated secondary antibody to label vinculin in cells expressing Rac-GFP chimeras and by using FITC-conjugated secondary antibody to label talin for samples in which endogenous Rac1 was identified using Cy5-conjugated antibodies. In cells transfected with PIX-Flag, FITC-conjugated secondary antibody was used to label Flag and identify transfected cells, whereas Cy3- and Cy5-conjugated secondary antibodies were used to label 1 integrin and Rac1, respectively.
Rac1 Activity Assay and Western Blotting
The cDNA of the p21-binding domain (PBD) from human PAK1 (amino acids 67-150; generous gift from Dr. Keith Burridge, University of North Carolina at Chapel Hill) that had been cloned into the bacterial expression vector pGEX-4T3 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. This PBD-GST fusion protein was purified and immobilized on glutathione-Sepharose beads (Benard et al., 1999). Cells were washed with ice-cold HEPES buffer and then lysed with lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 0.5 mM MgCl2, 1% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, 0.5 mM sodium vanadate). Cell lysates were clarified by centrifugation at 20,000 x g at 4°C for 4 min. GST-PBD, 50 µg, immobilized on glutathione-Sepharose beads was added to 500 µg of protein from cell lysates and incubated at 4°C with rotation for 60 min. The beads were then washed three times with lysis buffer and boiled in Laemmli sample buffer (Laemmli et al., 1970). GST-PBD bound active Rac1 (in the GTP-bound form) was detected by Western blotting using an mAb against Rac1. Total Rac1 was detected by immunoblotting in samples from corresponding cell lysates. For Western blot analysis, after blocking with 1% BSA in TBST (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Tween 20) overnight, membranes were incubated with appropriate primary antibodies for 1 h at room temperature, washed three times with TBST, and incubated with horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit IgG. Proteins were visualized using chemiluminescence (Amersham Biosciences, Piscataway, NJ) and exposure to x-ray film (Eastman Kodak, Rochester, NY). To quantify relative Rac1 activity, a clear image of the autoradiograph was taken using an Epson Perfection scanner (model 2450; Long Beach, CA). The intensity of the bands was then analyzed and normalized to the total cellular Rac protein band using a software-based algorithm in NIH image (version 1.62). Rac1 activity of FAK– cells at 0 min was designated as 100%.
In Vitro FAK Kinase Assay
Full-length murine PIX cDNA (generous gift of Peter Hordijk, Sanquin Research and Landsteiner Laboratorium, University of Amsterdam, The Netherlands) was cloned into the bacterial expression vector pGET-4T3 and expressed in E. coli BL21 (Stratagene, La Jolla, CA). The PIX-GST fusion protein was purified from bacterial lysates, immobilized on glutathione-Sepharose beads, and then released with elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM reduced glutathione; Sigma). Cells were transfected with either full-length FAK or the truncated carboxy-terminus FAK mutant Dter that lacks the kinase domain. Both FAK constructs were EGFP-tagged. 48 h after transfection protein lysates were obtained using modified RIPA buffer (0.1% DOC, 0.1% Triton X-100, 2 mM EDTA, 1 mM PMSF, 2 mM sodium vanadate, 20 µg/ml leupeptin, 20 µg/ml aprotinin in PBS). EGFP-FAK or EGFP-Dter were immunoprecipitated with anti-GFP antibody from 500 µg of protein lysates for 2 h and captured on protein A-Sepharose by rotation for 1 h at 4°C. After four washes in lysis buffer EGFP-FAK or EGFP-Dter immunoprecipitates were resuspended in kinase buffer (50 mM Tris, pH 7.4, 5 mM MnCl2, and 5 mM MgCl2). Approximately 2 µg of purified PIX-GST and 20 µM of ATP (final concentration) were added to the beads as substrate to a total volume of 60 µl and incubated at 37°C for 30 min on a shaker to keep the beads in suspension. In other experiments, aliquots of GST- wtFAK411-686 or GST-FAK411-686K454R (1, 3, or 6 µg) were added to 2 µg of purified GST-PIX or 4 µg of purified GST in kinase buffer with or without 20 µM of ATP (final concentration) in a total volume of 60 µl and incubated at 37°C for 30 min. The kinase reactions were stopped by the addition of sample buffer and analyzed using SDS-PAGE, transfer to nitrocellulose, and serial immunoblotting with antibodies against GFP (or GST), PIX, and phosphotyrosine. Some experiments were done in the presence of the Src inhibitor PP2 (10 µM; EMD Biosciences, San Diego, CA).
In Vitro Binding Assay for PIX and Rac1
FAK– cells were cotransfected with PIX-Flag and either EGFP-Dter, EGFP-FAK, or myristylated FAK. Two days after transfection, cells were washed with ice-cold HEPES buffer and then lysed with modified RIPA buffer. Cell lysates were clarified by centrifugation at 20,000 x g at 4°C for 4 min. Lysate volumes were then normalized for equal protein content using the bicinchoninic assay (Pierce, Rockford, IL). To immunoprecipitate PIX-Flag, lysates containing 500 µg of protein were equalized for volume with lysis buffer and incubated at 4°C with rotation first with anti-Flag antibody and then with protein A-Sepharose beads (Jackson). The beads with the bound, immunoprecipitated PIX-Flag were then washed four times with lysis buffer before adding 5 µg of purified GST-Rac1 (gift of Ian Macara, University of Virginia at Charlottesville). These mixtures were then incubated for 3 h or overnight at 4°C with rotation. Beads were then washed again with lysis buffer. Proteins were released from the beads by boiling in Laemmli sample buffer and subjected to SDS-PAGE using 4–15% gradient polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes for Western blotting (Towbin et al., 1979). The membranes were probed serially using primary antibodies against Rac1, phosphotyrosine, and the Flag epitope tag. Each of these was followed by the appropriate horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG secondary antibody. Processing for chemiluminescence was as detailed above. Before reprobing with different antibodies, membranes were immersed in stripping buffer (2% SDS, 0.6% -mercaptoethanol 
, 62.5 mM Tris-Cl, pH 6.8) at 60°C for 30 min with frequent shaking, and washed thoroughly with TBST.
Statistical Analysis
The Student's t test was used to analyze Rac1 activation assay data and the effects of altered PIX expression on cell surface area. The Mann-Whitney test was used to evaluate maximum area (Amax) and time to 50% of maximum area (t50) data for cell spreading from individual curve fit analyses, and data on Rac1 localization to focal adhesions. p values are supplied in each figure legend, and significance was adjudged to be present at p < 0.05 for all data. All graphs include SE bars.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FAK-mediated Tyrosine Phosphorylation of PIX
To identify the mechanisms underlying FAK facilitation of both Rac1 activation and Rac1 targeting to focal adhesions, we studied interaction between Rac1-associated GEFs and FAK. To begin, nitrocellulose membranes used for Rac activity assays were reprobed with anti-phosphotyrosine antibody. Interestingly, PIX was the only protein that was tyrosine-phosphorylated and pulled down with Rac1 by PBD in the Rac1 activity assays.
In vitro kinase assays were done to ascertain whether FAK could directly tyrosine phosphorylate PIX (Figure 4A). FAK– cells were transfected with EGFP-tagged wild-type FAK or the carboxy-terminus truncation mutant of FAK Dter, which lacks a kinase domain. EGFP-Dter and EGFP-FAK were immunoprecipitated using anti-GFP antibody and protein A-Sepharose. Purified PIX-GST, 2 µg, that had been produced in E. coli was then added to the EGFP-Dter or EGFP-FAK immunoprecipitates together with ATP. These data demonstrated that PIX was tyrosine-phosphorylated by EGFP-FAK in the presence of ATP, but not by EGFP-Dter. The addition of 10 µM PP2, a Src inhibitor (lane 6), did not change the tyrosine phosphorylation of PIX by immunoprecipitated FAK. To be certain that the data observed in these in vitro kinase experiments were due to direct tyrosine phosphorylation of PIX by FAK, additional in vitro kinase experiments were performed using purified, baculovirus-derived FAK kinase domain (Figure 4, B and C). PIX, but not GST alone, was tyrosine-phosphorylated by FAK. PIX was not tyrosine-phosphorylated by kinase-dead (K454R) FAK (Figure 4B, lanes 4–6), or in the absence of ATP (Figure 4B, lane 8). These experiments demonstrated the direct, specific, dose-dependent tyrosine phosphorylation of PIX by FAK.
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PIX, we prepared three populations of FAK– cells that expressed PIX-Flag together with cotransfected EGFP-Dter (48 kDa; Figure 4D, lanes marked 1), EGFP-FAK (155 kDa; lanes marked 2), or constitutively active (membrane-targeted) myristylated FAK (myr-FAK, 125 kDa; lanes marked 3), respectively. An anti-FAK immunoblot of whole cell lysates (WCL lanes) was done to adjudge transfection efficiency. PIX-Flag was immunoprecipitated with anti-Flag antibody from lysates of each of the three doubly transfected cell populations that had been normalized for total protein and serially immunoblotted with anti-Flag (top row), and anti-phosphotyrosine (rows 2–4) antibodies. We noted that FAK coimmunoprecipitated with PIX as evidenced by both anti-phosphotyrosine (second set of blots) and anti-FAK (not shown) immunoblotting. In findings that confirmed our in vitro kinase assays, anti-phosphotyrosine immunoblotting also revealed that the low baseline levels of phosphotyrosine on PIX seen in the FAK– cells was increased in the presence of wild-type FAK expression (EGFP-FAK) and was further augmented in the presence of myristylated FAK ("IP lanes," bottom of the second blot, Figure 4D). To verify that the tyrosine phosphorylation of PIX observed here was not mediated by Src that might have associated with the FAK immunoprecipitates, this experiment was repeated in SYF cells (Figure 4E) with the same results.
To define the impact of FAK-mediated tyrosine phosphorylation of PIX on PIX interaction with Rac1, purified Rac1-GST was added to the immobilized purified PIX-Flag after the immunoprecipitations described above. The effect of tyrosine phosphorylation on the PIX-Rac1 interaction was studied by reprobing these immobilized PIX immunoprecipitates with an anti-Rac1 antibody (IP lanes, third blot, Figure 4D). These data showed that tyrosine-phosphorylated PIX bound Rac1-GST more effectively than non-tyrosine–phosphorylated PIX. PIX loading was comparable in all three lanes (IP lanes, top row, Figure 4D).
Subcellular Localization of PIX in FAK+ Fibroblasts
Because our experimental evidence indicated that PIX might have mediated the patterns of Rac1 activation and targeting seen in the FAK+ cells, we interrogated these cells by immunofluorescence labeling of 1 integrin in FAK+ cells to discern the subcellular localization of endogenous PIX (Figure 4F). Although PIX was not often found in focal adhesions, some cells revealed a pattern of antibody staining for endogenous PIX that corresponded with a portion of cell-matrix adhesions. In these cells PIX was found in peripheral adhesions. Further, in cells that exhibited PIX in focal adhesions, the subset of focal adhesion surface area targeted by Rac1 (panel c) reproducibly colocalized with PIX (panel d). These findings suggested that PIX interaction with Rac1 may occur in focal adhesions (as has been proposed by ten Klooster et al., 2006).
PIX Effects on Rac1 Targeting to Focal Adhesions
To further explore the role of PIX in Rac1 targeting to focal adhesions, FAK– cells were transfected with wild-type PIX-Flag. Cells were triple-labeled for Rac1, 1 integrin, and Flag. Expression of ectopic wild-type PIX rescued Rac1 targeting to focal adhesions in FAK– cells (Figures 5, A and B). Thus, increased PIX expression recovered Rac1 targeting in FAK– cells (p = 0.0007).
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PIX-SH3/DH-Flag (W43J, L238R, L239S) with absent GEF activity and absent PAK binding. Expression of this double mutant GEF-negative PIX decisively hampered Rac1 translocation to focal adhesions in FAK+ cells, causing a drop in Rac1 incorporation into focal adhesions (p < 0.0001). These data further implicate PIX as a specific molecular mediator of Rac1 trafficking by FAK.
Cell Spreading Responses to Changes in Rac1 and PIX Expression
The effects of Rac1 activation mutants were examined in FAK– and FAK+ cells (Figure 6A). Cells were prepared for immunofluorescence study on the second day after transfection, and 2 h after plating on fibronectin-coated coverslips. FAK– cells that were transfected with constitutively active L61-Rac1 had a mean surface area threefold greater than nontransfected controls (p < 0.0001). Conversely, FAK+ cells transfected with dominant negative N17-Rac1 had 35% the mean surface area of untransfected controls (p < 0.001). Thus, Rac1 had a profound effect on cell spreading behavior both in the presence and the absence of FAK expression.
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PIX might alter Rac1-mediated cell spreading was then examined. On the second day after transfection, cells were prepared for immunofluorescence analysis 2 h after plating on fibronectin. Increased wild-type PIX-Flag expression in FAK– cells more than doubled cell size (p = 0.0007; Figure 6B). Ectopic PIX expression also caused morphological modifications in the actin cytoskeleton (Figure 6C). Thus, FAK– cells expressing wild-type PIX had an active leading edge with many membrane extensions and subcortical actin arrays that were perpendicular to the cell perimeter (Figure 6 Ca), features that are not characteristic of untransfected FAK– cells.
FAK+ cells transfected with PIX-SH3/DH-Flag showed decreased spreading (p = 0.0019; Figure 6B). FAK+ cells transfected with PIX-SH3/DH-Flag exhibited a smooth leading edge, a rounded morphology, and few membrane extensions (Figure 6C). Changes in the availability and functionality of PIX were therefore associated with dramatic differences in cell spreading efficiency.
Immunofluorescence analysis of transfected FAK– cells demonstrated that the exogenous Flag-tagged wild-type PIX colocalized with vinculin in some peripheral focal adhesions, focal complexes, and membrane ruffles and was associated with morphological features seen in rapidly spreading cells including an active ruffling membrane edge (Figure 6D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2000; Chen et al., 2002; Holinstat et al., 2006) and accelerate lamellipodial extension by adding a second small GTPase-regulated mechanism—the activation of Rac1. The quantitative characterization of cell spreading that is presented here affords some clues to the relative importance of Rho inhibition and Rac activation in the improved spreading efficiency seen in FAK-expressing fibroblasts. Taken together, data shown in Figures 1B and 6A indicate that Rac activation via FAK and PIX may account for approximately two thirds of the differences in cell spreading between the FAK– and FAK+ populations in this study. Our work demonstrates that FAK is involved in Rac1 activation and targeting, and that these two events occur in a similar time frame after integrin ligation.
After activation by specific GEFs, the Rho-family GTPase Rac1 promotes peripheral actin polymerization to induce the formation of lamellipodia and drive extension of the cell's leading edge (Hall, 2005). Membrane extension is made possible by Rac1 effects on peripheral F-actin organization. Rac1 can induce Arp2/3 complex formation (Smith and Li, 2004), and this can be spatially focused by vinculin to sites of new focal adhesions (DeMali et al., 2002). Formation of a stable lamellipodium in cells requires the activation and translocation of Rac to a subset of the cell perimeter (Ridley et al., 1992; Burridge and Wennerberg, 2004). Recent data indicate that Rac targeting to the membrane edge is due at least in part to preferential binding to lipid rafts (del Pozo et al., 2004). In addition to these positive targeting signals, Rac may also respond to negative molecular switches that reduce its activity in the trailing tails of migrating cells. Thus, the paxillin LD4 domain may mediate a reduction in Rac activity in these regions by recruiting an ADP-ribosylation factor GTPase-activating protein (Arf-GAP, e.g., GIT1 and/or PKL) that decreases Arf activity and inhibits Rac (Nishiya et al., 2005). Our data demonstrate that Rac1 can precisely localize to focal adhesions and focal complexes at the leading edges of actively evolving membrane ruffles and lamellipodia. This localization does not appear to be restricted to areas occupied by lipid rafts (data not shown). Further, we document that the activation state of Rac1 may strongly affect its localization. This finding is consonant with the work of other investigators who have reported both that constitutively active Rac1 may be found in focal adhesions (Manser et al., 1997), and that Rac inactivation is associated with its exclusion from the rear of polarized, motile fibroblasts (Nishiya et al., 2005). Rac1-targeting to focal adhesions in FAK+ cells may, therefore, be partly dependent on Rac1 activation.
Interactions between FAK and Rac1 are incompletely understood. Our work demonstrates that FAK is involved in Rac1 activation and targeting, with subsequent positive effects on cell spreading. FAK has been implicated as an indirect actor in various Rac1 activation mechanisms. First, a p130CAS-Crk-DOCK180 complex can activate Rac1 in 293T cells (Kiyokawa et al., 1998), and FAK may play a scaffolding role in the process of Src-mediated CAS phosphorylation (Ruest et al., 2001). In another cascade in NIH3T3 cells, PI 3-kinase can stimulate the Rac-specific GEF activity in Vav2 by providing membrane targeting through phosphatidylinositol 3,4,5-trisphosphate (Das et al., 2000). FAK activates PI 3-kinase after FAK Y397 interaction with the SH2 domain in the p85 regulatory subunit of PI 3-kinase (Chen and Guan, 1994; Chen et al., 1996). Third, focal adhesion targeting of the paxillin-PKL-PIX-PAK complex is dependent on tyrosine phosphorylation of p95PKL by FAK and Src in CHO.K1 cells (Brown et al., 2005; ten Klooster et al., 2006).
The third of these scenarios is most germane to the data presented in this current report. PIX is an activator of Rac that associates with Rac1 in our activity assays and may target Rac1 to focal adhesions (ten Klooster et al., 2006). Additionally, PIX has been shown to play a key role in focal adhesion formation, cell migration, cell polarity, and cytoskeletal rearrangement. Recent studies have shown that disruption of PAK/PIX binding in the human breast cancer cell lines SK-BR-3 and ZR-75-1 altered cell-matrix adhesion and motility (Stofega et al., 2004). In addition, overexpression of PIX drives the formation of membrane ruffles via activation of Rac1, and it has been shown that LPA-induced cell motility in NIH-3T3 cells requires PIX (Lee et al., 2005). These observations strongly suggest that PIX may accelerate leading edge extension.
Our studies suggest direct and specific roles for PIX in FAK-mediated Rac1 targeting to focal adhesions and in FAK-facilitated cell spreading. We report that FAK associates with and tyrosine-phosphorylates PIX and that this phosphorylation event is associated with an increased binding of PIX to Rac1. An interaction between PIX and FAK has also been demonstrated in NIH3T3 mouse fibroblasts (Lee et al., 2005). Tyrosine phosphorylation has been shown to be an important regulatory event for other GEFs that are Rac-specific. For example, the activity of Vav is positively regulated by phosphorylation via the tyrosine kinase Lck (Han et al., 1997). Ras-GRF1 has also been shown to be tyrosine-phosphorylated by Src (Kiyono et al., 2000), and this induces its Rac-specific GEF activity.
Spatial regulation of PIX has yet to be precisely defined. It is possible that FAK-dependent tyrosine phosphorylation of PIX has a role in dynamic shuttling between cytoplasmic complexes, endosomal sequestration, and membrane targeting at ruffles and lamellipodial leading edges (Manabe et al., 2002; Rosenberger and Kutsche, 2005). GIT1 and PKL may also be part of the PIX trafficking between these various destinations (Manabe et al., 2002; Brown et al., 2005). Our observations that PIX localizes to a subset of focal adhesions and that its presence at these sites may be challenging to document correlate well with the findings of other investigators (ten Klooster et al., 2006; Wiggan et al., 2006). It may therefore be reasonable to conclude at this juncture that direct binding to PIX only partly accounts for Rac1 targeting to focal adhesions.
We propose a tripartite process that synthesizes our data on Rac1 activation and PIX effects into a coherent model linking Rac1 and PIX to focal adhesions. The three components of this model may go forward in the same time frame while mutually reinforcing one another. In the first component, adhesion-mediated activation of FAK is followed by FAK-mediated augmentation of PIX-induced Rac1 activation. The second component of this model involves Rac1 targeting to focal adhesions. This targeting is favored by the interaction of PIX with Rac1 that may be augmented by tyrosine phosphorylation of PIX by FAK and by the specific intermolecular binding interaction that has just recently been described between the PIX SH3 domain and the proline-rich sequence near the carboxy-terminus of Rac1 (ten Klooster et al., 2006). Increased GTP loading of Rac1 may also favor targeting to focal adhesions. The third and final component of this proposed schema is that elevated levels of GTP loading on activated Rac1 may stabilize intermolecular interactions with its downstream effectors (del Pozo et al., 2002). This effect would favor membrane extension and cell spreading.
The case for PIX as a key player in FAK-facilitated lamellipodial extension and stability is made stronger by the aggregate of our findings: The GEF-deficient and PAK-binding incompetent PIX-SH3/DH mutant antagonized Rac1 focal adhesion targeting and cell spreading efficiency in FAK+ cells, whereas wild-type PIX rescued both of these processes in FAK– cells. The effectiveness of the PIX-SH3/DH construct as a dominant negative reagent in our assays may be due in part to the abrogation of the function of key intermolecular complexes that incorporate PIX, PAK, and Rac. We have observed dimerization of exogenous PIX-Flag with native PIX. Others have noted that this dimerization increases activation of, and specificity for the substrate Rac, whereas the PIX SH3 domain stabilizes the interaction of these dimers with both Rac and PAK (Feng et al., 2002, 2004). We propose that the double mutant PIX construct inhibits Rac-binding by these complexes, making the double mutant PIX a more effective dominant negative than either of the single PIX mutants in which either the DH or SH3 domain alone is defective.
Rescue of the FAK– phenotype for Rac1 targeting and cell spreading may be due to several alternative or complementary mechanisms. First, optimal PIX GEF function is apparently dependent on both serine phosphorylation via PAK and tyrosine phosphorylation by FAK. If the size of the PIX pool is a limiting factor for Rac1 activation and cell spreading, the elevation of PIX availability in WT-PIX–transfected FAK– cells may overcome the functional deficit from the lack of tyrosine phosphorylation and sufficiently boost Rac1 activation levels to restore cell spreading. It is also possible that the increased level of PIX in these cells augments Rac1 recruitment to focal adhesions more efficiently and that this spatial focusing substantially increases the functional impact of the added PIX expression on cell phenotype (ten Klooster et al., 2006).
Finally, several critically important cellular behaviors require spatial focusing of Rac1 and directed, persistent extension of lamellipodia. These include fibroblast motility during wound repair and endothelial metamorphosis during angiogenesis (Cascone et al., 2003; Harms et al., 2005). The FAK-PIX-Rac1 pathway that is defined in this work may be a critical determinant of success in these endeavors by providing the targeting and assembly of Rac1-containing molecular networks at focal complex and focal adhesion sites.
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ACKNOWLEDGMENTS |
|---|
PIX-GST, to Silvio Gutkind for the myristylated FAK, to Keith Burridge and Mike Schaller for valuable discussions, and to Patricia Arauz for comments on the manuscript. This work was supported by the National Institutes of Health (P60DE13079 and AI061042
[GenBank]
), the Fogarty International Center (F06TW02341), and the Johns Hopkins University Funds for Medical Discovery to L.R. |
Footnotes |
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The online version of this contains supplemental material at MBC Online (http://www.molbiolcell.org). 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-03-0207) on November 8, 2006.
Address correspondence to: Lewis Romer (Lromer{at}jhmi.edu)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, n = 11) and FAK– cells (
, n = 9) by phase-contrast microscopy. After initial attachment (
