Association of rasGAPSH3 Binding Protein 1, G3BP1, and rasGAP120 with Integrin Containing Complexes Induced by an Adhesion Blocking Antibody Xiaobo Meng,† Jaykumar Krishnan†,‡ Yemen She,§ Werner Ens,§,| Kenneth Standing,§,| and John A. Wilkins*,†,‡,| Manitoba Centre for Proteomics, Rheumatic Diseases Research Laboratory and Departments of Medicine, Immunology, University of Manitoba, Time-of-Flight Laboratory, Department of Physics and Astronomy, University of Manitoba Received November 3, 2003
The adhesion blocking antibody 3S3 was used to probe the regulation of R5β1 integrin mediated adhesion in K562 cells. This antibody prevented cellular adherence but it did not interfere with ligand binding by cells or purified integrin. Interaction with 3S3 induced change in the cytoskeletal organization resulting in extensive filopodia formation. The antibody also prevented ligand and anti-integrin antibody induced phosphorylation of FAK in a trans acting fashion. MS based analysis of 3S3 induced integrin containing complexes identified rasGAP SH3 binding protein 1, G3BP1, as a component of these structures. The G3BP1 binding molecule, rasGap120, was also identified in the complexes. Microscopic examination confirmed the recruitment of a component of cellular G3BP1 and rasGap120 pools to sites of integrin cross-linking. G3BP1 was also observed in the 3S3 induced filopodia. In untreated cells, G3BP1 was shown to associate with submembranous regions involved in cellular polarization. Collectively, these results suggest that G3BP1 and rasGap120 can be recruited to sites of integrin ligation where they may play a role in cytoskeletal reorganization. Such changes may result in reduced adhesive potential and account for the 3S3 effects on cellular adhesion. It should be emphasized that these results do not necessarily indicate a direct interaction of integrin with G3BP1 and rasGap120. Keywords: integrins • MALDI • rasGAP120 • G3BP1 • adhesion • cytoskeleton • adhesion complexes
Introduction Integrins are one of the major mediators of cellular adhesion to components of the extracellular matrix.1 These heterodimeric type I membrane proteins are also responsible for the initiation of a number of cellular responses to their microenvironment such as migration, differentiation and mitosis. Such processes involve ligand recognition, integrin redistribution, and the recruitment of a number of adapter and signaling molecules to sites of adhesion.2-4 Although the extracellular domains of the integrin β chains have recently been reported to express thiol isomerase activity,5 the cytoplasmic domains of the integrins are apparently devoid of enzymatic activity. It is thought that the recruitment of other molecular species to the adhesion sites is responsible for the integrin initiated signaling responses.4,6,7 The functional responses mediated by integrin ligation are dependent upon the recruitment and interactions of other * To whom correspondence should be addressed. Dr. J. A. Wilkins, Rheumatic Diseases Research Laboratory, Rm. 805 John Buhler Research Centre, 715 McDermot Ave., Winnipeg, Manitoba R3E 3P4, Canada. Phone: (204) 789-3835. Fax: (204) 789-3987. E-mail:
[email protected]. † Rheumatic Diseases Research Laboratory and Department of Medicine, University of Manitoba. ‡ Department of Immunology, University of Manitoba. § Time-of-Flight Laboratory, Department of Physics and Astronomy, University of Manitoba. | Manitoba Centre for Proteomics, University of Manitoba.
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molecular species. Microscopic studies have demonstrated the sequential accumulation of a number of signaling and adaptor proteins to areas of integrin substrate contact.7-9 The composition and lifetime of these complexes varies dependent on the types of stimuli associated with the adhesive events.10,11 Even structures such as focal adhesions which indicate areas of strong integrin ligand contact have been shown to turn over at a rapid rate.12 Similarly there can be distinct differences in the topographical distribution of integrin complexes with different adhesive strengths for example. Integrins on the leading edges of migrating cells display an increase in adhesive strength relative to molecules dorsal and distal to the leading edge.13 These observations highlight the complex and dynamic nature of integrin medicated adhesion. Furthermore, they demonstrate the need to develop approaches to examine the molecular species involved in these responses.14-17 Several antibodies to integrins have been shown to alter cellular adhesion.18-21 Although the modes of action are still controversial, such antibodies have been used extensively to probe the mechanisms of integrin regulation. It has been suggested that integrin activating antibodies stabilize their high affinity, which facilitates cellular adherence.9,22 Inhibitory antibodies that function as ligand mimetics block ligand binding by steric inhibition of access to the ligand binding sites.20,21,23 However, there is another class of antibodies that 10.1021/pr0340983 CCC: $27.50
2004 American Chemical Society
rasGAPSH3 Binding Protein 1, G3BP1, and rasGAP120
appears to act as allosteric inhibitors of ligand binding.24,25 These antibodies cause conformational changes, which reduce access to the integrin ligand binding site. During our analysis of the adhesion blocking antibody 3S3 we made several observations, which suggested the existence of other mechanisms to prevent cellular adhesion. Treatment of cells with 3S3 blocked adhesion but id did not prevent ligand cell binding by cell associated or purified integrins. The present studies were initiated to examine the cellular responses to interaction with an adhesion blocking antibody, 3S3,19 to the β1 integrin chain. The results suggest a novel mechanism of trans inhibition of cell adhesion, which causes down regulation of focal adhesion kinase, FAK, phosphorylation. Integrin containing complexes were induced by the clustering with 3S3 bound to latex beads. This approach has been shown to induce the association of a number of adhesion complex proteins both microscopically and biochemically.7,9,26,27 Mass spectrometric analysis of the separated components of the 3S3-induced complexes identified the rasGAP SH3 binding protein, G3BP1. Microscopic analysis suggests a possible role of these molecules in the reorganization of the cytoskeleton associated with cell polarization and migration.
Materials and Methods Cell and Reagents. The human erythroleukemia cell line, K562, was obtained from the ATCC, and low passage human foreskin fibroblasts were maintained in RPMI 1640 medium (GIBCO BRL, Grand Island, NY) supplemented with 10% FBS (GIBCO BRL). In the soluble ligand binding experiments, the cells were cultured overnight in serum free medium AIM-V (GIBCO BRL). Purified human fibronectin was obtained from Chemicon (Temecula, CA). EZ-Link Sulfo-NHS-LC-Biotin was purchased from Pierce Chemicals (Rockford, IL). All the other chemical reagents were bought from Sigma Chemicals (St. Louis, MO) or Fisher Scientific (Fair Lawn, NJ). Antibodies. The murine monoclonal antibodies to the human β1 integrin, 3S3, N29, B44, and B3B11 were produced in this laboratory,18,19,27 12G1028 was purchased from Chemicon. Other antibodies employed were to G3BP1 (Transduction Laboratories), RasGAP120, rabbit anti-FAK antibody (K-17) (Santa Cruz Biotechnology), and anti-CD3 (PharMingen). Avidin-FITC, goat anti-mouse FITC, biotin conjugated mouse antiphosphotyrosine (clone PT66), avidin-HRP, alkaline phosphatase conjugated goat anti-rabbit antibody were purchased from Sigma. Biotinylation of Proteins. Antibodies or fibronectin were labeled with biotin according to the manufacturer’s protocols. Briefly, 10 mg/mL of fibronectin or 1 mg/mL of antibody was reacted with 50 µg/mL EZ-Link Sulfo-NHS-Biotin in sodium bicarbonate buffer (pH 8.0) for 1 h at room temperature. Unbound biotin was removed by dialysis against PBS. Cell Adhesion Assay. Nontissue culture 96-wells plates were coated with purified plasma fibronectin (10 µg/mL) in PBS at 4 °C overnight. The plates were blocked with 1% BSA for 1 h at room temperature and washed 3 times with PBS. K562 cells were washed and suspended in serum free RPMI medium alone or in the presence of the indicated concentrations of antibodies for 30 min at room temperature and transferred to wells coated with fibronectin. The cells were incubated for 30 min at 37 °C and the nonadherent cells were removed by centrifugation of the inverted plates for 5 min at 200 × g. In some experiments, cells were allowed to adhere to fibronectin coated surfaces for 30 min prior to the addition of the indicated antibodies. The
research articles cells were then incubated for an additional 30 min at 37 °C and centrifuged as described above. The level of adherence was assessed colorimetrically. The adherent cells were stained with 0.5% crystal violet, washed with water and the bound dye was extracted in methanol. The absorbance was determined at 550 and 690 nm. The cell binding to BSA (1%) coated wells was used as a background control in all the assays and this value was subtracted from that obtained in fibronectin coated wells. All of the assays were repeated at least three times with quadruplicate wells for each group. Ligand Binding to Intact Cells. Cells were grown in serum free media, AIM V (Gibco) for 24 h prior to the experiments and pretreated with 3S3 or control antibody for 30 min. After washing in media biotin labeled fibronectin (100 µg/mL) was added for 30 min at 37 °C and the cells were washed in media. FITC conjugated avidin was added to the cells for 30 min after which the cells were washed. Fluorescence analysis was performed with a BD FACScaliber. All assays included cells treated with avidin-FITC alone as a control for nonspecific binding. This experiment was performed 3 times with similar results. Ligand Binding to Purified Integrin. Human R5β1 integrin was immunoaffinity purified from placenta and assayed for ligand binding as previously described.29 The purity and identity of the integrin was confirmed by mass spectrometric analysis of in gel digests of SDS-PAGE separated purified integrins. For studies examining the effects of 3S3, the antibody was added to the immobilized integrin 30 min prior to the addition biotinylated fibronectin. The level of non specific binding was assessed by determining the level of biotinylated fibronectin binding in the presence of 100 fold excess of unlabeled fibronectin. This value (less than 15% of the specific binding value) was subtracted from all values to obtain the specific ligand binding levels. Analyses were performed in sextuplicate and the experiments were repeated 3 times. The intra group variation was less than 12% for all groups. FAK Immunoprecipitation and Phosphotyrosine Detection. Nontissue culture dishes (6 cm) were coated overnight at 4 °C with fibronectin (10 µg/mL) or antibody (10 µg/mL). The coated dishes were blocked with 1% BSA for 1 h at room temperature. K562 cells were washed and resuspended in AIM-V serum free medium. Equal numbers of cells were added to the dishes and the cells were precultured for 1 h at 37 °C. Where indicated, soluble antibody (10 µg/mL) was added the incubation was continued for an additional 30 min. The cells were recovered by scraping in lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 5mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 10mM NaF, and a protease inhibitor cocktail (Roche). The lysates were clarified by centrifugation at 12 000 g for 10 min. Protein-G beads (Pharmacia, Sweden) preloaded with rabbit anti-FAK were added to the supernatants and incubated with end-over-end mixing for 6 h at 4 °C. The beads were washed 3 times with lysis buffer. Proteins were eluted and reduced by boiling the beads in 2% 2-mercaptoethanol in Laemelli sample buffer. Proteins were resolved by SDS-PAGE in 8% gels. After electrophoresis, proteins were electro transferred to nitrocellulose membranes (Bio-Rad). The membranes were blocked overnight with 1% BSA in PBS. The blots were reacted with biotinylated anti-phosphotyrosine antibody. Avidin-HRP was used to detect bound antibody and visualized with the ECL chemiluminescent system (Amersham, UK). The nitrocellulose membranes were then stripped and reprobed to detect FAK. Journal of Proteome Research • Vol. 3, No. 3, 2004 507
research articles After reacting with rabbit anti-FAK antibody (K-17), the blots were treated with goat anti-rabbit alkaline phosphatase and visualized by reaction with alkaline phosphatase substrates, BCIP/NBT. Procedure for Coating of Dynabeads and Cell Treatment. A total of 5 × 108 M-450 Dynabeads (Dynal, Norway) were coated with 50 µg antibody (3S3 or anti-CD3) for 24 h in 0.1M phosphate buffer (pH 7.4). The beads were blocked with 0.1% BSA and washed to remove free antibody. The antibody-coated beads were added to pre-adherent cells at the ratio of (5 beads: 1 cell) and incubated for 30 min. The FAK phosphorylation was analyzed as described above. Isolation of Adhesion Complexes. Adhesion complexes were isolated using a modification of the method Plopper and Ingber.26 Briefly, 1.5 × 107 cells were incubated with a 10-fold excess of antibody coated beads at 37 °C for 30 min. The beads and associated cells were isolated with a magnet and washed with detergent free ice cold CSK buffer (50mM NaCl, 300mM sucrose, 3mM MgCl2, 10mM NaF, 1mM sodium vanadate, complete protease cocktail (Boehringer) and 10 mm PIPES buffer pH 6.8). The cells/beads were resuspended in complete CSK buffer containing 0.5%Triton X-100, sonicated for 10 s, and homogenized in a Wheaton homogenizer. The beads were magnetically collected and washed 5 times in complete buffer. The sample was recovered by boiling the beads in Laemelli sample buffer with DTT and separated on 7% SDS-PAGE gel. The gels were silver stained according to the method of Shevchenko et al.30 and selected bands were excised. The bands were digested with trypsin. The peptides were extracted with 0.1% trifluoroacetic acid, concentrated on ziptips (Millipore) and eluted with 50% acetonitrile. Mass Spectrometry and Analysis. The samples were mixed with an equal volume of matrix, DHB dihydroxybenzoic acid, and analyzed in an orthogonal injection QqTOF developed at the University of Manitoba. This instrument has a resolution of approximately 10 000 and mass accuracy of 10 ppm.31 The spectra were analyzed using M/Z and Prowl (Proteometrics Canada) to search the NR databases. In the case of MS/MS analysis the peaks were labeled and analyzed using Sonar (Proteometrics). Western Blot Analysis. Adhesion complexes were isolated and separated as described above, transferred to nitrocellulose membranes, probed with the indicated antibodies and visualized with an alkaline phosphatase conjugated anti-mouse immunoglobulin.32 Microscopic Analysis. Microscopic analysis was performed with an Olympus BX60 microscope equipped with a Xenon lamp and light pipe for epifluorescence. Images were collected with an Optikon CCD camera and processed using ImagePro software. For the induction of filopodia formation K562 cells (3 × 105/ ml) were incubated at 37 °C with 3S3 (10 µg/mL) for 2 h. The cells were then washed and transferred to polylysine-coated cover slips and processed for staining. In some cases K562 cells were incubated for the indicated times in chamber slides (Nunc International) that were precoated with the 3S3 or 12G10 antibody (20 µg/mL) for 12 h at 4 °C. The cells were, fixed with fresh 4% paraformaldehyde, permeabilised and stained with Oregon green phalloidin (Molecular Probes). For the demonstration of bead induced recruitment of G3BP, latex beads were used because of the auto-fluorescence of the Dynabeads. Latex beads were coated with anti-β1 or anti-CD3.9 Cells were incubated with latex beads at a ratio of 1:4 and gently 508
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rotated at 37 °C for 30 min. The cells were gently transferred to polylysine-coated cover slips (10 µg/mL). The cells were fixed with 4% paraformaldehyde PBS containing 5% sucrose, permeabilised and stained as per LaFlamme et.33 Cells were incubated with biotinylated antibody to G3BP1 or β1 for 60 min at 4 °C, washed and stained avidin FITC and Oregon green phalloidin (Molecular Probes). Antibodies to G3BP1 and B3B11 were biotinylated using a kit (Pierce Chemical) as per the company’s instructions.
Results 3S3 Effects on Cellular Adherence. K562 cells spontaneously adhere to fibronectin in an R5β1 dependent fashion.34 However, following a 30 min pretreatment of the cells with 3S3 there was almost complete inhibition of this adherence (Figure 1A). The anti-β1 monoclonals, N29 and 12G10, have previously been shown to increase integrin mediated adherence. 12G10 recognizes a cation sensitive, ligand induced binding site on β1.28 N29 interacts with a region near the N terminal region of the β1 chain that can be exposed by activation or treatment with dithiothreitol.18 Treatment of K562 cells with either of these antibodies resulted in an increase in cellular adherence. In contrast, exposure to the control antibody to CD3 did not influence cellular adherence. Following K562 contact with fibronectin the cells spread and adhere firmly. It was questioned if 3S3 could alter this process. Cells were allowed adhere to fibronectin coated surfaces for 30 min and then treated with antibodies to β1. The addition of 3S3 resulted in a 75-80% reduction in the number of bound cells (Figure 1B). Under these conditions, N29 and 12G10 caused a slight enhancement of adherence suggesting that near maximal binding had already been achieved in the absence of antibodies. Effects of 3S3 on Ligand Binding. The above results indicated that 3S3 could prevent β1 dependent cellular adherence. The most obvious potential mechanism involves interference with integrin fibronectin ligand interaction. To test this possibility, the effects of 3S3 on the binding of soluble fibronectin were examined. The binding of soluble biotinylated fibronectin by K562 cells was readily demonstrable by flow cytometry (Figure 1C). The specificity of the binding was demonstrated by the observation that a 100-fold excess of unlabeled fibronectin fully reversed this binding. However, the addition of 3S3 at concentrations up to the highest level tested, 50 µg/mL, did not interfere with fibronectin binding. This concentration of antibody was 5-10 times the minimum amount required to achieve maximal inhibition of cell adhesion. These data suggested that that 3S3 did not interfere with integrin fibronectin interactions on living cells. Although R5β1 is the only known functional receptor for fibronectin on K562 cells, it was possible that binding of soluble fibronectin might involve other cell surface associated receptors (ref 34, unpublished data JAW, XM). To exclude this possibility, R5β1 was affinity purified from placenta and the effects of 3S3 on fibronectin binding were examined. The identity of the R5β1 was confirmed by mass spectrometry and western blot. Biotinylated fibronectin bound to immobilized R5β1 in a concentration dependent fashion that reached saturation at concentrations of greater than 50 µg/mL (Figure 1D). The specificity of the fibronectin binding was confirmed by addition of a 100 fold excess of unlabeled fibronectin which resulted in ∼90% inhibition of biotinylated fibronectin binding. Pretreat-
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Figure 1. Effects of 3S3 on integrin activity. (A) Cell Adhesion: Cells were treated with the indicated concentration of antibodies to β1 integrin, 3S3 (b), N29 (O), 12G10 (0) or control anti-CD3 (2). Antibody was added to the cells 30 min prior to addition to the fibronectin coated wells. (B) Cells were allowed to adhere to fibronectin coated wells for 30 min after which the indicated antibodies were added and incubated for an additional 30 min. The level of adherence was determined as described in the Materials and Methods section. (C) Cell Ligand Binding: Cells were treated with biotinylated-fibronectin, alone (Con) or in the presence of 3S3 (3S3). The levels of fibronectin binding were assessed by flow cytometry. The specificity of binding was demonstrated by the ability of a 100-fold excess of unlabeled fibronectin to block the binding of biotinylated fibronectin (unlabeled thin line). (D) Purified Integrin Ligand Binding: Immobilized human R5β1 integrin was incubated in the presence or absence of 3S3 for 1 h at 20 °C after which the indicated concentrations of biotinylated fibronectin were added. After 3 h the plates were washed and the amount of binding was assessed.
ment of immobilized R5β1 with 3S3 at concentrations as high as 20 µg/mL did not interfere with the binding of fibronectin (Figure 1D). These results suggested that 3S3 did not interfere with the ability of integrin to interact with fibronectin contacts. Morphological Changes Associated with 3S3 Contact. Cells treated with 3S3 were observed to round up prior to detachment. This contrasted with the situation when cells were treated with 12G10. These observations raised the possibility that binding of 3S3 was influencing aspects of cell spreading. These morphological changes were also observed when K562 cells were bound to immobilized 12G10 or 3S3 (Figure 2). Although the cells were captured with equal efficiency by the two antibody coated surfaces, there marked differences in the patterns of F actin staining with Oregon green phalloidin. At early time points, both 3S3 and 12G10 induced extensive filopodia formation. However, after 90 min cells plated on 12G10 spread and contained an extensive F actin network (Figure 2A,B). In contrast, 3S3 treated cells were rounded with an extensive array of filopodia with little organized F actin other than in the periphery of these cells (Figure 2C,D). The 3S3 treated cells continued to express high levels of filopodia formation on their surface at the later time points. These results
suggested that both 3S3 and 12G10 stimulated filopodia formation but that only the latter antibody induced cell spreading. The possibilities were that 3S3 failed to induce spreading or that it actively suppressed spreading. As a direct test of the latter possibility, cells were allowed to adhere and spread on immobilized 12G10 and then treated with soluble 3S3 for 30 min. Following treatment with 3S3 the cells acquired features more characteristic of cells immobilized on 3S3 (i.e., filopodia and cell body retraction) (Figure 2E,F). These results indicated that 3S3 interaction with β1 integrins resulted in the inhibition of 12G10 induced actin polymerization and cell spreading suggesting an active mechanism of suppression. Loss of Adhesion is Associated with Active Suppression of FAK Phosphorylation. Integrin ligation and aggregation result in the recruitment and activation of a number of adapter and signaling molecules to the sites of substrate contact.35,36 One such molecule is focal adhesion kinase, FAK.35-40 Previous studies from several groups have demonstrated that one of the earliest consequences of integrin aggregation is the accumulation and phosphorylation of FAK at adhesion sites.36,38 As 3S3 did not interfere with ligand recognition it was unclear what the effects of 3S3 would be on FAK phosphorylation. Journal of Proteome Research • Vol. 3, No. 3, 2004 509
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Figure 2. Effects of 3S3 on cell morphology: K562 cells were incubated on chamber slides precoated with antibodies to human β1 integrin 12G10 (A,B) or 3S3 (C,D). (E,F) K562 cells were incubated on immobilized 12G10 as in A) after which soluble 3S3, 10 µg/mL was added for the final hour of incubation. Cells were stained with Oregon green phalloidin to visualize F actin. Cells were examined at 60× (A,C,E) or 100× (B,D,F).
Following adherence to fibronectin, K562 cells expressed significant levels of phosphorylated FAK (Figure 3A, lane 3). This contrasted with the situation for nonadherent cells, where very little FAK phosphorylation is observed (Figure 3A, lane 1). The addition of 3S3 to adherent cells resulted in almost complete inhibition of FAK phosphorylation (Figure 3A, lane 4). Under these conditions, the cells had not yet retracted and rounded up, indicating that the loss of FAK phosphorylation preceded these events. The levels of FAK were not altered by 3S3 treatment implying that the changes in phosphorylation reflected the modification status of the FAK rather than a loss in the overall levels of this molecule (Figure 3A, lower panel). Antibody mediated aggregation of integrins can also induce FAK phosphorylation.9,37,38,40 Furthermore, both inhibitory and noninhibitory antibodies have been reported to induce FAK phosphorylation, suggesting that aggregation is sufficient to induce this event.7,9 It was therefore questioned if 3S3 interaction with integrin could also induce FAK phosphorylation. Cells 510
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were bound to culture dishes coated with fibronectin or antibodies, 12G10 or 3S3, and the levels of FAK phosphorylation were determined. The cells that bound to the 3S3 coated plates did not express detectable levels of FAK phosphorylation (Figure 3B, lane 2). In contrast, cells adherent to fibronectin and the 12G10 coated surfaces displayed an increase in FAK phosphorylation relative to cells in suspension (Figure 3B, lanes 3 and 5, respectively). The above results suggested that either 3S3 did not activate FAK or alternatively that 3S3 interaction with integrin suppressed FAK phosphorylation. As a direct test of the latter possibility, cells adherent to fibronectin or immobilized 12G10 were treated with soluble 3S3 and the levels of FAK phosphorylation determined. In both cases, there was a marked reduction in the levels of FAK phosphorylation following 3S3 treatment (Figure 3B, lanes 4 and 6, respectively). However, the 3S3 treatment did not alter the total amount of FAK present in the cells (Figure 3B, lower panel). These changes were
rasGAPSH3 Binding Protein 1, G3BP1, and rasGAP120
research articles either form resulted in a marked reduction in the levels of FAK phosphorylation (Figure 3C, lanes 5, 6). These results suggested that 3S3 interactions with integrins on the dorsal surface of the cell leads to the generation of signals that interfere with the maintenance of FAK phosphorylation induced by 12G10 on the ventral surface of the cell. These effects were not observed with other antibodies to the integrins suggesting a differential response pattern following integrin ligation by 3S3.
Figure 3. Effects of 3S3 on FAK phosphorylation. (A) Cells in suspension (1,2) or on immobilized fibronectin (3,4) were untreated (1,3) or exposed to 3S3 (2,4). The cells were lysed and immunoprecipitated with anti-FAK. The FAK phosphorylation (upper row) and the amount of FAK (lower row) were determined by western blot. 3S3 prevented the phosphorylation of FAK on fibronectin adherent cells. (B) Cells in suspension (1) or on immobilized 3S3 (2), fibronectin (3,4), 12G10 (5,6), were untreated (1,2,3,5) or incubated in the presence of soluble 3S3 for 15 min (4,6) and the levels of FAK phosphorylation were determined as in A). (C) Cells were bound to plates coated with 12G10 (2-6) for 30 min. The cells were then left untreated (2), or treated for 30 min with soluble JB1 (3), beads coated with JB1 (4), soluble 3S3 (5), or with beads coated with 3S3 (6). The levels of FAK and FAK phosphorylation were determined as in (A). For comparative purposes the levels of cells maintained in suspension are shown lane 1.
detected prior to any apparent effects of 3S3 on cellular adherence. These results indicate that binding of integrins by 3S3 inhibits FAK phosphorylation induced by antibody or ligand interactions with integrins. The effects of 3S3 on FAK phosphorylation could arise either from 3S3 acting directly on the 12G10,antibody bound integrins and preventing the transduction of signal(s) via these integrins or from indirect effects mediated by a negative signal that interfered with FAK phosphorylation at sites distal to the 3S3 binding. To differentiate between these two possibilities, 3S3 was coupled to beads in order to restrict interactions between 3S3 and integrins to the contact sites between the beads and cells (i.e., on the dorsal surface of the cells away from the sites of integrin interaction that were inducing FAK phosphorylation). Cells were allowed to adhere to 12G10 coated plates for 30 min after which 3S3 or JB1 coated beads were added to the cells for an additional 30 min at a ratio of 5 beads/cell. Microscopic examination of the cells indicated that the beads were almost exclusively (>99%) localized to the upper surface of the cell. The antibody JB1 was used as a control for the effects of bead associated integrin interactions on cell FAK phosphorylation. This antibody recognizes the β1 chain but it does not influence cellular adhesion.18 The addition JB1in solution or on beads did not interfere with the levels of FAK phosphorylation (Figure 3C, lanes 3 and 4) relative to untreated cells bound to 12G10 (lane 2). In contrast treatment with 3S3 in
Isolation of ras-GTPase-Activating Protein SH3-DomainBinding Protein-1, G3BP1, with Integrin Associated Complexes. As an approach to defining the composition of 3S3 induced integrin containing complexes, K562 cells were incubated with anti-CD3 or 3S3 coated magnetic beads. The associated proteins were then separated by SDS-PAGE, silver stained, and the patterns of staining were compared. The resulting immunoprecipitates revealed a number of common bands between the control and experimental immunoprecipitates and 23 unique bands to the integrin isolates. Particular attention was focused on one band with a molecular weight of approximately 68kDa that was consistently observed in the cells exposed to the beads coated with 3S3 (Figure 4A). This band was excised from the gel and digested with trypsin. The resulting peptides were analyzed by MALDI QqTOF mass spectrometry. The peptides from this band were identified as components of the rasGAP120 SH3 binding protein, G3BP1. Six peptides were identified giving 22% coverage of the total protein (Figure 4B). Although there are three-members in the G3BP family, all of the fragments were predicted to specifically derive from G3BP1.41 To directly address this point, three tryptic fragments, predicted to derive uniquely from G3BP1 (i.e., m/z 1209.6, 1231.7, and 1572.8), were analyzed by tandem mass spectrometry (Figure 4C,D). The results from these analyses confirmed the identities of these peptides (i.e., residues 124-132, 394403, and 358-370 of G3BP1). The presence of G3BP1 in integrin containing complexes was further demonstrated by western blot with an antibody to G3BP1. Both β1 integrin and G3BP1 were readily visualized in the 3S3 isolates but there was no evidence of this molecule in the control precipitates (Figure 5A). This association was observed in isolates from human foreskin fibroblasts and K562 cells indicating that this interaction occurred in both fibroblastic and haematopoietic cells. (Data not shown). Identification of ras-GTPase-Activating Protein, (rasGAP120) in Integrin Containing Complexes. G3BP1 is one of a family of proteins that interact with the SH3 domain of rasGAP120.41,42 However, rasGAP120 was not detected by mass spectrometry in the G3BP1 containing precipitates and it was questioned if the G3BP1 association with integrin containing complexes was independent of this interaction. Integrin containing complexes were isolated as described above and probed by western blot with anti-rasGAP120. Consistent with the previous results, G3BP1 was isolated with 3S3 coated beads but not with control beads (Figure 5B). Probing of the same precipitates with anti- rasGAP120 revealed that this protein was also present in the integrin containing complexes and absent in the control preparations. Thus both G3BP1 and rasGAP120 were associated with integrin containing complexes (Figure 5B). Isolates with beads coated with another antibody to β1, JB1, did not contain G3BP1 suggesting that this was a specific effect of 3S3 (Data not shown). Journal of Proteome Research • Vol. 3, No. 3, 2004 511
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Figure 4. Identification of G3BP1 in integrin containing complexes. (A) Lysates of K562 cells complexes were incubated with control, anti-CD3 (lane 1) or anti-β1 (lane 2) coated Dynabeads and the resulting immunoprecipitates were separated by SDS-PAGE and the proteins were silver stained. A unique band in the integrin precipitate (arrowhead, lane 2) was excised and digested in gel with trypsin. (B) MS spectrum of the digested band from (A). The labeled peaks were identified as corresponding to human rasGAPSH3 binding protein 1. (C) and (D) MS/MS spectra of singly charged parent ions with m/z 1210.627 and 1232.704, respectively. The deduced sequences of these peptides are indicated in each frame.
β1 integrins were readily demonstrated at the sites of contact with the 3S3 coated beads (Figure 6A). Similarly an accumulation of G3BP1 and rasGAP120 were also observed (Figure 6B, C). These results contrasted with those in which cells in contact with the control beads. In this case none of the proteins, β1 integrins, G3BP1 and rasGAP120 were found to accumulate at the contact interface between the cells and the beads (Figure 6D-F).
Figure 5. G3BP1 and ras GAP120 are present in integrin containing complexes. (A) Complexes isolated with anti-β1 (lanes 1, 3) or control anti-CD3 (lanes 2, 4) coated beads were separated by SDS-PAGE and examined for the presence of β1 (lanes 1, 2) or G3BP1 (lanes 3, 4). Integrin (lane 1 arrow) and G3BP1 was detected in the integrin complexes (arrow lane 3) but not in the control isolate probed with the same antibodies (lanes 2 and 4). (B) Adhesion complexes (lane 2) or control isolates (lane 1) were probed for the presence of G3BP1 or ras GAP120. The upper section of the blot was probed with anti- ras GAP120 and the lower section with anti-G3BP1.
Recruitment of G3BP1 and rasGAP120 to 3S3 Coated Beads. The above results suggested that G3BP1 and rasGAP120 could be recruited to sites of 3S3 integrin interaction. As a test of this prediction, K562 cells were incubated with 3S3 coated or control polylysine coated beads. The distributions of intracellular G3BP and rasGAP120 were then compared microscopically. 512
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3S3 Induced Recruitment of G3BP1 to Filopodia. The above results suggested that G3BP1 was recruited to sites of 3S3 integrin contact integrin. Preliminary studies indicated that the treatment of K562 cells with 3S3 induced the persistent formation of filipodia. However, control cells (i.e., untreated cells) did not display this type of organization (Figure 7A,B left panel). It was therefore questioned what the G3BP1 distribution was in 3S3 treated cells. K562 cells were incubated with 3S3 for 2 h to induce filopodia formation. The cells were permeabilised and co stained with antibody to G3BP1 and Oregon green phalloidin to demonstrate the location of filamentous actin. A significant component of the staining for G3BP1 was observed in the filipodia and around the cell periphery (Figure 7A, right panel). F actin staining was present in similar regions (Figure 7B, right panel). The distribution of G3BP1 and F actin fully co-localized at the cell surface and in the filipodia (Figure 7C, right panel). However, there was no apparent correlation of the distribution of these molecules in the cytoplasm. Control cells did not display filipodia and G3BP1 was almost exclusively cytosolic in its
research articles
rasGAPSH3 Binding Protein 1, G3BP1, and rasGAP120
Figure 7. Recruitment of G3BP to 3S3 Induced Filopodia. Left Panel: K562 cells were incubated (A) alone or (B) in the presence of 3S3 for 2 h. The cells were permeabilised and stained with Oregon green phalloidin and examined microscopically. There was a marked and sustained induction of filipodia formation in the presence of 3S3 that is not observed in control cells. Right Panel: K562 cells were treated with 3S3 for 2 h to induce filipodia formation. The cells were stained for (A) G3BP1 and (B) F actin. (C) Merged image of (A) and (B) demonstrates the co-localization of G3BP1 and F actin.
Figure 6. Recruitment of β1 integrins, G3BP1 and ras GAP120 to sites of 3S3 contact. K562 cells were incubated with 3S3 (A-C) or control anti-CD3 (D-F) coated latex beads. The cells were permeabilised and probed with biotin conjugated anti-β1 (A), -G3BP1 (B), or -ras GAP120 (C). Cells were examined in fluorescent (A-F) and DIC (A′-F′) modes. Integrin, G3BP1 and -ras GAP120 staining are demonstrable around the 3S3 beads but not the control beads.
distribution (data not shown). These results suggested that the interaction of integrin of with 3S3 induced the recruitment G3BP1 to membrane proximal sites where actin polymerization and filipodia formation were occurring. The staining of adherent fibroblasts with antibodies to β1 integrin and G3BP1 suggested that there was not a strong colocalization between these two molecules (Figure 8 upper panel). In contrast, when subconfluent migrating cells were examined there was an alteration in the distribution of a
component of the G3BP1. The majority of G3BP1 staining was still observed in the cell cytoplasm. However, a component of the G3BP1 was recruited to a region just posterior to the leading edge of the cell (Figure 8 A lower panel). Integrin staining was diffuse in linear arrays that often ended at the cell periphery (Figure 8B lower panel). There was colocalization of staining for integrin and G3BP1 in the membranous region, but it was clear that the majority of these two molecules did not co localize (Figure 8C lower panel).
Discussion The present study was undertaken to examine the regulation of integrin function using the inhibitory antibody to β1 integrin, 3S3, as a probe. This antibody inhibited the strength of cell adhesion but it did not appear to prevent cellular interactions with soluble or immobilized ligand. Binding of 3S3 stimulated the prolonged generation of filopodia formation with an associated depolymerization of F actin within the cytoplasm. Under these conditions, the cells failed to spread and remained rounded. These effects appeared to involve active inhibition as the addition of 3S3 to cells immobilized on 12G10 coated surfaces reversed cell spreading and actin polymerization. The binding of 3S3 also inhibited fibronectin or anti-integrin antibody induced FAK phosphorylation. These effects involve Journal of Proteome Research • Vol. 3, No. 3, 2004 513
research articles
Figure 8. Distribution of Integrins and G3BP1 in Human Fibroblasts. Cells were cultured for 18 h under subconfluent conditions. The cells were fixed, stained for β1 integrin, permeabilised and stained for G3BP1. Upper Panel. Adherent cells displaying distribution of integrin A,C and G3BP B,D in the corresponding cells. Lower Panel. Motile cell moving toward the bottom of the frames. Distribution of G3BP1 frame A, integrin frame B, and merged images frame C.
a transacting inhibitory pathway as 3S3 coated beads interacting on the dorsal side of the cell can inhibit activating events on the ventral cell surface. These data suggested that 3S3 inhibits adhesion by a novel mechanism involving integrin cross talk. As an initial approach to characterizing the molecular basis for these effects, we attempted to identify some of the proteins that associate with integrin complexes following 3S3 binding. Mass spectrometric-based analysis identified the rasGAP120 SH3 binding, G3BP1, as a component of these complexes. This conclusion was further confirmed by western blot and microscopic analysis. These results suggest that G3BP1 and rasGAP120are involved in the regulation of integrin mediated cell adhesion. Although MS did not detect rasGAP120, one of the known interaction partners of G3BP1, in these complexes, it was demonstrated to be present by immunological and microscopic approaches. These results highlight how MS derived information can be used to develop targeted strategies for the identification of components of protein complexes. Two types of antibody mediated inhibition of integrin function have been described.23,25 Antibodies, designated ligand mimetics, interact with the ligand binding sites of integrins and function as steric inhibitors of ligand integrin interactions.21,23 A second group of antibodies appear to function as allosteric inhibitors of integrin function.24,25,43 In the latter group, antibody and ligand function as reciprocal allosteric inhibitors. The binding of ligand induces conformational changes that result in the loss of the epitopes recognized by these antibodies. Conversely, the binding of the inhibitory antibody results in a loss of ligand accessibility to the integrin. A number of inhibitory antibodies to the R5 or the β1 chain display such properties suggesting that this may be a common mode of action.25 514
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The mode of action of 3S3 appears to be distinct from either of the above categories of antibodies. The expression of the 3S3 epitope is not influenced by either ligand or activating divalent cations, indicating that access to the 3S3 epitope is not influenced by ligand binding (unpublished data XM, JAW). Furthermore, the level of 3S3 binding appears to be invariant and independent of integrin functional status, implying that the epitope is not a reporter of integrin functionality or occupancy.18,29,32 3S3 binding does not appear to prevent low affinity ligand binding by cell associated or isolated integrins. These results suggested that events after receptor ligand interactions are involved in the inhibitory effects. One of the most proximal events following integrin clustering is the recruitment and phosphorylation of FAK to the sites of integrin accumulation.37,38 Ligands and antibodies to integrins can induce this response. Indeed, the cross linking of integrins with antibodies has been used several groups as an approach to examining this process.38,40,44,45 Studies using antibody coated latex beads suggested that cross linking of integrins, independent of the functional effects of the antibodies on adhesion, was sufficient to induce FAK recruitment and phosphorylation.9 It was therefore an unanticipated result when immobilized 3S3 failed to induce FAK phosphorylation. However, our studies could not differentiate between the possibilities that 3S3 did not induce FAK phosphorylation or that 3S3 binding induced dephosphorylation of FAK. The use of immobilized ant-integrin, 12G10, to induce FAK activation ruled out the possibility of 3S3 induced changes in the degree of integrin clustering as an explanation for the inhibition. Similarly, the immobilization of 3S3 on beads eliminated the possibility of any 3S3 effects being related to interference with signal generation by 12G10 bound integrins. These results suggested that engagement of integrins by 3S3 resulted in the inhibition of FAK phosphorylation. The observations that Fab′ of 3S3 also inhibited the phosphorylation suggested that integrin clustering was not required for inhibition (unpublished data XM, JAW). The ability of 3S3 coated beads restricted to the dorsal surface of the 12G10 bound cells to prevent FAK phosphorylation indicated that the signals induced by 3S3 could function in a trans fashion and act on sites distal to their origin. At this time it is not possible to differentiate between 3S3 mediated signals preventing induction of FAK phosphorylation by 12G10 bound integrins or 3S3 enhancing the rate of dephosphorylation of FAK. Thus, it should be emphasized that the current results do not necessarily indicate that FAK is a target of 3S3 mediated inhibition. Rather FAK status has merely been used as a downstream reporter of events at sites of integrin occupancy or cross linking. Functional heterogeneity within members of the integrin family on a single cell have been described.12,13 These differences have been suggested to be important in processes such as migration, trans migration and focal adhesion turnover. The effects of 3S3 identify a pathway for crosstalk between integrins of the same type on the same cell. This may be important as it could suggest mechanisms whereby interactions with the extracellular domains of a limited number of integrins could regulate the functional status of the entire integrin population of a cell. Such pathways could be envisaged to be of importance in changes in integrin utilization patterns in response to chemotactic stimuli or transitions to new adhesive substrates. Integrin mediated adhesion and the subsequent intracellular responses are dependent upon the recruitment of other
research articles
rasGAPSH3 Binding Protein 1, G3BP1, and rasGAP120
molecules to sites of integrin ligation. The resulting molecular complexes are responsible for the integrin mediated cellular response patterns and morphological changes. Such complexes are transient with their distribution and lifetimes being largely determined by the degree and term of aggregation of integrins. Thus, to study such complexes it is necessary to use isolation methods that incorporate an event capable of inducing their formation. Plopper and Ingbar described a method incorporation the use of ligand coated magnetic beads, which effectively induces the recruitment of a number of known components of integrin containing adhesion complexes.26 It should be emphasized that the isolation conditions used for this approach employ buffers, which stabilize cytoskeletal structure. Thus, the isolation of supramolecular complexes could be anticipated with this method. The results of the present studies indicate that binding of integrin with 3S3 induces the recruitment of G3BP1 and rasGAP120 to integrin containing complexes. However, these observations do not necessarily infer direct binding of these molecules to integrins. G3BP1 belongs to a protein family with three members, G3BP1, G3BP2a, and G3BP2b.41,42 These proteins are characterized by the presence of several minimal SH3 binding, PxxP, domains, an RNA recognition motif (RRM), RRG domains (G3BP1 and G3BP2a) and nuclear transport factor-2 like, NTF2like, domain. While initial studies suggested that rasGAP120 was the sole binding partner for G3BP1, the latter has recently been shown to also bind to ubiquitin specific protease 10, USP10.46 This interaction is not dependent on the ubiquitination of G3BP1 and it is specific for USP10. G3BP1 also displays phosphorylation dependent RNase activity for the 3′ UTR of c-myc.47 Several recent studies indicate changes of expression levels of G3BP1 in tumor cells implying a role in the regulation of cellular proliferation and association with metastatic potential.48,49 G3BP1 and rasGAP120 are cytosolic proteins. However, following activation, a fraction of these proteins are recruited as co complexes to the plasma membrane.42 Previously, this recruitment had only been observed in proliferating or transformed cells suggesting a linkage between these two events. However, the results of the present study indicate that integrin initiated events can also induce recruitment under conditions that do not require proliferation. The association with integrin does not appear to be constitutive as immunoprecipitates of integrins from cells that were lysed prior to integrin capture did not contain G3BP1. This suggests that binding of integrins can initiate processes that induce the translocation of these molecules from the cytosol. G3BP1 is normally diffusely distributed but it accumulated at sites of 3S3 induced filopodia suggesting a possible role in actin reorganization. The Drosophila homologue of G3BP, Rasputin (Rin), has been implicated in the generation of cell polarity.50 Disruption of Rin leads to defects in photoreceptor recruitment and the establishment of ommatidial polarity. These mutants displays phenotypic similarities with animals expressing defects in Rho A. The genetic interaction between Rin and Rho A mutants indicated that Rin might provide a linkage between Ras and Rho A dependent processes. Although it was apparent that Rin played a role in cell migration and polarity, the involvement seemed to be distal to the chemotactic process. RasGAP120 has also been implicated in the generation of cell polarity.51 Murine cells deficient in rasGAP120 display a reduced capacity to establish cell polarity and directed migration. The
results of the latter study were consistent with a role for rasGAP120 in cytoskeletal remodeling. It was suggested that rasGAP120 influenced aspects of stress fiber and focal adhesion orientation rather than cell motility per se. These effects were dependent on SH2 interactions, and it is not clear what role G3BP1 would play in these processes. However, there is evidence of rasGAP120 SH3 dependent regulation of cytoskeletal assembly. Antibody to the SH3 domain of rasGAP120inhibits serum induced stress fiber formation in fibroblasts.52 Activated Rho or Cdc42 were both shown to reverse these effects. On the basis of these studies it appears that rasGAP120 may provide multiple contributions to the regulation of cytoskeletal remodeling through SH2 and SH3 dependent interactions. The conditions under which G3BP1 co-localizes with integrins appear to be complex. There was no evidence of colocalization of G3BP1 with integrins on untreated adherent or suspended cells. Under conditions of cell polarization and migration there was an accumulation of G3BP1 in the membrane proximal region. This was most notable in filopodia and small lamellapodia. This selective association of G3BP1 with smaller membrane protrusions raises the possibility that G3BP1 and rasGAP120 are recruited to sites of cytoskeletal remodeling and assembly. It still remains to be determined what the temporal relationship is between changes in FAK phosphorylation and the recruitment of G3BP1 and rasGAP120. It is also important to consider that although the latter two molecules were coisolated with integrin containing complexes, the results do not necessarily indicate that these molecules are directly interacting with integrins. There could, for example, be several other molecular species that indirectly physically link integrin with G3BP1 and rasGAP120. However, the results of the present study clearly indicate that, under some conditions, ligation of integrin can induce the recruitment of G3BP1 and rasGAP120 to integrin associated complexes. However, they also suggest that these may be very specialized and perhaps transient associations. Abbreviations. BSA, bovine serum albumen; DHB, dihydroxybenzoic acid, FA, focal adhesion; FAK, focal adhesion kinase; G3BP, rasGAP120 binding protein; MALDI, matrix assisted laser desorption ionization; MS, mass spectrometry; MS/ MS, tandem mass spectrometry; QqTOF, quadrupole time-offlight; rasGAP120, ras GTPase activating protein; SDS PAGE, sodium dodecyl sulfate polyacrylamide electrophoresis.
Acknowledgment. J.A.W. thanks Dr. R. Beavis for his helpful discussions on protein identification and spectral analysis and Ms. Sheryl Hagenstein for her role in the preparation of the manuscript. This research was supported by Grants from CIHR (J.A.W.). References (1) Hynes, R. O. Cell 1992, 69, 11-25. (2) Critchley, D. R.; Holt, M. R.; Barry, S. T.; Priddle, H.; Hemmings, L.; Norman, J. Biochem. Soc. Symp. 1999, 65, 79-99. (3) Hemler, M. E. Curr. Opin. Cell Biol. 1998, 10, 578-585. (4) Giancotti, F. G.; Ruoslahti, E. Science 1999, 285, 1028-1032. (5) O’Neill, S.; Robinson, A.; Deering, A.; Ryan, M.; Fitzgerald, D. J.; Moran, N. J. Biol. Chem. 2000, 275, 36 984-36 990. (6) Schwartz, M. A.; Shattil, S. J. Trends Biochem. Sci. 2000, 25, 388391. (7) Miyamoto, S.; Teramoto, H.; Coso, O. A.; Gutkind, J. S.; Burbelo, P. D.; Akiyama, S. K.; Yamada, K. M. J. Cell Biol. 1995, 131, 791805. (8) Geiger, B.; Bershadsky, A. Curr. Opin. Cell Biol. 2001, 13, 584592. (9) Miyamoto, S.; Akiyama, S. K.; Yamada, K. M. Science 1995, 267, 883-885.
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PR0340983
