COMMUNICATION
Activation of beta 1 Integrin Signaling Stimulates Tyrosine Phosphorylation of p190RhoGAP and Membrane-protrusive Activities at Invadopodia*

Hirokazu NakaharaDagger §, Susette C. MuellerDagger , Motoyoshi Nomizu, Yoshihiko Yamada, Yunyun YehDagger , and Wen-Tien ChenDagger par

From the Dagger  Lombardi Cancer Center & Department of Cell Biology, Georgetown University Medical Center, Washington, D. C. 20007 and the  Craniofacial Developmental Biology and Regeneration Branch, NIDR, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The ligation of available alpha 6beta 1 integrin in adherent LOX melanoma cells by laminin G peptides and integrin stimulatory antibodies induced cell invasiveness, independent of adhesion activity of integrins that were pre-bound to extracellular matrix (Nakahara, H., Nomizu, M., Akiyama, S. K., Yamada, Y., Yeh, Y., and Chen, W.-T. (1996) J. Biol. Chem. 271, 27221-27224). Here, we show that this induced invasion involves an increase in tyrosine phosphorylation of a 190-kDa GTPase-activating protein for Rho family members (p190RhoGAP; p190) and membrane-protrusive activities at invadopodia. This tyrosine phosphorylation does not occur when the adherent cells are treated with non-activating antibody against beta 1 integrin, control laminin peptides, or tyrosine kinase inhibitors genistein and herbimycin A. Although p190 and F-actin co-distribute in all cell cortex extensions, tyrosine-phosphorylated proteins including p190 appear to associate with F-actin specifically in invadopodia. In addition, the localized matrix degradation and membrane-protrusive activities were blocked by treatment of LOX cells with tyrosine kinase inhibitors as well as microinjection of antibodies directed against p190 but not by non-perturbing antibodies or control buffers. We suggest that activation of the alpha 6beta 1 integrin signaling regulates the tyrosine phosphorylation state of p190 which in turn connects downstream signaling pathways through Rho family GTPases to actin cytoskeleton in invadopodia, thus promoting membrane-protrusive and degradative activities necessary for cell invasion.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Integrin receptors play important roles in organizing the actin-containing cytoskeleton and in signal transduction from the extracellular matrix (ECM)1 upon cell adhesion to immobilized ECM components (1-3). The assembly of integrin adhesion complexes requires both ECM and Rho/Rac family signaling proteins (4-6). However, integrin's role in cell invasiveness remains to be determined. Invasive cells express a specialized surface structure for invasion, termed invadopodia, which exhibit dynamic motility, adhesion to, and degradation of the ECM (7). Invadopodia show the following biochemical properties: localization of oncogenic variants of pp60src (8), dramatic association of cytoskeleton talin-beta 1 integrin with ECM (9), the elevated tyrosine phosphorylation state of a subset of proteins (10), and the localization of ECM-degrading proteases (11) including gelatinase A (12), seprase (13), and membrane type-1 matrix metalloproteinase (14).

Activated Src may associate with the state of tyrosine phosphorylation of a major RasGAP-associated protein p190RhoGAP (p190) (15-17) or p190B (5), which in turn creates a binding site for the SH2 domains of other signaling molecules to promote actin cytoskeletal motility. p190 contains a GTPase domain at its amino terminus and a GTPase-activating protein (GAP) domain at its carboxyl terminus. It forms a stable association with the p120RasGAP that down-regulates Rho and Rac activity (16, 18, 19). Specifically, pp60c-src regulates the simultaneous rearrangement of actin cytoskeleton and p190 following epidermal growth factor stimulation (17). Microinjection of the p190 into Swiss 3T3 cells blocks Rho-mediated changes in membrane ruffling (20) and the decrease in fibronectin adhesion (21). These findings suggest a role for p190 in connecting actin motility to downstream signaling pathways mediated through different Rho family GTPases.

Recently, the laminin alpha 1 chain carboxyl-terminal globular domain (G domain amino acid residues 2111-3060) has been shown to have adhesion activities involving integrins (22-24). We showed that laminin G peptide AG-10 acts on the alpha 6beta 1 integrin signaling of invasion by stimulating localized ECM degradation at invadopodia that are distinct from their direct effects on cell adhesion on immobilized ECM (25). Here we have examined tyrosine phosphorylation of signaling molecules in beta 1 integrin-ligation using this recently developed model of melanoma invasion that can be induced by laminin G peptides and anti-beta 1 integrin antibodies (25). Soluble peptides and antibodies specific for beta 1 integrins were added to melanoma cells that had already adhered to immobilized fibronectin and gelatin films and the state of protein tyrosine phosphorylation and membrane-protrusive activities at invadopodia were assessed.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- The human amelanotic melanoma cell line LOX was obtained and cultured as described (25). Mouse laminin-1 and all laminin G peptides, including AG-10 (NPWHSIYITRFG) and control AG-10-s (scrambled peptide of AG-10, NYITRFGPWHSI), were prepared as described (23). Rat mAb 13 (against human beta 1 integrin subunit) was provided by Dr. S. K. Akiyama (NIH, Bethesda, MD). Monoclonal antibody K20 (against human beta 1 integrin subunit) was purchased from Immunotech (Westbrook, ME); anti-FAK antibody, anti-RasGAP, anti-p120 pp60src-s, anti-phosphotyrosine mAb 4G10 from Upstate Biotechnology (Lake Placid, NY); anti-p190, anti-cortactin, anti-p130cas from Transduction Laboratory (Lexington, KY); and genistein, herbimycin A, and cytochalasin D from Calbiochem Novabioch (San Diego, CA).

Culture of Cells on ECM Substrata, in Vitro Degradation/Invasion Assay, and Immunofluorescence-- Suspended cells (105/ml) were added to fluorescein-fibronectin-coated cross-linked gelatin films on a 15-mm round glass coverslip in a 12-well plate for in vitro degradation/invasion assay as described (25, 26). Cells were allowed to grow on the films for the indicated times, fixed and stained with rhodamine conjugate of phalloidin (against F-actin, Molecular Probes), and antibodies were directed against p190 or other proteins. The cells were further stained with fluorescein conjugate of secondary antibodies. Stained samples were photographed using the Planapo 63/1.4 or 25/1.2 objectives on a Zeiss Photomicroscope III (Carl Zeiss, Inc., Thornwood, NY) under epifluorescence as described (13).

Preparations of Cell Lysate, Immunoprecipitation, and Immunoblotting-- After treatment with laminin G peptides or anti-beta 1 integrin antibodies, cell monolayers were washed with ice-cold PBS and lysed with a modified immunoprecipitation buffer A (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 10 mM sodium fluoride, 0.5 mM orthovanadate, 10 mg/ml aprotinin, 5 mg/ml leupeptin) as described (27). Lysates were clarified by centrifugation at 15,000 × g for 15 min. Antibodies were added to the lysate (1:100 antibody:protein concentration), and samples were rotated at 4 °C for 1 h. To precipitate the antibody-antigen complexes, Gammabind-Sepharose (Life Technologies, Inc.) was added to the lysates, and rotation was continued for 2 h. The immunoprecipitates, pelleted by low speed centrifuging, were washed three times in wash buffer (buffer A without sodium deoxycholate). For anti-phosphotyrosine immunoblotting, the antibody-antigen pellet was boiled in SDS buffer, electrophoresed on 7.5% SDS-polyacrylamide gels, transferred to nitrocellulose, and visualized by incubation with horseradish peroxidase-conjugated anti-phosphotyrosine 4G10 antibody followed by enhanced chemiluminescence detection (ECL, Amersham Corp.). To detect other proteins shown in Fig. 2, the YPP blot was stripped and reprobed with antibodies against individual proteins, followed by horseradish peroxidase-conjugated secondary antibodies.

Microinjection-- LOX cells were seeded on fibronectin/gelatin films that had been coated on a gridded coverslip (CELLocate, Eppendorf, Hamburg, Germany). Prior to microinjection, anti-p190 antibodies were dialyzed against PBS for 1 day. Antibody solutions at the concentration of 1 mg/ml were microinjected into the cytoplasm with Femtotip microcapillaries (Eppendorf) using the Eppendorf microinjection system (Transjector 5246/micromanipulator 5171). Mouse immunoglobulin G (IgG) at 1 mg/ml was used as a control and in some experiments the injected IgG was localized with fluorescein-labeled goat anti-mouse IgG. In all cases, cells showing a response to the microinjected antibodies contained the mouse IgG inside the cells.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

To examine tyrosine phosphorylation of proteins in cells during the induced matrix degradation, we seeded a human melanoma cell line LOX on immobilized fibronectin-cross-linked gelatin films in the presence of serum and allowed the cells to adhere for 1 h. Serum-free media containing soluble peptides or anti-integrin antibodies were then added to cells for an additional 5 h to assess the induced ECM degradation/invasion. As shown in Fig. 1A (lanes marked m13, AG-10), ligation of beta 1 integrin by stimulatory anti-beta 1 integrin mAb 13 or laminin G peptide AG-10 resulted in elevated tyrosine phosphorylation of proteins of 190, 120-130, and 60-90 kDa in molecular mass as revealed by immunoblotting using anti-phosphotyrosine mAb 4G10. When lysates obtained from cells treated with a non-activating anti-beta 1 integrin mAb K20, non-immune IgG, or control laminin peptide AG-10-s, major bands around 120-130 and 60-90 kDa could be detected. These results suggest that major 120-130- and 60-90-kDa proteins are tyrosine-phosphorylated during continued cell adhesion as described (1-3, 28), whereas the 190-kDa protein may be involved in the formation of invadopodia.


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Fig. 1.   Soluble AG-10 laminin G peptide and beta 1 integrin antibody m13 promote tyrosine phosphorylation of cellular proteins at invadopodia and enhance the local ECM degradation by LOX cells. A, YPP profile of LOX cells in response to treatment with soluble laminin G peptide and beta 1 integrin antibodies. Antibodies (10 µg of antibodies per ml): non-immune IgG (IgG), non-activating anti-beta 1 integrin mAb (K20), or activating beta 1 integrin mAb (m13), and laminin G peptides (50 µg/ml): AG-10 or AG-10-s, control scrambled peptide of AG-10, were added to adherent cells 1 h after seeding and incubated for 5 h. Cell lysates were extracted with Triton X-100 in the presence of vanadate. The soluble material was separated by SDS-PAGE. The protein bands were transferred onto a nitrocellulose paper and immunoblotted with mouse mAb 4G10 to identify the YPP. Each lane contains an aliquot representing an equal volume from the original extract. Molecular masses (kDa) are given at left. B, local degradation of the fibronectin film by LOX (left panel) and YPP localization at focal adhesions (arrowheads) and invadopodia (arrows) of the cell (right panel) in the presence of AG-10. C, lack of degradation but YPP localization at focal adhesions (arrowheads) and residual invadopodia (arrows) of the cell (right panel) in the presence of AG-10 and genistein (5 µg/ml). D, effect of the tyrosine kinase inhibitor genistein. Cell invasiveness was determined by counting the percentage of cells that produced ECM degradation spots on the film and that exhibited invadopodia (see Fig. 3). Each value represents the mean ± S.D. of three independent experiments, in which 200 cell counts were made.

To determine the effect of tyrosine kinase inhibitors on the formation of focal adhesions and invadopodia, LOX cells cultured on fluorescently labeled films and treated with laminin peptides or anti-beta 1 integrin antibodies were further treated with genistein and herbimycin A. Intense localization of YPPs was observed at invadopodia, which correspond to degradation sites of the fibronectin-gelatin substratum, and at focal adhesions of activated LOX cells (Fig. 1B). Local ECM degradation and tyrosine phosphorylation at invadopodia were blocked by genistein (5 µg/ml) (Fig. 1C) and herbimycin A (20 µg/ml), but cells remained attached to the substratum and retained tyrosine phosphorylation at focal adhesions (arrowheads, Fig. 1C). Following wash out of genistein, cells regained their ability to degrade the films (data not shown). As shown in Fig. 1D, laminin G peptide- or beta 1 integrin antibody-induced cell invasiveness can be blocked by genistein in a concentration-dependent manner. Thus, we suggest that a signaling pathway involving YPPs regulates these invadopodial activities.

To identify a YPP involved in cell invasion, samples from adherent cells stimulated by beta 1 integrin antibodies were immunoprecipitated with antibodies against potential molecules, and they were analyzed by immunoblotting with an anti-phosphotyrosine antibody (Fig. 2, upper panel). When immunoprecipitated from adherent cells treated with non-immune IgG or non-activating beta 1 integrin antibody K20, p190 exhibited very low levels of tyrosine phosphorylation (Fig. 2, left two lanes). Interestingly, p190 from cells treated with stimulatory beta 1 integrin mAb m13 shows elevated tyrosine phosphorylation (Fig. 2, right lane). Thus, p190 may be the 190-kDa protein that was seen to increase its tyrosine phosphorylation in response to laminin G peptides and beta 1 integrin antibody m13 (Fig. 1A). The 120-130- and 60-90-kDa YPPs (Fig. 1A) are major phosphotyrosine forms during early attachment of cells on the fibronectin-gelatin film (within 6 h). Among these molecules, p120RasGAP (RasGAP), p120src substrate (p120src-s), cortactin, and pp60src (Fig. 2) show enhanced tyrosine phosphorylation in cells treated with K20 and m13 antibodies as compared with that treated with non-immune IgG. However, treatment of beta 1 integrin antibodies had no apparent effect on the tyrosine phosphorylation of FAK and p130cas (Fig. 2), which already may be highly phosphorylated in cells adherent on ECM as previously shown (29). Therefore, additional beta 1 ligation may not produce any detectable change in tyrosine phosphorylation of FAK and p130cas (Fig. 2). We conclude that the increased tyrosine phosphorylation of p190 is implicated in invadopodial activities while that of p120RasGAP, p120src substrate, FAK, p130cas, cortactin, and pp60src may be due to adhesion and antibody binding to beta 1 integrins.


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Fig. 2.   Protein tyrosine phosphorylation in LOX cells treated with beta 1 integrin antibodies. Adherent LOX cells were stimulated with antibodies (10 µg of antibodies per ml): non-immune IgG (IgG) (lanes 1, 4, 7, 10, 13, 16, and 19), non-activating anti-beta 1 integrin mAb (K20) (lanes 2, 5, 8, 11, 14, 17, and 20) or activating beta 1 integrin mAb (m13) (lanes 3, 6, 9, 12, 15, 18, and 21). Immunoprecipitates of antigens, including p190RhoGAP (p190), p120RasGAP (rasGAP), p120src substrate (p120(src-s)), FAK, p130cas, cortactin, and pp60src, were analyzed by anti-phosphotyrosine immunoblotting (upper panel). The blots were stripped and reprobed with anti-p190, anti-RasGAP, anti-p120src substrate, anti-FAK, anti-p130cas, cortactin, and pp60src. No significant difference in the amount of these proteins was found in cells treated with these antibodies (lower panel).

To study the connection between p190 tyrosine phosphorylation and actin-driven membrane motility, activated LOX cells that had invaded fibronectin-coated gelatin films were labeled with phalloidin (for F-actin) (Fig. 3A) and anti-p190 (Fig. 3B). Intense F-actin (Fig. 3A) and p190 (Fig. 3B) localization was observed in membrane protrusions such as invadopodia and lamellipodia of activated LOX cells. Their localization corresponded to the YPP labeling (Fig. 1B) observed at the cell-ECM interface, the invadopodia. However, YPP labeling was quite low in lamellipodia (Fig. 1B) whereas p190 and F-actin were intense in these membranes (Fig. 3, short arrowheads). The data suggest that tyrosine-phosphorylated p190 and F-actin co-distribute in membrane protrusions during cell invasion.


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Fig. 3.   F-actin co-distributes with p190 in membrane protrusions. A and B, fluorescent microscopies of F-actin and p190 distribution on AG-10 induced cells. LOX cells were treated with AG-10 peptide (50 µg/ml). Cells were labeled with anti-p190 antibody that was detected with a fluorescein conjugate of secondary antibodies (panel A) and the rhodamine conjugate of phalloidin (to stain F-actin, panel B). Both p190 and F-actin were concentrated in membrane protrusions including invadopodia (large arrows) and lamellipodia in the periphery (short arrows) of the cell. Bar, 10 µm.

To further examine the role of p190 in cell invasion, we microinjected antibodies directed against p190 into LOX cells that were cultured on immobilized fibronectin-cross-linked gelatin films and assessed effects of antibodies on ECM degradation/invasion (Fig. 4). Effects of antibody injection were determined by counting the number of cells among a total of 25 injected cells in a designated area on fibronectin films. Injection of antibodies up to concentrations of 1 µg/µl or PBS into melanoma cells, which had been plated on fibronectin films for 3 h and cultured for another 5 h after injection, did not affect the number of cells that remained attached 5 h after injection. Microinjection of anti-p190 antibodies into LOX cells blocked their degradative activities (Fig. 4, A and B) whereas that of non-immune IgG did not (Fig. 4, C and D). There was a 10-fold decrease in invasiveness when cells were microinjected with anti-p190 antibodies (p < 0.005) compared with that of cells injected with control non-immune IgG or control saline alone (Fig. 4E). We conclude that p190 exerts its signaling effects on invadopodial activities through beta 1 integrins.


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Fig. 4.   Microinjection of antibodies directed against p190 into LOX cells inhibits localized matrix degradation. A, B, C, and D, cells microinjected with anti-p190 antibody (A and B) or non-immune IgG (C and D) were examined for the degradation of fibronectin substrata (A and C) by these cells shown in phase contrast images (B and D). Microinjection of anti-p190 antibodies into LOX cells completely blocks their degradative activities (A and B). Panel width, 150 µm. E, cell populations microinjected with anti-p190 antibodies were analyzed for their invasiveness. Five sets of 25 cells were used for each antibody or control buffer. The values are mean ± S.D. Cells microinjected with control mouse IgG degraded significantly more fibronectin-gelatin films (p <0.005) than those microinjected with antibodies against p190. Furthermore, the former had similar degradative potential as cells injected with buffer alone.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The biochemical mechanisms by which cell invasiveness is induced are poorly understood partly because of the difficulty involved in defining signaling molecules of invasion. Recently, we have shown that cell invasiveness can be triggered when alpha 6beta 1 integrin on ECM-free surfaces of the cell is ligated by soluble laminin G peptides or antibodies (25). Activated alpha 6beta 1 integrin was shown to induce the recruitment of seprase, a serine-type integral membrane protease, to invadopodia of invading cells, and a subsequent increase of local ECM degradation. In this report, we show that, when alpha 6beta 1 integrin is ligated by laminin G peptides or antibodies, p190 becomes tyrosine-phosphorylated and associates with F-actin in a specific membrane protrusion, invadopodia. In addition, the stimulated invasive phenotype was blocked by treatment of LOX cells with tyrosine kinase inhibitors and microinjection of antibodies directed against p190. Thus, activation of alpha 6beta 1 integrin may regulate the tyrosine phosphorylation state of p190 and its association with F-actin organization at invadopodia. The tyrosine-phosphorylated p190 may serve as a binding site for tyrosine kinases containing SH2 and link the Rho pathway to the actin cytoskeleton, thus promoting membrane-protrusive activities necessary for cell invasion.

Following activation of beta 1 integrins, the tyrosine kinase responsible for p190 phosphorylation remains to be identified. Src family tyrosine kinases are present and active in invadopodia of invasive cells (8, 10). Activated Src may create a binding site for SH2 domains of p190 (15-17) or p190B (5). Thus, c-Src may phosphorylate p190 that in turn may regulate actin polymerization mediated by different Rho family GTPases (16-19). Also, p190 associates with membrane-protrusive activities involving Rho-mediated changes in the organization of cytoskeletal actin (20) and the decrease in fibronectin adhesion (21). Microinjection and tyrosine kinase inhibition experiments present in this report thus support the role of tyrosine-phosphorylated p190 in invadopodial activities.

Cell adhesion to ECM promotes tyrosine phosphorylation of many signaling molecules. It has been shown recently that antibody-mediated clustering of alpha 5beta 1 integrin causes recruitment of p190B and Rho family members to the cell surface (29, 30). Antibody-mediated integrin clustering and adhesion of cells to fibronectin leads to an increase in the tyrosine phosphorylation of Src tyrosine kinases, FAK, p130cas, talin, paxillin, cortactin, and tensin (2, 6, 29-32). While we show a specific association of p190 tyrosine phosphorylation with invadopodial formation, we have confirmed the observation that antibody activation of integrin promotes tyrosine phosphorylation of pp60src, cortactin, FAK, p120RasGAP, and p120src substrate (Fig. 2). It is possible that tyrosine phosphorylation of these signaling molecules in response to adhesion is a prerequisite for the expression of cell invasiveness. Consistent with the inhibition of invasiveness in cells microinjected with anti-p190 antibody (Fig. 4E), there was a more than 3-fold decrease in invasive potential when cells were microinjected with anti-FAK, cortactin, or pp60src antibodies (data not shown). Thus, we suggest that FAK, cortactin, and pp60src may exert their primary signaling effects on cell invasion through integrin-mediated adhesion on ECM.

    ACKNOWLEDGEMENTS

We are most grateful to L. Howard, L. A. Goldstein (Georgetown University), and Steve Akiyama (NIH) for critical discussion.

    FOOTNOTES

* This work was supported by United States Public Health Service Grants R01 CA-39077 and HL-33711 (to W.-T. C.) and CA-61273 (to S. C. M.) and in part by United States Public Health Service Grant 2P30-CA-51008 from the Lombardi Cancer Center Microscopy & Imaging Shared Resource.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: The First Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Osaka University, Osaka, 565 Japan.

par To whom correspondence should be addressed: Lombardi Cancer Center & Department of Cell Biology, TRB E415 Georgetown University, 3970 Reservoir Rd. N.W., Washington, D. C. 20007. Tel.: 202-687-1769; Fax: 202-687-3300; E-mail: chenw{at}gunet.georgetown.edu.

1 The abbreviations used are: ECM, extracellular matrix; FAK, focal adhesion kinase; GAP, GTPase activating protein; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SH2, Src homology 2 domains; YPP, tyrosine-phosphorylated proteins.

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Abstract
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Discussion
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