From the 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
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ABSTRACT |
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The ligation of available 6
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
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
6
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.
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INTRODUCTION |
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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-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 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
6
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
1 integrin-ligation using this recently developed model of
melanoma invasion that can be induced by laminin G peptides and
anti-
1 integrin antibodies (25). Soluble peptides and antibodies
specific for
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.
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EXPERIMENTAL PROCEDURES |
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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 1 integrin subunit) was provided by
Dr. S. K. Akiyama (NIH, Bethesda, MD). Monoclonal antibody K20
(against human
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-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.
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RESULTS |
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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 1 integrin by
stimulatory anti-
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-
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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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-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
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 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
1 integrin antibody K20, p190 exhibited very low
levels of tyrosine phosphorylation (Fig. 2, left two lanes).
Interestingly, p190 from cells treated with stimulatory
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
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
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
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
1
integrins.
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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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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 1
integrins.
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DISCUSSION |
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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 6
1 integrin on ECM-free
surfaces of the cell is ligated by soluble laminin G peptides or
antibodies (25). Activated
6
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
6
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
6
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 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 5
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.
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ACKNOWLEDGEMENTS |
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We are most grateful to L. Howard, L. A. Goldstein (Georgetown University), and Steve Akiyama (NIH) for critical discussion.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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