Correspondence to: Arthur M. Mercurio, Beth Israel Deaconess Medical Center-Dana 601, 330 Brookline Ave., Boston, MA 02215. Tel:(617) 667-7714 Fax:(617) 975-5531 E-mail:amercuri{at}bidmc.harvard.edu.
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Abstract |
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We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the 6ß4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express
6ß4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the
6ß4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis (~1 ng/ml), the
6ß4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained
6ß4 in association with F-actin. Importantly, we demonstrate that this mobilization of
6ß4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-
and that it is associated with the phosphorylation of the ß4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actinrich cell protrusions that mediate
6ß4-dependent cell movement but also the disruption of
6ß4-containing hemidesmosomes by protein kinase C.
Key Words: integrins, cell movement, PKC, hemidesmosomes, cytoskeleton
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Introduction |
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CHEMOTACTIC migration is essential for embryonic development, tissue homeostasis, and the immune response
The 6ß4 integrin is ideal for studying differences in integrin function between stably adherent and migrating epithelial cells. This integrin, which is a receptor for the laminins (reviewed in
6ß4 mediates the formation of stable adhesive structures termed hemidesmosomes that link the intermediate filament cytoskeleton with the extracellular matrix
6ß4, the classical hemidesmosome contains at least three other known proteins: BPAG-11, BPAG-2, and HD1/plectin
6ß4 and HD1/plectin
6ß4 is linked to intermediate filaments through HD1/plectin, and that this interaction is critical for hemidesmosomal formation
6ß4 integrin in hemidesmosome function and epithelial architecture has been reinforced by the generation of ß4-nullizygous mice. The most obvious defect in these mice is a loss of hemidesmosomes and detachment of the epidermis
6ß4 can stimulate carcinoma migration and invasion through its ability to interact with the actin cytoskeleton and mediate the formation and stabilization of lamellae
6ß4 in enhancing the migration of invasive carcinoma cells is quite distinct from its role in maintaining stable adhesive contacts in normal epithelia by associating with intermediate filaments. In fact, we have established that the ability of
6ß4 to stimulate carcinoma migration and invasion depends upon its activation of distinct signaling pathways including PI3-K
6ß4 with F-actin and the activation of a specific signaling pathway by this integrin.
The above observations raise the important issue of whether migratory stimuli influence the localization and cytoskeletal interactions of 6ß4. Such changes could provide a mechanism to account for the dichotomy of
6ß4 function in stably adherent and migrating cells. To address this issue, we used squamous carcinoma-derived A431 cells for several reasons. A431 cells express
6ß4, as well as the EGF receptor that is known to stimulate their chemotactic migration and in vitro invasion
6ß4 integrin from hemidesmosomes and increases the formation of
6ß4-containing lamellipodia and membrane ruffles. Importantly, we also demonstrate that this mobilization of
6ß4 occurs by a protein kinase C (PKC)dependent mechanism, and that it involves the phosphorylation of the ß4 integrin subunit on serine residues.
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Materials and Methods |
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Cells and Reagents
The A431 squamous carcinoma cell line was obtained from the American Type Culture Collection and maintained in DMEM with 10% fetal calf serum, at 37°C in a humidified atmosphere containing 5% CO2.
The following antibodies were used in this study: mouse mAb 2B7 (integrin 6specific) was prepared in our laboratory
6specific) was purchased from Immunotech; rat mAb 439-9B (integrin ß4specific; reference 18) was provided by Dr. Rita Falcioni (Regina Elena Cancer Institute, Rome, Italy). A peptide-specific antiserum elicited against the last 20 amino acids of the carboxy terminus of the ß4 subunit was prepared commercially. A rabbit polyclonal antibody specific for the EGF receptor was purchased from Santa Cruz Biotechnology. The phosphotyrosine-specific antibodies PY20 and 4G10 were purchased from Transduction Labs and Upstate Biotechnology Incorporated, respectively. Mouse monoclonal antibodies specific for BPAG-1 (R185), BPAG-2 (1D1), and HD1/plectin (121)
Laminin-1, prepared from the EHS sarcoma was provided by Dr. Hynda Kleinman (NIDR, Bethesda, MD). Collagen type I was purchased from Collagen Corp. Human recombinant EGF was purchased from Sigma Chemical. The PKC inhibitor Gö6976 was obtained from Alexis Corp. PMA was obtained from Calbiochem-Novabiochem.
Chemotaxis Assays
Chemotaxis was analyzed using 6.5-mm TranswellTM chambers, 8-µm pore size (Costar). The separating membrane was coated with laminin-1 (20 ug/ml) on both sides for 2 h at room temperature and then blocked with 1% albumin in PBS for 30 min. A431 cells (3 x 104) were resuspended in DMEM containing 0.1% albumin. In some experiments, antibodies (10 µg/ml of 2B7 or mouse IgG control) were added to the resuspended cells. The cells were added to the top wells of the TranswellTM chambers and allowed to settle on the filters for 1 h at 37°C, before EGF (1 ng/ml) was added to the lower chamber. In some cases, Gö6976 (1 µM) or vehicle alone (DMSO) was added 30 min before EGF stimulation. After a 2-h incubation, cells that had not migrated from the upper surface of the membrane were removed using cotton swabs and the remaining cells on the lower side of the membrane were fixed in methanol, dried, and stained with a 0.2% solution of crystal violet in 2% ethanol. Migration was quantified by digital analysis as described below.
Analysis of Lamellar Area
A431 cells were plated on laminin-1 for 1 h and either not stimulated or stimulated with EGF (1 ng/ml) for 15 min in the presence or absence of antibody (2B7 or IgG, 10 µg/ml). The antibodies were added 30 min before EGF stimulation. The lamellar area, defined as a characteristic flat and thin protrusion of the cell containing no vesicles, was measured using a Nikon Diaphot 300 inverted microscope with phase contrast optics. This microscope was connected to a CCD camera (Dage-MTI), a frame-grabber (Scion), and a 7600 Power Macintosh computer to capture the images. Images were collected and analyzed with IPlab Spectrum image analysis software. Lamellar area was determined by tracing the lamellae contour and quantifying the area digitally. For each individual experiment 5080 cells were analyzed.
Indirect Immunofluorescence Microscopy
Bacteriological dishes were coated with 20 µg of laminin-1 or collagen type I for 2 h at room temperature and the dishes were then blocked with PBS containing 1% bovine serum albumin (BSA) for 30 min. A431 cells were resuspended in serum-free RPMI 1640 medium containing 10 mM Hepes and 0.1% BSA. The cells were plated at low density (2 x 104 cells/cm2) on the matrix-coated dishes and allowed to adhere for 12 h in a humidified atmosphere with 5% CO2 at 37°C. When indicated, Gö6976 (0.51 µM) in DMSO or vehicle alone was added 30 min before stimulation. The cells were then stimulated with either EGF (0.5100 ng/ml) for 15 min or PMA (2550 ng/ml) for 30 min.
Cells were fixed with a buffer containing 2% paraformaldehyde, 100 mM KCl, 200 mM sucrose, 1 mM EGTA, 1 mM MgCl2, 1 mM PMSF, and 10 mM Pipes at pH 6.8 for 15 min. In some cases, the cells were extracted before fixation with a buffer containing 0.2% Triton X-100, 100 mM KCl, 200 mM sucrose, 10 mM EGTA, 2 mM MgCl2, 200 µM sodium vanadate, 1 mM PMSF, and 10 mM Pipes at pH 6.8 for 1 min. After fixation, the cells were rinsed with PBS and incubated with a blocking solution that contained 1% albumin and 5% goat serum in PBS for 30 min. Cells that were to be stained for HD1/plectin, BPAG-1 or BPAG-2, were fixed with acetone/methanol 1:1 (vol/vol) instead of paraformaldehyde. Either primary antibodies or FITC phalloidin (20 µg/ml) in blocking solution were added to the fixed cells separately or in combination for 30 min. The cells were rinsed three times and either a fluorescein-conjugated donkey antimouse or a rhodamine-conjugated donkey antirat IgG (minimal cross-reaction inter-species; Jackson ImmunoResearch Laboratories) in blocking buffer (1:150) were used separately or in combination to stain the cells for 30 min. Cells were rinsed with PBS and mounted in a mixture (8:2) of glycerol and PBS (pH 8.5) containing 1% propylgallate. The dishes were cut into slides and examined by confocal microscopy.
Detergent Extractions
To obtain a fraction enriched in cytokeratins
Analysis of Protein Phosphorylation
To examine tyrosine phosphorylation using phosphotyrosine-specific antibodies, A431 cells were plated on laminin-1coated dishes for 1 h at 37°C as described above. Cells were then stimulated with EGF (1100 ng/ml) for 15 min, extracted with RIPA buffer containing 0.1% SDS, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM sodium vanadate, 1 mM PMSF, 10 ug/ml each of leupeptin, pepstatin A, and aprotinin, and 50 mM Tris-HCl, pH 7.5. The samples were immunoprecipitated using the 439-9B antibody, resolved by SDS-PAGE and immunoblotted using a combination of both the PY20 and 4G10 phospho-specific antibodies. Immune complexes were detected using a secondary antibody conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence (Amersham, Inc.). Subsequently, the membranes were stripped and immunoblotted with the ß4-specific polyclonal antibody.
For metabolic radiolabeling with 32PO4, A431 cells (2 x 106) were plated on laminin-1coated dishes for 30 min as described above. Subsequently, the medium was removed and replaced with a phosphate-deficient medium (GIBCO). After a 1-h incubation in this medium, 32PO4 [0.52.0 mCi/ml (NEN)] was added and the cells were incubated for an additional 2 h. When indicated, Gö6976 (0.51 µM) in DMSO or vehicle alone was added 30 min before stimulation. The cells were then stimulated with either EGF (0.5100 ng/ml) for 15 min or PMA (2550 ng/ml) for 30 min. The cells were extracted with RIPA buffer as described above and the extracts were immunoprecipitated with 439-9B antibody, resolved by PAGE (6% gels) and transferred to PVDF membranes (Immobilon-P; Micropore). The membranes were exposed to x-ray film, developed, and then immunoblotted with the ß4-specific polyclonal antibody to control for equivalent amounts of protein in the samples. A quantitative analysis of the relative intensities of the radioactive bands was made using a 2D electronic counter (Instant Imager; Packard, Meriden, CT). For phosphoamino-acid analysis, the area of the membrane that contained the ß4 subunit was excised with a razor, acid hydrolyzed, and the hydrolysate was separated using 2D-thin layer chromatography following standard techniques
Construction, Analysis, and Expression of PKC- cDNAs
Wild-type and myristoylated PKC- constructs were generated by PCR using a bovine PKC-
cDNA as a template. The wild-type PKC-
was subcloned into the pCMV5 mammalian expression vector using the EcoRI and XhoI sites. The FLAG epitope (DYKDDDDK) was added to the carboxy terminus of the PKC-
cDNA by PCR. The myristoylated PKC-
cDNA was constructed by adding the relevant PCR fragment of bovine PKC-
to the XbaI and EcoRI sites of pCMV6 and by adding the Src myristoylation site (MYPYDVPDYA) at the amino terminus. All sequences were confirmed by DNA sequencing.
To assess the activity of the PKC- cDNAs, human embryonic kidney 293T cells were transfected with 1 µg of either the vector alone (pCMV5), PKC-
-FLAG cDNA, or myristoylated PKC
-FLAG cDNA using calcium phosphate for 6 h. Subsequently, the cells were cultured in the absence of serum for 24 h and extracted in a 1% NP-40 buffer
proteins were immunoprecipitated using a FLAG M2 mAb (Sigma Chemical Co.) and a mixture of protein A and G beads. The beads were washed stringently and immune complex kinase assays were performed on the washed beads using MBP as the substrate (see reference 13 for details). No lipid cofactors were added to the reaction mix. The kinase assay was resolved on a 12.5% SDS gel and phosphorylated MBP was detected by autoradiography. Relevant expression of PKC-
was detected by immunoblotting total cell extracts with a PKC-
specific polyclonal Ab (Santa Cruz).
To analyze the effects of PKC- expression on A431 cells, these cells were transfected with 5 µg of each construct using Superfect (Qiagen) according to manufacturer's guidelines. After 24 h, the cells were fixed as described above and double stained with anti-FLAG mAb and either GoH3 mAb or mAb 121.
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Results |
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EGF Stimulation of A431 Cells Redistributes the Localization of the 6ß4 Integrin from Hemidesmosomes to Lamellipodia and Membrane Ruffles
Indirect immunofluorescence microscopy revealed that the 6ß4 integrin on the ventral surface of A431 cells plated on either laminin-1 (Figure 1AD) or collagen (not shown) is localized primarily in discrete structures and plaques in areas that exclude stress fibers. This pattern of staining for
6ß4 is characteristic of its localization in hemidesmosomes
6ß4 with the hemidesmosomal components BPAG-1 (Figure 1 A), BPAG-2 (Figure 1 B), and HD1/plectin (Figure 1 C) was evident in these structures. These data establish that
6ß4 is localized in structures that are characteristic of hemidesmosomes on the ventral surface of adherent A431 cells.
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Our previous data established that 6ß4 participates in carcinoma migration through its ability to interact with the actin cytoskeleton
6ß4 and no
6ß1 integrin (
6-subunit specific antibodies to examine the contribution of
6ß4 to A431 chemotaxis. Treatment of A431 cells with the 2B7 mAb inhibited chemotaxis toward EGF on laminin-1 by 60% (Figure 2 B). This mAb did not inhibit the attachment of the cells to laminin-1 (data not shown), indicating a distinct function for
6ß4 in the chemotactic migration of A431 cells. Lamellipodial protrusions are thought to be critical in cell migration and are the basis for generating lamellae, which are larger protrusions that are associated with the direction the cell migrates
6ß4 in the formation of such protrusions is supported by the fact that the lamellar area of A431 cells was substantially reduced by pretreatment with the 2B7 mAb before plating on laminin-1 (Figure 2 C).
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The findings that 6ß4 is localized in hemidesmosomes in adherent A431 cells and that EGF stimulated their
6ß4-dependent migration raised the possibility that EGF also altered the localization and cytoskeletal interactions of this integrin. Under these conditions of EGF stimulation, a striking change in the localization of
6ß4 was apparent. Specifically, this integrin was substantially reduced in hemidesmosomes on the ventral surface (Figure 1D and Figure E, and Figure 7, left panels), but it was readily apparent in the lamellipodia and ruffles that are formed in response to EGF stimulation (Figure 1 E). We observed also that EGF stimulation results in a reduction of HD1/plectin staining in hemidesmosomes, indicating a disassembly of hemidesmosome structure (Figure 1 F).
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Our observation that 6ß4 is redistributed from hemidesmosomes to lamellipodia and membrane ruffles in response to EGF stimulation prompted us to examine its association with cytokeratins and F-actin in more detail using an in situ extraction scheme that solubilizes proteins to an extent that correlates with their cytoskeletal associations
6ß4 present in each fraction was detected by immunoprecipitation and subsequent immunoblotting with ß4-specific antibodies. The relative distribution of actin and cytokeratin among the three fractions was also determined to assess the efficiency of the fractionation (Figure 3). As expected, the cytokeratins were present largely in fraction 3 and actin was distributed between fractions 1 and 2, which represent the G-actin and F-actin pools, respectively. Importantly, EGF stimulation did not alter this relative distribution of cytoskeletal proteins among the three fractions. However, as shown in Figure 3, EGF stimulation resulted in a substantial reduction in the amount of
6ß4 in fraction 3 (cytokeratin) and an increase in the amount of
6ß4 in the actin fraction in comparison to unstimulated cells. Densitometric analysis of the ß4-specific bands in this figure revealed an approximate 63% reduction of
6ß4 in the cytokeratin fraction and a 48% increase in the actin fraction. These observations provide evidence that the mobilization of
6ß4 from hemidesmosomes that we detected by indirect immunofluorescence microscopy is associated with a disruption in its association with cytokeratins and an increase in its association with F-actin.
The localization of 6ß4 in membrane ruffles and lamellipodia that form in response to EGF stimulation prompted us to explore the possibility of its association with F-actin in these structures because we had previously observed such an association in colon carcinoma cells
6ß4 with F-actin in cell protrusions detected by immunofluorescence was retained in a significant number of protrusions after extraction of EGF-stimulated cells with a Triton X-100 buffer that preserves the actin cytoskeleton (Figure 4A and Figure B). However, extraction of these EGF-stimulated cells with the Tween-40/DOC buffer described above eliminated both the F-actin and
6ß4 staining (data not shown). Taken together, these findings indicate that EGF stimulates a dissociation of
6ß4 from cytokeratin-associated hemidesmosomes, as well as the formation of lamellipodia and ruffles that contain
6ß4 in association with F-actin.
The 6ß4 Integrin Colocalizes with the EGF Receptor and Phosphotyrosine in Membrane Ruffles and Lamellipodia
Our findings that EGF stimulation mobilizes 6ß4 from hemidesmosomes and promotes
6ß4-dependent chemotaxis suggested a possible association between
6ß4 and the EGF receptor. To address this possibility, EGF-stimulated A431 cells were stained for both
6ß4 and the EGF receptor. As shown in Figure 5 A, a striking colocalization of these two receptors was evident in membrane ruffles and lamellipodia. The specificity of this colocalization is evidenced by the finding that another surface protein, the HLA antigen, was not present in these F-actinrich structures (data not shown). We were unable, however, to detect a specific, physical association between
6ß4 and the EGF receptor by coimmunoprecipitation (data not shown).
Given the fact that the EGF receptor is a tyrosine kinase and the report that EGF stimulation of A431 cells results in a substantial increase in the tyrosine phosphorylation of the ß4 integrin subunit 6ß4 (Figure 5 B) or EGFR (data not shown) with phosphotyrosine in these structures was not evident. Moreover, significant phosphotyrosine staining was not evident in hemidesmosomes (Figure 5 B). EGF stimulation, however, resulted in the colocalization of phosphotyrosine and
6ß4 in lamellipodia and ruffles (Figure 5 C). From these results, we can conclude that
6ß4 is associated with more phosphotyrosine-containing proteins in cell protrusions than in hemidesmosomes.
EGF Stimulation Induces the Phosphorylation of the ß4 Integrin Subunit on Serine Residues
The colocalization of 6ß4 with phosphotyrosine in the lamellipodia and ruffles of EGF-stimulated A431 cells prompted us to examine the phosphorylation of
6ß4 induced by EGF. For this purpose,
6ß4 was immunoprecipitated from stimulated A431 cells with the 439-9B mAb and the immunoprecipitates were blotted with two phosphotyrosine-specific Abs (PY20 and 4G10). In these experiments, the cells were extracted with RIPA buffer because we observed a nonspecific interaction between
6ß4 and the EGFR using a Triton X-100 buffer (data not shown). Under these conditions, we detected no phosphotyrosine in the ß4 subunit in response to the concentration of EGF (1 ng/ml) that induced
6ß4 redistribution to lamellipodia and ruffles, and that stimulated optimal A431 chemotaxis (Figure 6 A). Moreover, even high concentrations of EGF (100 ng/ml) that induced a rapid rounding-up of adherent A431 cells and did not stimulate chemotaxis (Figure 2 A) induced only a marginal increase, at best, in the phosphotyrosine content of the ß4 subunit as detected by these antibodies (Figure 6 A). Similar results were obtained with other
6 and ß4-specific mAbs including GoH3, 2B7, A9, as well as a ß4-specific polyclonal antibody (data not shown). These findings are in contrast to the report that EGF stimulation induces a substantial increase in the tyrosine phosphorylation of the ß4 subunit in A431 cells
To exclude the possibility that the phosphotyrosine-specific Abs we used were unable to detect significant tyrosine phosphorylation of the ß4 subunit after EGF stimulation, A431 cells were labeled metabolically with 32P-orthophosphate and then stimulated with EGF. As shown in Figure 6 B, EGF stimulation did increase the phosphorylation of the ß4 subunit substantially with half-maximal phosphorylation observed at ~1 ng/ml of EGF. The discrepancy between the phosphotyrosine-specific Ab results and the metabolic labeling results prompted us to do a phosphoamino acid analysis of the radiolabeled ß4 subunit. Surprisingly, we found that the ß4 subunit is phosphorylated almost exclusively on serine (Figure 6 C). Indeed, both the basal and EGF-induced increases in ß4 phosphorylation that we detected in Figure 6 B result from serine phosphorylation (Figure 6 C). This phosphoamino-acid analysis in conjunction with the phosphotyrosine antibody data provide convincing evidence that EGF stimulation induces significant phosphorylation of the ß4 subunit on serine but not tyrosine residues.
Activation of PKC Redistributes 6ß4 from Hemidesmosomes to Cell Protrusions and Induces Phosphorylation of the ß4 Subunit on Serine Residues
The above findings indicated that EGF activates a serine protein kinase that is involved in ß4 phosphorylation and that could also be involved in the redistribution of 6ß4 from hemidesmosomes to lamellipodia and membrane ruffles. We hypothesized that a likely candidate for this kinase is PKC because its activation by EGF is well documented
6ß4 localization in A431 cells. PMA stimulation mobilized
6ß4 from hemidesmosomes (Figure 7, left panels), and increases the formation of
6ß4-containing lamellipodia and ruffles (data not shown) as assessed by indirect immunofluorescence microscopy. These data were substantiated biochemically by analyzing the amount of ß4 that remains associated with the cytokeratin fraction after stimulation with PMA, using the detergent extraction procedure described above. PMA stimulation markedly reduced the amount of
6ß4 in the cytokeratin fraction in comparison to unstimulated cells (Figure 8 C). Consistent with a role of PKC-dependent phosphorylation in the redistribution of
6ß4, we found that PMA stimulation itself increased the phosphorylation of the ß4 subunit significantly as assessed by 32P-orthophosphate labeling (Figure 8 A). The fact that we detected no tyrosine phosphorylation of ß4 in response to PMA stimulation using the phosphotyrosine-specific antibodies (data not shown) indicates that the increase in 32P-orthophosphate labeling can be attributed to serine phosphorylation.
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If PKC activation is required for the mobilization of 6ß4 from hemidesmosomes, inhibition of PKC activity should inhibit this process. To establish this causality, we used Gö6976, an inhibitor of the conventional isoforms of PKC (
, ß,
)
6ß4 localization were profound. As shown in Figure 7Gö6976 blocked the release of
6ß4 from hemidesmosomes on the ventral surface of A431 cells in response to either EGF or PMA stimulation. Consistent with a role for serine phosphorylation in the release of
6ß4 from hemidesmosomes, we found that Gö6976 reduced the EGF-stimulated phosphorylation of the ß4 subunit by ~50% (Figure 8 B), a reduction that corresponds to the level of ß4 phosphorylation observed in the absence of EGF stimulation (see Figure 6 B). Moreover, Gö6976 inhibited the EGF-induced dissociation of
6ß4 from the cytokeratin fraction as assessed by the detergent extraction approach described above (Figure 8 C). This inhibition was evident for both EGF and PMA-stimulated cells (Figure 8 C).
The above data suggested the participation of a conventional PKC isoform in the disassembly of the hemidesmosome and mobilization of 6ß4 integrin. To obtain additional evidence for PKC involvement, we examined the possibility that activation of PKC-
, a widely distributed conventional PKC isoform, was sufficient to disassemble hemidesmosomes in the absence of EGF stimulation. For this purpose, we constructed a constitutively active PKC-
cDNA that contained the Src myristoylation site at its amino terminus. This myristoylated PKC-
exhibited a high level of in vitro kinase activity relative to the wild-type enzyme (Figure 9). Subsequently, we expressed both the myristoylated and wild-type PKC-
cDNAs in A431 cells and analyzed the effect of these cDNAs on hemidesmosome structure. As shown in Figure 10A431 cells that expressed myristoylated PKC-
, as evidenced by expression of the epitope tag (FLAG), showed a striking reduction in hemidesmosomes. More specifically, expression of both HD-1 and
6ß4 was markedly reduced on the basal surface of these cells. In contrast, the cells that expressed the wild-type PKC-
, showed little change in the formation of hemidesmosomes. These results suggest that activation of PKC-
is sufficient to cause redistribution of the
6ß4 integrin and other components of the hemidesmosome.
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PKC Is Essential for 6ß4-mediated Chemotaxis
A key implication of the above findings is that PKC activity is required for EGF-stimulated chemotaxis of A431 cells because the PKC-dependent redistribution of 6ß4 is a necessary event in the mechanism of chemotaxis. We tested this implication by analyzing the effect of Gö6976 on the chemotactic response of A431 cells to EGF. The results in Figure 8 D reveal that Gö6976 inhibited EGF-stimulated chemotaxis by >80%. It is important to note that Gö6976 at the concentrations used did not inhibit the attachment or spreading of A431 cells (see Figure 7). These results support the involvment of PKC in the chemotactic signal elicited by EGF, and they substantiate the importance of a regulated and dynamic redistribution of the
6ß4 integrin in chemotactic migration.
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Discussion |
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The data we present provide insight into the mechanism of cell migration, especially the migration of epithelial and carcinoma cells. Recent work by our group has established that 6ß4 participates in the chemotactic migration of carcinoma cells by interacting with F-actin at their leading edges and regulating essential signaling pathways
6ß4 function in the hemidesmosomes of epithelial-derived cells and its ability to promote the migration of these cells. Epithelial cells use hemidesmosomes to anchor to the basal lamina. These multi-protein structures connect the substratum with cytokeratins to form rigid adhesion complexes. The
6ß4 integrin is an essential component of hemidesmosomes and it is necessary for mediating their adhesive function
6ß4-containing hemidesmosomes to generate stable adhesive contacts, it is reasonable to assume that their function needs to be disrupted to facilitate cell migration similar to the disruption of focal contacts and reduction in the strength of cell-substratum adhesion that occurs during fibroblast migration
6ß4-associated hemidesmosomes in 804G bladder carcinoma cells
6ß4-containing lamellipodia and membrane ruffles, thus establishing a mechanism for the dichotomy of
6ß4 function in stably adherent and migrating epithelial-derived cells. An important implication of this model is that chemotactic factors can drive the migration of invasive carcinoma cells by mobilizing
6ß4 and disassembling hemidesmosomes.
The redistribution of 6ß4 from hemidesmosomes to lamellipodia and ruffles that we observed in response to EGF stimulation, implied the existence of EGF-mediated signaling events responsible for this redistribution. Indeed, an important finding in this study is that redistribution is dependent on the activity of PKC and that it is associated with serine phosphorylation of the ß4 subunit. The serine phosphorylation of ß4 by EGF stimulation provided an important clue in our identification of the kinase activity involved in the redistribution of
6ß4. It is well established that EGF can activate PKC through the PLC-
mediated formation of diacylglycerol and inositol trisphosphate
participates in EGF-stimulated cell migration
6ß4 from hemidesmosomes to lamellipodia and ruffles and stimulating the phosphorylation of the ß4 subunit on serine residues.
Most likely, the conventional PKC isoform, PKC-, is involved in the redistribution of
6ß4 and the disassembly of hemidesmosomes. Gö6976, a specific inhibitor of the conventional PKC isoforms (
, ß,
)
6ß4 from hemidesmosomes and inhibit the EGF-stimulated phosphorylation of the ß4 subunit. Indeed, hemidesmosomes were well preserved in EGF-stimulated A431 cells that had been pretreated with Gö6976. Consistent with the notion that the preservation of hemidesmosomes impedes cell migration, Gö6976 also inhibited the chemotactic response of A431 cells to EGF. In addition to these data, we observed that activation of PKC-
by expression of a constitutively active, myristoylated form of the enzyme, was sufficient to induce the redistribution of
6ß4 and disassembly of hemidesmosomes.
An issue that remains to be addressed is the nature of the signaling pathway that links the EGF receptor to activation of PKC- and the mobilization of
6ß4 from hemidesmosomes. As mentioned above, PLC-
is a likely intermediary
inhibitor U73122 (1 µM) had rather drastic effects on the morphology of A431 cells and could not be used to assess the involvement of this phospholipase in the dynamic behavior of
6ß4. Similarly, other widely used inhibitors such as wortmannin induced morphological changes in A431 cells even in the absence of EGF stimulation. We did observe, however, a partial inhibition of the EGF-induced redistribution of
6ß4 with the MEK inhibitor PD98059 (data not shown). Although this observation needs to be established more rigorously, it does suggest the possible involvement of the MAP kinase pathway in the EGF-induced mobilization of
6ß4 from hemidesmosomes. Interestingly, PD98059 can abrogate EGF-induced focal adhesion disassembly and cell motility in fibroblasts
Although we cannot exclude a role for tyrosine phosphorylation of the ß4 subunit in the EGF-induced chemotaxis of A431 cells, the data we obtained argue strongly against such a role primarily because we did not detect tyrosine phosphorylation of ß4 using EGF at concentrations that promote optimal chemotaxis. In fact, when we used much higher concentrations of EGF (100 ng/ml) we detected an additional increase in ß4 serine phosphorylation, but only a marginal increase, at best, in ß4 tyrosine phosphorylation. These results contrast with a previous study by Mainiero et al.
The EGF-stimulated phosphorylation of the ß4 subunit on serine residues that we identified is novel and it provides an impetus for investigating the nature of this phosphorylation and its role in regulating the cytoskeletal interactions of the 6ß4 integrin in more detail. For example, the extremely large ß4 cytoplasmic domain contains multiple consensus motifs for PKC phosphorylation based on our analysis of the human ß4 cDNA sequence using Prosite (data not shown). The presence of these motifs supports the possibility that PKC may phosphorylate
6ß4 directly. It is also worth considering the possibility that PKC may activate another downstream serine kinase that is involved in ß4 phosphorylation because there are also consensus sites in the ß4 sequence for other serine kinases such as casein kinase II. Another important issue to be studied is whether phosphorylation of the ß4 subunit is essential for either its mobilization from hemidesmosomes or its recruitment into lamellipodia and ruffles. The data we provide in this study provide a strong correlation of serine phosphorylation with these events. Definitive proof of involvement will require identification of the specific serine residue(s) in the ß4 subunit that are phosphorylated by EGF stimulation and the subsequent mutational analysis of these sites. The possibility that PKC-dependent serine phosphorylation also influences other components of hemidesmosomes should be considered. For example, there is evidence that PKC can mobilize BPAG-2 from hemidesmosomes
The EGF-induced release of 6ß4 from hemidesmosomes and its association with F-actin in lamellipodia and membrane ruffles are probably independent events. This idea is derived from our finding that Gö6976, an inhibitor of the conventional PKC isoforms, did not block the EGF-induced formation of
6ß4-containing membrane ruffles and lamellipodia (data not shown), even though it prevented
6ß4 mobilization from hemidesmosomes (Figure 7). This observation suggests that the PKC-dependent mobilization of
6ß4 from hemidesmosomes is independent of the recruitment of
6ß4 into the lamellipodia and ruffles that are formed in response to EGF stimulation. As we have shown, however, Gö6976 did inhibit EGF-induced chemotactic migration. Collectively, these findings underscore the hypothesis that the increased formation of cell protrusions in the form of ruffles and lamellipodia is not enough to generate movement and that the destabilization of hemidesmosome function mediated by PKC is an essential component of the migration process.
An interesting issue that arises is the role that EGF plays in the recruitment of 6ß4 to the lamellipodia and ruffles. Most likely, EGF stimulates the formation of new cell protrusions that contain
6ß4 rather than promoting the preferential incorporation of
6ß4 into such structures. This assumption is based on our finding that the few lamellipodia that form in the absence of EGF do incorporate
6ß4, and they have a similar intensity of
6ß4 expression as those lamellipodia that are formed in response to EGF stimulation (for example, see Figure 1). Thus, we suggest that
6ß4 is transported to and concentrated at the leading edges by mechanisms intrinsic to lamellipod formation, and that EGF increases the number of protrusions while providing a pool of
6ß4 liberated from hemidesmosomes. One possible mechanism by which
6ß4 could be recruited to the leading edge is exemplified by the transient association of small aggregates of ß1 integrins with the actin cytoskeleton that occurs in neuronal growth cones. These aggregates of ß1 integrins are transported on the dorsal surface in a directed way to the leading edge
6ß4 with the actin cytoskeleton, the fact that only a fraction of the
6ß4 in lamellipodia is resistant to extraction with a Triton X-100 buffer (e.g., compare the extracted lamellipodia of Figure 4 to the unextracted ones of Figure 5) may be the reflection of a dynamic equilibrium attained by the constant association and dissociation of
6ß4 with F-actin. Another model for the recruitment of membrane proteins into motile structures involves the concentration of recycling proteins in ruffles by directed exocytosis induced by EGF through a Rac-dependent pathway
6ß4 is recycled on the cell surface
6ß4-dependent migration and invasion of carcinoma cells
6ß4 that is released from hemidesmosomes by EGF could provide a pool for newly forming ruffles and lamellipodia.
If the mobilization of 6ß4 from hemidesmosomes constitutes one essential component of the EGF-stimulated migration of A431 cells, the second component of migration in which
6ß4 participates is the actual process of migration itself. Indeed, a function for
6ß4 in A431 migration is supported by its localization in lamellipodia and ruffles and, more directly, by our finding that an
6-specific mAb inhibited both chemotactic migration and lamellae formation on laminin-1. These results are consistent with our previous work on colon carcinoma cells that established a role for
6ß4 in the formation and stabilization of lamellae and filopodia
6ß4, are frequently followed by the extension of the lamella towards the anchoring point. This function of
6ß4 may relate to the concept of a molecular clutch that anchors actin bundles to the substrate providing traction tracks for myosin II motors
6ß4 that are essential for lamellae formation, chemotactic migration and invasion of carcinoma cells. These pathways involve PI3-kinase/Rac
6ß4 on lamellae formation and chemotactic migration. For example, expression of
6ß4 in a breast carcinoma cell line stimulates lamellae formation and chemotaxis on collagen in response to a chemoattractant, processes that are not inhibited by
6-specific Abs
6ß4 can regulate key signaling pathways independently of its adhesive functions.
The colocalization of the EGFR, 6ß4, and phosphotyrosine in membrane ruffles and lamellipodia suggests the presence of an active signaling complex involved in cell migration. In fact, there is evidence that the activated form of the EGFR is localized in ruffles and lammelipodia, and that these structures are also sites of PLC-
activation stimulated by EGF
6ß4 with the EGF receptor in lamellipodia and ruffles suggests that these two receptors may interact to facilitate their ability to signal chemotactic migration. This possibility, which remains to be demonstrated for
6ß4 and the EGFR, is supported by the recent finding that the
6ß4 and
6ß1 integrins associate with the erbB-2 receptor in several carcinoma cell lines
6ß4, and the close proximity of these two receptors in F-actinrich areas, along with the other signaling molecules mentioned above, suggests cooperation in their signaling of chemotactic migration.
In summary, our findings describe a mechanism for the chemotactic migration of carcinoma cells that assemble hemidesmosomes. An important implication of our findings is that the mobilization of the 6ß4 integrin from hemidesmosomes by a PKC-dependent mechanism, is an essential step in the migration process, presumably because it releases the strong and stable adhesion mediated by hemidesmosomes and allows for the dynamic adhesive interactions that are required for migration. Importantly, we also demonstrate that the
6ß4 integrin can associate with F-actin in lamellipodia and membrane ruffles, and participate in the migration process itself. Collectively, our results explain how this integrin can mediate both stable adhesion and cell migration. They also suggest that growth and motility factors that are known to promote tumor progression may function, in part, by changing the cytoskeletal interactions and localization of this unique receptor.
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Acknowledgements |
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We thank Bao-Kim Nguyen for technical help. Valuable discussions were had with Robin Bachelder, Rita Falcioni, Kathy O'Connor, and Leslie Shaw.
This work was supported by National Institutes of Health grants CA80789 and CA44704, and the Harvard Digestive Diseases Center.
Submitted: 8 February 1999
Revised: 20 July 1999
Accepted: 22 July 1999
1.used in this paper: BPAG, bullous pemphigoid antigen; EGFR, EGF receptor; PKC, protein kinase C; PLC, phospholipase C; PI3K, phosphoinositide 3-OH kinase
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