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Address correspondence to Martin J. Humphries, School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Rd., Manchester M13 9PT, UK. Tel.: 44 (0) 161-275-5071. Fax: 44 (0) 161-275-1505. E-mail: martin.humphries{at}man.ac.uk
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Abstract |
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Key Words: fibronectin; syndecan; PKC; cytoskeleton; vinculin
* Abbreviations used in this paper: BIM, bisindolylmaleimide; CCBD, central cellbinding domain; FN, fibronectin; HBD, heparin-binding domain; HepII, COOH-terminal heparin binding domain of fibronectin; IIICS, type III connecting segment; PIP2, phosphatidylinositol-4,5-bisphosphate.
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Introduction |
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Particularly distinctive cellular responses have been observed on substrates recognized by the fibronectin (FN)*-binding integrin 4ß1. For example,
4ß1 engagement promotes enhanced cell migratory activity while reducing spreading and focal adhesion formation. Elegant studies using integrin chimeras demonstrated that these functional properties were conferred by the
4 cytoplasmic domain (Chan et al., 1992; Kassner et al., 1995), which in turn suggested that this domain either modulates association of cytoskeletal and signaling molecules with its partner ß1 subunit differently to other ß1-associated
subunits, or that it interacts directly with cytoskeletal and signaling molecules. The latter hypothesis is supported by the fact that
4 can bind directly to paxillin, and that this association contributes to the reduction of spreading and promotion of migration (Liu et al., 1999, Liu and Ginsberg, 2000).
The signaling mechanisms that determine the specific functional properties of other integrins are largely unknown, although work from a number of laboratories has demonstrated that 5ß1-dependent focal adhesion formation requires engagement of (and signaling via) a syndecan coreceptor (Woods and Couchman, 2001; Bass and Humphries, 2002). Initially, plating of cells onto the isolated central cellbinding domain (CCBD) and heparin-binding domain (HBD) of FN was shown to promote attachment, but to be insufficient for focal adhesion formation (Izzard et al., 1986; Woods et al., 1986); however, addition of soluble HBD to cells pre-spread on a CCBD fragment triggered vinculin recruitment and actin stress fiber formation (Woods et al., 1986). The site responsible for this activity was narrowed down to a 29-amino acid sequence within the type III repeat 13 of FN (Bloom et al., 1999). Of the four known members of the syndecan family of proteoglycans, only syndecan-4 has been found in focal adhesions (Woods and Couchman, 1994; Baciu and Goetinck, 1995). Treatment of cells with anti-syndecan-4 antibody was also found to trigger focal adhesion formation in cells adherent to the CCBD of FN (Saoncella et al., 1999), and fibroblasts from syndecan-4 knockout mice were unable to respond to the HBD of FN (Ishiguro et al., 2000), implicating syndecan-4 as a key receptor for focal adhesion formation on FN. In serum-starved cells, syndecan-4 was absent from focal adhesions, but was recruited by activation of PKC (Woods and Couchman, 1992; Baciu and Goetinck, 1995). Overexpression of syndecan-4 increased focal adhesions (Echtermeyer et al., 1999), whereas a COOH-terminal truncation mutant acted as a dominant-negative inhibitor of focal adhesion formation (Longley et al., 1999).
Although the role of syndecans in 4ß1-mediated adhesion is unknown, it is intriguing that the binding sites for both molecules overlap within FN. The
4ß1-binding domain of FN is primarily located in the type III connecting segment (IIICS; Wayner et al., 1989), which is adjacent to the major HBD, the COOH-terminal heparin binding domain of fibronectin (HepII). Three sites each for integrin and heparin binding have been pinpointed within the HepII/IIICS region (Humphries et al., 1986, 1987; McCarthy et al., 1988, 1990; Wayner et al., 1989; Barkalow and Schwarzbauer, 1991; Mould and Humphries, 1991; Mould et al., 1991; Mostafavi-Pour et al., 2001). The overlapping locations of these sites, in part revealed by x-ray crystallography (Sharma et al., 1999), suggest a close coordination between integrin and proteoglycan binding. Here, using melanoma cells that express both
4ß1 and
5ß1, we have investigated the mechanisms underlying focal adhesion formation and cell migration mediated by engagement of the HepII/IIICS region of FN, and compared this to the CCBD.
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Results |
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Glycosaminoglycanbinding sites within the HepII/IIICS region are not needed for focal adhesion formation
As one approach to determine whether integrin 4ß1 required a syndecan coreceptor to trigger focal adhesion formation, site-directed mutagenesis was performed on H/120 to remove all of its potential GAG-binding sites (found in type III repeats 13 and 14, and the IIICS-B region; Fig. 1). All arginine and lysine residues in these regions were mutated to serine, and the mutant protein (termed H/120-GAG-ABC) was prepared as a GST fusion protein in bacteria. After purification, the protein was analyzed by SDS-PAGE under reducing conditions.
To assess folding of H/120-GAG-ABC, an ELISA assay was performed in which the binding of a panel of HepII/IIICS mAbs was determined. Some of these mAbs, including 9E9 and 16E6, do not Western blot, and therefore recognize conformational epitopes within FN. As shown in Fig. 3 A, all mAbs recognized H/120-GAG-ABC to a similar extent to native H/120 with the exception of 9E9, where binding was reduced by 50%. It is possible that one or more arginine or lysine residues contribute to the epitope for this mAb. The binding of biotinylated heparin to the mutant protein was then investigated. The level of binding to H/120-GAG-ABC was reduced by >98% compared with that of native H/120, and was not significantly different from the background, indicating that all key heparin-binding sites had been removed (unpublished data). Recombinant native and mutated proteins were then tested for their ability to support A375-SM melanoma cell adhesion. The proteins supported attachment in a dose-dependent manner (Fig. 3 B). Attachment to native H/120 variant was maximal at a level of 80%, and a coating concentration of 0.7 µg/ml was required for half-maximal attachment. H/120-GAG-ABC showed only slightly lower activity, with a maximal level of cell attachment of
65% and a coating concentration of 1.6 µg/ml being required for half-maximal attachment. Finally, H/120-GAG-ABC was unable to trigger vinculin recruitment to focal adhesions when A375-SM cells were prespread on 50K (unpublished data).
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To examine whether the differential dependence of 4ß1 and
5ß1 on PKC signaling was manifested during cell movement as well as during focal adhesion formation, the sensitivity of A375-SM cell migration to inhibitors of PKC was tested in a wounding assay. For these studies, the early phase of migration between 6 h and 12 h after wounding was examined. Initially, the effects of the broad spectrum PKC inhibitor, BIM, were tested. As shown in Fig. 7 (and Videos 14, available at http://www.jcb.org/cgi/content/full/jcb.200210176/DC1), addition of BIM 6 h after wounding prevented wound closure on a 50K + H/0 substrate, but had no discernible effect on H/120-mediated migration. A DMSO vehicle control showed no inhibition. Thus, the substrate-specific inhibition of focal adhesion formation by BIM was also seen for cell migration.
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Discussion |
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Initially, we were able to confirm previous reports that cells fail to form focal adhesions, or to migrate, on FN fragments spanning the CCBD unless a heparin-binding fragment is added in soluble form (Izzard et al., 1986; Woods et al., 1986). Using Western blotting to detect activated PKC and pharmacological or dominant-negative inhibition, we have also confirmed a requirement for PKC
for focal adhesion formation via
5ß1/syndecan-4. In contrast to these findings,
4ß1-binding ligands stimulated focal adhesion formation without addition of a heparin-binding fragment, and such supplementation did not augment focal adhesion formation. Furthermore, PKC
was not activated, and neither soluble heparin, BIM, nor dominant-negative PKC
blocked focal adhesion formation. These findings indicate that engagement of syndecan-4 with the HBD of FN is not needed for focal adhesion formation via
4ß1, and either that separate signaling mechanisms are used by the two integrins to recruit cytoskeletal proteins to focal adhesions, or
4ß1 is able to bypass the requirement for syndecan-4mediated signaling. Given the currently indistinguishable gross composition of
5ß1- and
4ß1-containing focal adhesions, we favor the latter explanation, and we hypothesize that some of the earliest events in integrin signaling differ between
4ß1 and
5ß1.
It is now well-established that 4ß1 has a number of unusual properties (Chan et al., 1992; Kassner et al., 1995), and it is conceivable that the "gearing" of
4ß1 to the cytoskeleton differs from other integrins. The recent finding that the cytoplasmic domain of
4 interacts directly and specifically with the signaling adaptor paxillin (Turner, 2000) may contribute to these different properties (Liu et al., 1999, Liu and Ginsberg, 2000; Han et al., 2001). PIX (Manser et al., 1998) is a nucleotide exchange factor for both Cdc42 and Rac1, and its recruitment by paxillin (through PKL) may potentially enhance membrane ruffling and protrusion through these Rho family GTPases. On the other hand, an integrin such as
5ß1, which does not bind paxillin but directly activates PKC
, may stimulate Rho GTPase activities, in a PKC-dependent manner. This hypothesis is consistent with several lines of evidence that Rac1 may act downstream of PKC in the control of cell migration. In melanoma cells, for instance, TPA-induced lamellipodium formation is Rac1-dependent (Ballestrem et al., 2000). By using dominant inhibitory constructs, the activity states of several small GTPases, including Rac1, have been shown to influence TPA-induced disassembly/reassembly of actin stress fibers and focal adhesions in MDCK cells (Imamura et al., 1998). Also, in NIH3T3 cells, growth factor-induced Rac1 activation is sensitive to PKC inhibition, suggesting a hierarchical relationship between the two signaling proteins (Buchanan et al., 2000).
In an attempt to identify the PKC isoform responsible for 5ß1-dependent migration, we used a dominant-negative overexpression approach. Overexpression of dominant-negative pEGFP-PKC
(A25E-K368M double mutant) abrogated
5ß1-dependent migration in a TPA-dependent manner. Two additional dominant-negative constructs were tested in the same assay, pEGFP-PKC
-K376M and pEGFP-PKC
-K281M. As neither was able to perturb
5ß1-dependent migration, this provides a clear indication that PKC
plays a specific role. Interestingly, overexpression of wild-type PKC
did retard migration, a result that is consistent with a recent paper reporting a syndecan-4dependent inhibitory effect of PKC
on PKC
(Murakami et al., 2002). PKC
was shown to phosphorylate the cytoplasmic domain of syndecan-4 and thereby prevent PKC
binding. Therefore, it seems likely that the synergy between integrin
5ß1 and syndecan-4 for adhesion and migration relies on the same regulatory pathway.
The downstream targets of PKC phosphorylation during
5ß1-mediated adhesion and migration are not well defined; however, PKC
has been shown to bind directly to syndecan-4 in an interaction mediated by its catalytic domain (Oh et al., 1997a, b; Horowitz and Simons, 1998). In addition, phosphatidylinositol-4,5-bisphosphate (PIP2) appears to make a key contribution to syndecan function through its ability to bind to the variable region of the syndecan-4 cytoplasmic domain (Oh et al., 1998) and to stabilize a "twisted clamp" homodimer conformation discernible by nuclear magnetic resonance (Lee et al., 1998; Shin et al., 2001). This suggests that a ternary complex between syndecan-4, PIP2, and PKC
may form in cells. Integrin ligation has been shown to activate PI-5 kinase, the enzyme that catalyzes the production of PIP2 (McNamee et al., 1993), supporting the close functional link between syndecans and integrins. PIP2 has a wide variety of functions, including the conversion of several cytoskeletal proteins (ERM proteins, vinculin, and talin; Gilmore and Burridge, 1996; Hamada et al., 2000; Martel et al., 2001) from inactive to active forms, and it may be that PIP2 binding to syndecans is a necessary step for the recruitment of these molecules to
5ß1-containing focal adhesions. In addition, PKC
has been shown to associate with the ERM protein ezrin (probably subsequent to its release from membrane receptors such as CD44; Legg et al., 2002), phosphorylates its COOH-terminal phosphorylation on the T567 site, and thereby facilitates its conformational activation (Ng et al., 2001; Bretscher et al., 2002; Gautreau et al., 2002). Therefore,
5ß1 integrin-derived signals may stimulate ERM activities through both PIP2- and PKC-dependent mechanisms. By contrast, the direct binding of paxillin to
4ß1 provides a direct link to vinculin, and therefore talin.
It is also conceivable that the functions of PKC are required during trafficking of integrin receptors, as studies from our laboratories and others have previously demonstrated close intracellular associations in recycling compartments between integrin ß1 and PKC
(Ng et al., 1999a; Podar et al., 2002) and ß1 and PKC
(Ivaska et al., 2002) by biochemical and fluorescence resonance energy transfer analysis. These findings may be reconciled with the data in this report if either a stable integrinPKC
interaction at the plasma membrane requires coreceptor function, or if the integrinPKC
associations in the two compartments differ in their requirement for a coreceptor.
Finally, PKCintegrin interactions are likely to have important implications in growth factormediated cell migration. For instance, a constitutive PKCß1 integrin complex has been found in multiple myeloma cells and may play a significant role in the development of a VEGF-responsive migratory phenotype (Podar et al., 2002). Furthermore, perturbation of PKCintegrin interaction blocks carcinoma cell chemotaxis (Parsons et al., 2002). This may provide an important mechanism for explaining the well-documented cross-talk between growth factor receptor- and integrin-mediated processes (Wang et al., 1998).
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Materials and methods |
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Cloning and expression of recombinant FN fragments
cDNA clones encoding the H/120 and H/0 variants of the HepII/IIICS region of human FN (Fig. 1) were prepared as described previously (Makarem et al., 1994) with some modifications (Mostafavi-Pour et al., 2001). To create H/120-GAG-ABC, mutations (shown in bold) were introduced into H/120 cDNA by site-directed mutagenesis (Kunkel, 1985) as follows: GAG-A (residues 16971704 of FN using Swiss-Prot accession no. PO2751; PPRRARVT to PPSSASVT), GAG-B (two oligonucleotides for residues 19361944, HGFRRTTPP to HGFSSTTPP, and residues 19501958, IRHRPRPYP to ISHSPSPYP), and GAG-C (two oligonucleotides for residues 18171836, KYEKPGSPPREVVPRPRPRPGV to SYESPGSPPSEVVPSPSPGV, and residues 18581872, KNNQKSEPLIGRKKT to SNNQSSEPLIGSSST). The 50K CCBD of human FN was produced and purified as described previously (Mould et al., 1997).
Cell attachment
A375-SM cells, a human metastatic melanoma cell line (provided by Josh Fidler, M.D. Anderson Hospital and University of Texas, Houston, TX) were cultured as described previously (Kozlowski et al., 1984). Cell culture reagents were purchased from GIBCO BRL. Cell attachment assays were performed as described previously (Mould et al., 1994; Mostafavi-Pour et al., 2001).
Focal adhesion formation
13-mm diam glass coverslips were derivatized for 30 min with 250 µl 1 mM m-maleimidobenzoyl-N-hydroxysuccinimide ester (Pierce Chemical Co.), washed three times with 1 ml Dulbecco's PBS lacking divalent cations (PBS-), and coated for 1 h at RT with 250 µl of adhesive substrate in PBS-. The solution was aspirated, and 250 µl 10 mg/ml heat-denatured BSA (Humphries et al., 1986) was added to each well for 30 min at RT. The BSA was then aspirated and the coverslips were washed three times with PBS-. In all experiments, A375-SM cells were treated with 25 µg/ml cycloheximide for 2 h to prevent de novo matrix synthesis and were then detached as described previously (Mostafavi-Pour et al., 2001). 10 µg/ml BIM (diluted 1:200 from a DMSO stock; Calbiochem) was added to cells 2 h before detachment. Cells were resuspended to 5 x 104 ml in DME/25 mM Hepes, 0.5 ml aliquots were added to the coverslips, and the cells were incubated for 2 h at 37°C. To test either the effect of soluble heparin-binding fragment (H/0) or clustering of syndecan-4 by anti-syndecan-4 antibody, cells were allowed to spread for 2 h on 50K before the addition of H/0 or antibody for 30 min. Cells were fixed either directly for 20 min at RT with 50 µl 37% (wt/vol) formaldehyde, or unattached cells were removed by two gentle washes with 1 ml PBS-, and remaining cells fixed for 20 min at RT with 250 µl 3% (wt/vol) formaldehyde diluted in PBS-. Coverslips were washed with PBS- and then the formaldehyde was quenched with 0.1 M glycine in PBS- for 20 min at RT. Cells were permeabilized for 4 min at RT with 0.5% (wt/vol) Triton X-100 diluted in PBS-, washed three times with PBS-, and then blocked for 1 h at RT or overnight at 4°C with 3% (wt/vol) BSA in PBS- (blocking buffer). Primary antibodies were diluted in blocking buffer and incubated for 1 h at RT. After washing, antibodies were detected using TRITC-conjugated goat antirat IgG (1:100 dilution) and/or FITC-conjugated donkey antimouse IgG (1:100 dilution; Jackson ImmunoResearch Laboratories) in blocking buffer for 30 min. F-actin was detected using rhodamine-conjugated phalloidin (1:1,000 dilution; Sigma-Aldrich) in blocking buffer for 30 min. Coverslips were washed three times with PBS- and stained with 250 µl 20 ng/ml DAPI (Sigma-Aldrich) in PBS- for 30 s. Coverslips were mounted face down onto microscope slides using 5 µl Vectashield® (Vector Laboratories), viewed on a microscope (Leica), and immunofluorescence images were taken in the green (FITC) and red (TRITC/rhodamine) channels using a CCD camera. Basic image acquisition and analysis was performed using IPLab software v3.2. Advanced image analysis was performed using Adobe Photoshop® v5.0.
PKC activation
3 x 106 A375-SM cells were resuspended in 300 µl 4x Laemmli SDS sample buffer, with 1 mM sodium vanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 5 µg/ml DNase, and were incubated on ice for 45 min. Cell debris was removed by centrifugation at 25,000 g for 30 min at 4°C. Supernatants were analyzed by 412% SDS-PAGE using the NuPAGE® Novex Bis-Tris gel system (Invitrogen). Gels were transferred to 0.45 µm nitrocellulose (Schleicher and Schuell) at 10 V (limit 0.5 A) for 30 min. Nonspecific binding sites on nitrocellulose membranes were blocked for 1 h at RT with 3% (wt/vol) BSA in 150 mM NaCl, 10 mM Tris-HCl, pH 7.4, containing 0.1% (wt/vol) Tween-20 (TBS-Tween) as described previously (Ng et al., 1999b; Parekh et al., 2000). Membranes were then incubated for 16 h at 4°C with either MC5 mouse antihuman PKC diluted 1:500 in 3% (wt/vol) BSA in TBS-Tween for 1 h, or PPA182 rabbit anti-activated human PKC
diluted 1:2,000 in 1% (wt/vol) skimmed milk powder, 1% (wt/vol) BSA, and TBS-Tween. PPA182 was incubated in the presence of 1 µg/ml cognate dephosphorylated peptide to block nonspecific binding of the pAb to the nonphosphorylated form of PKC
(Ng et al., 1999b; Parekh et al., 2000). After three washes with TBS-Tween for 10 min, HRP-conjugated goat antimouse (1:1,000 dilution in blocking buffer; Dako) or antirabbit IgG (1:4,000 dilution) were added and incubated for 1 h. After three 10-min washes with TBS-Tween, proteins were detected using ECL substrate (NEN Life Science Products).
Wound migration assay
35-mm dishes with 14-mm glass bottoms (MatTek) were coated with either H/120 (10 µg/ml in PBS-) or 50K (50 µg/ml in PBS-) for 1 h, and were blocked with 10 mg/ml heat-denatured BSA (Humphries et al., 1986) for 30 min at RT. A375-SM cells were detached as described previously (Mostafavi-Pour et al., 2001). 2 x 105 cells in complete medium (10% FCS-MEM) were seeded for 16 h at 37°C in a humidified chamber with 5% CO2 until the cells formed a confluent monolayer. 5 µg/ml H/0 was added to the cells at the time of wounding when 50K was used as a substrate. Before wounding, the cell layer was washed two times with PBS-, the medium was replaced, and then the cell monolayer was wounded along the center of the dish using a sterile P10 pipette tip. To test the effect of either anti-4 antibody (HP2/1), PKC inhibitor (BIM), or PKC activator (TPA) on migration, HP2/1 was added to the medium at a concentration of 10 µg/ml just before wounding, or 10 µg/ml BIM or 5 ng/ml TPA were added to the medium just before videomicroscopy, i.e., 6 h after wounding. The wound width was consistently between 270300 µm (285 ± 15 µm, n = 30) 1 h after wounding, when the wound had stabilized. Images were taken using a microscope (Axiovert 135; Carl Zeiss MicroImaging, Inc.) equipped with a 20x 0.3 NA objective and a CCD camera (Photometrics Quantix; Roper Scientific). Basic image acquisition and analysis was performed using IPLab software v3.2. The images were processed using Adobe Photoshop® v5.0. Movement was also studied by observing cells using time-lapse video microscopy. The images were taken using a 20x objective at 5-min intervals for 6 h and organized into time-lapse movies using the IPLab image software.
Transfection
To assess the isoform specificity of PKC signaling during integrin-mediated migration, A375-SM cells were transiently transfected with wild-type and dominant-negative pEGFP-PKC constructs. These were as follows: wild-type pEGFP-PKC, pEGFP-PKC
and pEGFP-PKC
, pEGFP-PKC
-A25E (pseudosubstrate site mutation), pEGFP-PKC
-A25E-K368M (pseudosubstrate site and kinase-dead, ATP-binding mutations), pEGFP-PKC
-T497A (kinase-dead, substrate-binding mutant), pEGFP-PKC
-K376M (kinase-dead, ATP-binding mutant), and pEGFP-PKC
-K281M (kinase-dead, ATP-binding mutant). pEGFP-PKC
was constructed by subcloning the SacII/StuI fragment from bovine PKC
into pEGFP-C2 (BD Biosciences; CLONTECH Laboratories, Inc.) digested with SacII/SmaI. The various pEGFP-PKC
mutants were constructed using the same strategy using the constructs as described previously (Bornancin and Parker, 1997). The pEGFP-PKC
and pEGFP-PKC
-K376M plasmids have been described previously (Srivastava et al., 2002). The pEGFP-PKC
plasmid was constructed in two steps. A blunted 1.2-kb NdeI/PmeI fragment from pcDNA3.1-PKC
was ligated into pEGFP-C1 (CLONTECH Laboratories, Inc.) cut with SmaI to make pEGFP-PKM
. PEGFP-PKC
was constructed by cutting pEGFP-PKM
with BspE1/EcoRV and ligating in a fragment from pcDNA3.1-PKC
cut with NgoMIV/EcoRV. The kinase-dead pEGFP-PKC
-K281M plasmid was constructed by cutting pEGFP-PKC
with NotI/XbaI and ligating in the same fragment from pcDNA3-PKC
-K281M (provided by Anne LeGood, Cancer Research UK Laboratories). All constructs were confirmed by sequencing and expression by Western blot. Transfection of A375-SM cells was conducted in 6-well culture plates (Costar). Cells were detached using 0.05% (wt/vol) trypsin, 0.02% (wt/vol) EDTA in PBS- and seeded at 105 cells/ml in 2 ml growth medium. Cells were transfected the next day using the LipofectAMINETM 2000 protocol (Invitrogen), when the culture reached 8090% confluence. Transfected cells were detached (as described under Wound migration assay) and seeded onto 35-mm dishes for wounding assays after 48 h of transfection.
Online supplemental material
Video 1 shows A375-SM melanoma cells on 50K with soluble H/0 in the presence of BIM. Video 2 shows A375-SM melanoma cells on 50K with soluble H/0 in the presence of DMSO. Video 3 shows A375-SM melanoma cells on H/120 in the presence of BIM. Video 4 shows A375-SM melanoma cells on H/120 in the presence of DMSO. Video 5 shows pEGF-PKC-WTtransfected cells on 50K with soluble H/0 in the presence of 5 ng/ml TPA. Video 6 shows pEGF-PKC
A25E-K368Mtransfected cells on 50K with soluble H/0 in the absence of TPA. Video 7 shows pEGF-PKC
A25E-K368Mtransfected cells on 50K with soluble H/0 in the presence of 5 ng/ml TPA. Video 8 shows pEGF-PKC
A25E-K368Mtransfected cells on H/120 in the presence of 5 ng/ml TPA. Video 9 shows pEGF-PKC
-K376Mtransfected cells on 50K with soluble H/0 in the presence of 5 ng/ml TPA. Video 10 shows pEGF-PKC
-K281Mtransfected cells on 50K with soluble H/0 in the absence of TPA. Video 11 shows pEGF-PKC
-WTtransfected cells on 50K with soluble H/0 in the presence of 5 ng/ml TPA. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200210176/DC1.
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Acknowledgments |
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This work was supported by grants from the Wellcome Trust (045225 to M.J. Humphries), the Iranian Government (to Z. Mostafavi-Pour), the Medical Research Council (to T.T.C. Ng in the form of a Clinician Scientist Grant), and the European Union (to S.J. Parkinson and P.J. Parker).
Submitted: 31 October 2002
Revised: 28 January 2003
Accepted: 10 February 2003
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