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First published online June 20, 2006
doi: 10.1242/10.1242/jcs.03020

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PKC-dependent activation of RhoA by syndecan-4 during focal adhesion formation

Division of Biomedical Sciences, Imperial College London, London, SW7 2AZ, UK

View larger version (46K): [in this window] [in a new window]   Fig. 1. Expression and intracellular localization of conventional and novel PKC isozymes in REF cells. (A) PKC isoforms expressed in REF cells. REF cell lysates were resolved by SDS-PAGE and probed with monoclonal antibodies against the PKC isoforms indicated. +ve, positive control; REF, REF cell lysate. (B) Differential localization of endogenous PKCs , and with respect to the actin cytoskeleton. PKC is localized at the ends of stress fibres (focal adhesions) and in a juxtanuclear pool, whereas PKC is mostly cytoplasmic with some staining associated with cortical actin (arrows). PKC is perinuclear and shows a Golgi-like staining. Bar, 50 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 2. Overexpression of dominant-negative PKC results in stress fibre disassembly under serum-free conditions. (A) REF cells were transfected with a plasmid encoding dnPKC-GFP fusion protein. 24 hours after transfection, cells were either grown with (+FCS) or without (–FCS) serum for a further 16 hours. Some serum-starved cultures were treated for 30 minutes with LPA (400 ng/ml), fixed and stained for F-actin. (B) REF cells were transfected with wtPKC-GFP and treated as described above. Transfected cells retain stress fibres in the presence or absence of serum. Bar, 50 µm. (C) Quantification of the percentage of cells expressing the PKC-GFP constructs undergoing stress fibre disassembly. Black bars, dnPKC-GFP; white bars, wtPKC-GFP. LPA stimulation was not performed on cells transfected with the wt construct and, therefore, quantitative results are not shown. Results were obtained from three independent experiments (± s.d.).

View larger version (104K): [in this window] [in a new window]   Fig. 3. PKC-deficient REF cells disassemble stress fibres and focal adhesions under serum-free conditions. (A) REF cells were transfected with control siRNA oligonucleotide (–ve) or siRNA oligonucleotides 2 and 4 at the indicated concentrations. 48 hours after transfection, cells were lysed and immunoblotted for PKC and actin. The amount of PKC relative to the control, normalized to the actin content, is indicated below each lane. (B) REF cells were transfected with control (–ve) or siRNA duplex 4 (oligo 4) and serum starved for 16 hours. Cells were subsequently left untreated (–) or treated with LPA for 30 minutes (+LPA), fixed and stained for F-actin and -actinin, as indicated in the Materials and Methods. Boxed areas have been enlarged to highlight the filamentous and focal adhesion localization of -actinin. Identical results were also observed with oligo 2 (not shown). Bar, 50 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 4. Rottlerin inhibits cell spreading of REFs on FN but does not interfere with focal adhesions of REFs pre-spread on FN. (A) REF cells were serum starved overnight, trypsinized and either treated with rottlerin at 1 µM (a,b) or 3 µM (c,d) or DMSO (e) in suspension for 20 minutes, then plated on FN-coated coverslips for 2.5 hours. For some cultures, rottlerin-containing medium was replaced with fresh serum-free medium 30 minutes after seeding on the FN substrate to ascertain whether the rottlerin effects were reversible (b,d). Cells were subsequently fixed and stained for F-actin. Bar, 50 µm. The graph (f) shows the cell area (mean ± s.d.) obtained from 40 cells selected at random from a representative experiment. (B) Serum-starved REF cells were spread on FN for 2 hours, then treated with rottlerin at 1 µM (a) or 3 µM (d) for 30 minutes. Cells were fixed, permeabilized and double stained for F-actin (red) and paxillin (green). Arrows indicate cells with a protrusive phenotype, arrowhead indicates a cell with no obvious filamentous actin organization. Bar, 50 µm. The graph (c) depicts the percentage of cells (± s.d.) with stress fibres, protrusive and no F-actin structures under the various rottlerin treatments. Black bars, DMSO; white bars, 1 µM rottlerin; hatched bars, 3 µM rottlerin. At least 30 cells were scored from each of two independent experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Pharmacological inhibition of PKC activity impairs stress fibre and focal adhesion formation but not cell spreading of REFs on FN. (A) REF cells growing under standard conditions were incubated with the indicated amounts of PKC inhibitors for 30 minutes and lysed for western blotting. Equal amounts of lysates were resolved by SDS-PAGE and probed with antibodies against pT-250, pT-638, pS-657 and total PKC as a loading control, as indicated. (B) Serum-starved REFs were trypsinized and treated with 0.5 µM Gö6976 in suspension for 20 minutes before plating on FN. At 2.5 hours cells were fixed, permeabilized and stained for F-actin and vinculin. (C) REFs were incubated with cell-permeable C3 transferase (TAT-C3) protein (45 µg/ml) in growth medium overnight at 37°C. Trypsinized REFs were replated on FN for 2.5 hours, fixed with 4% paraformaldehyde, permeabilized and stained for F-actin and paxillin. Bar, 50 µm.

View larger version (62K): [in this window] [in a new window]   Fig. 6. Loss of focal adhesions by pharmacological inhibition of PKC activity can be overcome by activation of RhoA by LPA. (A) REF cells were plated on FN-coated coverslips and allowed to adhere. At 1.5 hours following seeding, DMSO or Gö6976 (0.5 µM) was added. 30 minutes later some of the cells that had received Gö6976 were either left unstimulated or treated with LPA (400 ng/ml) for 30 minutes (Gö6976 +LPA). Cells were fixed, permeabilized and stained for F-actin and vinculin. Bar, 50 µm. (B) REF cells were transfected with plasmids encoding wt or dnPKD-GFP fusion proteins. 24 hours after transfection cells were either kept in normal growth conditions (+FCS) or serum-starved for 16 hours (–FCS). Cells were fixed and stained for F-actin. Bar, 50 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 7. Lack of focal adhesions resulting from PKC inhibition can be overcome by LPA treatment but not by HepII and results in recruitment of syndecan-4. (A) REF cells were plated on FN110-coated coverslips for 1.5 hours and either treated with DMSO or with 0.5 µM Gö6976. Cells were subsequently cultured without further treatment (–) or treated with LPA, recombinant HepII, or vehicle as indicated in the Materials and Methods for 30 minutes and cells were fixed, permeabilized and double stained for F-actin (red) and vinculin (green). The percentage of cells forming stress fibres and focal adhesions under the various treatments is indicated. Results are representative of at least three independent experiments. Bar, 50 µm. (B) REFs were plated on FN- or FN110-coated coverslips. At 2 hours, cells on FN110 were either left untreated (–) or treated with LPA, HepII, or pre-treated with Gö6976 for 30 minutes before LPA stimulation, as described above. Cells were subsequently fixed and stained for syndecan-4 and -actinin. Arrows indicate localization of syndecan-4 and -actinin to focal adhesions, arrowheads to focal complexes. Bar, 50 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 8. LPA depends on RhoA-ROCK, not PKC, in order to phosphorylate myosin light chain. REFs plated on FN110 for 1.5 hours were treated with Gö6976, Y-27632 or pre-treated with TAT-C3 before plating as indicated in the Materials and Methods, followed by stimulation with LPA at 2 hours. After a further 30 minutes, cells were fixed, permeabilized and stained for F-actin and phospho-MLC (pMLC2). Bar, 50 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Inhibition of PKC prevents activation of RhoA induced by syndecan-4 clustering. REF cells were plated on FN110-coated dishes for 1.5 hours and DMSO or Gö6976 (1 µM) were added for a further 30 minutes. Subsequently, cells were stimulated with recombinant HepII (1 µg/ml; A), or LPA (400 ng/ml; B). Cells were lysed at the indicated time points and GTP-bound RhoA was captured on GST-Rhotekin-RBD-conjugated glutathione-agarose beads. Samples were resolved by SDS-PAGE, immunoblotted for RhoA and the relative activities of the protein at the indicated time points under each condition determined densitometrically. , DMSO; , Gö6976. (A) Results were obtained from three independent experiments (mean + s.e.m.; n=3). **P<0.05 compared with levels in Gö6976-treated cells. (B) Results obtained from one of three independent experiments are shown in the graph. The inset shows the ratio of relative RhoA activity between Gö6976- and DMSO-treated cells at various time points following LPA stimulation (mean ± s.e.m.; n=3).