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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02584

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E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function

¹ Department of Genetics, Biology and Biochemistry, University of Torino, Via Santena 5/bis, Torino, 10126, Italy
² IMBA–Institute of Molecular Biotechnology, Dr Bohr-Gasse 3-5, 1030 Vienna, Austria
³ Department of Biochemistry, University of Pavia, Strada Nuova 65, 27100, Pavia, Italy

View larger version (32K): [in a new window]   Fig. 1. Rap1 activity is gradually down-regulated during cell adhesion independently of integrin activation and function. (A) Confluent cells kept overnight in serum-free medium were either lysed directly onto tissue culture dishes (Ad, adherent cells), or harvested by treatment with 5 mM EDTA in PBS and resuspended in serum-free medium before lysis (Su, suspension cells). Active, GTP-bound Rap1 (GTP-Rap1) was detected by a pull-down assay using a GST-tagged RalGDS-RBD protein prebound to glutathione-Sepharose beads (see Materials and Methods); western blotting of whole lysates ensured that relative equal amounts of total Rap1 proteins (Total Rap1) were analyzed. Notice that cell detachment from the substratum promoted a strong activation of Rap1 in all cell lines tested. (B-D) Resuspended cells were either lysed (Su, suspension cells), or replated at high density and allowed to adhere for the indicated times (in minutes) to tissue culture dishes coated with 10 µg/ml of the substratum proteins, fibronectin (FN) (B), poly-L-lysine (PL) (C) or an activating mAb to ß1 integrin (TS2/16) (D), before measurement of active Rap1. Notice that cell adhesion induced a progressive down-regulation of Rap1 activity, which was independent of the adhesive substratum used and reached the maximal level at about 1 hour.

View larger version (60K): [in a new window]   Fig. 2. The inhibition of Rap1 activity during cell adhesion does not require the integrity of the actin cytoskeleton, but is significantly influenced by cell density. (A) Confluent cells were harvested using 5 mM EDTA treatment, resuspended in serum-free medium in the presence (+) or absence (–) of 1 µM cytochalasin D (CytD), and either lysed (Su, suspension cells) or allowed to adhere for 1 hour at 37°C to FN-coated tissue culture dishes before measurement of active Rap1 (GTP-Rap1) by the pull-down assay. Western blotting of whole cell lysates was used to assess total levels of Rap1 (Total Rap1). Notice that the cell adhesion-dependent down-regulation of Rap1 activity did not require an intact actin cytoskeleton. (B) In parallel experiments, cells were seeded onto FN-coated glass coverslips either in the absence (Ctrl) or presence (CytD) of 1 µM CytD for 1 hour at 37°C to disrupt the actin cytoskeleton, and processed for immunofluorescence microscopy analysis to detect filamentous actin (F-actin). Notice the effectiveness of CytD treatment in disrupting the actin cytoskeleton. (C) Cells were harvested by trypsinization, extensively pipetted to generate a maximal dispersed cell suspension, replated at various densities (0.25x105 to 1.5x105 cells/cm2) in serum-free medium, and allowed to recover overnight at 37°C before measurement of Rap1 activity. Notice that the down-regulation of Rap1 activity during cell adhesion was clearly dependent on cell density.

View larger version (45K): [in a new window]   Fig. 3. The disruption of AJs by distinct mechanisms leads to a strong activation of Rap1. (A) Serum-starved epithelial FRT cell monolayers were either left untreated and lysed (Ctrl), or incubated in serum-free DMEM supplemented with 4 mM EGTA for 30 minutes at 37°C to disrupt E-cadherin-mediated cell-cell contacts. Treated cells were then either lysed (EGTA) or allowed to reform adherens junctions by further incubation with fresh medium containing 1.8 mM Ca2+ for 45 minutes at 37°C before lysis (EGTA+Ca2+). Cell lysates were then processed for measurement of active Rap1 levels by the pull-down assay. Notice that the disruption of adherens junctions by EGTA treatment led to a strong activation of Rap1, while the amounts of active Rap1 returned to the basal level as E-cadherin-mediated cell-cell adhesion was re-established by Ca2+ restoration. Relative levels of total Rap1 in whole cell lysates are showed in the lower blot as control. (B) In parallel experiments, cells were subjected to the above calcium switch procedure while plated onto glass coverslips, and further processed for immunofluorescence microscopy analysis. The staining of cortical actin with FITC-conjugated phalloidin confirms the effectiveness of the calcium switch procedure. (C) Serum-starved epithelial FRT cell monolayers were either left untreated (–) or treated with 5 µM PAO for the indicated times at 37°C to disrupt cell-cell contacts. Cells were then processed for measurement of active Rap1 levels by the pull-down assay. Notice that PAO treatment clearly affected the GTP-loading of Rap1 (GTP-Rap1) within 30 minutes, while total Rap1 levels did not change. (D) FRT cells plated onto glass coverslips were either left untreated (Ctrl) or treated with 5 µM PAO for 30 minutes (PAO), and processed for immunofluorescence microscopy analysis. Notice the effectiveness of PAO treatment in disrupting cell-cell adhesion, as detected by staining F-actin with FITC-conjugated phalloidin, and the now fibroblast-like morphology of the treated cells. (E) Serum-starved epithelial FRT cell monolayers were subjected to the calcium switch procedure, and further processed for measurement of the active levels of both Rap1 and Rac1 through specific pull-down assays. Notice that the changes in Rap1 activity that occurred during the calcium switch were closely paralleled by inverse changes in Rac1 activity.

View larger version (52K): [in a new window]   Fig. 4. E-cadherin-mediated cell-cell adhesion inhibits Rap1 activity both in adherent and suspended cells. (A) Serum-starved FRT cell monolayers were subjected to the Ca2+ switch procedure either in the absence or presence of DECMA-1, a neutralizing antibody against E-cadherin, and further processed for measurement of active Rap1 by the pull-down assay. Notice that the decrease in Rap1 activity induced by restoration of cell-cell adhesion was inhibited by DECMA-1, indicating an E-cadherin function requirement. (B,C) Serum-starved FRT cells were harvested from tissue culture dishes and completely dissociated into single cells by 5 mM EDTA treatment and pipetting, and were then subjected to an aggregation assay (see Materials and Methods) either in the absence or in the presence of DECMA-1 to block E-cadherin function. Cells were then either analyzed by phase-contrast microscopy (B), or processed for measurement of active Rap1 (C). Notice the effectiveness of DECMA-1 in blocking both cell aggregation (B) and the E-cadherin function-dependent down-regulation of Rap1 activity (C) induced by Ca2+ restoration.

View larger version (30K): [in a new window]   Fig. 5. E-cadherin adhesive function modulates Src activity, which in turn is required for Rap1 activation in response to the disruption of AJs. (A,B) FRT cell monolayers kept overnight in serum-free medium were either subjected to the Ca2+ switch procedure [(Ctrl), (EGTA), (EGTA+Ca2+)], resuspended by 5 mM EDTA treatment in PBS (Su), or pretreated for 1 hour at 37°C with the specific SFK inhibitor PP2 (10 µM) before EGTA treatment (PP2+EGTA). Cell extracts were then prepared, and Src was immunoprecipitated from lysate supernatants with a c-Src-specific mAb. Src immunoprecipitates were then washed and divided equally; one half was subjected to an in vitro autophosphorylation assay (A), and the other half was used for a quantitative western blotting assay to confirm equal loading of immunoprecipitated Src proteins (B). Notice that the Ca2+ switch procedure clearly affected the kinase activity of endogenous Src. The effectiveness of cell resuspension in activating, and of PP2 treatment in inhibiting, Src activity is also shown. (C) FRT cells were either left untreated (Ctrl), treated with 4 mM EGTA for 30 minutes (EGTA), or pretreated with PP2 (10 µM) for 1 hour before EGTA treatment (PP2+EGTA). Active Rap1 levels were then measured by the pull-down assay. Notice that SFK inhibition by PP2 completely blocked the activation of Rap1 induced by EGTA treatment.

View larger version (33K): [in a new window]   Fig. 6. The disruption of cell-cell adhesion promotes the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. FRT cell monolayers kept overnight in serum-free medium were either left untreated (Ctrl), treated with 4 mM EGTA for 30 minutes (EGTA), pretreated with 10 µM PP2 for 1 hour before EGTA treatment (PP2+EGTA), or resuspended by treatment with 5 mM EDTA in PBS (Su), and then processed for immunoprecipitation and western blotting. (A) Rap1 immunoprecipitates (IP Rap1) were immunoblotted sequentially with antibodies against the indicated proteins (left panels). Equivalent loading of immunoprecipitated Rap1 was demonstrated by reprobing the blot for Rap1. In parallel, western blots of whole cell lysates were reprobed sequentially to assess total levels of the analyzed proteins (right panels). The multiple bands of p120ctn represent distinct isoforms. Notice that immunoprecipitation of Rap1 from EGTA-treated cells resulted in the enhanced coprecipitation of a subset of E-cadherin-catenin complexes that did not contain p120ctn. (B) Immunoprecipitations of Rap1 (IP Rap1) and E-cadherin (IP E-cadh) were immunoblotted sequentially with antibodies against the indicated proteins. Notice that PP2 blocked the increased formation of Rap1/E-cadherin-catenin complexes induced by EGTA treatment (left panels). The p120ctn protein that coprecipitated with E-cadherin (p120ctn, right panel) is the smaller but most abundant of the four p120ctn isoforms expressed in FRT cells. #, E-cadherin full length; *, major E-cadherin cleavage products.

View larger version (98K): [in a new window]   Fig. 7. Rap1 activation promoted by the disassembly of AJs requires E-cadherin internalization and endocytic trafficking. (A) FRT cell monolayers kept overnight in serum-free medium were either left untreated (Ctrl), treated with CytD (1 µM) for 30 minutes to disrupt the actin cytoskeleton (CytD), or EGTA (4 mM) for 30 minutes to disrupt cell-cell adhesion (EGTA), or pretreated with CytD for 30 minutes before EGTA treatment (CytD+EGTA). Cells were then fixed, permeabilized, double stained for E-cadherin (E-cadh, a-d) and filamentous actin (F-actin, e-h), and analyzed by immunofluorescence microscopy. The insets in c and g shown magnified images of the outlined areas. Notice that CytD pretreatment prevented E-cadherin internalization induced by EGTA. Scale bar: 25 µm. (B) Lysates from FRT cells treated as above were used to measure the GTP-loading of Rap1 by the pull-down assay. Notice that the activation of Rap1 induced by EGTA treatment was clearly prevented by CytD. (C) Confluent FRT cells were either left untreated (Ctrl), treated with 1 µM bafilomycin A1 (BAF) or 1 mM N-ethylmaleimide (NEM) for 1 hour and 30 minutes, respectively, or EGTA (4 mM) for 30 minutes (EGTA), or pretreated with BAF or NEM before EGTA treatment (BAF+EGTA or NEM+EGTA). Cells were then fixed, permeabilized, stained for E-cadherin, and analyzed by immunofluorescence microscopy. Notice that cell pretreatment with NEM resulted in a complete block of EGTA-induced E-cadherin endocytosis (e), while the disruption of cell-cell contacts and the endocytosis of E-cadherin induced by EGTA clearly occurred in BAF-pretreated cells (f). (D) Cells treated as in C were used to measure Rap1 activity by the pull-down assay. Notice that both BAF and NEM clearly blocked the activation of Rap1 induced by EGTA treatment. (E) Confluent FRT cells were either left untreated (Ctrl), treated with EGTA (4 mM) for 30 minutes to disrupt cell-cell adhesion (EGTA), or pretreated with CytD for 30 minutes before EGTA treatment (CytD+EGTA) to prevented E-cadherin internalization. Cells were then fixed, permeabilized, double stained with antibodies to E-cadherin (E-cadh) and Rap1 (Rap1) followed by FITC- and RITC-conjugated secondary antibodies, respectively, and analyzed by confocal immunofluorescence microscopy. E-cadherin and Rap1 distribution was compared by merging images (Merge). Notice that both E-cadherin and Rap1 were redistributed to and partially colocalized at the perinuclear cytoplasm upon depletion of extracellular Ca2+ by EGTA (g,h,i). By contrast, in cells pretreated with CytD prior to exposure to EGTA, a majority of E-cadherin remained at the cell surface despite the loss of cell-cell adhesion, whereas Rap1 mostly remained diffuse in the cytoplasm (d,e,f) as it was in untreated cells (a,b,c). (F) FRT cells were transiently cotransfected with EGFP-tagged Rab11, a marker for perinuclear recycling endosomes, and mRFP-Rap1, and grown to confluence for 12 hours on glass coverslips. Cells were then either left untreated (Ctrl) or treated with EGTA (4 mM) for 30 minutes to disrupt cell-cell adhesion (EGTA), and analyzed by confocal immunofluorescence microscopy. Notice that the perinuclear pool of mRFP-Rap1 significantly colocalized with EGFP-Rab11.

View larger version (92K): [in a new window]   Fig. 8. AJ disassembly induces a Rap1 activity-dependent assembly of focal adhesions. FRT cells transiently transfected with EGFP-zyxin, as a marker for focal adhesions, were plated on FN-coated coverslips and grown to confluence for 12 hours. Living cells were then analyzed by time-lapse microscopy during disruption of cell-cell junctions by EGTA treatment in serum-free medium. (A) The two selected frames of the video sequence show the subcellular distribution of EGFP-zyxin before cell treatments (Ctrl), and after 30 minutes of EGTA treatment (EGTA). Notice the assembly of EGFP-zyxin-containing focal adhesions upon EGTA treatment. (B) Cells expressing EGFP-zyxin were microinjected with GST-Rap1GAP (left cell) or with GST alone (right cell) 30 minutes before EGTA treatment. The two selected frames of the video sequence show the subcellular distribution of EGFP-zyxin in microinjected cells before (Ctrl) and after 30 minutes of EGTA treatment (EGTA). Notice that GST-Rap1GAP injection clearly affected the formation of focal adhesions upon disruption of cell-cell junctions. Phase contrast images of the treated cells are also shown. QuickTime movies are available in the supplementary material.

View larger version (31K): [in a new window]   Fig. 9. Hypothetical model for the E-cadherin endocytosis-mediated activation of Rap1. In confluent epithelial cells both Src kinase and Rap1 activity are reduced to basal levels. Rap1 is diffusely distributed into the cytoplasm, where it associates with endosomal compartments, while AJs are stabilized by p120ctn binding to the E-cadherin juxtamembrane domain. Upon stimuli that induce the weakening of cell-cell adhesion, Src kinase activity is enhanced and may in turn activate the endocytic machinery underling E-cadherin internalization. Major known targets for Src tyrosine kinase activity in response to stimuli that weaken AJs are E-cadherin itself and p120ctn. However, at least two Rap1GEFs, one of which (C3G) is known to be regulated by tyrosine phosphorylation, have been reported to bind to either the E-cadherin cytoplasmic domain or ß-catenin, suggesting that Rap1GEF directly or indirectly linked to the E-cadherin cytoplasmic domain may also be activated upon cell-cell adhesion weakening. Internalized E-cadherin must proceed through the endocytic pathway in order to induce Rap1 activation, which probably occurs upon E-cadherin transit from early to recycling endosomes where a protein complex containing E-cadherin and Rap1 may form. Activated Rap1 may in turn control the polarized redistribution of integrins and/or integrin regulators to new adhesion sites, leading to the formation of enhanced integrin-mediated cell-matrix adhesion (see text for details).

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