Phosphorylation of VE-cadherin controls endothelial phenotypes via p120-catenin coupling and Rac1 activation

Abstract

To establish the role of vascular endothelial (VE)-cadherin in the regulation of endothelial cell functions, we investigated the effect of phosphorylation of a VE-cadherin site sought to be involved in p120-catenin binding on vascular permeability and endothelial cell migration. To this end, we introduced either wild-type VE-cadherin or Y658 phosphomimetic (Y658E) or dephosphomimetic (Y658F) VE-cadherin mutant constructs into an endothelial cell line (rat fat pad endothelial cells) lacking endogenous VE-cadherin. Remarkably, neither wild-type- nor Y658E VE-cadherin was retained at cell-cell contacts because of p120-catenin preferential binding to N-cadherin, resulting in the targeting of N-cadherin to cell-cell junctions and the exclusion of VE-cadherin. However, Y658F VE-cadherin was able to bind p120-catenin and to localize at adherence junctions displacing N-cadherin. This resulted in an enhanced barrier function and a complete abrogation of Rac1 activation and lamellipodia formation, thereby inhibiting cell migration. These findings demonstrate that VE-cadherin, through the regulation of Y658 phosphorylation, competes for junctional localization with N-cadherin and controls vascular permeability and endothelial cell migration.

Fig. 1.

Y658F mutants localize at adherens junctions. Rat fat pad endothelial cells (RFPECs) expressing wild-type vascular endothelial (VE)-cadherin (VEC-WT), Y658E, or Y658F mutants were fixed, and the expression patterns of green fluorescent protein (GFP)-tagged VE-cadherin and N-cadherin were determined by immunofluorescence. Cells were stained for VE-cadherin (red) and N-cadherin (blue). A: in RFPECs, endogenous VE-cadherin was not detected and N-cadherin formed cell-cell contacts. B: VEC-WT did not concentrate at cell-cell junctions. C and D: Y658F mutants clearly localized at cell-cell junctions, whereas Y658E mutants did not homogenously line on the cell borders with increased cytoplasmic distribution. Scale bars = 10 μm. E: VEC-WT was proteolytically degraded in RFPECs. Total cell lysates of confluent RFPECs expressing VEC-WT, Y658E, or Y658F mutants were subjected to SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. The antibody against VE-cadherin recognizes the cytoplasmic tail of VE-cadherin.

Fig. 2.

Knockdown of N-cadherin restores VE-cadherin localization at adherens junctions. A: RFPECs expressing VEC-WT was treated with N-cadherin small interfering RNA (siRNA). Forty eight hours later, total cell lysates were harvested with radioimmunoprecipitation assay buffer and subjected to SDS-PAGE followed by immunoblotting with VE-cadherin antibody that recognizes the cytoplasmic tail of VE-cadherin. Y658F VE-cadherin was loaded as a control. B and C: RFPECs expressing VEC-WT were treated with N-cadherin siRNA and fixed, and the expression patterns of GFP-tagged VE-cadherin, N-cadherin, and p120-catenin were determined by immunofluorescence. Cells were stained for VE-cadherin (red), N-cadherin (blue; B), and p120-catenin (blue; C). Scale bars = 10 μm.

Fig. 3.

Y658F mutation of VE-cadherin keeps cell monolayer integrity. Endothelial monolayer permeability was evaluated with the electric cell substrate impedance sensing (ECIS) system. Impedance was measured every 5 min for 8 h after the cells became fully confluent. Representative data (A and C) and quantification of 6 separate measurements of ECIS (B and D) are shown. A and B: Y658E mutation decreased the cell monolayer impedance. C and D: after cells were treated with N-cadherin siRNA, cell monolayer impedance was measured. Note that Y658F mutant maintains high impedance. Data are means ± SD for n = 6. *P < 0.05 by t-test.

Fig. 4.

Y658F mutant, but not Y658E, is associated with p120-catenin. A–D: RFPECs expressing VEC-WT, Y658E, or Y658F mutants were fixed, and the expression patterns of GFP-tagged VE-cadherin and p120-catenin were determined by immunofluorescence. Cells were stained for VE-cadherin (red) and p120-catenin (blue). Note that Y658F mutants clearly colocalized with p120-catenin (arrows in D). Scale bars = 10 μm. E and F: RFPECs expressing VEC-WT, Y658E, or Y658F mutants were lysed, and p120-catenin (E) or VE-cadherin (F) was immunoprecipitated (IP). Immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. G: human umbilical vein endothelial cells expressing VE-cadherin-GFP (WT, Y658E, or Y658F) were lysed, and VE-cadherin-GFP was immunoprecipitated using anti-GFP antibody. Immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. Note that Y658E mutation disrupted VE-cadherin-p120-catenin association.

Fig. 5.

Y658F mutation impairs cell migration, whereas Y658E mutation is sufficient to promote the migratory response. Cells in 6-well plates were cultured confluent. A scratch was made using a 200-μl tip, and the cells were allowed to migrate for 16 h. A: representative pictures at 0 and 16 h. B: relative gap closure was measured using ImageJ software. Data are means ± SD for n = 8. *P < 0.01 by t-test compared with RFPECs or Y658E. C and D: Y658F impaired lamellipodia formation. C: RFPECs expressing Y658E or Y658F at the migrating front were stained with actin (red) using Alexa Fluor 568-conjugated phalloidin. Scale Bars = 10 μm. D: the number of cells with lamellipodia at the wound edge was counted. Data are means ± SD for n = 8 *P < 0.01 by t-test.

Fig. 6.

Rac1 activity is impaired in Y658F cells. A, top: RFPECs were cultured in 0.5% FBS-DMEM for 24 h before Rac1 activity assay. Cells were treated with 50 ng/ml IGF for 10 min before lysis. Rac1 was activated by IGF in control cells, and Y658E VE-cadherin-expressing cells, but not in Y658F cells. A, bottom: densitometric analysis of immunoblots. Data are means ± SD for n = 4. *P < 0.05 by t-test. B, top: RFPECs cultured in 10% FBS-DMEM were subjected to Rac1 activity assay when they reached at 50% confluency. Cells expressing Y658F VE-cadherin showed reduced Rac1 activity compared with control cells or Y658E cells. (RFPECs transfected with an empty vector were used as a control.) B, bottom: densitometric analysis of immunoblots. Data are means ± SD for n = 4. *P < 0.05 by t-test. C: cells at the migrating front were stained with the antibody against GTP-bound Rac1 (red). Note that active Rac1 did not localize at the migrating front in Y658F cells. White line indicates the cell edge at the leading front. Scale Bars = 5 μm.