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. 2013 Jun 21;288(25):18290-9.
doi: 10.1074/jbc.M112.432757. Epub 2013 May 7.

Interaction of p190RhoGAP with C-terminal domain of p120-catenin modulates endothelial cytoskeleton and permeability

Noureddine Zebda  1 , Yufeng Tian, Xinyong Tian, Grzegorz Gawlak, Katherine Higginbotham, Albert B Reynolds, Anna A Birukova, Konstantin G Birukov
Affiliations

Interaction of p190RhoGAP with C-terminal domain of p120-catenin modulates endothelial cytoskeleton and permeability

Noureddine Zebda et al. J Biol Chem. .

Abstract

p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820-843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.

Keywords: Adherens Junction; Catenin; Endothelial Cell; GTPase; Signal Transduction.

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Figures

FIGURE 1.
FIGURE 1.
Binding of p190RhoGAP to GST-tagged p120 catenin deletion mutants. A, GST-tagged full-length p120 catenin 1A (GST-p120 1A) and related deletion mutants were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. B, interacting complexes were pulled down with His-tagged Dynabeads, and p120 catenin domains bound to p190RhoGAP were detected by Western blot with anti-GST and anti-p120-catenin antibodies. The bottom panel shows the protein content of recombinant p120-catenin and its deletion mutants in total whole cell lysates (WCL). Asterisks indicate the bands corresponding to p120–1A-catenin constructs expressed. MW, molecular weight.
FIGURE 2.
FIGURE 2.
Binding of p120 catenin truncation mutants to His-tagged p190RhoGAP. GST-tagged full-length p120 catenin 1A (GST-p120 1A) and its deletion mutants were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. Interacting complexes were pulled down with glutathione magnetic beads, and their content was analyzed by Western blotting using antibodies to the GST tag, p120 catenin, or p190RhoGAP as indicated. The bottom panel depicts the protein content of recombinant His-tagged p190RhoGAP in whole cell lysates (WCL). MW, molecular weight.
FIGURE 3.
FIGURE 3.
Identification of the C-terminal polypeptide sequence in p120-catenin responsible for recruitment of p190RhoGAP. GST-tagged full-length p120-catenin 1A (GST-p120 1A), related deletion mutants as well as truncated mutants of p120-catenin created by site specific mutagenesis (A) were cotransfected in HEK 293T cells together with His-tagged p190RhoGAP. Interacting complexes were pulled down with His-tagged Dynabeads (B), and their content was analyzed by Western blotting using antibodies to the His tag, GST tag, p120-catenin, or p190RhoGAP as indicated. Asterisks in B indicate the bands corresponding to p120-catenin constructs expressed. The bottom panel depicts the protein content of recombinant GST-tagged p120-catenin and its deletion mutants in whole cell lysates (WCL).
FIGURE 4.
FIGURE 4.
Role of the p120-catenin CRAD domain responsible for p190RhoGAP recruitment in the OxPAPC-induced barrier enhancement. GST-tagged full length p120 catenin (p120 1A) and truncated mutants p120(1–843) and p120(1–820) catenin were expressed in HPAEC followed by OxPAPC treatment. Time-resolved TER measurements of barrier integrity across transiently transfected HPAEC monolayers are shown in A–C. Norm. Resistance, normalized resistance; non-TF, non-transfected. D, statistical analysis of TER changes at 30 min and 2 h of OxPAPC stimulation. *, p < 0.05 versus p120 1A (n = 5 independent experiments).
FIGURE 5.
FIGURE 5.
p120(1–820) catenin mutant colocalizes with VE-cadherin to the adherens junctions but fails to accumulate at the plasma membrane upon OxPAPC treatment. GST-tagged full-length p120 catenin (p120 1A) and truncated mutant p120(1–820) catenin were transiently transfected in HPAEC. A, double immunostaining with antibodies to GST tag to visualize ectopically expressed p120-catenin (green) and endogenous VE-cadherin (red) was carried out 48 h after transfection as described under “Experimental Procedures.” B, effect of OxPAPC treatment (20 μg/ml, 30 min) on the membrane localization of p120 1A and p120(1–820) mutant. Expressed proteins were analyzed by immunofluorescence using anti-GST antibody. Cell nuclei were visualized by DAPI counterstaining.
FIGURE 6.
FIGURE 6.
p120(1–820) catenin attenuates OxPAPC-induced p190RhoGAP membrane translocation and activation of Rac signaling. GST-tagged full length p120 catenin and truncation mutants were expressed in HPAEC followed by OxPAPC treatment (15 μg/ml) for the indicated periods of time. A, Western blot analysis of p190RhoGAP in the membrane fractions from control and OxPAPC-treated HPAEC expressing full-length or p120(1–820)-catenin mutant (A, top panel). Total p190RhoGAP content in the cell lysates (middle panel) and expression levels of the recombinant p120 proteins detected by Western blot with anti-GST antibody (bottom panel) were used as normalization controls. B, Rho-GTP pulldown assay from unstimulated HPAEC and HeLa cells showing basal Rho activation (top panel) and total (bottom panel) Rho levels in cells expressing p120 1A, p120(1–820), or p120(1–843) mutants. C, Western blot analysis of basal phospho-MLC levels in HPAEC expressing p120 1A, p120(1–820), or p120(1–843) mutants. Samples for each condition are presented in duplicates. Western blot detection of GST tag (middle panel) was used to monitor recombinant p120-catenin expression; detection of β-tubulin (bottom panel) was used as normalization control. RDU, relative density units. D, Rac-GTP pulldown assay showing active Rac (top panel) and total (bottom panel) Rac levels. E, Western blot analysis of phospho-PAK1 and phospho-cortactin levels in OxPAPC-treated HPAEC expressing full-length and p120(1–820)-catenin mutant using phospho-site-specific PAK1 and cortactin antibodies. GST and β-tubulin staining were included to account for construct expression levels and sample loading, respectively. Bar graphs represent quantitative densitometry analysis of Western blot data. *, p < 0.05 versus p120 1A (n = 5 independent experiments).
FIGURE 7.
FIGURE 7.
p120(1–820) catenin delays thrombin-induced barrier restoration and prolongs thrombin-induced activation of Rho signaling. GST-tagged full-length p120 catenin (p120 1A) and its truncation mutant p120(1–820) were expressed in HPAEC followed by thrombin treatment (0.2 units/ml). A, TER measurements showing thrombin-induced permeability and barrier restoration in HPAEC monolayers. Thrombin-induced barrier dysfunction was markedly prolonged by expression of p120(1–820) catenin. Norm. Resistance, normalized resistance. B, Rho-GTP pulldown assay on thrombin-treated HPAEC expressing full-length or p120(1–820) catenin mutant during the recovery phase. C, Western blot analysis of phospho-MYPT, phospho-MLC, and phospho-cortactin levels in HPAEC expressing full-length and p120(1–820) catenin mutant upon thrombin stimulation for the indicated periods of time. GST and β-tubulin staining were included in C to account for construct expression levels and sample loading, respectively.
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