Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers

Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2010; 285(10):7045-55. DOI: 10.1074/jbc.M109.079277
Source: PubMed


Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4-6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and beta-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.

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    • "In all, these findings show not only that VE-cadherin phosphorylation at particular tyrosines is an important step in the loss of endothelial cell-cell adhesion leading to an increase in permeability and TEM, but also that VE-cadherin can be phosphorylated in the absence of vascular leakage, demonstrating in vivo that other signals must be activated concurrently. In vitro, the effect of SFK activation on human dermal microvascular cells depends on the method of activation [94]. Consistent with the findings in epithelial cells, overexpression of a constitutively active form (Y530A) of Src promoted VEcadherin phosphorylation, monolayer gap formation, and loss of TEER. "
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    ABSTRACT: Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing.
    Mediators of Inflammation 11/2015; 2015(4):272858. DOI:10.1155/2015/272858 · 3.24 Impact Factor
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    • "The cytoplasmic domain of VE-cadherin binds to several protein partners, including β-catenin, plakoglobin, and p120 and tyrosine phosphorylation of VE-cadherin prevents association of catenins with VE-cadherin thereby disorganizing the cadherin complex and reducing the strength of the junctions [6]. Recent studies suggest that phosphorylation of VE-cadherin is necessary but not sufficient to induce dissolution of endothelial junctions [10]; thus, the coordinated induction of multiple signaling cascades is likely key to the opening of endothelial junctions. "
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    PLoS ONE 09/2014; 9(9):e108092. DOI:10.1371/journal.pone.0108092 · 3.23 Impact Factor
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    • "Src activation in turn mediated phosphorylation of VE-cadherin at Tyr 658, the phosphorylation site known to promote the dissociation of p120-catenin from VE-cadherin and the subsequent internalization of VE-cadherin (Xiao et al., 2003; Potter et al., 2005; Hatanaka et al., 2011). Although the role of Src and resulting VE-cadherin phosphorylation at Tyr658 in mediating disassembly of AJs has been previously proposed (Adam et al., 2010), our results are the first to show that in the context of Src activation induced by G13–VE-cadherin interaction, Src phosphorylation of VE-cadherin at Tyr656 is an important mechanism of AJ disassembly. This contention is reinforced by the finding that endothelial cell expression of phosphorylation-resistant Y658F VE-cadherin not only restored endothelial AJ integrity but also protected AJs from disruption induced by multiple inflammatory mediators. "
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    ABSTRACT: The heterotrimeric G protein Gα13 transduces signals from G protein-coupled receptors (GPCRs) to induce cell spreading, differentiation, migration, and cell polarity. Here, we describe a novel GPCR-independent function of Gα13 in regulating the stability of endothelial cell adherens junctions (AJs). We observed that the oxidant H2O2, which is released in response to multiple proinflammatory mediators, induced the interaction of Gα13 with VE-cadherin. Gα13 binding to VE-cadherin in turn induced Src activation and VE-cadherin phosphorylation at Tyr 658, the p120-catenin binding site thought to be responsible for VE-cadherin internalization. Inhibition of Gα13-VE-cadherin interaction using an interfering peptide derived from the Gα13 binding motif on VE-cadherin abrogated the disruption of AJs in response to inflammatory mediators. These studies identify a unique role of Gα13 binding to VE-cadherin in mediating VE-cadherin internalization and endothelial barrier disruption and inflammation.
    Journal of Experimental Medicine 03/2014; 211(3). DOI:10.1084/jem.20131190 · 12.52 Impact Factor
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