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

ABSTRACT 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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    • "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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    • "However , the precise mechanisms through which Src exerts its action are far from being completely clear. In vitro studies have shown that phosphorylation of VE-cadherin by Src is not enough to increase endothelial permeability (Adam et al., 2010). Accordingly , a recent study by our group (Orsenigo et al., 2012) showed that in vivo, under resting conditions, VE-cadherin is phosphorylated on tyrosine in the veins, but not in the arteries. "
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