Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers
Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers
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DOI：
10.1074/jbc.m109.079277
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发表时间：
2010-03-05
影响因子：
4.8
通讯作者：
Vincent, Peter A.
中科院分区：
文献类型：
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作者：
Adam, Alejandro P.;Sharenko, Amy L.;Vincent, Peter A.
Src家族激酶（SFK）的激活和随后的VE-钙粘蛋白的磷酸化被认为是导致血管通透性响应于炎症介质和生长因子而增加的主要调节步骤。为了研究Src信号传导在缺乏由生长因子或炎症介质启动的平行信号传导途径的情况下，我们通过表达显性负性Csk、表达组成性活性Src或敲低Csk来激活Src和SFKs。通过显性负性Csk的过度表达激活SFK诱导VE-钙粘蛋白在酪氨酸658、685和731处磷酸化。然而，显性负Csk表达不能诱导单层通透性的变化。相反，组成性活性Src的表达降低屏障功能，促进酪氨酸658和731上的VE-钙粘蛋白磷酸化，尽管VE-钙粘蛋白磷酸化的增加先于渗透性增加4-6 h。Csk敲低诱导VE-钙粘蛋白磷酸化的网站658和731，但没有引起屏障功能的损失。免疫共沉淀和免疫荧光研究表明，这些网站的磷酸化并没有损害VE-钙粘蛋白的能力，结合p120和β-连环蛋白或这些蛋白质定位在质膜的能力。综上所述，我们的数据表明，Src诱导的VE-钙粘蛋白酪氨酸磷酸化不足以促进内皮细胞单层通透性的增加，并表明导致血管通透性变化的信号传导对炎症介质或生长因子的反应可能需要VE-钙粘蛋白酪氨酸磷酸化与其他信号传导途径同时促进屏障功能的丧失。
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.