Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208, USA.
J Biol Chem. 2010 Mar 5;285(10):7045-55. doi: 10.1074/jbc.M109.079277. Epub 2010 Jan 4.
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.
Src 家族激酶（SFK）的激活以及随后的 VE-钙黏蛋白磷酸化被认为是导致血管通透性增加的主要调节步骤，以响应炎症介质和生长因子。为了在没有由生长因子或炎症介质引发的平行信号通路的情况下研究 Src 信号，我们通过表达显性负性 Csk、表达组成性激活的 Src 或敲低 Csk 来激活 Src 和 SFK。通过表达显性负性 Csk 过度表达 SFK 诱导 VE-钙黏蛋白在酪氨酸 658、685 和 731 处磷酸化。然而，显性负性 Csk 的表达无法诱导单层通透性的变化。相比之下，组成性激活的 Src 的表达降低了屏障功能，并促进了 VE-钙黏蛋白在酪氨酸 658 和 731 处的磷酸化，尽管 VE-钙黏蛋白磷酸化的增加比通透性的增加提前了 4-6 小时。Csk 敲低诱导 VE-钙黏蛋白在 658 和 731 位点磷酸化，但不会诱导屏障功能丧失。共免疫沉淀和免疫荧光研究表明，这些位点的磷酸化不会损害 VE-钙黏蛋白结合 p120 和β-连环蛋白的能力，也不会损害这些蛋白在质膜上定位的能力。综上所述，我们的数据表明，Src 诱导的 VE-钙黏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以响应炎症介质或生长因子的信号可能需要 VE-钙黏蛋白酪氨酸磷酸化与其他信号通路同时进行，以促进屏障功能的丧失。
