Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability
Occludin Phosphorylation and Ubiquitination Regulate Tight Junction Trafficking and Vascular Endothelial Growth Factor-induced Permeability
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DOI：
10.1074/jbc.m109.016766
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发表时间：
2009-07-31
影响因子：
4.8
通讯作者：
Antonetti, David A.
中科院分区：
文献类型：
--
作者：
Murakami, Tomoaki;Felinski, Edward A.;Antonetti, David A.
血管内皮生长因子 (VEGF) 会改变紧密连接 (TJ) 并促进许多视网膜和脑部疾病的血管通透性。然而，人们对屏障调节的分子机制知之甚少。在这里，我们证明了 occludin 磷酸化和泛素化调节 VEGF 诱导的 TJ 蛋白运输和伴随的血管通透性。 VEGF 处理诱导 TJ 断裂和 occludin 从细胞边界运输到早期和晚期内体，同时增加 occludin Ser-490 磷酸化和泛素化。此外，免疫共沉淀和免疫细胞化学均表明，VEGF 治疗增加了 occludin 与含有泛素相互作用基序的细胞内运输调节剂之间的相互作用，包括 Epsin-1、表皮生长因子受体途径底物 15 (Eps15) 和肝细胞生长因子调节的酪氨酸激酶底物 （小时）。通过将 Ser-490 突变为 Ala 来抑制 occludin 磷酸化，可抑制 VEGF 诱导的泛素化，抑制 TJ 蛋白的运输，并防止内皮通透性增加。此外，occludin-泛素嵌合体在没有 VEGF 的情况下破坏了 TJ，并增加了通透性。这些数据证明了 VEGF 诱导的 occludin 磷酸化和泛素化的新机制，有助于 TJ 运输和随后的血管通透性。
Vascular endothelial growth factor (VEGF) alters tight junctions (TJs) and promotes vascular permeability in many retinal and brain diseases. However, the molecular mechanisms of barrier regulation are poorly understood. Here we demonstrate that occludin phosphorylation and ubiquitination regulate VEGF-induced TJ protein trafficking and concomitant vascular permeability. VEGF treatment induced TJ fragmentation and occludin trafficking from the cell border to early and late endosomes, concomitant with increased occludin phosphorylation on Ser-490 and ubiquitination. Furthermore, both co-immunoprecipitation and immunocytochemistry demonstrated that VEGF treatment increased the interaction between occludin and modulators of intracellular trafficking that contain the ubiquitin interacting motif, including Epsin-1, epidermal growth factor receptor pathway substrate 15 (Eps15), and hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Inhibiting occludin phosphorylation by mutating Ser-490 to Ala suppressed VEGF-induced ubiquitination, inhibited trafficking of TJ proteins, and prevented the increase in endothelial permeability. In addition, an occludin-ubiquitin chimera disrupted TJs and increased permeability without VEGF. These data demonstrate a novel mechanism of VEGF- induced occludin phosphorylation and ubiquitination that contributes to TJ trafficking and subsequent vascular permeability.