Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo.
Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo.
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DOI：
10.2337/db11-1367
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发表时间：
2012-06
期刊：
影响因子：
7.7
通讯作者：
Antonetti DA
中科院分区：
文献类型：
--
作者：
Murakami T;Frey T;Lin C;Antonetti DA
血管内皮生长因子（VEGF）诱导的血视网膜屏障破坏需要蛋白激酶C（PKC）β激活。然而，与此过程相关的分子机制仍然知之甚少。在这项研究中，occludin磷酸化和下游的PKCβ激活的泛素化的作用，在紧密连接（TJ）的运输和内皮通透性进行了研究。通过处理牛视网膜内皮细胞和玻璃体内注射PKCβ抑制剂以及显性负性激酶的表达来确定PKCβ对内皮通透性的贡献和用位点特异性抗体检测的Ser490处的occludin磷酸化。采用体外激酶试验证明PKCβ直接磷酸化occludin。在闭合蛋白免疫沉淀后通过免疫印迹测量泛素化。共聚焦显微镜显示组织的TJ蛋白。结果表明，抑制VEGF诱导的PKCβ活化阻断了视网膜血管内皮细胞中occludin Ser490磷酸化、泛素化和TJ运输，并阻止了VEGF刺激的血管通透性。封闭蛋白Ser490是PKCβ的直接靶点，将Ser490突变为Ala（S490A）可阻断PKCβ下游的通透性。因此，PKCβ激活使Ser490上的occludin磷酸化，导致VEGF诱导的渗透性所需的泛素化。这些数据证明了PKCβ靶向抑制剂调节血管通透性的新机制。
Vascular endothelial growth factor (VEGF)–induced breakdown of the blood-retinal barrier requires protein kinase C (PKC)β activation. However, the molecular mechanisms related to this process remain poorly understood. In this study, the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation in tight junction (TJ) trafficking and endothelial permeability was investigated. Treatment of bovine retinal endothelial cells and intravitreal injection of PKCβ inhibitors as well as expression of dominant-negative kinase was used to determine the contribution of PKCβ to endothelial permeability and occludin phosphorylation at Ser490 detected with a site-specific antibody. In vitro kinase assay was used to demonstrate direct occludin phosphorylation by PKCβ. Ubiquitination was measured by immunoblotting after occludin immunoprecipitation. Confocal microscopy revealed organization of TJ proteins. The results reveal that inhibition of VEGF-induced PKCβ activation blocks occludin Ser490 phosphorylation, ubiquitination, and TJ trafficking in retinal vascular endothelial cells both in vitro and in vivo and prevents VEGF-stimulated vascular permeability. Occludin Ser490 is a direct target of PKCβ, and mutating Ser490 to Ala (S490A) blocks permeability downstream of PKCβ. Therefore, PKCβ activation phosphorylates occludin on Ser490, leading to ubiquitination required for VEGF-induced permeability. These data demonstrate a novel mechanism for PKCβ targeted inhibitors in regulating vascular permeability.
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影响因子：
4.4
作者：
Phillips, Brett E.;Cancel, Limary;Antonetti, David A.
通讯作者：
Antonetti, David A.
影响因子：
13.7
作者：
Abraham, Prema;Adelman, Ron A.;Zhi, Xin (Eric)
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Zhi, Xin (Eric)
影响因子：
7.8
作者：
Nitta, T;Hata, M;Tsukita, S
通讯作者：
Tsukita, S
影响因子：
4.8
作者：
Morimoto, S;Nishimura, N;Sasaki, T
通讯作者：
Sasaki, T
DOI：
10.1084/jem.169.6.1977
发表时间：
1989-06-01
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Brett J;Gerlach H;Nawroth P;Steinberg S;Godman G;Stern D
通讯作者：
Stern D