Protein Kinase Cβ Phosphorylates Occludin Regulating Tight Junction Trafficking in Vascular Endothelial Growth Factor–Induced Permeability In Vivo
- Department of Cellular and Molecular Physiology and Ophthalmology, Penn State College of Medicine, Hershey, Pennsylvania
- Corresponding author: David A. Antonetti, .
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.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db11-1367/-/DC1.
T.M. is currently affiliated with the Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Sakyo, Kyoto, Japan.
C.L. and D.A.A. are currently affiliated with the Department of Ophthalmology and Visual Sciences, Kellogg Eye Center, Ann Arbor, Michigan.
- Received September 29, 2011.
- Accepted January 28, 2012.
- © 2012 by the American Diabetes Association.
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