title: Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth factor-induced permeability in vivo.
abstract: 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.
authors.affiliation: Department of Cellular and Molecular Physiology and Ophthalmology, Penn State College of Medicine, Hershey, Pennsylvania, USA.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Murakami
authors.forename: Tiffany
authors.initials: T
authors.lastname: Frey
authors.forename: Chengmao
authors.initials: C
authors.lastname: Lin
authors.forename: David A
authors.initials: DA
authors.lastname: Antonetti
mesh_terms: D000818:Animals; D001813:Blood-Retinal Barrier / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002417:Cattle; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D062793:Occludin; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D064546:Protein Kinase C beta; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D008565:Membrane Proteins; D062793:Occludin; C568987:Ocln protein, rat; D047428:Protein Kinase Inhibitors; D042461:Vascular Endothelial Growth Factor A; D011493:Protein Kinase C; D064546:Protein Kinase C beta