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Protein kinase cβ phosphorylates occludin regulating tight junction trafficking in vascular endothelial growth

Tomoaki Murakami1, Tiffany Frey, Chengmao Lin

  • 1Department of Cellular and Molecular Physiology and Ophthalmology, Penn State College of Medicine, Hershey, Pennsylvania, USA.

Diabetes
|March 23, 2012
PubMed

Insights

Vascular endothelial growth factor (VEGF) increases blood-retinal barrier permeability via protein kinase C beta (PKCβ) activation. This study shows PKCβ phosphorylates occludin, causing its ubiquitination and breakdown of the barrier.

Area of Science:

  • Ocular biology
  • Molecular mechanisms of vascular permeability
  • Cell signaling

Background:

  • Vascular endothelial growth factor (VEGF) triggers blood-retinal barrier (BRB) breakdown.
  • Protein kinase C beta (PKCβ) activation is essential for VEGF-induced BRB permeability.
  • The precise molecular events linking PKCβ activation to barrier dysfunction are not fully elucidated.

Purpose of the Study:

  • To investigate the role of occludin phosphorylation and ubiquitination downstream of PKCβ activation.
  • To determine how these modifications affect tight junction (TJ) trafficking and endothelial permeability.
  • To explore the therapeutic potential of targeting this pathway.

Main Methods:

  • Utilized bovine retinal endothelial cells and in vivo models.
  • Employed PKCβ inhibitors and dominant-negative kinase expression.
  • Assessed occludin phosphorylation at Ser490 using site-specific antibodies and in vitro kinase assays.
  • Measured occludin ubiquitination via immunoprecipitation and immunoblotting.
  • Analyzed TJ protein organization using confocal microscopy.

Main Results:

  • Inhibition of VEGF-induced PKCβ activation prevented occludin Ser490 phosphorylation and ubiquitination.
  • PKCβ directly phosphorylates occludin at Ser490.
  • Mutating occludin Ser490 to Ala blocked VEGF-induced permeability.
  • PKCβ inhibition disrupted TJ trafficking and prevented VEGF-stimulated vascular permeability in vitro and in vivo.

Conclusions:

  • PKCβ activation phosphorylates occludin on Ser490, initiating a cascade of ubiquitination.
  • This phosphorylation and subsequent ubiquitination are critical for VEGF-induced endothelial permeability.
  • Targeting PKCβ-mediated occludin phosphorylation offers a novel therapeutic strategy for controlling vascular permeability.

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