The Role of PKCAlpha-Mediated p120-Catenin Phosphorylation on Endothelial Permeability
thesisposted on 21.02.2013, 00:00 by Emily E. Vandenbroucke St Amant
Adherens junctions are the primary cell-cell junction that maintains the integrity of the endothelium. Vascular leakage resulting from inflammation contributes to a variety of pathological conditions. This thesis investigates the role of protein kinase C-α (PKCα) phosphorylation of p120-catenin p120) on adherens junction stability and regulation of endothelial permeability during inflammation. Vascular endothelial cadherin (VEC) is the major structural protein comprising adherens junctions. VEC requires binding of p120 to maintain intact adherens junctions; the loss of bound p120 results in VEC internalization and disassembly of adherens junctions. PKCα, a serine/threonine kinase, phosphorylates p120 on S879. We found that phosphorylation of this site causes p120 to dissociate from VEC and results in increased endothelial permeability. p120 phosphorylated on S879 showed no colocalization with VEC, indicating that once p120 is phosphorylated on this site, it can no longer bind VEC. We created point mutations of p120 on S879, and expressed these mutants using a liposome-based cDNA transfection in endothelial cells and mouse lungs. The pro-inflammatory agents thrombin and LPS were used to stimulate increased permeability in endothelial cells, and similarly PAR-1 agonist peptide or LPS was used in mice. The phosphomimetic p120 (S879D) did not associate with VEC in endothelial cells, whereas the phosphodefective p120 (S879A) remained bound to VEC even after thrombin stimulation. Endothelial cells expressing phosphodefective p120 exhibited less interendothelial gap formation after thrombin stimulation, suggesting that expression of phosphodefective p120 protected against adherens junction disassembly. Mice expressing phosphodefective p120 showed less of an increase in lung vessel permeability and less edema formation following in vivo stimulation with PAR-1 agonist peptide as compared to mice expressing wild-type p120. This further confirms that phosphorylation of p120 on S879 by PKCα is a critical signaling mechanism involved in regulation of adherens junction stability and endothelial barrier function. We also created a peptide that blocked S879 phosphorylation in endothelial cells by competitively binding p120. Cells incubated with the blocking peptide showed significantly less monolayer disruption after thrombin stimulation, indicating that blocking S879 may be a potential therapeutic target used to block edema.