Pyk2 phosphorylation destabilizes cell-cell adhesion 1 PROLINE-RICH TYROSINE KINASE 2 (PYK2) MEDIATES VE-CADHERIN- BASED CELL-CELL ADHESION BY REGULATING β -CATENIN TYROSINE PHOSPHORYLATION. Jaap D. van Buul *# , Eloise C. Anthony * , Mar Fernandez-Borja * , Keith Burridge # and Peter L. Hordijk * * Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. # Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill. Running title: Pyk2 phosphorylation destabilizes cell-cell adhesion Address correspondence to: Dr. Peter L. Hordijk, Department of Molecular Cell Biology, Sanquin Research at CLB and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX, Amsterdam, The Netherlands. Phone: +31205123263; Fax: +31205123474; E-mail: p.hordijk@sanquin.nl VE-cadherin controls endothelial cell-cell adhesion and preserves endothelial integrity. In order to maintain endothelial barrier function, VE-cadherin function is tightly regulated through mechanisms that involve protein phosphorylation and cytoskeletal dynamics. Here, we show that loss of VE - cadherin function results in intercellular gap formation and a drop in electrical resistance of monolayers of primary human endothelial cells. Detailed analysis revealed that loss of endothelial cell-cell adhesion, induced by VE-cadherin- blocking antibodies, is preceded by and dependent on a rapid activation of Rac1 and increased prodution of reactive oxygen species. Moreover, VE-cadherin- associated β -catenin is tyrosine phosphorylated upon loss of cell-cell contact. Finally, the redox-sensitive, proline -rich tyrosine kinase 2 (Pyk2) is activated and recruited to cell-cell junctions following the loss of VE - cadherin homotypic adhesion. Conversely, inhibition of Pyk2 activity in endothelial cells by expression of CRNK, an N-terminal deletion mutant that acts in a dominant negative fashion, not only abolishes the increas e in β-catenin tyrosine phosphorylation but also prevents loss of endothelial cell-cell contact. These results implicate Pyk2 in the reduced cell-cell adhesion induced by the Rac-mediated production of ROS, through the tyrosine phosphorylation of β -catenin. This signaling is initiated upon loss of VE-cadherin function, and is important for our insight in the modulation of endothelial integrity. Vascular-Endothelial Cadherin (VE- cadherin, Cadherin-5) is a transmembrane, calcium-dependent, homophilic adhesion molecule that connects adjacent endothelial cells. Loss of VE-cadherin function results in unstable endothelial junctions and a decrease in endothelial monolayer electrical resistance, despite the fact that several other adhesion proteins, such as claudin, occludin and PECAM-1, are also concentrated at sites of endothelial cell-cell contact (1,2). Thus, VE-cadherin function is indispensable for the maintenance of the endothelial barrier function. VE-cadherin is linked to the actin cytoskeleton via the armadillo-family members β- and γ -catenin that bind the actin-binding protein α-catenin (3,4). VE- cadherin function is controlled by cytoskeletal dynamics and by protein phosphorylation events. Lampugnani and colleagues showed that tyrosine JBC Papers in Press. Published on March 18, 2005 as Manuscript M500898200 Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on February 5, 2016 http://www.jbc.org/ Downloaded from