The Influence Of PKCδActivity On RhoA Interaction With Its Effector Proteins

The balance of RhoA activation is vital in maintaining endothelial function in part due to its role in cell contraction, a function which regulates vascular permeability. Previous studies have implicated PKCδ activity in the regulation of endothelial barrier function via modulation of RhoA activity. To understand the relationship between PKCδ and RhoA in the pulmonary endothelium, we used co‐precipitation experiments to identify Rho effectors that bind RhoA in the presence and absence of the PKCδ inhibitor, rottlerin. We noted PKCδ binding with both constitutively inactive, RhoA G17A , as well as the constitutively active RhoA Q63L , forms of RhoA. Chemical inhibition of PKCδ resulted in increased PKCδ binding to RhoA G17A but decreased PKCδ binding to RhoA Q63L . Altered binding of RhoA Q63L and RhoA G17A with p190RhoGAP and p115RhoGEF, respectively, was observed upon PKCδ inhibition. Using electric cell‐substrate impedance sensing (ECIS) system, we noted that similar to PKCδ inhibition, Rho kinase inhibition with Y27632 promoted endothelial barrier dysfunction. The data demonstrates that PKCδ influences RhoA activity in pulmonary endothelial cells and the decline in PKCδ or Rho kinase signaling correlated with dysfunction of the endothelial barrier. We propose that PKCδ functions as a regulator of vascular permeability and thus presents an interesting target in the regulation of the pulmonary endothelium.