Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src)

Inflammatory diseases of the lung are characterized by increases in vascula r permeability and enhanced leukocyte infiltration, reflecting compromise o f the endothelial cell (EC) barrier. We examined potential molecular mechan isms that underlie these alterations and assessed the effects of diperoxova nadate (DPV), a potent tyrosine kinase activator and phosphatase inhibitor, on EC contractile events. Confocal immunofluorescent microscopy confirmed dramatic increases in stress-fiber formation and colocalization of EC myosi n light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings con sistent with activation of the endothelial contractile apparatus. DPV produ ced significant time-dependent increases in MLC phosphorylation that were s ignificantly attenuated but not abolished by EC MLCK inhibition with KT-592 6. Pretreatment with the Rho GTPase-inhibitory C-3 exotoxin completely abol ished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC pho sphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipi tation of EC MLCK after DPV challenge revealed dramatic time-dependent tyro sine phosphorylation of the kinase in association with increased MLCK activ ity and a stable association of MLCK with the p85 actin-binding protein cor tactin and p60(src). Translocation of immunoreactive cortactin from the cyt osol to the cytoskeleton was noted after DPV in concert with cortactin tyro sine phosphorylation. These studies indicate that DPV activates the endothe lial contractile apparatus in a Rho GTPase-dependent fashion and suggests t hat p60(src)-induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.