Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src).
Inflammatory diseases of the lung are characterized by increases in vascular permeability and enhanced leukocyte infiltration, reflecting compromise of the endothelial cell (EC) barrier. We examined potential molecular mechanisms that underlie these alterations and assessed the effects of diperoxovanadate (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 myosin light chain (MLC) kinase (MLCK) with the actin cytoskeleton, findings consistent with activation of the endothelial contractile apparatus. DPV produced significant time-dependent increases in MLC phosphorylation that were significantly attenuated but not abolished by EC MLCK inhibition with KT-5926. Pretreatment with the Rho GTPase-inhibitory C3 exotoxin completely abolished DPV-induced MLC phosphorylation, consistent with Rho-mediated MLC phosphatase inhibition and novel regulation of EC MLCK activity. Immunoprecipitation of EC MLCK after DPV challenge revealed dramatic time-dependent tyrosine phosphorylation of the kinase in association with increased MLCK activity and a stable association of MLCK with the p85 actin-binding protein cortactin and p60(src). Translocation of immunoreactive cortactin from the cytosol to the cytoskeleton was noted after DPV in concert with cortactin tyrosine phosphorylation. These studies indicate that DPV activates the endothelial contractile apparatus in a Rho GTPase-dependent fashion and suggests that p60(src)-induced tyrosine phosphorylation of MLCK and cortactin may be important features of contractile complex assembly.
Garcia
J G
JG
Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. drgarcia@welchlink.welch.jhu.edu
Verin
A D
AD
Schaphorst
K
K
Siddiqui
R
R
Patterson
C E
CE
Csortos
C
C
Natarajan
V
V
D000199:Actins / Q000502:physiology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002417:Cattle; D051356:Cortactin; D003599:Cytoskeleton / Q000378:metabolism; D004727:Endothelium / Q000166:cytology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D008840:Microfilament Proteins / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D015688:Oncogene Protein pp60(v-src) / Q000502:physiology*; D010545:Peroxides / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000502:physiology
D000199:Actins; C495868:CTTN protein, human; D051356:Cortactin; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D010545:Peroxides; C091767:diperoxovanadate; D019000:Phosphotyrosine; D014638:Vanadates; D015688:Oncogene Protein pp60(v-src); D009219:Myosin-Light-Chain Kinase; D020558:GTP Phosphohydrolases; C503256:RHOQ protein, human; D009218:Myosins; D020741:rho GTP-Binding Proteins