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

Joe G.N. Garcia, Alexander D. Verin, Kane Schaphorst, Rafat Siddiqui, Carolyn E. Patterson, Csilla Csortos, Viswanathan Natarajan

Research output: Contribution to journalArticle

151 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)L989-L998
JournalAmerican Journal of Physiology - Lung Cellular and Molecular Physiology
Volume276
Issue number6 20-6
StatePublished - Jun 1 1999
Externally publishedYes

Keywords

  • Endothelial cell contraction
  • Myosin phosphorylation
  • Permeability
  • Src kinases

ASJC Scopus subject areas

  • Physiology
  • Pulmonary and Respiratory Medicine
  • Physiology (medical)
  • Cell Biology

Fingerprint Dive into the research topics of 'Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src)'. Together they form a unique fingerprint.

  • Cite this

    Garcia, J. G. N., Verin, A. D., Schaphorst, K., Siddiqui, R., Patterson, C. E., Csortos, C., & Natarajan, V. (1999). Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60(src). American Journal of Physiology - Lung Cellular and Molecular Physiology, 276(6 20-6), L989-L998.