Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function

S. Shi, A. D. Verin, K. L. Schaphorst, L. I. Gilbert-Mcclain, C. E. Patterson, R. P. Irwin, V. Natarajan, J. G.N. Garcia

Research output: Contribution to journalArticle

52 Scopus citations

Abstract

Thrombin-induced endothelial cell (EC) barrier dysfunction is highly dependent upon phosphorylation of serine and threonine residues present on myosin light chains (MLC) catalyzed by a novel EC myosin light chain kinase (MLCK) isoform. In this study, we examined the participation of tyrosine protein phosphorylation in EC contraction, gap formation and barrier dysfunction. We first determined that thrombin significantly increases protein tyrosine kinase activity and protein tyrosine phosphorylation in bovine pulmonary artery EC. Tyrosine kinase inhibitors, genistein and 2,5 DHC, reduced EC tyrosine kinase activities, however, only genistein significantly attenuated thrombin-mediated increases in albumin clearance and reductions in transendothelial electrical resistance. Similarly, genistein but not 2,5 DHC, decreased basal and thrombin-induced Ca2+ increases and MLC phosphorylation in the absence of alterations in Type 1 or 2A serine/threonine phosphatase activities. Immunoprecipitation of the EC MLCK isoform revealed a 214 kD immunoreactive phosphotyrosine protein and genistein pretreatment significantly reduced MLCK activity in MLCK immunoprecipitates. Although thrombin induced the translocation of p60src from the cytosol to the EC cytoskeleton, a detectable increase in the level of MLCK tyrosine phosphorylation was not noted after thrombin challenge. Taken together, our data suggest that genistein-sensitive tyrosine kinase activities are involved in thrombin-mediated EC MLCK activation, MLC phosphorylation, and barrier dysfunction.

Original languageEnglish (US)
Pages (from-to)153-171
Number of pages19
JournalEndothelium: Journal of Endothelial Cell Research
Volume6
Issue number2
DOIs
StatePublished - Jan 1 1998
Externally publishedYes

Keywords

  • Endothelial cell permeability
  • MLCK
  • Tyrosine kinases
  • Tyrosine phosphatases

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Fingerprint Dive into the research topics of 'Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function'. Together they form a unique fingerprint.

  • Cite this