Myosin Light Chain Kinase in Endothelium: Molecular Cloning and Regulation

Joe G.N. Garcia, Virginie Lazar, Lydia I. Gilbert-McClain, Patricia J. Gallagher, Alexander D. Verin

Research output: Contribution to journalArticle

167 Scopus citations

Abstract

The phosphorylation of myosin light chains by myosin light chain kinase (MLCK) is a key event in agonist-mediated endothelial cell gap formation and vascular permeability. We now report the cloning and expression of a nonmuscle MLCK isoform in cultured endothelium. Screening of a human endothelial cell cDNA library identified a 7.7 kb cDNA with substantial (> 95%) homology to the coding region of the rabbit and bovine smooth muscle (SM) MLCK (amino acid #923-1913) as well as with the reported avian nonmuscle MLCK (65-70% homology). Sequence analysis also identified, however, a 5′ stretch of novel sequence (amino acids #1-922) which is not contained in the open reading frame of mammalian SM MLCK, and is only 58% homologous to the avian fibroblast MLCK sequence. Immunoprecipitation with NH2-specific antisera revealed a 214 kD high molecular weight MLCK in bovine and human endothelium which exhibits MLC phosphorylation properties. Amino acid sequence analysis revealed endothelial MLCK consensus sequences for a variety of protein kinases including highly conserved potential phosphorylation sites for cAMP-dependent protein kinase A (PKA) in the CaM-binding region. Augmentation of intracellular cAMP levels markedly enhanced MLCK phosphorylation (2.5-fold increase) and reduced kinase activity in MLCK immunoprecipitates (4-fold decrease). These data suggest potentially novel mechanisms of endothelial cell contraction and barrier regulation.

Original languageEnglish (US)
Pages (from-to)489-494
Number of pages6
JournalAmerican journal of respiratory cell and molecular biology
Volume16
Issue number5
DOIs
StatePublished - 1997

ASJC Scopus subject areas

  • Molecular Biology
  • Pulmonary and Respiratory Medicine
  • Clinical Biochemistry
  • Cell Biology

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