Biochemical regulation of the nonmuscle myosin light chain kinase isoform in bovine endothelium

Alexander Dmitriyevich Verin, Lydia I. Gilbert-McClain, Carolyn E. Patterson, Joe G.N. Garcia

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

Specific models of vascular permeability are critically dependent on myosin light chain phosphorylation, a reaction catalyzed by a novel high molecular-weight (214 kD) Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To evaluate mechanisms of endothelial cell (EC) barrier dysfunction evoked by the serine protease thrombin, we studied the regulation of the 214-kD EC MLCK isoform expressed in bovine endothelium. The EC MLCK isoform bound biotinylated CaM in a Ca2+-dependent manner and co-immunoprecipitated in a functional complex with myosin, actin, and CaM. Thrombin rapidly increased MLCK activity in concert with time-dependent translocation of the enzyme to the actin cytoskeleton. To evaluate whether EC MLCK activity was regulated by direct phosphorylation, amino acid sequence analysis identified multiple potential EC MLCK sites for Ser/Thr phosphorylation, including highly conserved phosphorylation sites for cyclic adenosine monophosphate-dependent protein kinase A (PKA) adjacent to the CaM-binding region. EC MLCK activity was attenuated by either PKA-mediated MLCK phosphorylation or inhibition of Ser/Thr phosphatase activity (fluoride or calyculin), which significantly increased MLCK phosphorylation while decreasing MLCK activity (3-to 4-fold decrease). In summary, although the EC MLCK isoform exhibits multiple features intrinsic to this family of kinases, thrombin-mediated EC contraction and barrier dysfunction requires increased EC MLCK-actin interaction and MLCK translocation to the cytoskeleton. EC MLCK activity appears to be highly dependent upon the phosphorylation status of this key contractile effector.

Original languageEnglish (US)
Pages (from-to)767-776
Number of pages10
JournalAmerican journal of respiratory cell and molecular biology
Volume19
Issue number5
DOIs
StatePublished - Jan 1 1998
Externally publishedYes

Fingerprint

Myosin-Light-Chain Kinase
Endothelium
Protein Isoforms
Endothelial cells
Phosphorylation
Endothelial Cells
Calmodulin
Thrombin
Actins
Cyclic AMP-Dependent Protein Kinases
Myosin Light Chains
Protein Sequence Analysis
Capillary Permeability
Serine Proteases
Myosins
Cytoskeleton
Fluorides
Actin Cytoskeleton
Phosphoric Monoester Hydrolases
Cyclic AMP

ASJC Scopus subject areas

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

Cite this

Biochemical regulation of the nonmuscle myosin light chain kinase isoform in bovine endothelium. / Verin, Alexander Dmitriyevich; Gilbert-McClain, Lydia I.; Patterson, Carolyn E.; Garcia, Joe G.N.

In: American journal of respiratory cell and molecular biology, Vol. 19, No. 5, 01.01.1998, p. 767-776.

Research output: Contribution to journalArticle

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