Myosin light chain kinase in endothelium: molecular cloning and regulation.
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.
Garcia
J G
JG
Department of Medicine, Indiana University School of Medicine, and Richard Roudebush Veterans Administration Center, Indianapolis, USA.
Lazar
V
V
Gilbert-McClain
L I
LI
Gallagher
P J
PJ
Verin
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011651:Pulmonary Artery; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014471:Umbilical Veins
D018076:DNA, Complementary; D002772:Cholera Toxin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase