Expression of a novel high molecular-weight myosin light chain kinase in endothelium.
Myosin light chain phosphorylation results in cellular contraction and is a critical component of agonist-mediated endothelial cell (EC) junctional gap formation and permeability. We have shown that this reaction is catalyzed by a novel high molecular-weight Ca2+/calmodulin-dependent nonmuscle myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To characterize EC MLCK expression further in cultured and adult tissues, we employed immunoblotting techniques and reverse transcriptase-polymerase chain reaction to demonstrate that freshly isolated and cultured human macro- and microvascular EC express only the EC MLCK isoform (214 kD), which is distinct from smooth-muscle MLCK isoforms (130 to 150 kD). Immunocytochemical studies demonstrated the presence of the high molecular-weight MLCK isoform in adult human cardiac endothelium using anti-MLCK antibodies, which preferentially recognize the high molecular-weight EC MLCK isoform. Monitoring of MLCK expression in different cell types with antibodies generated against a unique human EC MLCK N-terminal sequence revealed a high level of expression of the 214-kD enzyme in endothelium, minimal level of expression in smooth muscle, and no expression in skeletal muscle. These data suggest that the novel 214-kD kinase, the only MLCK isoform found in endothelium, may be preferentially expressed in this nonmuscle tissue.
Verin
A D
AD
Department of Medicine, Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Lazar
V
V
Torry
R J
RJ
Labarrere
C A
CA
Patterson
C E
CE
Garcia
J G
JG
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D009206:Myocardium / Q000166:cytology / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
D007527:Isoenzymes; D009219:Myosin-Light-Chain Kinase