Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium.
The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.
Wadgaonkar
Raj
R
Department of Medicine, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA.
Dudek
Steven M
SM
Zaiman
Ari L
AL
Linz-McGillem
Laura
L
Verin
Alexander D
AD
Nurmukhambetova
Saule
S
Romer
Lewis H
LH
Garcia
Joe G N
JG
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism*; D006801:Humans; D008263:Macrophage Migration-Inhibitory Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques; D014470:Umbilical Cord / Q000378:metabolism
D000199:Actins; D008263:Macrophage Migration-Inhibitory Factors; D009219:Myosin-Light-Chain Kinase