Regulation of leukotriene A4 hydrolase activity in endothelial cells by phosphorylation.
Endothelial cells contain leukotriene (LT) A4 hydrolase (LTA-H) as detected by Northern and Western blotting, but several studies have been unable to detect the activity of this enzyme. Since LTA-H could play a key role in determining what biologically active lipids are generated by activated endothelium during the inflammatory process, we studied possible mechanisms by which this enzyme may be regulated. We find that LTA-H is phosphorylated under basal conditions in human endothelial cells and in this state does not exhibit epoxide hydrolase activity (i.e. conversion of LTA4 to LTB4). LTA-H purified from endothelial cells is efficiently dephosphorylated by incubation with protein phosphatase-1 in the presence of an LTA-H peptide substrate and not at all in the absence of substrate. Under conditions that lead to dephosphorylation, protein phosphatase-1 activates the epoxide hydrolase activity of LTA-H. Using peptide mapping and site-directed mutagenesis, we have identified serine 415 as the site of phosphorylation of LTA-H by a kinase found in endothelial cell cytosol. In parallel, we have studied a human lung carcinoma cell line that expresses active LTA-H. Although these cells have cytosolic kinases that phosphorylate recombinant LTA-H, they do not target serine 415 and thus do not inhibit LTA-H activity. We believe that LTA-H is regulated in intact cells by a kinase/phosphatase cycle and further that the kinase in endothelial cells specifically recognizes and phosphorylates a regulatory site in the LTA-H.
Rybina
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IV
Department of Pharmacology, College of Physicians & Surgeons, Columbia University, New York, New York 10032, USA.
Liu
H
H
Gor
Y
Y
Feinmark
S J
SJ
D019943:Amino Acid Substitution; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007975:Leukotriene B4 / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation
D007975:Leukotriene B4; D004851:Epoxide Hydrolases; C043134:leukotriene A4 hydrolase