Abstract
5-Lipoxygenase catalyzes the first two steps in the biosynthesis of leukotrienes, potent extracellular mediators of inflammation and allergic disorders. The unanticipated observation of 5-lipoxygenase in the nucleus of some cell types including bone marrow-derived mast cells (Chen, X. S., Naumann, T. A., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1995) J. Biol. Chem. 270, 17993-17999) has raised speculation about intranuclear actions of leukotrienes or the enzyme itself. To explore the entry of 5-lipoxygenase into the nucleus we have transfected various cell types with expression vectors encoding native 5-lipoxygenase and green fluorescent protein/5-lipoxygenase (GFP-5LO) fusion proteins. 5-Lipoxygenase and green fluorescent protein/5-lipoxygenase co-localized with the nuclear DNA stain Hoechst 33258 in each cell type. The three main basic regions of 5-lipoxygenase were incapable of acting as "classical" nuclear localization signal sequences. Mutations that abolished enzyme activity/non-heme iron resulted in proteins that would no longer enter the nucleus. An NH2-terminal 5-lipoxygenase fragment of 80 residues was sufficient for directing nuclear localization of green fluorescent protein but not cytosolic pyruvate kinase. The combined data suggest that 5-lipoxygenase enters the nucleus not by a classical nuclear localization signal but by a non-conventional signal located in the predicted beta-barrel domain that may be masked by structural alterations.
Highlights
5-Lipoxygenase is a non-heme iron enzyme found primarily in white blood cells, macrophages, and mast cells that converts arachidonic acid first to 5-hydroperoxyeicosatetraenoic acid (5HPETE)1 and to leukotriene (LT)A4 (5,6-oxido-7,9,11,14eicosatetraenoic acid)(1)
More recent work based on immunofluorescence techniques and cellular fractionation demonstrated that certain cell types capable of leukotriene formation express 5-lipoxygenase completely or partially in the nucleus (8 –11)
Transfection of cDNA Encoding 5-Lipoxygenase into Various Cell Types Results in Nuclear Localization—Since 5-lipoxygenase is situated in the cytosol of resting neutrophils, the nucleus of bone marrow-derived mast cells (BMMC) and in both compartments in rat basophilic leukemia-1 cells and macrophages, we sought to determine the cellular localization of 5-lipoxygenase in various cell types transfected with 5-lipoxygenase expression vectors prior to detailed analysis of nuclear localization signal (NLS) sequences
Summary
5-HPETE, 5-hydroperoxyeicosatetraenoic acid; 5LO, 5-lipoxygenase; GFP, green fluorescent protein; GFP5LO, green fluorescent protein-5-lipoxygenase fusion protein; 5-HETE, 5-hydroxyeicosatetraenoic acid; 5LOϪ/Ϫ, 5-lipoxygenase deficient; NLS, nuclear localization signal; PK, pyruvate kinase; BMMC, bone marrowderived mast cells; HEK, human embryonic kidney; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography. More recent work based on immunofluorescence techniques and cellular fractionation demonstrated that certain cell types capable of leukotriene formation (alveolar macrophages, rat basophilic leukemia cells, and mouse bone marrow-derived mast cells) express 5-lipoxygenase completely or partially in the nucleus (8 –11). The discovery of 5-lipoxygenase in the nucleus was a surprising observation since it is well known that leukotrienes must exit the cell once synthesized to act on cell surface G proteincoupled receptors to exert their actions on neutrophils or bronchiole smooth muscle [13, 14]. Proteins enter the nucleus by nuclear localization signal (NLS) sequences that are recognized by specific importins, prior to nuclear pore docking, translocation through the pore and release from the pore’s inner side [16, 17]. We demonstrate primarily with the use of green fluorescent protein (GFP)/5-lipoxygenase fusion proteins the complexity of events for 5-lipoxygenase nuclear entry
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