The N-terminal "beta-barrel" domain of 5-lipoxygenase is essential for nuclear membrane translocation.
5-Lipoxygenase is the key enzyme in the formation of leukotrienes, which are potent lipid mediators of asthma pathophysiology. This enzyme translocates to the nuclear envelope in a calcium-dependent manner for leukotriene biosynthesis. Eight green fluorescent protein (GFP)-lipoxygenase constructs, representing the major human and mouse enzymes within this family, were constructed and their cDNAs transfected into human embryonic kidney 293 cells. Of these eight lipoxygenases, only the 5-lipoxygenase was clearly nuclear localized and translocated to the nuclear envelope upon stimulation with the calcium ionophore. The N-terminal "beta -barrel" domain of 5-lipoxygenase, but not the catalytic domain, was necessary and sufficient for nuclear envelope translocation. The GFP-N-terminal 5-lipoxygenase domain translocated faster than GFP-5-lipoxygenase. beta-Barrel/catalytic domain chimeras with 12- and 15-lipoxygenase indicated that only the N-terminal domain of 5-lipoxygenase could carry out this translocation function. Mutations of iron atom binding ligands (His550 or deletion of C-terminal isoleucine) that disrupt nuclear localization do not alter translocation capacity indicating distinct determinants of nuclear localization and translocation. Moreover, data show that GFP-5-lipoxygenase beta-barrel containing constructs can translocate to the nuclear membrane whether cytoplasmic or nuclear localized. Thus, the predicted beta-barrel domain of 5-lipoxygenase may function like the C2 domain within protein kinase C and cytosolic phospholipase A(2) with unique determinants that direct its localization to the nuclear envelope.
Chen
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Department of Pharmacology, Center for Experimental Therapeutics University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Funk
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D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D020134:Catalytic Domain; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D049452:Green Fluorescent Proteins; D006801:Humans; D007501:Iron / Q000378:metabolism; D008024:Ligands; D008164:Luminescent Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism*; D019913:Nuclear Localization Signals; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D008024:Ligands; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; D000001:Calcimycin; D007501:Iron; D001094:Arachidonate 5-Lipoxygenase