Determinants of 5-lipoxygenase nuclear localization using green fluorescent protein/5-lipoxygenase fusion proteins.
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.
Chen
X S
XS
Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Zhang
Y Y
YY
Funk
C D
CD
D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000201:enzymology*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D019159:Nonheme Iron Proteins / Q000235:genetics / Q000378:metabolism*; D019913:Nuclear Localization Signals; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008164:Luminescent Proteins; D019159:Nonheme Iron Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001094:Arachidonate 5-Lipoxygenase