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.