Determinants of 5-Lipoxygenase Nuclear Localization Using Green Fluorescent Protein/5-Lipoxygenase Fusion Proteins*
- From the Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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 β-barrel domain that may be masked by structural alterations.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grant HL58464 (to C. D. F.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Present address: Boston University School of Medicine, Whitaker Cardiovascular Institute, Boston, MA 02118.
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↵§ To whom correspondence should be addressed: Center for Experimental Therapeutics, 805 Stellar-Chance Laboratories, 422 Curie Blvd., University of Pennsylvania, Philadelphia, PA 19104. Tel.: 215-898-0254; Fax: 215-573-9004; E-mail: colin{at}spirit.gcrc.upenn.edu.
- Abbreviations:
- 5-HPETE
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5-hydroperoxyeicosatetraenoic acid
- 5LO
-
5-lipoxygenase
- GFP
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green fluorescent protein
- GFP-5LO
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green fluorescent protein-5-lipoxygenase fusion protein
- 5-HETE
-
5-hydroxyeicosatetraenoic acid
- 5LO−/−
-
5-lipoxygenase deficient
- NLS
-
nuclear localization signal
- PK
-
pyruvate kinase
- BMMC
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bone marrow-derived mast cells
- HEK
-
human embryonic kidney
- CHO
-
Chinese hamster ovary
- PBS
-
phosphate-buffered saline
- HPLC
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high performance liquid chromatography.
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- Received August 5, 1998.
- Revision received September 4, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
