The N-terminal “β-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 A23187. The N-terminal “β -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. β-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 β-barrel containing constructs can translocate to the nuclear membrane whether cytoplasmic or nuclear localized. Thus, the predicted β-barrel domain of 5-lipoxygenase may function like the C2 domain within protein kinase C and cytosolic phospholipase A2 with unique determinants that direct its localization to the nuclear envelope. 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 A23187. The N-terminal “β -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. β-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 β-barrel containing constructs can translocate to the nuclear membrane whether cytoplasmic or nuclear localized. Thus, the predicted β-barrel domain of 5-lipoxygenase may function like the C2 domain within protein kinase C and cytosolic phospholipase A2 with unique determinants that direct its localization to the nuclear envelope. human embryonic kidney green fluorescent protein lipoxygenase rat basophilic leukemia polymerase chain reaction reverse transcription hydroxyeicosatetraenoic acid nuclear localizing signal Leukotrienes are potent lipid mediators of inflammation and anaphylaxis (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2312) Google Scholar). They are generated by an initial reaction with the enzyme 5-lipoxygenase from arachidonic acid that has been liberated from membrane lipids (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2312) Google Scholar, 2Samuelsson B. Funk C.D. J. Biol. Chem. 1989; 264: 19469-19472Abstract Full Text PDF PubMed Google Scholar, 3Lewis R.A. Austen K.F. Soberman R.J. N. Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1168) Google Scholar). 5-Lipoxygenase is a 78-kDa protein found predominantly within inflammatory cell types (e.g.macrophages, mast cells, neutrophils, and eosinophils). The location of the enzyme is cell-type specific. In unstimulated neutrophils, it is found in the cytoplasm, whereas in alveolar macrophages and bone marrow-derived mast cells it is situated mainly in the nucleus (4Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 5Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 6Chen X.S. Naumann T.A. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. J. Biol. Chem. 1995; 270: 17993-17999Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Woods J.W. Coffey M.J. Brock T.G. Singer I.I. Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2046Crossref PubMed Scopus (158) Google Scholar). Transfection of 5-lipoxygenase cDNA leads to nuclear localized protein in HEK1293, COS, NIH-3T3, and Chinese hamster ovary cells, as well as bone marrow-derived mast cells and RAW macrophages (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Healy A. Peters-Golden M. Yao J. Brock T. J. Biol. Chem. 1999; 274: 29812-29818Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).Regardless of the cellular localization, 5-lipoxygenase undergoes a calcium-dependent translocation event to the nuclear membrane in activated inflammatory cells (5Brock T.G. McNish R.W. Peters-Golden M. J. Biol. Chem. 1995; 270: 21652-21658Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Early studies documented a reversible, soluble to membrane compartment transition in neutrophils challenged with the calcium ionophore A23187 (10Rouzer C.A. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7393-7397Crossref PubMed Scopus (95) Google Scholar, 11Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar). Experiments with RBL-2H3 cells demonstrated that the translocation to membranes required influx of extracellular calcium, which could be induced either with ionomycin or cross-linking of IgE receptors (12Wong A. Hwang S.M. Cook M.N. Hogaboom G.K. Crooke S.T. Biochemistry. 1988; 27: 6763-6769Crossref PubMed Scopus (65) Google Scholar, 13Wong A. Cook M.N. Foley J.J. Sarau H.M. Marshall P. Hwang S.M. Biochemistry. 1991; 30: 9346-9354Crossref PubMed Scopus (74) Google Scholar, 14Wong A. Cook M.N. Hwang S.M. Sarau H.M. Foley J.J. Crooke S.T. Biochemistry. 1992; 31: 4046-4053Crossref PubMed Scopus (38) Google Scholar). The neutrophil 5-lipoxygenase cytosol to membrane translocation was associated with a loss of activity (11Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar). However, in alveolar macrophages and RBL cells the translocation to membrane fractions activated 5-lipoxygenase activity (12Wong A. Hwang S.M. Cook M.N. Hogaboom G.K. Crooke S.T. Biochemistry. 1988; 27: 6763-6769Crossref PubMed Scopus (65) Google Scholar, 15Coffey M. Peters-Golden M. Fantone J.C. Sporn P.H. J. Biol. Chem. 1992; 267: 570-576Abstract Full Text PDF PubMed Google Scholar). It is generally regarded that the translocation event to the nuclear envelope is a necessary event in leukotriene formation. At this site there is an apparent transfer of substrate to 5-lipoxygenase by unknown mechanisms, via the integral membrane protein referred to as 5-lipoxygenase activating protein (16Miller D.K. Gillard J.W. Vickers P.J. Sadowski S. Leveille C. Mancini J.A. Charleson P. Dixon R.A. Ford-Hutchinson A.W. Fortin R. Gauthier J.Y. Rodkey J. Rosen R. Rouzer C. Sigal I.S. Strader C.D. Evans J.F. Nature. 1990; 343: 278-281Crossref PubMed Scopus (379) Google Scholar, 17Mancini J.A. Abramovitz M. Cox M.E. Wong E. Charleson S. Perrier H. Wang Z. Prasit P. Vickers P.J. FEBS Lett. 1993; 318: 277-281Crossref PubMed Scopus (180) Google Scholar).5-Lipoxygenase was recently shown to directly bind two calcium ions (18Hammarberg T. Radmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (67) Google Scholar). It has long been known that this enzyme is unique among lipoxygenases in its ability to have its activity stimulated by calcium (19Rouzer C.A. Shimizu T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7505-7509Crossref PubMed Scopus (74) Google Scholar, 20Hogaboom G.K. Cook M. Newton J.F. Varrichio A. Shorr R.G. Sarau H.M. Crooke S.T. Mol. Pharmacol. 1986; 30: 510-519PubMed Google Scholar). Although this calcium stimulatory effect is not an absolute requirement for the purified enzyme, it is essential when the enzyme is present in intact cells, or in isolated preparations incubated with membranes or phospholipids (21Skorey K.I. Gresser M.J. Biochemistry. 1998; 37: 8027-8034Crossref PubMed Scopus (24) Google Scholar, 22Reddy K.V. Hammarberg T. Radmark O. Biochemistry. 1999; 39: 1840-1848Crossref Scopus (35) Google Scholar). 5-Lipoxygenase also possesses a nucleotide-binding site of unknown function and ATP is known to stimulate activity (23Zhang Y. Hammarberg T. Radmark O. Samuelsson B. Ng C. Funk C.D. Loscalzo J. Biochem. J. 2000; 351: 697-707Crossref PubMed Scopus (31) Google Scholar, 24Ueda N. Kaneko S. Yoshimoto T. Yamamoto S. J. Biol. Chem. 1986; 261: 7982-7988Abstract Full Text PDF PubMed Google Scholar).There has been little, if any, insight to document the domain(s) within mammalian lipoxygenases that govern membrane translocation. The lipoxygenases are known to possess two domains based on the crystal structures of two soybean enzymes and the rabbit reticulocyte 15-lipoxygenase (25Boyington J.C. Gaffney B.J. Amzel L.M. Science. 1993; 260: 1482-1486Crossref PubMed Scopus (453) Google Scholar, 26Minor W. Steczko J. Stec B. Otwinowski Z. Bolin J.T. Walter R. Axelrod B. Biochemistry. 1996; 35: 10687-10701Crossref PubMed Scopus (392) Google Scholar, 27Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar, 28Skrzypczak-Jankun E. Amzel L.M. Kroa B.A. Funk Jr., M.O. Proteins. 1997; 29: 15-31Crossref PubMed Scopus (149) Google Scholar). The N terminus contains a β-barrel region of ∼110–115 (mammals) and 150 (plants) amino acids, in addition to the large non-heme iron containing catalytic domain at the C terminus. Sequence alignments between mammalian 15- and 5-lipoxygenases indicate ∼33% identity in the predicted N-terminal domain and only around 10% homology between the corresponding region of plants and mammals. The function of the β-barrel of lipoxygenases is unknown but it has been suggested that this domain, which bears resemblance to the C-terminal domain of certain lipases, is important for lipid binding (27Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar). In fact, a recent study showed that this region within a cucumber lipoxygenase is important for binding to liposomes and lipid bodies (29May C. Hohne M. Gnau P. Schwennesen K. Kindl H. Eur. J. Biochem. 2000; 267: 1100-1109Crossref PubMed Scopus (38) Google Scholar).Here, we show strong proof that the 5-lipoxygenase putative 2Since the crystal structure of 5-lipoxygenase has not been determined, it can only be assumed that this enzyme has a two-domain structure similar to other lipoxygenases with elucidated structures. Instead of using “putative” throughout the manuscript it is assumed that there is a N-terminal β-barrel-like domain and a C-terminal catalytic domain and the word putative will not be used for the remainder of the text for simplicity.2Since the crystal structure of 5-lipoxygenase has not been determined, it can only be assumed that this enzyme has a two-domain structure similar to other lipoxygenases with elucidated structures. Instead of using “putative” throughout the manuscript it is assumed that there is a N-terminal β-barrel-like domain and a C-terminal catalytic domain and the word putative will not be used for the remainder of the text for simplicity. β-barrel domain is unique among the mammalian lipoxygenase members in its ability to direct nuclear membrane translocation. Using green fluorescent protein-lipoxygenase fusions we present evidence for its necessity and sufficiency in nuclear translocation in real time using transfected cells.EXPERIMENTAL PROCEDURESPlasmid Constructs5-Lipoxygenase ConstructsThe coding region for green fluorescent protein (GFP) from the vector pEGFP-C2 (CLONTECH) was ligated with the coding region for 5-lipoxygenase to make pEGFP-5LO as described previously (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). pEGFP-5LO was used for preparation of chimeric molecules and truncated 5-lipoxygenase cDNA constructs. GFP-tagged truncated 5LO constructs (see Fig. 2 C), including pEGFP-5LO-(1–114), pEGFP-5LO-(115–673), pEGFP-5LO (N6-deletion), and pEGFP-5LO (N17-deletion) were prepared using polymerase chain reaction (PCR) and standard subcloning techniques (30Davis L.G. Dibner M.D. Battey J.F. Basic Methods in Molecular Biology. Elsevier, New York1986Google Scholar). Chimeric lipoxygenases (see Fig.4 B) were engineered from pEGFP-5LO (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), pEGFP-12LO, and pEGFP-15LO (see below for preparation) and pEGFP-truncated 5LO templates. To prepare a GFP-tagged chimera, generally a restriction enzyme site was incorporated in a region of interest in the lipoxygenase DNA sequence by using the QuikChange Site-directed Mutagenesis kit (Stratagene) or PCR. Subsequently, ligation of restriction fragments was carried out using a Rapid DNA Ligation kit (Roche Molecular Chemicals). For example, to obtain pEGFP-N-15LO-(1–111)-C-5LO-(115–673), an EcoRI site was incorporated between the region encoding amino acid residue 111 and 112 of 15-LO through site-directed mutagenesis. EcoRI-cut fragment DNA encoding N-terminal 111 amino acids of 15-LO was cloned in EcoRI-digested pEGFP-5LO-(115–673). Ligation boundaries and PCR-generated DNA sequences in the above constructs were verified by automated DNA sequencing using the facilities of the Department of Genetics, University of Pennsylvania. More detailed information of these DNA constructs and mutagenic primers may be obtained upon request. The pEGFP-5LO-(1–80), pEGFP-5LO-(1–127), pEGFP-5LO-(1–166), pEGFP-5LO-(81–673), pEGFP-5LO-(C6-deletion), pEGFP-5LO-(H367Q), and pEGFP-5LO-(H550Q) were prepared previously (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Figure 4A23187-induced translocation of various GFP-lipoxygenase chimeras to the nuclear envelope requires the β-barrel region of 5-lipoxygenase. A, the results from four different chimeras, N5LO-(1–127)-CP12LO-(126–663) (a 12-lipoxygenase enzymatically active chimera containing the N-terminal β-barrel and interdomain connecting region of 5-lipoxygenase plus theC-terminal catalytic domain of platelet-type12-lipoxygenase; top panels), NP12LO-(1–75)-C5LO-(81–673) (a 5-lipoxygenase enzymatically active chimera; second level panels), N5LO-(1–114)-C15LO-(112–663) (containing the β-barrel of 5-lipoxygenase and catalytic domain of 15-lipoxygenase type 1;third level panels), and N15LO-(1–111)-C5LO-(115–673) (containing the β-barrel of 15-lipoxygenase type 1 and catalytic domain of 5-lipoxygenase; bottom panels), respectively.B, summary of results in tabular form depicting these constructs and additional chimeras tested. The data are representative from experiments performed at least three times. 5-Lipoxygenase contains additional residues within the N-terminal region with respect to 12- and 15-lipoxygenases; thus, numbering may not always correspond in linear sequence.View Large Image Figure ViewerDownload Hi-res image Download (PPT)pEGFP-12LOThe XbaI insert from pcDNA1–6His12LX (31Chen X.S. Brash A.R. Funk C.D. Eur. J. Biochem. 1993; 214: 845-852Crossref PubMed Scopus (65) Google Scholar) was blunt end ligated into the blunted HindIII site of pEGFP-C2. The six-histidine tag was shown previously not to affect enzyme activity (31Chen X.S. Brash A.R. Funk C.D. Eur. J. Biochem. 1993; 214: 845-852Crossref PubMed Scopus (65) Google Scholar). The EGFP-tagged protein displayed high level 12-lipoxygenase activity in HEK 293-transfected cells (exclusive formation of 12-HPETE from arachidonic acid).pEGFP-15LO-1An EcoRI/BglII fragment of the coding region for human 15-lipoxygenase (32Funk C.D. Furci L. FitzGerald G.A. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 33-36PubMed Google Scholar) was cloned into the EcoRI and BamHI sites of pEGFP-C2. This construct yielded both 15-HETE and 12-HETE (9:1 ratio) from arachidonic acid in transfected cells similar to studies without the GFP tag (32Funk C.D. Furci L. FitzGerald G.A. Adv. Prostaglandin Thromboxane Leukotriene Res. 1991; 21A: 33-36PubMed Google Scholar).pEGFP-15LO-2The coding region for human 15-lipoxygenase-2 was amplified by RT-PCR using primers and conditions based on the published sequence (33Brash A.R. Boeglin W.E. Chang M.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6148-6152Crossref PubMed Scopus (350) Google Scholar) starting with human hair RNA template (34Sun D. McDonnell M. Chen X.S. Lakkis M.M. Li H. Isaacs S.N. Elsea S.H. Patel P.I. Funk C.D. J. Biol. Chem. 1998; 273: 33540-33547Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The PCR products were first cloned in pCR2.1 (Invitrogen) and sequences verified. Two fragments, a 0.9-kilobase EcoRI/AseI piece encoding the N-terminal region, and a 1.1-kilobase AseI/BamHI insert encoding the C terminus, were ligated in a three-fragment reaction with EcoRI/BamHI-digested pEGFP-C2. The construct when transfected into HEK 293 cells and subsequently incubated with arachidonic acid synthesized exclusively 15-HPETE.pEGFP-12(R)LOThe construction and characterization of this plasmid was described previously (34Sun D. McDonnell M. Chen X.S. Lakkis M.M. Li H. Isaacs S.N. Elsea S.H. Patel P.I. Funk C.D. J. Biol. Chem. 1998; 273: 33540-33547Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).pEGFP-e12LOThe original expression construct described previously (35Funk C.D. Keeney D.S. Oliw E.H. Boeglin W.E. Brash A.R. J. Biol. Chem. 1996; 271: 23338-23344Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) was cut with EcoRI, filled in with a Klenow reaction, and blunt end ligated into the SmaI site of pEGFP-C2.pEGFP-8LOThe coding region for murine 8-lipoxygenase was amplified by RT-PCR using primers and conditions based on the published sequence (36Jisaka M. Kim R.B. Boeglin W.E. Nanney L.B. Brash A.R. J. Biol. Chem. 1997; 272: 24410-24416Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) starting with phorbol ester-treated epidermal RNA template from a 6-day-old mouse (35Funk C.D. Keeney D.S. Oliw E.H. Boeglin W.E. Brash A.R. J. Biol. Chem. 1996; 271: 23338-23344Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The PCR products were first cloned in pCR2.1 (Invitrogen) and sequences verified. The EcoRI fragment with the correct sequence was first cloned into the expression vector pcDNA3 (Invitrogen) and then in pEGFP-C2. When transfected into HEK 293 cells 8-lipoxygenase activity was detected.pEGP-eLO-3The sequence for this novel lipoxygenase was cloned exactly as done for 8-lipoxygenase described above based on the published sequence (37Kinzig A. Heidt M. Furstenberger G. Marks F. Krieg P. Genomics. 1999; 58: 158-164Crossref PubMed Scopus (46) Google Scholar). No enzyme activity was detected with arachidonic acid substrate as described, presumably since this enzyme utilizes a different, as of yet undetermined, substrate.Cell Culture and TransfectionHEK 293 cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Cells growing in culture dishes, cover glass-bottomed culture dishes (for photography of living cells) or glass chamber slides were transfected with FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer's instructions. The transfected cells (15–20 h post-transfection) were used for translocation studies, fluorescence microscopy, and protein preparation for Western blot and lipoxygenase activity assays.Translocation Studies and Fluorescence MicroscopyThe HEK 293 cells on chamber slides, 15–20 h post-transfection, were washed twice with pre-warmed Dulbecco's modified Eagle's medium. For data documentation, the washed cells, 20–30 min post-incubation with 10 μmA23187 (Sigma) or 0.1% Me2SO (vehicle) in serum-free Dulbecco's modified Eagle's medium, were fixed with 2% paraformaldehyde in phosphate-buffered saline for 20 min. Slides were mounted with Gel/Mount and kept at 4 °C. Cell images were examined as described previously (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) for conventional fluorescence microscopy or using a Nikon E600 upright microscope equipped with Bio-Rad 1024 confocal imaging system. For time course analysis of translocation, the GFP fluorescence of living cells incubated in a 37 °C stage chamber, 15–20 h post-transfection, was recorded before and after addition of 10 μmA23187 using a Nikon TE300 inverted microscope equipped with Bio-Rad 1024 confocal system. Raw data photoimages were acquired by LazerSharp software (Bio-Rad) and processed further by Confocal Assistant and Adobe Photoshop programs.Western Blot and Activity AssaysCytosol proteins from transfected cells for Western blot and lipoxygenase activity assay were prepared as described previously (6Chen X.S. Naumann T.A. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. J. Biol. Chem. 1995; 270: 17993-17999Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). A mouse monoclonal antibody against GFP (Berkeley Antibody Co., 1:1000 dilution) was used for enhanced chemiluminescence detection. Activity assay of 12- and 15-lipoxygenases was carried out with 10,000 ×g supernatant proteins of transfected cells in phosphate-buffered saline. The supernatants were incubated with 100 μm arachidonic acid for 15 min at 37 °C and the reactions were terminated with 2 volumes of stop solution (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Activity assay for 5-lipoxygenase with addition of Ca2+ and ATP and reverse phase-high pressure liquid chromatography analysis of arachidonate metabolites (HETEs and HPETEs) by lipoxygenases were done as described previously (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) using an acetonitrile:H2O:acetic acid (65:35:0.1) mobile phase with additional assessment of leukotriene B4 production in transfected cells using UV detection at 270 or 280 nm. Authentic 5-H(P)ETE, LTB4, and 6-trans-12-epi-LTB4 standards (Cayman Chemical Co.) were chromatographed in parallel.DISCUSSIONWe have demonstrated using GFP-lipoxygenase fusion constructs transfected into HEK 293 cells and stimulated with calcium ionophoreA23187 the following conclusions: 1) 5-lipoxygenase is unique in its nuclear localization and ability to translocate to the nuclear envelope when compared with other human and murine lipoxygenase family members; 2) the β-barrel region of 5-lipoxygenase is necessary and sufficient for nuclear membrane translocation; 3) determinants at both ends of the β-barrel region are important for translocation; 4) catalytically active functional chimeras of 5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase type 1 can be created by correct association of the two domains at or close to the junction of the two domains; 5) 5-lipoxygenase nuclear targeting and translocation determinants are distinct; and 6) nuclear membrane translocation can occur from either the nuclear or cytoplasmic side.Lipoxygenases possess a two-domain structure (25Boyington J.C. Gaffney B.J. Amzel L.M. Science. 1993; 260: 1482-1486Crossref PubMed Scopus (453) Google Scholar, 26Minor W. Steczko J. Stec B. Otwinowski Z. Bolin J.T. Walter R. Axelrod B. Biochemistry. 1996; 35: 10687-10701Crossref PubMed Scopus (392) Google Scholar, 27Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nat. Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar, 28Skrzypczak-Jankun E. Amzel L.M. Kroa B.A. Funk Jr., M.O. Proteins. 1997; 29: 15-31Crossref PubMed Scopus (149) Google Scholar). The N-terminal β-barrel domain of rabbit reticulocyte 15-lipoxygenase revealed striking homology with the C-terminal domain of various lipases. These domains on the distinct proteins share approximately the same size (115–125 amino acids) and both need to access lipid substrates at a membrane surface. A detailed characterization of the role of the β-barrel in mammalian lipoxygenase function for substrate or membrane interaction has not been addressed previously. However, a recent report with a cucumber lipoxygenase indicated that the β-barrel was necessary for transport from cytosol to lipid bodies (29May C. Hohne M. Gnau P. Schwennesen K. Kindl H. Eur. J. Biochem. 2000; 267: 1100-1109Crossref PubMed Scopus (38) Google Scholar). Among the mammalian lipoxygenase members identified so far, only the 5-lipoxygenase has a definite binding requirement for calcium ions (18Hammarberg T. Radmark O. Biochemistry. 1999; 38: 4441-4447Crossref PubMed Scopus (67) Google Scholar). Calcium is essential for membrane association and has recently been shown to bind to the β-barrel of 5-lipoxygenase (40Hammarberg T. Provost P. Persson B. Radmark O. J. Biol. Chem. 2000; 275: 38787-38793Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). 3X-S. Chen and C. D. Funk, unpublished observations. Thus, in many respects this domain appears to mimic the C2 domains of protein kinase C and cytosolic phospholipase A2, both which bind calcium and translocate to membranes (41Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 42Perisic O. Paterson H.F. Mosedale G. Lara-Gonzalez S. Williams R.L. J. Biol. Chem. 1999; 274: 14979-14987Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). What directs the 5-lipoxygenase, and not other lipoxygenases, specifically to the nuclear membrane is not clear. Besides the clear calcium binding and dependence on this cation in vivo, the 5-lipoxygenase appears unique among other lipoxygenases in having an accessory protein (5-lipoxygenase activating protein) that is important for substrate presentation. Moreover, certain interactions with other cytoskeletal and signaling molecules may represent another mode of directing 5-lipoxygenase translocation specifically to the nuclear envelope (43Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar, 44Lepley R.A. Muskardin D.T. Fitzpatrick F.A. J. Biol. Chem. 1996; 271: 6179-6184Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 45Provost P. Samuelsson B. Radmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (106) Google Scholar). The 5-lipoxygenase contains an “insertion” sequence of 5 residues in the β-barrel that is not present in most mammalian 12- and 15-lipoxygenases (46Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (236) Google Scholar). An aspartic acid residue within this stretch, as well as other determinants at both ends of the domain, could be important for calcium binding, and proper protein folding for optimum interactions with membrane sites analogous to cytosolic phospholipase A2 (47Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).Although other lipoxygenases did not translocate to the nuclear envelope with A23187 stimulation in the transfected HEK 293 cells, this finding does not exclude translocation to other membrane sites for access of substrate. It is likely these other lipoxygenases interact in defined ways with membranes that is not evident in this system. Indeed, it has been shown that rat and human 12-lipoxygenases in platelets and tumor cells, as well as 15-lipoxygenase in reticulocytes and interleukin 4-treated monocytes translocate from cytosol fractions to undefined membrane sites (48Baba A. Sakuma S. Okamoto H. Inoue T. Iwata H. J. Biol. Chem. 1989; 264: 15790-15795Abstract Full Text PDF PubMed Google Scholar, 49Hagmann W. Gao X. Zacharek A. Wojciechowski L.A. Honn K.V. Prostaglandins. 1995; 49: 49-62Crossref PubMed Scopus (24) Google Scholar, 50Brinckmann R. Schnurr K. Heydeck D. Rosenbach T. Kolde G. Kuhn H. Blood. 1998; 91: 64-74Crossref PubMed Google Scholar).In this study and a previous one (8Chen X.-S. Zhang Y. Funk C.D. J. Biol. Chem. 1998; 273: 31237-31244Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), we were able to visualize 5-lipoxygenase cellular localization in living cells in real time o