Determinants of 5-Lipoxygenase Nuclear Localization Using Green Fluorescent Protein/5-Lipoxygenase Fusion Proteins
1998; Elsevier BV; Volume: 273; Issue: 47 Linguagem: Inglês
10.1074/jbc.273.47.31237
ISSN1083-351X
AutoresXinsheng Chen, Yingyi Zhang, Colin Funk,
Tópico(s)Synthesis and Biological Evaluation
Resumo5-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. 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. 5-hydroperoxyeicosatetraenoic acid 5-lipoxygenase green fluorescent protein green fluorescent protein-5-lipoxygenase fusion protein 5-hydroxyeicosatetraenoic acid 5-lipoxygenase deficient nuclear localization signal pyruvate kinase bone marrow-derived mast cells human embryonic kidney Chinese hamster ovary phosphate-buffered saline high performance liquid chromatography. 5-Lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC1.13.11.34) is a non-heme iron enzyme found primarily in white blood cells, macrophages, and mast cells that converts arachidonic acid first to 5-hydroperoxyeicosatetraenoic acid (5-HPETE)1 and then to leukotriene (LT)A4 (5,6-oxido-7,9,11,14-eicosatetraenoic acid)(1). Subsequent conversion of leukotriene A4 by leukotriene A4 hydrolase yields the potent neutrophil chemoattractant leukotriene B4. Alternatively, conjugation of LTA4 with glutathione by leukotriene C4synthase plus downstream metabolism leads to the cysteinyl leukotrienes that influence airway reactivity and mucus secretion especially in asthmatics (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2312) Google Scholar, 2Samuelsson B. Dahlén S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1970) Google Scholar, 3Lewis R.A. Austen K.F. Soberman R.J. New Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1168) Google Scholar).5-Lipoxygenase was isolated originally from the cytosol fraction of human and porcine neutrophils (4Rouzer C.A. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6040-6044Crossref PubMed Scopus (212) Google Scholar, 5Ueda N. Kaneko S. Yoshimoto T. Yamamoto S. J. Biol. Chem. 1986; 261: 7982-7988Abstract Full Text PDF PubMed Google Scholar). The enzyme was shown subsequently to undergo a calcium-dependent translocation to the nuclear envelope upon ionophore A23187 stimulation and was dependent on the 5-lipoxygenase-activating protein situated in this location for leukotriene biosynthesis (6Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar, 7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (649) Google Scholar). More recent work based on immunofluorescence techniques and cellular fractionation demonstrated that certain cell types capable of leukotriene formation (alveolar macrophages, rat basophilic leukemia cells, and mouse bone marrow-derived mast cells) express 5-lipoxygenase completely or partially in the nucleus (8Brock T.G. Paine R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar, 9Brock 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, 10Chen 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, 11Woods J.W. Coffey M.J. Brock T.G. Singer II, Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2046Crossref PubMed Scopus (158) Google Scholar). Additionally, it was shown that cytosolic 5-lipoxygenase in rat neutrophils could enter the nucleus if they were first elicited in vivo with various inflammatory agents or subjected to adherence to glass in vitro (12Brock 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).The discovery of 5-lipoxygenase in the nucleus was a surprising observation since it is well known that leukotrienes must exit the cell once synthesized to act on cell surface G protein-coupled receptors to exert their actions on neutrophils or bronchiole smooth muscle (13Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar,14Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). The possibility of 5-lipoxygenase itself or leukotrienes acting in the nucleus was raised. The recent observation that LTB4could bind to the nuclear peroxisomal proliferator-activated receptor-α indicated that intranuclear actions of leukotrienes are feasible (15Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1201) Google Scholar).Nothing is known about control of 5-lipoxygenase entry into the nucleus in some cell types but not others. Proteins enter the nucleus by nuclear localization signal (NLS) sequences that are recognized by specific importins, prior to nuclear pore docking, translocation through the pore and release from the pore's inner side (16Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). The NLS is typically a short basic region or bipartite basic sequence (18Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1854) Google Scholar,19Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1240) Google Scholar). Increasingly, however, novel NLS sequences are being recognized for import of particular classes of proteins; for example, the 38-amino acid M9 domain of heterogeneous nuclear ribonucleoprotein A1 (20Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (437) Google Scholar, 21Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Here, we demonstrate primarily with the use of green fluorescent protein (GFP)/5-lipoxygenase fusion proteins the complexity of events for 5-lipoxygenase nuclear entry.DISCUSSIONWe have carried out a study to investigate the nuclear targeting of 5-lipoxygenase. More than 25 fusion protein constructs were prepared and analyzed by immunofluorescence microscopy in various transfected cell lines. The combined data indicate that 5-lipoxygenase nuclear targeting is dependent on a number of complex factors. First, 5-lipoxygenase does not possess a "classical" basic region that functions as a NLS although the protein does contain at least three potential basic cluster regions that could act in this function. Second, the proper folding of the enzyme is critical for nuclear localization. This result is based on the fact that fusion proteins, which completely lack or partially retain the non-heme iron atom, show a predominantly cytosolic localization. Third, a short NH2-terminal region (first 80 amino acids) appears sufficient for directing the enzyme to the nucleus but when this segment is extended the recognition sequence(s) are lost. Fourth, any NLS sequence(s) in the NH2-terminal region of 5LO are relatively weak since only one of two proteins tested could be transported to the nucleus (e.g. GFP versuspyruvate kinase).The nuclear import of proteins bearing the classical NLS such as those for the SV40 large T antigen and nucleoplasmin begins by binding to karyopherin-α which acts as the NLS receptor (17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). Karyopherin-β1 interacts further with the NLS-bound karyopherin-α to form the ternary complex, which is targeted to the nucleoporins in the nuclear pore complex. Subsequent translocation into the nucleus through the nuclear pore complex depends on GTPase Ran and its modulators (16Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). Novel import pathways have been identified recently. For instance, heterogeneous nuclear ribonucleoprotein A1 interacts directly with karyopherin-β2/transportin, which is one of the members in the β-karyopherin superfamily, through the substrate's distinct NLS known as M9 or NLS2 to target to the nuclear pore complex (20Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (437) Google Scholar, 21Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Common features of the novel nuclear import pathways appear to be NLS sequences distinct from the classical pathway and direct interaction with an individual karyopherin-β form independent of interaction with the adapter karyopherin-α. Perhaps 5-lipoxygenase is using a novel means of nuclear entry in this respect. 5-Lipoxygenase does not contain an M9 domain and it is not yet known if it can interact with karyopherin-α.5-Lipoxygenase is the only known mammalian lipoxygenase that resides in the nucleus. The NH2 termini of lipoxygenases differ to the greatest extent in this region (33Funk C.D. Prog. Nucleic Acids Res. Mol. Biol. 1993; 45: 67-98Crossref PubMed Scopus (84) Google Scholar, 34Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (236) Google Scholar). Thus, this region appears to be the most important for 5LO nuclear targeting since there was one fragment (1–80) that could direct GFP to the nucleus. This was not a nonspecific result since both a COOH-terminal fragment from 5-lipoxygenase (5LO(574–673), see Fig. 6) of similar size and a fragment corresponding to the same residues of platelet 12-lipoxygenase (1–75; in the NH2-terminal sequence five residues are not present in this lipoxygenase and several other mammalian lipoxygenases) did not direct nuclear localization. Platelet 12-lipoxygenase is known to reside in the cytosol, either soluble or membrane-bound, of human erythroleukemia cells, A431 cells, and epidermal homogenates (23Chen X.S. Brash A.R. Funk C.D. Eur. J. Biochem. 1993; 214: 845-852Crossref PubMed Scopus (65) Google Scholar, 35Mahmud I. Suzuki T. Yamamoto Y. Suzuki H. Takahashi Y. Yoshimoto T. Yamamoto S. Biochim. Biophys. Acta. 1993; 1166: 211-216Crossref PubMed Scopus (17) Google Scholar,36Hagmann W. Gao X. Timar J. Chen Y.Q. Strohmaier A.R. Fahrenkopf C. Kagawa D. Lee M. Zacharek A. Honn K.V. Exp. Cell Res. 1996; 228: 197-205Crossref PubMed Scopus (29) Google Scholar). The basic cluster within the 5LO(1–80) sequence was not essential for nuclear localization.Based on the three-dimensional x-ray crystal structures of soybean lipoxygenases and rabbit reticulocyte 15-lipoxygenase (29Boyington J.C. Gaffney B.J. Amzel L.M. Science. 1993; 260: 1482-1486Crossref PubMed Scopus (453) Google Scholar, 30Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nature Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar, 37Funk C.D. Loll P.J. Nature Struct. Biol. 1997; 4: 966-968Crossref PubMed Scopus (16) Google Scholar), the lipoxygenase family members possess two domains; a short β-barrel NH2-terminal domain of unknown function and a major catalytic domain that includes the non-heme iron atom. Gillmor et al. (30Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nature Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar) have hypothesized that the β-barrel NH2-terminal region, with homology to lipoprotein lipase, may participate in binding lipid membranes to gain access to the source of substrate. In the case of 5-lipoxygenase, they suggested a possible site of interaction with 5-lipoxygenase-activating protein, a co-accessory protein in leukotriene biosynthesis that may help to "transfer" arachidonic acid substrate to the enzyme (7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (649) Google Scholar, 38Abramovitz M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Crossref PubMed Scopus (180) Google Scholar). The data here could extend the possible list of functions for this domain, in particular for 5-lipoxygenase, as aiding in nuclear localization. The putative β-barrel domain of 5-lipoxygenase is about 125 amino acid residues in length. A construct with this domain still directed nuclear localization of GFP. However, if it was extended to 166 amino acids the fusion protein remained cytosolic. This result suggests that the folding of the extra portion beyond the β-barrel may have masked any potential NLS. The context within which the NLS is situated is important for nuclear localization (39Roberts B.L. Richardson W.D. Smith A.E. Cell. 1987; 50: 465-475Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 40Nelson M. Silver P. Mol. Cell. Biol. 1989; 9: 384-389Crossref PubMed Scopus (84) Google Scholar).Precedents for weak NLS sequences in proteins are prevalent in the literature and 5-lipoxygenase seems to fit in this class of proteins. For example, a 29-amino acid stretch of GAL4 could direct cytosolic invertase to the nucleus but not β-galactosidase (40Nelson M. Silver P. Mol. Cell. Biol. 1989; 9: 384-389Crossref PubMed Scopus (84) Google Scholar). Likewise, a NLS motif near the NH2 terminus of fibroblast growth factor 3 conferred nuclear localization to cytoplasmic β-galactosidase but not pyruvate kinase (41Antoine M. Reimers K. Dickson C. Kiefer P. J. Biol. Chem. 1997; 272: 29475-29481Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Signals in 5-lipoxygenase were capable of directing the GFP reporter protein to the nucleus but not pyruvate kinase. Complex patterns of nuclear targeting that may involve weak additive signals from opposite ends of the protein are known (e.g. fibroblast growth factor 3) (41Antoine M. Reimers K. Dickson C. Kiefer P. J. Biol. Chem. 1997; 272: 29475-29481Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Examples of NLS "masking" by structural alterations and/or other cellular proteins, a prime example being the transcription factor NF-κB/Rel bound by its inhibitor IκB are known (42Latimer M. Ernst M.K. Dunn L.L. Drutskaya M. Rice N.R. Mol. Cell. Biol. 1998; 18: 2640-2649Crossref PubMed Google Scholar). The data herein could be consistent with some sort of unmasking of an NLS to gain nuclear entry and also do not rule out the possibility that 5-lipoxygenase is "piggybacked" to the nucleus by some other chaperone protein. A somewhat surprising result was that 5-lipoxygenase could be localized to the nucleus in four different transfected cell types. Perhaps, in human and rat neutrophils, 5-lipoxygenase is specifically bound by an inhibitor protein that prevents nuclear transport since the enzyme in these resting cells is exclusively cytosolic. Upon in vivoactivation or adherence to glass in vitro (12Brock 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), therefore, these cells would lose the capacity to behind the inhibitor and enter the nucleus. Alternatively, some sort of post-translational modification such as phosphorylation of 5-lipoxygenase, particularly in neutrophils, may influence NLS recognition. Phosphorylation of a nuclear 5-lipoxygenase fraction has been detected in HL-60 cells but the site of phosphorylation has not been identified (43Lepley 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). We are currently studying potential interactions of 5-lipoxygenase with other proteins using the yeast two-hybrid system with relevance to the nuclear targeting paradigm.The use of 5-lipoxygenase inhibitors and leukotriene receptor antagonists has made its widespread debut in the clinical arena for asthma treatment in the last few years (44Tan R.A. Curr. Opin. Pulm. Med. 1998; 4: 25-30Crossref PubMed Scopus (13) Google Scholar). The recent discoveries of 5-lipoxygenase in the nucleus, that LTB4 can bind and activate a nuclear transcription factor (peroxisomal proliferator-activated receptor-α), and the potential for other nuclear functions make it essential to understand the mechanisms for nuclear localization of 5-lipoxygenase. This study has raised new questions in beginning to answer these issues. 5-Lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC1.13.11.34) is a non-heme iron enzyme found primarily in white blood cells, macrophages, and mast cells that converts arachidonic acid first to 5-hydroperoxyeicosatetraenoic acid (5-HPETE)1 and then to leukotriene (LT)A4 (5,6-oxido-7,9,11,14-eicosatetraenoic acid)(1). Subsequent conversion of leukotriene A4 by leukotriene A4 hydrolase yields the potent neutrophil chemoattractant leukotriene B4. Alternatively, conjugation of LTA4 with glutathione by leukotriene C4synthase plus downstream metabolism leads to the cysteinyl leukotrienes that influence airway reactivity and mucus secretion especially in asthmatics (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2312) Google Scholar, 2Samuelsson B. Dahlén S.E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (1970) Google Scholar, 3Lewis R.A. Austen K.F. Soberman R.J. New Engl. J. Med. 1990; 323: 645-655Crossref PubMed Scopus (1168) Google Scholar). 5-Lipoxygenase was isolated originally from the cytosol fraction of human and porcine neutrophils (4Rouzer C.A. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6040-6044Crossref PubMed Scopus (212) Google Scholar, 5Ueda N. Kaneko S. Yoshimoto T. Yamamoto S. J. Biol. Chem. 1986; 261: 7982-7988Abstract Full Text PDF PubMed Google Scholar). The enzyme was shown subsequently to undergo a calcium-dependent translocation to the nuclear envelope upon ionophore A23187 stimulation and was dependent on the 5-lipoxygenase-activating protein situated in this location for leukotriene biosynthesis (6Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Abstract Full Text PDF PubMed Google Scholar, 7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (649) Google Scholar). More recent work based on immunofluorescence techniques and cellular fractionation demonstrated that certain cell types capable of leukotriene formation (alveolar macrophages, rat basophilic leukemia cells, and mouse bone marrow-derived mast cells) express 5-lipoxygenase completely or partially in the nucleus (8Brock T.G. Paine R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Abstract Full Text PDF PubMed Google Scholar, 9Brock 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, 10Chen 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, 11Woods J.W. Coffey M.J. Brock T.G. Singer II, Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2046Crossref PubMed Scopus (158) Google Scholar). Additionally, it was shown that cytosolic 5-lipoxygenase in rat neutrophils could enter the nucleus if they were first elicited in vivo with various inflammatory agents or subjected to adherence to glass in vitro (12Brock 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). The discovery of 5-lipoxygenase in the nucleus was a surprising observation since it is well known that leukotrienes must exit the cell once synthesized to act on cell surface G protein-coupled receptors to exert their actions on neutrophils or bronchiole smooth muscle (13Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Abstract Full Text PDF PubMed Google Scholar,14Yokomizo T. Izumi T. Chang K. Takuwa Y. Shimizu T. Nature. 1997; 387: 620-624Crossref PubMed Scopus (847) Google Scholar). The possibility of 5-lipoxygenase itself or leukotrienes acting in the nucleus was raised. The recent observation that LTB4could bind to the nuclear peroxisomal proliferator-activated receptor-α indicated that intranuclear actions of leukotrienes are feasible (15Devchand P.R. Keller H. Peters J.M. Vazquez M. Gonzalez F.J. Wahli W. Nature. 1996; 384: 39-43Crossref PubMed Scopus (1201) Google Scholar). Nothing is known about control of 5-lipoxygenase entry into the nucleus in some cell types but not others. Proteins enter the nucleus by nuclear localization signal (NLS) sequences that are recognized by specific importins, prior to nuclear pore docking, translocation through the pore and release from the pore's inner side (16Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). The NLS is typically a short basic region or bipartite basic sequence (18Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1854) Google Scholar,19Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1240) Google Scholar). Increasingly, however, novel NLS sequences are being recognized for import of particular classes of proteins; for example, the 38-amino acid M9 domain of heterogeneous nuclear ribonucleoprotein A1 (20Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (437) Google Scholar, 21Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Here, we demonstrate primarily with the use of green fluorescent protein (GFP)/5-lipoxygenase fusion proteins the complexity of events for 5-lipoxygenase nuclear entry. DISCUSSIONWe have carried out a study to investigate the nuclear targeting of 5-lipoxygenase. More than 25 fusion protein constructs were prepared and analyzed by immunofluorescence microscopy in various transfected cell lines. The combined data indicate that 5-lipoxygenase nuclear targeting is dependent on a number of complex factors. First, 5-lipoxygenase does not possess a "classical" basic region that functions as a NLS although the protein does contain at least three potential basic cluster regions that could act in this function. Second, the proper folding of the enzyme is critical for nuclear localization. This result is based on the fact that fusion proteins, which completely lack or partially retain the non-heme iron atom, show a predominantly cytosolic localization. Third, a short NH2-terminal region (first 80 amino acids) appears sufficient for directing the enzyme to the nucleus but when this segment is extended the recognition sequence(s) are lost. Fourth, any NLS sequence(s) in the NH2-terminal region of 5LO are relatively weak since only one of two proteins tested could be transported to the nucleus (e.g. GFP versuspyruvate kinase).The nuclear import of proteins bearing the classical NLS such as those for the SV40 large T antigen and nucleoplasmin begins by binding to karyopherin-α which acts as the NLS receptor (17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). Karyopherin-β1 interacts further with the NLS-bound karyopherin-α to form the ternary complex, which is targeted to the nucleoporins in the nuclear pore complex. Subsequent translocation into the nucleus through the nuclear pore complex depends on GTPase Ran and its modulators (16Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). Novel import pathways have been identified recently. For instance, heterogeneous nuclear ribonucleoprotein A1 interacts directly with karyopherin-β2/transportin, which is one of the members in the β-karyopherin superfamily, through the substrate's distinct NLS known as M9 or NLS2 to target to the nuclear pore complex (20Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (437) Google Scholar, 21Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Common features of the novel nuclear import pathways appear to be NLS sequences distinct from the classical pathway and direct interaction with an individual karyopherin-β form independent of interaction with the adapter karyopherin-α. Perhaps 5-lipoxygenase is using a novel means of nuclear entry in this respect. 5-Lipoxygenase does not contain an M9 domain and it is not yet known if it can interact with karyopherin-α.5-Lipoxygenase is the only known mammalian lipoxygenase that resides in the nucleus. The NH2 termini of lipoxygenases differ to the greatest extent in this region (33Funk C.D. Prog. Nucleic Acids Res. Mol. Biol. 1993; 45: 67-98Crossref PubMed Scopus (84) Google Scholar, 34Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (236) Google Scholar). Thus, this region appears to be the most important for 5LO nuclear targeting since there was one fragment (1–80) that could direct GFP to the nucleus. This was not a nonspecific result since both a COOH-terminal fragment from 5-lipoxygenase (5LO(574–673), see Fig. 6) of similar size and a fragment corresponding to the same residues of platelet 12-lipoxygenase (1–75; in the NH2-terminal sequence five residues are not present in this lipoxygenase and several other mammalian lipoxygenases) did not direct nuclear localization. Platelet 12-lipoxygenase is known to reside in the cytosol, either soluble or membrane-bound, of human erythroleukemia cells, A431 cells, and epidermal homogenates (23Chen X.S. Brash A.R. Funk C.D. Eur. J. Biochem. 1993; 214: 845-852Crossref PubMed Scopus (65) Google Scholar, 35Mahmud I. Suzuki T. Yamamoto Y. Suzuki H. Takahashi Y. Yoshimoto T. Yamamoto S. Biochim. Biophys. Acta. 1993; 1166: 211-216Crossref PubMed Scopus (17) Google Scholar,36Hagmann W. Gao X. Timar J. Chen Y.Q. Strohmaier A.R. Fahrenkopf C. Kagawa D. Lee M. Zacharek A. Honn K.V. Exp. Cell Res. 1996; 228: 197-205Crossref PubMed Scopus (29) Google Scholar). The basic cluster within the 5LO(1–80) sequence was not essential for nuclear localization.Based on the three-dimensional x-ray crystal structures of soybean lipoxygenases and rabbit reticulocyte 15-lipoxygenase (29Boyington J.C. Gaffney B.J. Amzel L.M. Science. 1993; 260: 1482-1486Crossref PubMed Scopus (453) Google Scholar, 30Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nature Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar, 37Funk C.D. Loll P.J. Nature Struct. Biol. 1997; 4: 966-968Crossref PubMed Scopus (16) Google Scholar), the lipoxygenase family members possess two domains; a short β-barrel NH2-terminal domain of unknown function and a major catalytic domain that includes the non-heme iron atom. Gillmor et al. (30Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nature Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar) have hypothesized that the β-barrel NH2-terminal region, with homology to lipoprotein lipase, may participate in binding lipid membranes to gain access to the source of substrate. In the case of 5-lipoxygenase, they suggested a possible site of interaction with 5-lipoxygenase-activating protein, a co-accessory protein in leukotriene biosynthesis that may help to "transfer" arachidonic acid substrate to the enzyme (7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (649) Google Scholar, 38Abramovitz M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Crossref PubMed Scopus (180) Google Scholar). The data here could extend the possible list of functions for this domain, in particular for 5-lipoxygenase, as aiding in nuclear localization. The putative β-barrel domain of 5-lipoxygenase is about 125 amino acid residues in length. A construct with this domain still directed nuclear localization of GFP. However, if it was extended to 166 amino acids the fusion protein remained cytosolic. This result suggests that the folding of the extra portion beyond the β-barrel may have masked any potential NLS. The context within which the NLS is situated is important for nuclear localization (39Roberts B.L. Richardson W.D. Smith A.E. Cell. 1987; 50: 465-475Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 40Nelson M. Silver P. Mol. Cell. Biol. 1989; 9: 384-389Crossref PubMed Scopus (84) Google Scholar).Precedents for weak NLS sequences in proteins are prevalent in the literature and 5-lipoxygenase seems to fit in this class of proteins. For example, a 29-amino acid stretch of GAL4 could direct cytosolic invertase to the nucleus but not β-galactosidase (40Nelson M. Silver P. Mol. Cell. Biol. 1989; 9: 384-389Crossref PubMed Scopus (84) Google Scholar). Likewise, a NLS motif near the NH2 terminus of fibroblast growth factor 3 conferred nuclear localization to cytoplasmic β-galactosidase but not pyruvate kinase (41Antoine M. Reimers K. Dickson C. Kiefer P. J. Biol. Chem. 1997; 272: 29475-29481Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Signals in 5-lipoxygenase were capable of directing the GFP reporter protein to the nucleus but not pyruvate kinase. Complex patterns of nuclear targeting that may involve weak additive signals from opposite ends of the protein are known (e.g. fibroblast growth factor 3) (41Antoine M. Reimers K. Dickson C. Kiefer P. J. Biol. Chem. 1997; 272: 29475-29481Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Examples of NLS "masking" by structural alterations and/or other cellular proteins, a prime example being the transcription factor NF-κB/Rel bound by its inhibitor IκB are known (42Latimer M. Ernst M.K. Dunn L.L. Drutskaya M. Rice N.R. Mol. Cell. Biol. 1998; 18: 2640-2649Crossref PubMed Google Scholar). The data herein could be consistent with some sort of unmasking of an NLS to gain nuclear entry and also do not rule out the possibility that 5-lipoxygenase is "piggybacked" to the nucleus by some other chaperone protein. A somewhat surprising result was that 5-lipoxygenase could be localized to the nucleus in four different transfected cell types. Perhaps, in human and rat neutrophils, 5-lipoxygenase is specifically bound by an inhibitor protein that prevents nuclear transport since the enzyme in these resting cells is exclusively cytosolic. Upon in vivoactivation or adherence to glass in vitro (12Brock 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), therefore, these cells would lose the capacity to behind the inhibitor and enter the nucleus. Alternatively, some sort of post-translational modification such as phosphorylation of 5-lipoxygenase, particularly in neutrophils, may influence NLS recognition. Phosphorylation of a nuclear 5-lipoxygenase fraction has been detected in HL-60 cells but the site of phosphorylation has not been identified (43Lepley 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). We are currently studying potential interactions of 5-lipoxygenase with other proteins using the yeast two-hybrid system with relevance to the nuclear targeting paradigm.The use of 5-lipoxygenase inhibitors and leukotriene receptor antagonists has made its widespread debut in the clinical arena for asthma treatment in the last few years (44Tan R.A. Curr. Opin. Pulm. Med. 1998; 4: 25-30Crossref PubMed Scopus (13) Google Scholar). The recent discoveries of 5-lipoxygenase in the nucleus, that LTB4 can bind and activate a nuclear transcription factor (peroxisomal proliferator-activated receptor-α), and the potential for other nuclear functions make it essential to understand the mechanisms for nuclear localization of 5-lipoxygenase. This study has raised new questions in beginning to answer these issues. We have carried out a study to investigate the nuclear targeting of 5-lipoxygenase. More than 25 fusion protein constructs were prepared and analyzed by immunofluorescence microscopy in various transfected cell lines. The combined data indicate that 5-lipoxygenase nuclear targeting is dependent on a number of complex factors. First, 5-lipoxygenase does not possess a "classical" basic region that functions as a NLS although the protein does contain at least three potential basic cluster regions that could act in this function. Second, the proper folding of the enzyme is critical for nuclear localization. This result is based on the fact that fusion proteins, which completely lack or partially retain the non-heme iron atom, show a predominantly cytosolic localization. Third, a short NH2-terminal region (first 80 amino acids) appears sufficient for directing the enzyme to the nucleus but when this segment is extended the recognition sequence(s) are lost. Fourth, any NLS sequence(s) in the NH2-terminal region of 5LO are relatively weak since only one of two proteins tested could be transported to the nucleus (e.g. GFP versuspyruvate kinase). The nuclear import of proteins bearing the classical NLS such as those for the SV40 large T antigen and nucleoplasmin begins by binding to karyopherin-α which acts as the NLS receptor (17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). Karyopherin-β1 interacts further with the NLS-bound karyopherin-α to form the ternary complex, which is targeted to the nucleoporins in the nuclear pore complex. Subsequent translocation into the nucleus through the nuclear pore complex depends on GTPase Ran and its modulators (16Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 17Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (211) Google Scholar). Novel import pathways have been identified recently. For instance, heterogeneous nuclear ribonucleoprotein A1 interacts directly with karyopherin-β2/transportin, which is one of the members in the β-karyopherin superfamily, through the substrate's distinct NLS known as M9 or NLS2 to target to the nuclear pore complex (20Siomi H. Dreyfuss G. J. Cell Biol. 1995; 129: 551-560Crossref PubMed Scopus (437) Google Scholar, 21Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Common features of the novel nuclear import pathways appear to be NLS sequences distinct from the classical pathway and direct interaction with an individual karyopherin-β form independent of interaction with the adapter karyopherin-α. Perhaps 5-lipoxygenase is using a novel means of nuclear entry in this respect. 5-Lipoxygenase does not contain an M9 domain and it is not yet known if it can interact with karyopherin-α. 5-Lipoxygenase is the only known mammalian lipoxygenase that resides in the nucleus. The NH2 termini of lipoxygenases differ to the greatest extent in this region (33Funk C.D. Prog. Nucleic Acids Res. Mol. Biol. 1993; 45: 67-98Crossref PubMed Scopus (84) Google Scholar, 34Funk C.D. Biochim. Biophys. Acta. 1996; 1304: 65-84Crossref PubMed Scopus (236) Google Scholar). Thus, this region appears to be the most important for 5LO nuclear targeting since there was one fragment (1–80) that could direct GFP to the nucleus. This was not a nonspecific result since both a COOH-terminal fragment from 5-lipoxygenase (5LO(574–673), see Fig. 6) of similar size and a fragment corresponding to the same residues of platelet 12-lipoxygenase (1–75; in the NH2-terminal sequence five residues are not present in this lipoxygenase and several other mammalian lipoxygenases) did not direct nuclear localization. Platelet 12-lipoxygenase is known to reside in the cytosol, either soluble or membrane-bound, of human erythroleukemia cells, A431 cells, and epidermal homogenates (23Chen X.S. Brash A.R. Funk C.D. Eur. J. Biochem. 1993; 214: 845-852Crossref PubMed Scopus (65) Google Scholar, 35Mahmud I. Suzuki T. Yamamoto Y. Suzuki H. Takahashi Y. Yoshimoto T. Yamamoto S. Biochim. Biophys. Acta. 1993; 1166: 211-216Crossref PubMed Scopus (17) Google Scholar,36Hagmann W. Gao X. Timar J. Chen Y.Q. Strohmaier A.R. Fahrenkopf C. Kagawa D. Lee M. Zacharek A. Honn K.V. Exp. Cell Res. 1996; 228: 197-205Crossref PubMed Scopus (29) Google Scholar). The basic cluster within the 5LO(1–80) sequence was not essential for nuclear localization. Based on the three-dimensional x-ray crystal structures of soybean lipoxygenases and rabbit reticulocyte 15-lipoxygenase (29Boyington J.C. Gaffney B.J. Amzel L.M. Science. 1993; 260: 1482-1486Crossref PubMed Scopus (453) Google Scholar, 30Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nature Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar, 37Funk C.D. Loll P.J. Nature Struct. Biol. 1997; 4: 966-968Crossref PubMed Scopus (16) Google Scholar), the lipoxygenase family members possess two domains; a short β-barrel NH2-terminal domain of unknown function and a major catalytic domain that includes the non-heme iron atom. Gillmor et al. (30Gillmor S.A. Villasenor A. Fletterick R. Sigal E. Browner M.F. Nature Struct. Biol. 1997; 4: 1003-1009Crossref PubMed Scopus (390) Google Scholar) have hypothesized that the β-barrel NH2-terminal region, with homology to lipoprotein lipase, may participate in binding lipid membranes to gain access to the source of substrate. In the case of 5-lipoxygenase, they suggested a possible site of interaction with 5-lipoxygenase-activating protein, a co-accessory protein in leukotriene biosynthesis that may help to "transfer" arachidonic acid substrate to the enzyme (7Dixon R.A. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Crossref PubMed Scopus (649) Google Scholar, 38Abramovitz M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Crossref PubMed Scopus (180) Google Scholar). The data here could extend the possible list of functions for this domain, in particular for 5-lipoxygenase, as aiding in nuclear localization. The putative β-barrel domain of 5-lipoxygenase is about 125 amino acid residues in length. A construct with this domain still directed nuclear localization of GFP. However, if it was extended to 166 amino acids the fusion protein remained cytosolic. This result suggests that the folding of the extra portion beyond the β-barrel may have masked any potential NLS. The context within which the NLS is situated is important for nuclear localization (39Roberts B.L. Richardson W.D. Smith A.E. Cell. 1987; 50: 465-475Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 40Nelson M. Silver P. Mol. Cell. Biol. 1989; 9: 384-389Crossref PubMed Scopus (84) Google Scholar). Precedents for weak NLS sequences in proteins are prevalent in the literature and 5-lipoxygenase seems to fit in this class of proteins. For example, a 29-amino acid stretch of GAL4 could direct cytosolic invertase to the nucleus but not β-galactosidase (40Nelson M. Silver P. Mol. Cell. Biol. 1989; 9: 384-389Crossref PubMed Scopus (84) Google Scholar). Likewise, a NLS motif near the NH2 terminus of fibroblast growth factor 3 conferred nuclear localization to cytoplasmic β-galactosidase but not pyruvate kinase (41Antoine M. Reimers K. Dickson C. Kiefer P. J. Biol. Chem. 1997; 272: 29475-29481Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Signals in 5-lipoxygenase were capable of directing the GFP reporter protein to the nucleus but not pyruvate kinase. Complex patterns of nuclear targeting that may involve weak additive signals from opposite ends of the protein are known (e.g. fibroblast growth factor 3) (41Antoine M. Reimers K. Dickson C. Kiefer P. J. Biol. Chem. 1997; 272: 29475-29481Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Examples of NLS "masking" by structural alterations and/or other cellular proteins, a prime example being the transcription factor NF-κB/Rel bound by its inhibitor IκB are known (42Latimer M. Ernst M.K. Dunn L.L. Drutskaya M. Rice N.R. Mol. Cell. Biol. 1998; 18: 2640-2649Crossref PubMed Google Scholar). The data herein could be consistent with some sort of unmasking of an NLS to gain nuclear entry and also do not rule out the possibility that 5-lipoxygenase is "piggybacked" to the nucleus by some other chaperone protein. A somewhat surprising result was that 5-lipoxygenase could be localized to the nucleus in four different transfected cell types. Perhaps, in human and rat neutrophils, 5-lipoxygenase is specifically bound by an inhibitor protein that prevents nuclear transport since the enzyme in these resting cells is exclusively cytosolic. Upon in vivoactivation or adherence to glass in vitro (12Brock 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), therefore, these cells would lose the capacity to behind the inhibitor and enter the nucleus. Alternatively, some sort of post-translational modification such as phosphorylation of 5-lipoxygenase, particularly in neutrophils, may influence NLS recognition. Phosphorylation of a nuclear 5-lipoxygenase fraction has been detected in HL-60 cells but the site of phosphorylation has not been identified (43Lepley 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). We are currently studying potential interactions of 5-lipoxygenase with other proteins using the yeast two-hybrid system with relevance to the nuclear targeting paradigm. The use of 5-lipoxygenase inhibitors and leukotriene receptor antagonists has made its widespread debut in the clinical arena for asthma treatment in the last few years (44Tan R.A. Curr. Opin. Pulm. Med. 1998; 4: 25-30Crossref PubMed Scopus (13) Google Scholar). The recent discoveries of 5-lipoxygenase in the nucleus, that LTB4 can bind and activate a nuclear transcription factor (peroxisomal proliferator-activated receptor-α), and the potential for other nuclear functions make it essential to understand the mechanisms for nuclear localization of 5-lipoxygenase. This study has raised new questions in beginning to answer these issues. We thank Dr. M. Malim for helpful discussions and Dr. H. Siomi for important suggestions and for providing pcDNA3/myc-PK vector and anti-Myc antibody.
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