Identification of Two Novel Nuclear Import Sequences on the 5-Lipoxygenase Protein*

Sandra M. Jones, Ming Luo, Marc Peters-Golden, and Thomas G. BrockDagger

From the Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Health System, Ann Arbor, Michigan 48109-0642

Received for publication, October 28, 2002, and in revised form, January 9, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The nuclear import of 5-lipoxygenase modulates its capacity to produce leukotrienes from arachidonic acid. However, the molecular determinants of its nuclear import are unknown. Recently, we used structural and functional criteria to identify a novel import sequence at Arg518 on human 5-lipoxygenase (Jones, S. M., Luo, M., Healy, A. M., Peters-Golden, M., and Brock, T. G. (2002) J. Biol. Chem. 277, 38550-38556). However, this analysis also indicated that other import sequences must exist. Here, we identify two additional sites, at Arg112 and Lys158, as nuclear import sequences. Both sites were found to be common to 5-lipoxygenases from different species but not found on other lipoxygenases. Both sites also appeared to be a part of structures that were predominantly random loops. Peptide sequences at these sites were sufficient to direct nuclear import of green fluorescent protein. Mutation of basic residues in these sites impaired nuclear import and combinations of mutations at different sites were additive in effect. Mutations in all three sites were required to disable nuclear accumulation of 5-lipoxygenase in all cells. Significantly, mutation in these sites did not inhibit catalytic function. Taken together, these results indicate that nuclear import of 5-lipoxygenase may reflect the combined functional effects of three discrete import sequences. Mutation of individual sites can, by itself, impair nuclear import, which in turn could impact arachidonic acid metabolism.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Leukotrienes (LTs)1 are lipid mediators derived from arachidonic acid. They are synthesized primarily by leukocytes and orchestrate a variety of physiological responses in both host defense and inflammatory disease states (reviewed in Ref. 1). The enzyme 5-lipoxygenase (5-LO) catalyzes the rate-limiting first two steps of LT synthesis. Therefore, the regulation of 5-LO action and how it might be modulated in disease have been a focus of interest.

The cellular locale of 5-LO differs in different cell types. 5-LO is localized in the cytoplasm of peripheral blood neutrophils (2, 3), eosinophils (4, 5), and peritoneal macrophages (6). However, it is found predominantly in the nucleoplasm of rat basophilic leukemia cells (7), alveolar macrophages (8), mouse bone marrow-derived mast cells (9) and monocyte-derived dendritic cells (10). Subsequent observations have further indicated that nuclear import of 5-LO is a regulated process. Nuclear import can be triggered by adherence (2, 4, 11), by recruitment (2, 3), or by cytokines (5, 12). Conditions that cause nuclear import of 5-LO can enhance (2, 5, 12) or suppress (4) LT production. Thus, the nuclear import of the 5-LO enzyme is linked to its ability to synthesize LTs.

The molecular components that regulate nuclear import of 5-LO remain to be fully elucidated. Commonly, a nuclear import sequence (NIS), rich in the basic amino acids Arg and Lys, mediates nuclear import of proteins, and three such basic regions (BR) have been identified (13). Truncation of 5-LO suggested the presence of an NIS in the amino-terminal region of 5-LO (13). However, limited mutagenesis of a BR at Lys72 did not prevent nuclear import, suggesting that a non-conventional NIS may exist in the amino terminus of 5-LO. Another site, at Arg651, resembles a bipartite NIS. Mutational analysis of this region demonstrated that most basic residues could be replaced without affecting nuclear import (13-15), unless Arg651 was replaced (14, 15). However, replacement of Arg651 also caused loss of catalytic activity (15), suggesting that mutagenesis caused protein misfolding, which can also impair import (13). Consistent with this, analysis of 5-LO secondary structure indicated that Arg651 serves a critical structural role, through its association with Asp473 (16).

Recently, we developed novel structural and functional criteria to identify functional NIS on 5-LO (16). First, we sought basic residues that were common to 5-LO from different species but not shared by other LO, since nuclear import has been observed in 5-LO from all species but not in 15-LO and 12-LO. Second, we sought BRs having a predominantly random coil/loop secondary structure, which appears to be necessary for binding to importin-alpha proteins (17-19). Finally, mutations that altered nuclear import should not also inactivate the enzyme, since failed import may result from mutation-induced changes in protein structure (13); loss of activity, then, would be used as an indirect indication of such a false positive result. Application of these rigorous criteria to 5-LO revealed a novel site at Arg518, designated as BR518 (16). This BR alone was sufficient to drive nuclear import, and replacement of basic residues impaired import without inactivating the enzyme, indicating that BR518 is a functional NIS. Interestingly, however, mutations in BR518 could not totally abolish nuclear import in all cells, suggesting that additional NIS(s) must exist on 5-LO.

This study applies the same structural and functional criteria to search for the unidentified NIS(s) on 5-LO. Our results support the conclusion that BR68, the only basic region in the beta -barrel region of 5-LO, is unlikely to be a functional NIS. However, a novel site at Arg112, which links the beta -barrel region to the catalytic domain, meets these criteria and appears to act as an NIS. However, mutation of basic residues in both BR518 and BR112 did not eliminate nuclear import of 5-LO. A third site at Lys158, which also meets structural and functional criteria, was found to be, by itself, sufficient for nuclear import. Mutation of all three sites eliminated nuclear accumulation of 5-LO without loss of function, indicating that 5-LO contains three functional NISs.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence and Structural Analysis-- Amino acid sequences were obtained from Swiss-Prot from the ExPaSy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics. Primary accession numbers for proteins are: 5-LOs, human P09917, mouse P48999, rat P12527, hamster P51399; 15-LOs, rabbit P12630, and human P16050; human platelet-type 12-LO P18054; Clostridium perfringens alpha -toxin P15310. Alignment of protein sequences was performed using ClustalW (20). Structural analysis utilized the resolved structures of rabbit 15-LO (PDB: 1lox) and C. perfringens alpha -toxin (PDB: 1qmd), as well as published theoretical models of the 5-LO beta -barrel region (21) and the 5-LO catalytic region (22).

Plasmids and Mutagenesis-- To construct a fusion peptide joining BR112 or BR158 to green fluorescent protein (GFP), complementary oligonucleotides encoding the basic regions (indicated below) were annealed and ligated to the BamHI and HindIII sites of pEGFP-C1. BR112 peptide was Leu111-Asp121 (LRDGRAKLARD); BR158 peptide was Asp156-Asp166 (DAKCHKDLPRD).

Specific amino acids within the putative 5-LO NISs were substituted in the pEGFP-C1/5-LO template (14) using the QuikChange site-directed mutagenesis kit (Stratagene). Briefly, two complementary primers (125 ng each) containing the desired mutation and 20 ng of template in 1× reaction buffer were denatured at 95 °C for 30 s and annealed at 55 °C for 30s, and DNA synthesis was carried out by Pfu polymerase at 68 °C for 14 min. This cycle was repeated 12-18 times, depending on the number of bases substituted, according to the manufacturer's directions. The methylated template was removed by incubation with 10 units of DpnI at 37 °C for 1 h. The mutation BR518 was R518Q/R520Q/K521Q/K527Q/K530Q; mutation BR112 was R115Q/K117Q/R120Q; mutation BR158 was K158N/H160Q/K161N. All substitutions and constructs were verified by DNA sequence analysis (DNA Sequencing Core, University of Michigan). Oligonucleotides (sequences available upon request) were synthesized and PAGE-purified by Integrated DNA Technologies Inc. (Coralville, IA).

Cell Culture, Transfection, and Imaging-- NIH 3T3 cells were obtained from American Type Culture Collection (Manassas, VA) and grown under 5% CO2 in Dulbecco's Modified Eagle's Medium (Invitrogen) supplemented with 10% calf serum, 100 units/ml penicillin, and 100 units/ml streptomycin. Cells were transfected using Polyfect (Qiagen, Inc.) transfection reagents according to the manufacturer's specifications. Transient transfectants were evaluated microscopically, live, or after fixation with 4% paraformaldehyde, 16-20 h after transfection. Comparable results were obtained when cells were examined as early as 9 h after transfection.

Immunoblotting-- As described previously (14), cells were disrupted by sonication on ice, and protein concentrations were determined by a modified Coomassie Blue dye binding assay (Pierce). Samples containing 10 µg of protein were separated by SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to nitrocellulose. Membranes were probed with a rabbit polyclonal antibody raised against purified human leukocyte 5-LO (a generous gift from Dr. J. Evans, Merck Research Laboratories, Rahway, NJ) (23) or with rabbit polyclonal anti-GFP (Santa Cruz Biotechnology, Inc.; titer 1:500) followed by peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection (Amersham Biosciences).

Cell Stimulation and Analysis of Leukotriene Synthesis-- To stimulate 5-LO activity, cells transfected with various 5-LO constructs were washed, then incubated for 30 min at 37 °C in serum-free medium containing 10 µM calcium ionophore A23187 and 10 µM arachidonic acid. Immunoreactive LTB4 in conditioned media was quantitated by enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) according to the supplier's instructions. For each sample, the measured value was taken as the average of duplicate determinations. Media from non-transfected, mock-transfected, or GFP-transfected cells did not contain detectable LTB4. LTB4 determinations were standardized for transfection efficiency: cells were washed following stimulation, harvested by scraping, sonicated on ice, assayed by immunoblot analysis (using 10 µg of protein/sample) for expression using anti-GFP, with expression quantitated by densitometry. LTB4 synthesis, adjusted for construct expression, was evaluated for all constructs in at least two independent experiments. The detection limit for LTB4 was 4 pg/ml; cross-reactivity for AA, 5-HETE, LTC4, LTD4 and LTE4 was <0.01%.

Alternatively, activity of constructs was evaluated by cell-free assay: 5 × 106 COS-1 cells were transfected with 5 µg of plasmid DNA using Polyfect; 40-h post-transfection, cells were harvested, washed with phosphate-buffered saline once, and sonicated for 90 s in 10-s bursts on ice in cell lysis buffer containing 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 1 mM dithiothreitol, and protease inhibitor mixture (CompleteTM EDTA-free, Roche Molecular Biochemicals). After sonication, lysates were centrifuged (5000 rpm, 8 min, 4 °C) to remove cell debris and protein expression for each construct was confirmed first by Western blot using anti-GFP. The 5-LO activity of cell lysates (200 µg of total protein) was determined in 0.25 ml reaction mixtures containing 50 mM Tris-HCl (pH 7.5), 0.6 mM CaCl2, 0.1 mM EDTA, 0.1 mM ATP, 12 µg/ml phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL), 20 µM AA (Cayman Chemicals, Ann Arbor, MI), including ~100,000 dpm [3H]AA (PerkinElmer Life Sciences) and 10 µM 13(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (Cayman Chemicals). After a 30-min incubation at room temperature, the reaction was stopped by adding 1 ml of ether/methanol/1 M citric acid (30:4:1, v/v/v). After vortexing thoroughly, the mixture was centrifuged at 5000 rpm for 5 min. The upper phase was removed, evaporated under nitrogen, and stored at -70 °C. Lipid residues were dissolved in 250 µl of 50% acetonitrile/trifluoroacetic acid (1000:1, v/v) and 50% water/trifluoroacetic acid (1000:1, v/v), analyzed by reverse-phase high performance liquid chromatography (HPLC) on a 5-µm Bondapak C18 column (30 × 0.4 cm; Waters Associates, Milford, MA) using a mobile phase of acetonitrile/trifluoroacetic acid at a flow rate of 2 ml/min. 5-LO metabolites were eluted during a series of linear gradient increases of acetonitrile from initial conditions of 50:50 (v/v) to 73:27 (v/v) at 7 min, then to 85:15 (v/v) at 9 min, and finally to 100:0 (v/v) at 15 min. Radioactivity in 1-ml eluted fractions was quantitated by on-line radiodetection. There were no LTB4/LTB4 isomers detected in cell lysates; 5-HPETE and 5-HETE co-eluted as a single peak, clearly separated from un-metabolized AA. The 5-LO specific activity of different mutants was calculated and compared based on the ratio of conversion of radiolabeled AA to 5-HPETE/5-HETE.

Quantitation of Subcellular Distribution-- As an initial approach to quantitation, slides were fixed 16 h after transfection, and 100 positive cells were scored as to whether nuclear fluorescence was greater than, equal to or less than cytosolic fluorescence. Care was taken to avoid damaged, dead or autofluorescent cells. Results from at least three independent transfections per construct were used for statistical analysis. As a second approach, 100 individual cells per construct were scored for cytosolic and nuclear fluorescence intensity: using Adobe Photoshop 5.5, grayscale digital images were adjusted to include the full black-to-white range, and representative gray values, from 0 (white) to 100 (black), were obtained for the cytoplasm and nucleoplasm. Cytoplasmic and nuclear values for each cell were summed to give total cellular fluorescence, and the percent fluorescence values for the nuclear compartment were calculated.

Statistical Analysis-- Statistical significance was evaluated by one-way analysis of variance, using p < 0.05 as indicative of statistical significance. Pairs of group means were analyzed using the Tukey-Kramer post-test.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reassessment of BR68 as a Functional NIS-- As outlined above, cells expressing GFP·5-LO with mutated BR518 displayed either no import or significant import, indicating the existence of at least one other functional NIS on 5-LO. Since the BR518 NIS was identified using structural criteria noted above, these criteria were applied to other candidate sites. Previous work indicated that an NIS in the amino-terminal region of 5-LO, potentially at BR68, might function as an NIS (13). This site was a good candidate because its primary structure, RXXKRK, fulfills the criteria of a monopartite NIS, being a cluster of 4 of 6 basic residues. Using the structural and functional criteria, BR68 was evaluated further. Comparison with the primary sequences of other lipoxygenases, however, indicated that this BR was not unique to 5LO (Fig. 1A). Thus, if it were a functional NIS on 5-LO, it might also be expected to direct the import of 15-LO and, perhaps, 12-LO. Regarding the secondary structure of BR68, no resolved structure for 5-LO is available. However, predicted structures for the beta -barrel domain have been published using C. perfringens alpha -toxin (21) or 15-LO (24) as templates, and the structure of the 15-LO beta -barrel domain has been published (25). The majority of the amino acids in BR68 were found to be involved in the fifth beta -sheet, in the predicted structure of 5-LO patterned after alpha -toxin (Fig. 1B) and in the resolved structure of 15-LO (Fig. 1C), as well as in the 5-LO structure patterned after 15-LO (Ref. 24 and not shown here). This suggests that this region serves a critical structural role in the amino-terminal beta -barrel and is not available for binding importin. These results indicate that BR68 is unlikely to be a functional NIS.


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Fig. 1.   Primary and secondary structural evaluation of the amino-terminal BR68. A, sequence alignments performed using ClustalW, with asterisks and colons indicating identical and similar residues, respectively. Shaded areas on rabbit 15-LO and alpha -toxin indicate residues involved in beta -sheets from resolved structures. Region on 5-LO corresponds to amino acids 68-73. B, secondary structure of BR68 (shaded black) in the beta -barrel region of human 5-LO, using the theoretical model developed using alpha -toxin as template (21). C, secondary structure of the corresponding region on the rabbit 15-LO protein.

Evaluation of BR112 as a Functional NIS-- Alignment of LO primary sequences revealed a novel basic region, beginning at R112 on human 5-LO, which was conserved across 5-LOs and not found in 12- or 15-LOs (Fig. 2A). Correct alignment was suggested by high levels of amino acid similarity on both sides of the region as well as alignment of the alpha -helix in the catalytic domain. This region, designated BR112, contained 4 basic amino acids over a stretch of 9 residues. The region was located on a random coil between the beta -barrel and catalytic domains of 5-LO (Fig. 2B). The presence of a conserved glycine, which can serve as a "helix breaker," also indicated that this region would retain a random coil structure. Since the region was conserved across different 5-LOs, not found on other LOs and was on a coiled structural element, it met our primary and secondary structural criteria for a good candidate NIS.


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Fig. 2.   Primary and secondary structural evaluation of BR112. A, sequence alignments performed using ClustalW. Residues involved in beta -sheets (amino-terminal) and alpha -helices (carboxyl-terminal) from resolved and theoretical structures of 15-LO and 5-LO, respectively. B, secondary structure of the corresponding region on rabbit 15-LO (black) between the last sheet of the beta -barrel region and the first helix of the catalytic domain.

To test whether BR112 was sufficient to cause nuclear import, oligonucleotides were synthesized and inserted into the GFP vector to produce GFP with the peptide LRDGRAKLARD fused to the carboxyl terminus. As has been frequently described (e.g. (13)), GFP alone distributed evenly between nuclear and cytoplasmic compartments in transfected cells (Fig. 3), because it is small enough to diffuse freely through the nuclear pore. However, the GFP·BR112 fusion protein showed distinct nuclear accumulation (Fig. 3), indicating that this peptide alone is sufficient to drive nuclear import against a diffusion gradient.


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Fig. 3.   Effect of the BR112 peptide on the subcellular distribution of GFP. 3T3 cells were transfected with GFP alone or with GFP fused to the BR112 peptide (LRDGRAKLARD) and after 16 h were fixed, stained with 4,6-diamidino-2-phenylindole (DAPI), and imaged under blue (GFP) or ultraviolet (DAPI) light.

To determine whether BR112 was necessary for nuclear import, the effect of basic residue replacement in BR112, in the context of GFP·5-LO, was evaluated. Three residues were replaced by site-directed mutagenesis: R115Q/K117Q/R120Q. As described previously (13, 14), the wild type (WT) GFP·5-LO fusion protein showed strong nuclear accumulation in most cells (Fig. 4, A and B). Cells expressing GFP·5-LO with mutation of BR112 included two distinct phenotypes: some cells had little or no nuclear fluorescence, while others showed clear nuclear accumulation of the expressed protein (Fig. 4, C and D). This result was first quantitated by scoring individual cells as having nuclear fluorescence greater than, equal to or less than the cytosolic fluorescence. Representative images and numbers for one experiment are given in Fig. 5. While the majority (65%) of cells expressing WT GFP·5-LO had nuclear accumulation, a significant number (31%) had a balanced distribution. A balanced distribution of 5-LO, associated with nuclear envelope breakdown during mitosis, has been described (7) and quantitated (15). Mutation at the BR112 site reduced the number of cells with nuclear import and increased those with cytosolic fluorescence. These results indicated that mutation of BR112 in the context of GFP·5-LO impaired nuclear import of the fusion protein in some 3T3 cells.


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Fig. 4.   Effect of mutations within the BR112 region of 5-LO. A, representative field showing the subcellular distribution of WT GFP·5-LO. Cells typically showed strong nuclear accumulation of 5-LO. B, DNA staining of field shown in A. C, representative field showing heterogeneity of subcellular distribution of GFP·5-LO with mutation of BR112. Some cells showed predominantly cytosolic fluorescence, while other showed predominantly nuclear fluorescence. D, DNA staining of field shown in C.


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Fig. 5.   Initial evaluation of the distribution phenotypes observed with WT or mutBR112 GFP·5-LO. 3T3 cells were transfected with GFP·5-LO that was unmodified (WT) or mutated at BR112. After 16 h, 100 positive cells were scored for either nuclear accumulation (Nuc > Cyto), balanced (Nuc = Cyto), or cytosolic (Nuc < Cyto) distribution and representative cells were imaged. Numbers indicate results from one experiment.

While the above results demonstrated that mutation of the BR112 site affected the subcellular distribution of 5-LO, they do not clearly define the nature of that effect. In particular, they did not clearly indicate whether the mutation simply reduced the efficiency of nuclear import, or whether the mutation resulted in distinct subpopulations of cells. To address this question, nuclear and cytoplasmic fluorescence levels in individual cells were quantitatively analyzed as described under "Experimental Procedures." By this analysis, there were (at least) two subpopulations exhibiting nuclear import of WT GFP·5-LO, when expressed in 3T3 cells (Fig. 6A). A major peak, consistently found in multiple transfections, consisted of cells with 60-70% of total fluorescence in the nucleus (peak N1). A shoulder, associated with 70-90% nuclear fluorescence, also was consistently observed (peak N2). Mutation of BR112, as shown in Fig. 6B, reduced the number of strongly importing cells (peak N2) and reduced the rate of nuclear import, as indicated by the shift of peak N1 to the left (i.e. to 55%). More significantly, this mutation produced a new population with only 30-40% nuclear fluorescence, designated peak C1. It should be noted that these cells, shown in Figs. 4 and 5, had little or no nuclear import; the relatively high value of 35% nuclear fluorescence reflects the conservative scoring of this quantitative method. In general terms, these results indicated that mutation of BR112 reduced the capacity for the strong nuclear import that produced the N2 peak. The resulting protein was still capable of pronounced nuclear import in some cells, as evidenced by the persistent N1 peak. However, the resulting protein did not direct nuclear import in those cells comprising the C1 peak.


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Fig. 6.   Quantitative analysis of the subcellular distribution of fluorescence of WT and mutBR112 5-LO. Cells were transfected with either WT GFP·5-LO (A) or GFP·5-LO (B) with mutBR112. After 16 h, cells were fixed and the fluorescence in cytosolic and nuclear compartments was calculated as percent of total cell fluorescence, as described under "Experimental Methods." Results are presented as the percentage of cells scored with the indicated nuclear fluorescence. Nuclear (N) and cytosolic (C) populations defined arbitrarily.

The finding that mutation in BR112 produced discrete import competent and non-importing populations was very similar to results previously found with mutations in BR518 (16). This suggested the possibility that these sites might overlap in function. To address this possibility, mutations in both sites were performed. The changes in BR518 were R518Q/R520Q/K521Q/K527Q/K530Q. Sample subcellular distributions and frequencies, for mutation in BR518 alone or in BR112 plus BR518, are given in Fig. 7A. Mutation of the BR518 site alone resulted in ~20% of the cells having impaired import, with about half of the cells still having significant nuclear accumulation of GFP·5-LO, as reported previously (16). The combined mutations of BR112 plus BR518 had an additive effect, with over half of the cells showing a failure to import GFP·5-LO.


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Fig. 7.   Evaluation of the distribution phenotypes observed with mutBR518 with or without mutBR112 GFP·5-LO. A, 3T3 cells were transfected with GFP·5-LO with mutBR518 alone or with mutBR112 plus mutBR518. After 16 h, 100 positive cells were scored as in Fig. 5. Numbers indicate results from one experiment. B, immunoblot showing protein expression in 3T3 cells transfected with WT GFP·5-LO or GFP·5-LO with mutations at BR112 or BR518 or both sites. All lanes contain 10 µg of total cellular protein.

Statistical analysis of results from multiple transfections showed that, for each of the two mutants, both the reduction of cells with nuclear import and the increase in cells with cytosolic fluorescence were statistically significant (Table I). Moreover, mutation of both basic regions produced statistically greater changes in the two distribution groups than did either mutation alone. No statistically significant change in the group showing balanced distribution was found for any mutation. The additive nature of the mutations indicated that these sites represent distinct NISs.


                              
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Table I
Effect of mutations in the BR112 region on the subcellular distribution of GFP·5-LO and GFP·5-LO with mutBR518 in 3T3 cells
Cells were transfected, incubated for 16 h and fixed. 100 cells/transfection were scored. Results are reported as percent of cells with the given distribution of green fluorescence. Data are means (S.E.) of n = 7 experiments. *, p < 0.05 versus WT; **, p < 0.05 versus mutBR518. Activity was assessed as amount of LTB4 produced by transfected cells, adjusted for protein expression, as measured by enzyme immunoassay.

Significantly, all mutants were functionally active, producing LTB4 when stimulated with the calcium ionophore A23187 in the presence of 10 µM arachidonic acid (Table I). The double mutant produced marginally less LTB4 than WT GFP·5-LO. Furthermore, mutated proteins of the appropriate size were expressed in similar amounts as wild type GFP·5-LO in transfected 3T3 cells (Fig. 7B). This result was obtained using antibodies to either GFP (Fig. 7B) or 5-LO (data not shown). Thus, the changes in nuclear import were unlikely to result from altered protein expression, conformational folding, or protein degradation.

Evaluation of BR158 as a Functional NIS-- Because mutations of both the BR518 and BR112 did not completely impair nuclear import of 5-LO, the protein sequence was further evaluated using the structural and functional criteria described previously. Another novel basic region was identified, beginning at Lys158 on human 5-LO. Correct alignment was supported by amino acid similarity on both sides of the region as well as alignment of the DLP core (Fig. 8A). This region, BR158 (KCHKDLPR), contained 3 basic residues, which were conserved across 5-LOs and not found in 12- or 15-LOs. This region was predicted to form a random coil on the catalytic domain of 5-LO (22), although a helix-like turn involving Leu-Thr on 15-LO (replaced by His-Lys on 5-LO) was evident (Fig. 8B). The presence of the conserved proline within the region, which can serve as helix breaker, also indicates that this region would retain a random coil structure. Since the region was conserved across different 5-LOs, not found on other LOs and was on a largely coiled structural element, it met our primary and secondary structural criteria for a good candidate NIS.


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Fig. 8.   Primary and secondary structural evaluation of BR158. A, sequence alignments performed using ClustalW, with asterisks indicating identical residues. B, secondary structure of the corresponding region on rabbit 15-LO (black). C, effect of the BR158 peptide on the subcellular distribution of GFP. 3T3 cells were transfected with GFP fused to the BR158 peptide (DAKCHKDLPRD) and imaged after 16 h. D, DNA staining, using 4,6-diamidino-2-phenylindole, of field shown in C.

A vector was constructed to express the GFP·BR158 fusion protein, with DAKCHKDLPRD representing BR158. This fusion protein showed nuclear accumulation (Fig. 8, C and D). Quantitative analysis of nuclear/cytosolic fluorescence ratios for 100 cells revealed that nuclear accumulation of the GFP·BR158 fusion protein was greater than that for the GFP·BR112 fusion protein (data not shown). Thus, the BR158 peptide is sufficient to drive nuclear import.

To determine whether the BR158 region was necessary for nuclear import, site-directed replacement of basic residues on GFP·5-LO was performed. The mutation mutBR158 was K158N/H160Q/K161N. Mutated proteins of the appropriate size were expressed in similar amounts as wild type GFP·5-LO in transfected 3T3 cells (data not shown). When the mutBR158 construct was expressed in 3T3 cells, the majority of cells showed nuclear accumulation, with only 10% of the cells clearly indicating a failure to import (Fig. 9). However, when this mutation was combined with mutations at the other two NIS sites, no cells showed nuclear accumulation (Fig. 9).


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Fig. 9.   Representative images of the effects of mutBR158, alone or combined with mutBR112 plus mutBR158, on the subcellular distribution of GFP·5-LO.

Statistical analysis of results from multiple transfections showed that mutation of BR158 produced a small but statistically significant increase in cells with impaired nuclear import (Table II). As described above in Table I, the combination of mutBR112 + mutBR518 significantly but incompletely decreased nuclear import. When all three BRs were altered, no cells showed nuclear accumulation of GFP·5-LO. Significantly, all mutants were functionally active, producing LTB4 when stimulated with the calcium ionophore A23187 in the presence of 10 µM arachidonic acid (Table II). The double and triple mutants produced marginally less LTB4 than the single mutants or WT GFP·5-LO. Further analysis of mutants by cell-free assay confirmed that, although the multiple amino acid substitutions did indeed reduce activity relative to wild type GFP·5-LO, even the mutation of all three NISs did not abolish activity (Table III). These results indicated that all three basic regions, BR518, BR112, and BR158, were necessary for nuclear accumulation of 5-LO in all cells within a population.


                              
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Table II
Effect of mutations in the BR158 region on the subcellular distribution of GFP·5-LO and GFP·5-LO with mutBR112 and mutBR518 in 3T3 cells
Cells were transfected, incubated for 16 h and fixed. 100 cells/transfection were scored. Results are reported as percent of cells with the given distribution of green fluorescence. Data are means (S.E.) of n = 3 experiments. *, p < 0.05 versus WT; **, p < 0.05 versus mutBR112 + mutBR518. Activity was assessed as amount of LTB4 produced by transfected cells, adjusted for protein expression, as measured by enzyme immunoassay.


                              
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Table III
Comparison of the cell-free activity of GFP·5-LO fusion proteins
Cell lysates from transfected COS-1 cells were assayed for enzymatic activity and protein expression. Enzymatic activity was evaluated as capacity of cell lysates to convert AA to 5-HPETE in vitro, as described under "Experimental Procedures." Protein expression was quantitated by densitometric analysis of immunoblots of samples. Results are presented as relative 5-LO activity, defined as the percent activity relative to wild type GFP · 5-LO after enzymatic activity was standardized for protein expression. Enzymatic activity of wild type GFP·5-LO, evaluated as percent conversion/30 min, was 19.35%.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previously, we used novel structural and functional criteria to identify an NIS, designated BR518, on 5-LO, but predicted that at least one other NIS must also exist (16). In the present study, we continued to search for potential NISs on 5-LO using the same criteria. This work revealed two novel basic regions, BR112 and BR158. Subsequent analysis showed that these sites were both sufficient and necessary for normal import. The body of work presented in this and the previous study confirms that three NISs exist on 5-LO and that all three are functional in determining the subcellular localization of 5-LO in 3T3 cells. Moreover, these studies indicate that the different NISs act independently from one another and that they can be activated and inactivated. Finally, these results demonstrate how mutations at different NISs will differ in their impact on the subcellular distribution of 5-LO.

Our mutagenesis data showed that the first basic region, BR112, serves a relatively strong import role, since mutation of the basic residues in this region impaired 5-LO nuclear import to at least the same extent as the mutation of BR518. Although mutation of BR112 significantly impaired 5-LO nuclear import, enzyme immunoassay showed that the catalytic activity of 5-LO was not compromised. Thus, the mutation specifically impaired nuclear import without changing the general enzyme secondary structure. This further substantiates that BR112 is a functional NIS. Mutations of both BR112 and BR518 had additive effects in reducing nuclear import (Table I), indicating that these sites act independently from one another.

The fact that mutations on both BR112 and BR518 could not totally eliminate nuclear import implied the existence of an additional import sequence. The third site, BR158, was found to be unique to 5-LO, structurally appropriate for binding importin, and sufficient to import GFP. Because mutation of BR158 alone only slightly impaired nuclear import, we speculate that this region may act as a weak import sequence that has low affinity to the receptor protein importin. Alternatively, BR158 alone may not function as an independent import sequence; it may coordinate other NISs to mediate 5-LO nuclear import. Supporting this idea, when BR112, BR158 and BR518 were all mutated at once, nuclear import of 5-LO was totally eliminated. Combined with the finding that the multiple mutant was still active, these studies strongly indicate that BR112, BR158, and BR518 are functional NISs and that multiple NISs orchestrate 5-LO nuclear import.

Previously, it was reported that a peptide containing the first 80 amino acids of 5-LO could drive import, leading to the suggestion that this region contains an NIS (13). The role of secondary structure in determining import capacity, as stressed in this study, may help explain this result. The region BR68, as shown in Fig. 1 and in Ref. 21, normally forms the fifth sheet of the beta -barrel domain. However, removal of residues 81-111 will also remove the last three beta -sheets of the barrel. In the LOs, sheet 5 is positioned between sheets 2 and 8, across from sheets 6 and 7; loss of sheets 6-8 might allow the fifth sheet, and BR68, to reform as a random coil. In this conformation, BR68 would be able to bind importin and, misleadingly, act as an NIS.

As we have found for 5-LO, an increasing number of proteins have been described as having multiple NISs. These include BRCA1 (26), Epstein-Barr virus DNase (27), herpes simplex virus products ICP22 (28), ICP27 (29), and XPG nuclease (30). The importance of having multiple NISs is unclear. In some cases, the individual NISs are weak, and the actions of multiple NISs can be additive or synergistic, as appears to be the case for 5-LO (this study) and XPG nuclease (30). Alternatively, the isoforms of importin-alpha are differentially expressed in different cell types and may bind each NIS with varied specificity (31). Thus, the NIS that is actually functional in a given cell type may depend on the isoform(s) of importin-alpha that is present.

Finally, each NIS may be regulated independently from the others. The observation of two distinct populations, one with import and one without, in cells expressing GFP·5-LO with either mutBR112 (Fig. 4) or mutBR518 (16) suggests that the remaining NISs may be subject to regulation. Protein phosphorylation in the vicinity of NISs has been repeatedly shown to play a role in regulating nuclear import (e.g. Refs. 32 and 33). There is evidence that 5-LO can be modulated by different kinases, such as protein kinases A (34) and C (35, 36), protein tyrosine kinases (37), and by mitogen-activated protein kinase kinase (38, 39), but these studies have not shown direct phosphorylation of 5-LO. More recently, two groups have been able to show direct phosphorylation of 5-LO by a tyrosine kinase (40) and by MAPKAP kinase 2 (41). It is not known which, if any, of these phosphorylation events regulate the three NISs of 5-LO.

In certain leukocytes, such as neutrophils and eosinophils in circulating blood, 5-LO is found exclusively in the cytoplasm (2-5,12). It seems reasonable that, in cells under these conditions, no NIS is activated. As noted above, nuclear import of 5-LO can be induced by different cues, including adherence, recruitment and cytokines. Each of these cues might activate distinct kinase pathways and, in turn, activate specific NISs on 5-LO. This suggests that the purpose for having multiple NISs, combined with multiple activation pathways, would be to ensure the import of 5-LO in response to a range of conditions. Activation of a single NIS might be sufficient for significant accumulation of 5-LO in the nucleus, whereas activation of multiple NISs might drive even greater accumulation.

Elucidation of the functional NISs in 5-LO represents a first step toward our understanding of 5-LO nuclear import. Future work regarding how these nuclear targeting sequences are recognized and modulated in normal and diseased cell types may reveal the mechanisms as well as functional consequences of nuclear import of 5-LO.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants R29 AI43574 and R21 AI48141.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.

Dagger To whom correspondence should be addressed: Dept. of Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, MI 48109-0642. Tel.: 734-763-9077; Fax: 734-764-4556; E-mail: brocko@umich.edu.

Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M211021200

    ABBREVIATIONS

The abbreviations used are: LT, leukotriene; LO, lipoxygenase; NIS, nuclear import sequence; BR, basic region; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; WT, wild type; mut, mutant; PDB, protein data bank.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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