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INTRODUCTION |
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-
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
-barrel region of 5-LO, is unlikely to be a functional NIS. However,
a novel site at Arg112, which links the
-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.
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EXPERIMENTAL PROCEDURES |
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
-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
-toxin (PDB: 1qmd), as well as published theoretical models of the 5-LO
-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.
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RESULTS |
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
-barrel domain have been published
using C. perfringens
-toxin (21) or 15-LO (24) as
templates, and the structure of the 15-LO
-barrel domain has been
published (25). The majority of the amino acids in BR68
were found to be involved in the fifth
-sheet, in the predicted structure of 5-LO patterned after
-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
-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 -toxin
indicate residues involved in -sheets from resolved structures.
Region on 5-LO corresponds to amino acids 68-73. B,
secondary structure of BR68 (shaded black) in
the -barrel region of human 5-LO, using the theoretical model
developed using -toxin as template (21). C, secondary
structure of the corresponding region on the rabbit 15-LO
protein.
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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
-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
-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 -sheets (amino-terminal) and
-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 -barrel region and
the first helix of the catalytic domain.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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 |
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
-barrel
domain. However, removal of residues 81-111 will also remove the last
three
-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-
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-
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.