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J Biol Chem, Vol. 274, Issue 42, 29812-29818, October 15, 1999
From the Division of Pulmonary and Critical Care Medicine,
Department of Internal Medicine, University of Michigan,
Ann Arbor, Michigan 48109-0642
5-Lipoxygenase catalyzes the synthesis of
leukotrienes from arachidonic acid. This enzyme can reside either
in the cytoplasm or the nucleus; its subcellular distribution is
influenced by extracellular factors, and its nuclear import correlates
with changes in leukotriene synthetic capacity. To identify sequences responsible for the nuclear import of 5-lipoxygenase, we transfected NIH 3T3 cells and RAW 264.7 macrophages with expression vectors encoding various 5-lipoxygenase constructs fused to green fluorescent protein. Overexpression of wild type 5-lipoxygenase with or without fusion to green fluorescent protein resulted in a predominantly intranuclear pattern of fluorescence, similar to the distribution of
native 5-lipoxygenase in primary alveolar macrophages. Within the
5-lipoxygenase protein is a sequence
(Arg638-Lys655) that closely resembles a
bipartite nuclear localization signal. Studies using deletion mutants
indicated that this region was necessary for nuclear import of
5-lipoxygenase. Analysis of mutants containing specific amino acid
substitutions within this sequence confirmed that it was this sequence
that was necessary for nuclear import of 5-lipoxygenase and that a
specific arginine residue was critical for this function. As nuclear
import of 5-lipoxygenase may regulate leukotriene production, natural
or induced mutations in this bipartite nuclear localization sequence
may also be important in affecting leukotriene synthesis.
Leukotrienes (LTs)1 are
lipid mediators with important roles in immune responsiveness and
antimicrobial host defense. However, overproduction of LTs can
contribute to a variety of pathophysiological inflammatory processes,
including asthma and allergic responses (1). Understanding the
regulation of LT synthesis is critical to understanding both normal
immune responses and the underlying pathophysiology of a variety of diseases.
The synthesis of LTs from arachidonic acid (AA) is initiated by the
enzyme 5-lipoxygenase (5-LO). Activation of 5-LO, which is
characterized by its translocation to the nuclear envelope (2-5),
positions 5-LO close to its substrate and 5-LO activating protein
(6-8) and is essential for LT synthesis. Changes in substrate availability or in the level of expression of proteins essential for AA
metabolism (5-LO and/or 5-LO activating protein) have been correlated
with the modulation of LT synthetic capacity (9-14). However, changes
in these variables do not account for all observed changes in LT
synthetic capacity (15-17).
Evidence is accumulating that, even in resting or unstimulated cells,
the subcellular distribution of 5-LO is dynamic and may influence LT
synthetic capacity. In resting cells, the subcellular distribution of
5-LO is cell type-dependent; 5-LO is predominantly cytoplasmic in peripheral blood neutrophils (18-20), peripheral blood
monocytes (4), and peritoneal macrophages (3), whereas it is found in
both the nucleus and cytoplasm of alveolar macrophages (4, 5), mast
cells (21), and the mast cell-like rat basophilic leukemia cell line
(20). However, these subcellular distributions are not necessarily
fixed. For example, differentiation of monocytes to alveolar
macrophages (22), but not peritoneal macrophages (3), is associated
with nuclear accumulation of 5-LO. Conversely, removal of macrophages
from the alveolar milieu results in subsequent accumulation of 5-LO to
the cytoplasm (22), suggesting that factors in the alveolus drive
nuclear import of 5-LO. Furthermore, adherence or recruitment of
peripheral blood neutrophils (23) or eosinophils (24) induces nuclear
import of 5-LO from the cytoplasm. In each case, a change in the
subcellular distribution of 5-LO is associated with a change in LT
synthetic capacity.
All transport into the nucleus occurs through the nuclear pore complex
(25-27). The nuclear pore allows small molecules (<45 kDa) to diffuse
freely into and out of the nucleus (25-27). Targeting of larger
proteins to the nucleus usually requires the presence of a nuclear
localization signal (NLS) (25). NLSs are defined as sequences
sufficient and necessary for nuclear import of their respective
proteins. Two major types of NLSs are recognized: 1) a single cluster
of basic amino acids exemplified by the SV40 large T antigen NLS; and
2) a bipartite NLS composed of two basic amino acids, a spacer region
of 10-12 amino acids, and a basic cluster in which three of five amino
acids must be basic as found in nucleoplasmin (27, 28). For
NLS-mediated nuclear import to occur, the NLS must be recognized by and
associate with specific importins, which allows docking at the nuclear
pore and translocation through the pore via an
energy-dependent process (25-27).
A recent study of nuclear import of 5-LO failed to identify a classical
NLS as responsible for targeting this enzyme to the nucleus (29).
Rather, that study concluded that an as yet unidentified nonconventional signal located in the amino terminus targets 5-LO to
the nucleus. In the current study, we have addressed this same question
and have reached a completely different conclusion. Our studies have
identified a basic region in the carboxyl terminus that resembles a
classical bipartite NLS, have demonstrated that specific basic residues
within this region are necessary for targeting 5-LO to the nucleus, and
have provided evidence that the amino terminus is not sufficient to
direct nuclear import.
Cell Culture and Transfection--
RAW 264.7 cells and NIH 3T3
cells were obtained from American Type Culture Collection (Manassas,
VA) and grown under 5% CO2 in RPMI 1640 medium (Life
Technologies, Inc.) or Dulbecco's modified Eagle's medium (Life
Technologies, Inc.), respectively, supplemented with 10% fetal bovine
serum, 100 units/ml penicillin, and 100 units/ml streptomycin. Cells
were transfected using FuGENE 6 transfection reagent (Roche Molecular
Biochemicals) according to the manufacturer's specifications.
Transient transfectants were evaluated microscopically, live or after
fixation with 4% paraformaldehyde, 16-24 h after transfection.
cDNA, Plasmids, and Mutagenesis--
The 5-LO cDNA (30)
was obtained from Dr. M. Abramovitz (Merck Frosst Center for
Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada). To
construct a plasmid that would express 5-LO, a 2.2-kb fragment
containing the 5-LO cDNA was subcloned into the EcoRI
and HindIII sites of pcDNA3.1(-) (Invitrogen, San Diego, CA). To construct a plasmid that would express 5-LO fused to green fluorescent protein (GFP), a 2.2-kb fragment containing the 5-LO cDNA was subcloned into the EcoRI and SalI
sites of pEGFP-C1 (CLONTECH, Palo Alto, CA).
pEGFP-C1 allows fusion of a sequence of interest to the carboxyl
terminus of EGFP. All pEGFP-C1 constructs resulted in in-frame fusions,
as determined by DNA sequence analysis (DNA Sequencing Core, University
of Michigan). For simplicity, EGFP will be referred to as GFP.
GFP/5LO-N was constructed by deleting the 0.5-kb BamHI
fragment, encoding amino acids 563-673, from GFP/5LO. GFP/5LO-C was constructed by subcloning the 1.2-kb SmaI-SalI
fragment of GFP/5LO, encoding amino acids 332-673, into pEGFP-C1.
GFP/5LO (1-80) was constructed by deleting the 2.0-kb KpnI
fragment, encoding amino acids 81-673, from GFP/5LO. To construct
GFP/SV40-NLS, the complementary oligonucleotides
5'-CCTCCAAAAAAGAAGAGAAAGGTAGGCC-3' and
5'-TACCTTTCTCTTCTTTTTTGGAGGAGCT-3', encoding the SV40 NLS
(KKKRK) (31), were annealed and ligated to the SacI and
ApaI sites of pEGFP-C1. To construct GFP/BR3, the
complementary oligonucleotides
5'-GATCTCGCAAGAACCTCGAGGCCATTGTCAGCGTGATTGCTGAGCGCAACAAGAAGAAG-3' and
5'-AATTCTTCTTCTTGTTGCGCTCAGCAATCACGCTGACAATGGCCTCGAGGTTCTTGCGA-3', encoding Arg638-Lys655
(RKNLEAIVSVIAERNKKK), were annealed and
ligated to the BglII and EcoRI sites of
pEGFP-C1.
Specific amino acids within the putative 5-LO NLS were substituted
using the QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA) and verified by DNA sequence analysis. Briefly, two
complementary primers (125 ng each) containing the desired mutation and
50 ng of template in 1× reaction buffer were denatured at 95 °C for
30 s and annealed at 55 °C for 1 min, and DNA synthesis was
carried out by Pfu polymerase at 68 °C for 14 min; this
cycle was repeated 18 times. The methylated template was removed by incubation with 10 units of DpnI at 37 °C for 1 h.
Three types of alanine substitutions were constructed:
Lys653 and Lys654 were substituted with alanine
(R(K/R)NAAK) using 5'-GCGTGATTGCTGAGCGCAACGCGGCGAAGCAGCTGCC-3' and 5'-GGCAGCTGCTTCGCCGCGTTGCGCTCAGCAATCACGC-3' as mutagenic primers; Arg638 and Lys639 were substituted with alanine
(AA...RNKKK) using 5'-GCCATGGCCCGATTCGCCGCGAACCTCGAGGCC-3' and
5'-GGCCTCGAGGTTCGCGGCGAATCGGGCCATGGC-3' as primers; and
Arg651 was substituted with alanine (RK...ANKKK) using
5'GCGTGATTGCTGAGGCGAACGCGGCGAAGCAGCTGCC-3' and
5'-GGCAGCTGCTTCGCCGCGTTCGCCTCAGCAATCACGC-3' as primers. Plasmids containing combinations of these mutations (AA...RNAAK, AA...ANKKK, RK...ANAAK, and AA...ANAAK) were constructed by carrying out serial mutagenesis reactions. Two types of glutamine substitutions were constructed: Lys653 and Lys654 were substituted
with glutamine (RK...RNQQK) using
5'-GCGTGATTGCTGAGCGCAACCAGCAGAAGCAGCTGC-3' and
5'-GCAGCTGCTTCTGCTGGTTGCGCTCAGCAATCACGC-3' as mutagenic primers; and
Arg651 was substituted with glutamine (RK...QNKKK) using
5'-GCGTGATTGCTGAGCAAAACAAGAAGAAGCAGCTGC-3' and
5'-GCAGCTGCTTCTTCTTGTTTTGCTCAGCAATCACGC-3' as mutagenic primers. All
oligonucleotides were synthesized by the DNA Synthesis Core at the
University of Michigan.
Indirect Immunofluorescent Staining and Microscopy--
GFP
fluorescence in transfected cells was detected with a Zeiss Aristoplan
microscope equipped for epifluorescence using a 495-nm bandpass filter.
Indirect immunofluorescent microscopy was carried out as described
previously (20). Briefly, adherent cells were fixed in methanol at
Immunoblot Analysis--
Cells were disrupted by sonication (10 bursts at 20% duty cycle) in ice-cold homogenizing buffer (50 mM Tris-HCl, 25 mM KCl, 5 mM
MgCl2, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonyl fluoride, 1 mM leupeptin, pH 7.4), and
protein concentrations were determined by a modified Coomassie dye
binding assay (Pierce). Samples containing 20 µg of protein were
separated by SDS-polyacrylamide gel electrophoresis under reducing
conditions and transferred to nitrocellulose membranes. Membranes were
probed with the 5-LO antibody (titer, 1:5000) followed by
peroxidase-conjugated goat anti-rabbit antibody (titer, 1:5000) or with
a GFP monoclonal antibody (CLONTECH; titer, 1:5000)
followed by peroxidase-conjugated sheep anti-mouse antibody (titer,
1:5000) and treated with the ECL chemiluminescence detection system
(Amersham Pharmacia Biotech).
Cell Stimulation and Enzyme Immunoassay of Eicosanoids--
To
stimulate 5-LO activity and translocation, cells were incubated for 30 min at 37 °C in serum-free medium containing 10 µM of
the 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.
5-LO Nuclear Import in NIH 3T3 and RAW 264.7 Cells--
We
analyzed 5-LO nuclear import in the mouse cell line NIH 3T3 and in the
mouse macrophage cell line RAW 264.7. Because 5-LO is predominantly
expressed in cells of leukocytic origin, we preferred to analyze 5-LO
nuclear import in RAW cells. However, the low transfection efficiency
in these cells precluded their use in techniques requiring large
numbers of transfectants (e.g. metabolic studies); the
higher transfection efficiency of 3T3 cells permitted these analyses.
Western blot analysis of cell lysates revealed little or no detectable
5-LO expression in untransfected RAW and 3T3 cells (Fig.
1A). However, substantial 5-LO
expression was detected in 3T3 cells transiently transfected with
pcDNA/5LO, a plasmid that supports constitutive expression of human
5-LO (Fig. 1A). Immunofluorescent microscopy revealed that
5-LO colocalized with DNA and thus was localized predominantly in the
nucleus in both 3T3 (Fig. 1B) and RAW (Fig. 1C)
cells transfected with pcDNA/5LO. Nontransfected cells were evident
by their lack of staining for 5-LO and positive staining for DNA. After
stimulation of transfected 3T3 cells with the calcium ionophore A23187,
appreciable LTB4 synthesis (1356 ± 96 pg/ml,
n = 3) was detected by enzyme immunoassay. LT synthesis
could not be detected in untransfected 3T3 cells. These data indicate
that these cell lines are useful models for the analysis of 5-LO
nuclear import.
Use of GFP Fusions to Study Nuclear Import--
Next, we
determined whether a GFP tag would facilitate the study of 5-LO nuclear
import. Transiently transfected RAW or 3T3 cells expressing only the
~27-kDa GFP exhibited fluorescence distributed evenly between nucleus
and cytoplasm (Fig. 2A), in
agreement with results observed in other cell lines (e.g.
Ref. 32). This lack of a gradient in distribution between nucleus and
cytoplasm was presumably because of the small size of GFP, which would
allow it to move freely through the nuclear pore. To determine the
subcellular localization of a GFP/5LO fusion protein, we inserted the
full-length human 5-LO cDNA into pEGFP-C1 (fusing the GFP to the
carboxyl terminus of 5-LO was not attempted as the carboxyl terminus is known to be necessary for binding the iron in 5-LO). The resulting GFP/5LO construct would express an ~105-kDa fusion protein, which would be too large to diffuse through the nuclear pore. Upon transient transfection with this construct, a strong intranuclear fluorescence pattern was observed (Fig. 2B) in both cell lines. Thus,
both 5-LO and GFP/5LO accumulate within the nucleus in RAW and 3T3 cells, suggesting the presence of a functional NLS in 5-LO. Upon stimulation with A23187, the GFP/5LO fusion protein translocated to the
nuclear envelope and endoplasmic reticulum in both cell types (Fig.
2C). In addition, significant LTB4 production
(3667 ± 1365 pg/ml, n = 4) could be detected in
transfected 3T3 cells, but not in untransfected cells, after
stimulation with A23187. Thus, the GFP tag does not interfere with
subcellular localization, translocation, or catalytic activity of 5-LO.
Taken together, these results indicate that the use of a GFP tag is an
appropriate approach for analysis of 5-LO nuclear import.
Analysis of the Nuclear Import of GFP/5LO Constructs--
As
reported previously (29), 5-LO contains three regions of basic amino
acids that are candidate NLSs (Fig. 3).
The first 80 amino acids of 5-LO, which contains the first basic region (BR1), have been reported to be sufficient to target GFP to the nucleus
(29). However, images supporting that conclusion were unconvincing; the
nuclei were many times larger than those in any other image, and the
staining pattern for both DNA and the GFP/1-80 construct was much more
clumped than the patterns of DNA or GFP/5-LO in other images. To
re-examine these results, we also constructed GFP/5LO (1-80) in which
the first 80 amino acids of 5-LO were fused to GFP. Sequence analysis
indicated that this construct placed the 1-80 sequence in frame with
GFP (not shown); immunoblot analysis confirmed that the produced
protein was of the expected size (~36 kDa) (Fig. 3). Upon
transfection, fluorescence was observed to be evenly distributed
between the nucleus and cytoplasm in both cell types (Fig.
3B) and was distinctly different from the pattern produced
by the intact 5-LO protein (Fig. 3A). This pattern of
fluorescence is likely because of the passive diffusion of this
~36-kDa fusion protein and indicates that the first 80 amino acids of
5-LO are not sufficient to direct nuclear import of GFP in these cell
lines. A larger segment of 5-LO, encoding amino acids 1-562 and
including both BR1 and BR2, was also fused to GFP (5LO-N, Fig.
3C). This truncated ~92-kDa protein was completely
excluded from the nucleus in both cell types (Fig. 3C),
indicating that the putative BR1 and BR2 were insufficient to drive
nuclear import of this size-excluded protein. These results also
pointed to a possible role for the carboxyl terminus in nuclear import.
Analysis of the predicted amino acid sequence of 5-LO revealed an
excellent candidate bipartite NLS at amino acids 638-655 (BR3, Fig.
3). This region contains two basic amino acids, an 11-residue spacer
region, and a basic cluster in which four of the five amino acids are
basic. To determine if the carboxyl terminus of 5-LO could direct
nuclear import, we fused the region containing amino acids 332-673 to
GFP (GFP/5LO-C; Fig. 3D). In cells transfected with this
construct, which encoded an ~67-kDa fusion protein, fluorescence was
observed to be distributed evenly between nucleus and cytoplasm (Fig.
3D). Because this fusion protein is unlikely to freely
diffuse through the nuclear pore because of its size, these results
suggest that this construct is being targeted, albeit inefficiently, to
the nucleus. Immunoblot analysis confirmed that each fusion protein was
of the expected size (Fig. 3). Nonspecific bands at approximately 50, 54, 68, and 105 kDa were found in all lanes when this commercial
antibody to GFP was used.
Many NLSs are able to direct nuclear import of a heterologous protein.
GFP alone, again, is a small protein which, like GFP/5LO (1-80),
distributed equally between the nucleus and cytoplasm (Fig.
4A). The fusion of the SV40
large T antigen NLS to GFP, which results in a protein of ~28 kDa,
produced strong intranuclear fluorescence and weak cytoplasmic
fluorescence in 3T3 cells (GFP/SV40-NLS, Fig. 4B) as well as
RAW cells (data not shown). These results are in agreement with those
observed in other cell lines (e.g. Ref. 29) and reflect that
the GFP/SV40 NLS fusion protein is strongly targeted to the nucleus,
despite being small enough (~28 kDa) to move freely through the
nuclear pore. To determine if BR3 (amino acids 638-655) alone could
direct nuclear import of an unrelated protein, we fused the sequence
encoding BR3 to GFP (GFP/BR3). In cells expressing this fusion,
slightly more intranuclear fluorescence than cytoplasmic fluorescence
was observed (Fig. 4C). In multiple experiments in both 3T3
cells and RAW cells, nuclear accumulation of GFP/BR3 was greater than
GFP alone but less than either GFP/SV40-NLS or GFP/5LO (Fig.
4D). These results suggest that this sequence is able to
weakly target a heterologous protein to the nucleus.
Identification of Amino Acids Necessary for 5-LO Nuclear
Import--
To determine if the 5-LO BR3 is necessary for nuclear
import, we performed site-directed mutagenesis on specific residues of
this sequence, changing positively charged arginine and/or lysine
residues to alanine, which has a small uncharged side chain. The
effects of these mutations were evaluated in the context of the entire
GFP/5-LO fusion protein. As described above, the 5-LO BR3 possesses two
clusters of basic amino acids separated by a spacer region. Mutagenesis
of two basic residues in either the first (AA...RNKKK, Fig.
5A) or second basic cluster
(RK...RNAAK, Fig. 5B) did not disrupt nuclear import in
either cell type. Mutations in both basic clusters (AA...RNAAK, Fig.
5C) also did not disrupt nuclear import. However,
mutagenesis of a single residue, the arginine at 651 (RK...ANKKK),
resulted in an equal distribution of fluorescence between nucleus and
cytoplasm (Fig. 5D), suggesting a partial disruption of
nuclear import. Combining this single mutation with mutations in the
first basic cluster (AA...ANKKK, Fig. 5E) or the second
basic cluster (RK...ANAAK, Fig. 5F) resulted in an
extranuclear pattern of fluorescence, indicating a complete loss of
nuclear import. Combining all the mutations in both clusters (AA...ANAAK, Fig. 5G) also resulted in a failure to import.
Immunoblot analysis confirmed that each mutant fusion protein was of
comparable size and expression as GFP/5LO (Fig. 5); no breakdown
products were found in any sample.
These results indicated a critical role for Arg651 in
nuclear import. However, the side chain of the substituted amino acid, alanine, differs both in size and charge from that of arginine. To
eliminate size differences, Arg651 was also substituted
with glutamine. Surprisingly, the R651Q substitution alone was
sufficient to completely eliminate nuclear import in 3T3 cells (Fig.
6A). In contrast, substituting
Lys653 and Lys654 with glutamine had no effect
on nuclear import (Fig. 6B), as previously reported
(29).
To determine whether these NLS mutations resulted in similar
localization patterns when expressed in the context of 5-LO alone rather than as a GFP fusion protein, mutant 5-LO constructs were subcloned into pcDNA3.1. After transfection of 3T3 cells with these
pcDNA constructs, immunoblot analysis (Fig.
7) confirmed that each construct was of
comparable size to pcDNA/5LO (lane A). Immunofluorescent
microscopy revealed subcellular distributions identical to those
observed with the GFP fusion constructs (Fig. 7). These results showed
that GFP did not influence 5-LO subcellular localization, confirmed the
central role of Arg651, and supported the additive effects
of deletions at basic residues other than Arg651.
The nuclear import of 5-LO appears to be a critical regulatory
component of its function. The current study identifies a key group of
amino acids at positions 638-655 in the human protein, which are
essential for nuclear import. Substitution of basic residues within
this bipartite NLS completely block nuclear import of 5-LO, and the NLS
alone can direct nuclear import of a heterologous protein (GFP) albeit
poorly. We further demonstrate that these features are shared by both a
myeloid cell line (RAW 264.7 macrophages) and a nonmyeloid cell line
(NIH 3T3 cells), which is significant because 5-LO is largely limited
in distribution to myeloid cells. This study also presents evidence
that alternative candidate NLSs within the amino terminus are not
sufficient to direct nuclear import. Together, these results provide a
foundation for understanding the molecular regulation of nuclear import
of 5-LO.
The altered production of LTs can contribute to many inflammatory
processes. Recent evidence correlates altered nuclear import of 5-LO
with altered leukotriene synthetic capacity (22-24). Our site-directed
mutagenesis experiments point to Arg651 as being critical
for nuclear import of 5-LO with partial or complete loss of import when
replaced with alanine or glutamine, respectively. Substitution of basic
residues around Arg651 could enhance, but not mimic, this
effect. This arginine is conserved across species, including human,
rat, mouse, and hamster 5-LO (Fig. 8).
These results may have significant clinical implications: they predict
a site within the 5-LO gene where a mutation could alter localization
of the 5-LO enzyme and, conceivably, alter leukotriene synthetic
capacity. Studies are currently underway to determine the effect of
these mutations on leukotriene synthetic capacity in relevant
cells.
Both the bipartite NLS alone as well as the much larger carboxyl
terminus of 5-LO could target GFP to the nucleus; the nuclear import of
the carboxyl fragment, which should be excluded because of its size,
supports the presence of an NLS. However, import was less efficient
than that driven by the complete 5-LO protein fused to GFP. The results
obtained using these fragments must be interpreted with caution,
foremost because fragments may exclude key elements or include
conflicting elements. For example, the weakness of import observed with
the carboxyl fragment may indicate that a deleted sequence might
augment the action of BR3. Similarly, the amino-terminal fragments may
fail to be imported either because they lack an NLS or because they
contain a dominant nuclear export sequence. These possibilities will
require further study.
Our findings also indicate that nuclear import of 5-LO is constitutive,
but incomplete, in both cell lines, that is, 5-LO accumulates within
the nucleus without cell stimulation, but a significant pool of 5-LO
remains in the cytoplasm. Such a pattern of distribution may arise
simply by protein overexpression to the extent that the capacity for
import becomes saturated. Alternatively, the distribution may indicate
that import is faster than export or that import is slower than
synthesis. In any case, this pattern of a large nuclear pool with a
lesser cytoplasmic pool is characteristic of many cell types; it has
been reported for primary alveolar macrophages (4), recruited
neutrophils (23), adherent eosinophils (24), and the mast cell-like rat
basophilic cell (20). However, 5-LO is found largely in the cytoplasm
of several cell types, including monocytes (4), peripheral blood
neutrophils (20) and eosinophils (24, 33), and peritoneal macrophages
(3). These results suggest that the nuclear import of 5-LO can be
regulated. For many proteins, nuclear import is modulated by
phosphorylation (25). 5-LO reportedly can be phosphorylated (34), and a
comparison of the 5-LO sequence with a Prosite data base found at least
16 consensus phosphorylation sites scattered through the length of the
protein. Phosphorylation at a site distinct from the bipartite NLS site
may affect 5-LO localization and may be important in explaining some of
the current findings.
A previous study (29) was the first to examine the molecular regulation
of the nuclear import of 5-LO, and those results, combined with the
current study, give a more complete picture of our current
understanding. The region BR1 (AA 68-73) was analyzed in the previous
work (29) by site-directed mutagenesis of two of four basic residues
and found not to be necessary for import. As we have shown for BR3, a
more complete mutagenesis study is necessary to ascertain that BR1 is
not necessary for import. Chen et al. (29) also found the
1-80 fragment was sufficient for import. As noted above, the images
presented to support import were unusual; the nuclei were much larger
than those presented in any other image, and the clumped patterns of
nuclear and GFP fluorescence were also unlike other images.
Furthermore, the conclusions from the previous study were based on
preparations that were fixed prior to examination. Fixation can result
in a disproportionate loss of soluble cytosolic proteins relative to
soluble nuclear proteins (23), and this may have contributed to the
impression that the 1-80 fragment had accumulated in the nucleus. Our
findings, using live cells, contradict this conclusion, because neither the smaller 1-80 fragment nor the larger amino terminus fragment promoted nuclear import in either cell line; this site apparently requires further study. Previous mutagenesis of region BR2 (AA 128-133) blocked import, suggesting that this element contains an NLS
(29). However, in both this study and the previous work (29), fragments
that included this region do not direct import of size-excluded
proteins, indicating that this region is not sufficient for import.
Finally, Chen et al. (29) found that limited mutagenesis of
BR3 (AA 638-655) did not block import. We performed the same mutations
and obtained the same results. However, our more complete mutational
strategy revealed that replacement of Arg651 with alanine
diminished nuclear import and that additional replacements of other
basic residues augmented this effect. Chen et al. (29) also
reported that fragments that included BR3 were localized to the
cytoplasm. However, the only image presented for the short carboxyl
terminus (AA 564-673) showed an equal nuclear and cytoplasmic distribution as would be expected for a small protein, although they
reported it to be exclusively cytoplasmic. In our hands, larger
fragments, which should be size-excluded from the nucleus, are able to
enter the nucleus if they contain BR3. However, BR3 is insufficient to
drive import comparable to the intact 5-LO protein. Together, these
results indicate that, although this basic region may have an essential
role in the nuclear import of 5-LO, other parts of the protein must
also contribute to the regulation of import.
Understanding the molecular determinants regulating the subcellular
localization of 5-LO and the consequences of its compartmentalization will be critical to understanding the metabolic function of this important component of the LT synthetic pathway. In addition, because
the subcellular distribution of 5-LO may affect LT synthetic capacity,
the 5-LO NLS is a potential site for mutations having clinical
implications. Finally, the 5-LO protein may modulate cellular functions
independent of its catalytic capacity by e.g. protein-protein interactions (35, 36). Therefore, the potential nonmetabolic consequences of nuclear localization of 5-LO will need to
be addressed. Stable cell lines overexpressing 5-LO exclusively in the
nucleus or in the cytoplasm will be invaluable for dissecting the
mechanisms regulating the subcellular distribution of 5-LO and
determining its impact on LT synthesis as well as other cellular properties.
*
This work was supported by Specialized Programs of Research
Excellence in Prostate Cancer Grant CA695568, NIAID, National Institutes of Health Grant R29 AI43574 (to T. G. B.), NHLBI, National Institutes of Health Grant R01 HL47391, National Institutes of Health
Training Grant T32 HL07749, and Specialized Center of Research Grant
P50 HL46487 (to M. P. G.).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.
The abbreviations used are:
LT, leukotriene;
5-LO, 5-lipoxygenase;
AA, arachidonic acid;
BR, basic region;
EGFP, enhanced green fluorescent protein;
GFP, green fluorescent protein;
NLS, nuclear localization signal;
kb, kilobase(s).
Identification of a Bipartite Nuclear Localization Sequence
Necessary for Nuclear Import of 5-Lipoxygenase*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C for 30 min, permeabilized in acetone at 20 °C for 3 min, and air-dried. Cells were rehydrated and blocked with 0.1% bovine
serum albumin in phosphate-buffered saline containing nonimmune goat
serum. Rabbit polyclonal antibody raised against purified human
leukocyte 5-LO (a generous gift from Dr. J. Evans, Merck Frosst Center
for Therapeutic Research) (2) was prepared in 0.1% bovine serum
albumin in phosphate-buffered saline (titer, 1:200) and applied for
1 h at 37 °C. Mounts were washed, incubated with
rhodamine-conjugated goat anti-rabbit antibody (Sigma; titer, 1:200),
and washed again. Rhodamine fluorescence was visualized as described
above using a 585-nm bandpass filter. DNA was stained by
diamidino-2-phenylindole and visualized using ultraviolet excitation.
Differential interference contrast microscopy was also performed with
the Zeiss Aristoplan microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of 5-LO protein expression
in 3T3 and RAW cells. A, immunoblot analysis of 5-LO
protein expression in RAW 264.7 cells, NIH 3T3 cells, and NIH 3T3 cells
transfected with pcDNA/5LO. 5-LO was detectable in RAW, but not 3T3
cells, upon longer exposure. Equivalent amounts of protein (20 µg)
were loaded in each lane. B and C,
immunofluorescent localization of 5-LO in 3T3 (B) and RAW
(C) cells transfected with pcDNA/5LO. 5-LO and DNA
localization are shown for each field. As was commonly found, RAW cells
were multinucleate. All images were photographed using identical
settings and are presented at identical magnification. Bar,
10 µm. Results are representative of three independent
experiments.
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Fig. 2.
Localization of GFP and GFP/5LO fusion
proteins in 3T3 and RAW cells. Cells were transiently transfected
with GFP alone (A) or GFP fused to 5-LO (B and
C). In C, 16 h after transfection cells were
stimulated with 10 µM calcium ionophore A23187 for 30 min
at 37 °C and imaged live. As noted, multinucleate RAW cells were
common; the activated RAW cell shown here has 3 nuclei. Results are
representative of five independent experiments.
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Fig. 3.
Subcellular localization of fusion proteins
containing GFP linked to portions of the 5-LO protein. The
sequence and location of the three BRs discussed in the text are
indicated as well as the portions of 5-LO included in each construct.
3T3 and RAW cells were transfected with GFP fused to 5-LO
(A), 5LO(1-80) (B), 5LO-N (C), or
5LO-C (D). 5LO(1-80) includes sequences extending from the
amino (H2N) terminus to the KpnI site
indicated. 5LO-N includes 5-LO sequences extending from the amino
terminus to the BamHI site indicated. 5LO-C includes 5-LO
sequences extending from the SmaI site indicated to the
carboxyl (COOH) terminus. Representative cells from
transient transfections were photographed using fluorescent and/or
differential interference contrast microscopy. Immunoblot analysis of
GFP fusion proteins expressed in 3T3 cells and probed with an antibody
against GFP is also shown. Arrows indicate the expected size
of each fusion protein. Results are representative of five independent
experiments.
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Fig. 4.
Subcellular localization of GFP fusion
proteins in 3T3 cells. Cells were transfected with the GFP alone
(A), GFP fused to the SV40 NLS (B), GFP fused to
BR3 (C), or GFP fused to 5-LO (D). Fluorescence
images from GFP (left) or from DAPI (right) of
identical fields are shown. Results are representative of five
independent experiments.
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Fig. 5.
Subcellular localization of GFP/5LO fusion
proteins containing specific amino acid substitutions. The 5-LO
bipartite NLS (RKNLEAIVSVIAERNKKK) was
abbreviated RK ... RNKKK. Seven different mutants
(A-G), containing substitutions of alanine (A)
for arginine (R) or lysine (K) at the indicated
sites within the putative NLS, were transiently expressed in 3T3 and
RAW cells. For 3T3 cells, 5-LO and DNA localization are shown. For RAW
cells, 5-LO localization and differential interference contrast
(DIC) images are shown. Immunoblot analysis of these
constructs expressed in 3T3 cells and probed with an antibody against
GFP is also shown. Results are representative of five independent
experiments. WT, wild type.
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Fig. 6.
Subcellular localization of GFP/5LO fusion
proteins containing glutamine substitutions. 3T3 cells were
transfected with R651Q (A) or K653Q,K654Q (B).
Results are representative of three independent experiments.
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Fig. 7.
Immunofluorescent localization of 5-LO and
5-LO constructs containing specific amino acid substitutions. 3T3
cells were transfected with pcDNA/5LO (A) or the
indicated mutant subcloned into pcDNA3.1 (B-E).
Immunoblot analysis of each construct expressed in 3T3 cells and probed
with an antibody against 5-LO is also shown. Results are representative
of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 8.
Comparison of bipartite NLSs in mammalian
5-LOs. Predicted amino acid sequences of 5-LO from human
(Arg638-Gln656) (30), rat
(Arg637-Lys655) (37), mouse
(Arg638-Lys656) (21), and hamster
(Arg637-Lys655) (38) are shown. The two
clusters of basic amino acids are boxed, and the arginine
residues discussed in the text are underlined.
FOOTNOTES
To whom correspondence should be addressed: 6301 MSRB III, Div. of
Pulmonary and Critical Care Medicine, Dept. of Internal Medicine,
University of Michigan, Ann Arbor, MI 48109-0642. Tel.: (734) 763-9077, Fax: (734) 764-4556, E-mail: brocko@umich.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Drazen, J. M.
(1998)
Am. J. Respir. Crit. Care Med.
158,
S193-S200 2.
Woods, J. W.,
Evans, J. F.,
Ethier, D.,
Scott, S.,
Vickers, P. J.,
Hearn, L.,
Charleson, S.,
Heibein, J. A.,
and Singer, I. I.
(1993)
J. Exp. Med.
178,
1935-1946 3.
Peters-Golden, M.,
and McNish, R.
(1993)
Biochem. Biophys. Res. Commun.
196,
147-153[CrossRef][Medline]
[Order article via Infotrieve]
4.
Woods, J. W.,
Coffey, M. J.,
Brock, T. G.,
Singer, I. I.,
and Peters-Golden, M.
(1995)
J. Clin. Invest.
95,
2035-2040
5.
Brock, T. G.,
McNish, R. W.,
and Peters-Golden, M.
(1995)
J. Biol. Chem.
270,
21652-21658 6.
Dixon, R. A. F.,
Diehl, R. E.,
Opas, E.,
Rands, E.,
Vickers, P. J.,
Evans, J. F.,
Gillard, J. W.,
and Miller, D. K.
(1990)
Nature
343,
282-284[CrossRef][Medline]
[Order article via Infotrieve]
7.
Miller, D. K.,
Gillard, J. W.,
Vickers, P. J.,
Sadowski, S.,
Leveille, C.,
Mancini, J. A.,
Charleson, P.,
Dixon, R. A. F.,
Ford-Hutchinson, A. W.,
Fortin, R.,
Gauthier, J. Y.,
Rodkey, J.,
Rosen, R.,
Rouzer, C.,
Sigal, I. S.,
Strader, C. D.,
and Evans, J. F.
(1990)
Nature
343,
278-281[CrossRef][Medline]
[Order article via Infotrieve]
8.
Abramovitz, M.,
Wong, E.,
Cox, M. E.,
Richardson, C. D.,
Li, C.,
and Vickers, P. J.
(1993)
Eur. J. Biochem.
215,
105-111[Medline]
[Order article via Infotrieve]
9.
Pueringer, R. J.,
Bahns, C. C.,
and Hunninghake, G. W.
(1992)
J. Appl. Physiol.
73,
781-786 10.
Stankova, J.,
Rola-Pleszczynski, M.,
and Dubois, C. M.
(1995)
Blood
85,
3719-3726 11.
Coffey, M. J.,
Wilcoxen, S. E.,
and Peters-Golden, M.
(1994)
Am. J. Respir. Cell. Mol. Biol.
11,
153-158[Abstract]
12.
Pouliot, M.,
McDonald, P. P.,
Borgeat, P.,
and McColl, S. R.
(1994)
J. Exp. Med.
179,
1225-1232 13.
Brock, T. G.,
McNish, R. W.,
Coffey, M. J.,
Ojo, T. C.,
Phare, S. M.,
and Peters-Golden, M.
(1996)
J. Immunol.
156,
2522-2527[Abstract]
14.
Nakamura, T.,
Lin, L. L.,
Kharbanda, S.,
Knopf, J.,
and Kufe, D.
(1992)
EMBO J.
11,
4917-4922[Medline]
[Order article via Infotrieve]
15.
Suzuki, K.,
Yamamoto, T.,
Sato, A.,
Murayama, T.,
Amitani, R.,
Yamamoto, K.,
and Kuze, F.
(1993)
Am. J. Resp. Cell. Mol. Biol.
8,
500-508
16.
Steinhilber, D.,
Hoshiko, S.,
Grunewald, J.,
Radmark, O.,
and Samuelsson, B.
(1993)
Biochim. Biophys. Acta
1178,
1-8[Medline]
[Order article via Infotrieve]
17.
Petersen, M. M.,
Steadman, R.,
and Williams, J. D.
(1992)
Immunology
75,
275-280[Medline]
[Order article via Infotrieve]
18.
Rouzer, C. A.,
and Kargman, S.
(1988)
J. Biol. Chem.
263,
10980-10988 19.
Ochi, K.,
Yoshimoto, T.,
Yamamoto, S.,
Taniguchi, K.,
and Miyamoto, T.
(1983)
J. Biol. Chem.
258,
5754-5758 20.
Brock, T. G.,
Paine, R., III,
and Peters-Golden, M.
(1994)
J. Biol. Chem.
269,
22059-22066 21.
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 22.
Covin, R. B.,
Brock, T. G.,
Bailie, M. B.,
and Peters-Golden, M.
(1998)
Am. J. Physiol.
275,
L303-L310 23.
Brock, T. G.,
McNish, R. W.,
Bailie, M. B.,
and Peters-Golden, M.
(1997)
J. Biol. Chem.
272,
8276-8280 24.
Brock, T. G.,
Anderson, J. A.,
Fries, F. P.,
Peters-Golden, M.,
and Sporn, P. H. S.
(1999)
J. Immunol.
162,
1669-1676 25.
Jans, D. A.,
and Hubner, S.
(1996)
Physiol. Rev.
76,
651-685 26.
Schlenstedt, G.
(1996)
FEBS Lett.
389,
75-79[CrossRef][Medline]
[Order article via Infotrieve]
27.
Gorlich, D.,
and Mattaj, I. W.
(1996)
Science
271,
1513-1518[Abstract]
28.
Dingwall, C.,
and Laskey, R. A.
(1991)
Trends Biochem. Sci.
16,
478-481[CrossRef][Medline]
[Order article via Infotrieve]
29.
Chen, X.-S.,
Zhang, Y.-Y.,
and Funk, C. D.
(1998)
J. Biol. Chem.
273,
31237-31244 30.
Dixon, R. A. F.,
Jones, R. E.,
Diehl, R. E.,
Bennett, C. D.,
Kargman, S.,
and Rouzer, C. A.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
416-420 31.
Kalderon, D.,
Roberts, B. L.,
Richardson, W. D.,
and Smith, A. E.
(1984)
Cell
39,
499-509[CrossRef][Medline]
[Order article via Infotrieve]
32.
Kain, S. R.,
Adams, M.,
Kondepudi, A.,
Yang, T. T.,
Ward, W. W.,
and Kitts, P.
(1995)
BioTechniques
19,
650-655[Medline]
[Order article via Infotrieve]
33.
Bozza, P. T., Yu, W.,
Penrose, J. F.,
Morgan, E. S.,
Dvorak, A. M.,
and Weller, P. F.
(1997)
J. Exp. Med.
186,
909-920 34.
Lepley, R. A.,
Muskardin, D. T.,
and Fitzpatrick, F. A.
(1996)
J. Biol. Chem.
271,
6179-6184 35.
Lepley, R. A.,
and Fitzpatrick, F. A.
(1994)
J. Biol. Chem.
269,
24163-24168 36.
Provost, P.,
Samuelsson, B.,
and Radmark, O.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
1881-1885 37.
Balcarek, J. M.,
Theisen, T. W.,
Cook, M. N.,
Varrichio, A.,
Hwang, S.-M.,
Strohsacker, M. W.,
and Crooke, S. T.
(1988)
J. Biol. Chem.
263,
13937-13941 38.
Kitzler, J. W.,
and Eling, T. E.
(1996)
Prostaglandins Leukotrienes Essent. Fatty Acids
55,
269-277[CrossRef][Medline]
[Order article via Infotrieve]
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