From the Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II, Karolinska Institute, S-171 77 Stockholm, Sweden and the § Max-Planck-Institut für Biochemie, D-82152, Martinsried, Federal Republic of Germany
Received for publication, December 12, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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We have recently identified coactosin-like
protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO)
as a bait. In this report, we demonstrate a direct interaction between
5LO and CLP. 5LO associated with CLP, which was expressed as a
glutathione S-transferase fusion protein, in a
dose-dependent manner. Coimmunoprecipitation experiments
using epitope-tagged 5LO and CLP proteins transiently expressed in
human embryonic kidney 293 cells revealed the presence of CLP in 5LO
immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP
immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis
and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar
stoichiometry in a Ca2+-independent manner. Site-directed
mutagenesis suggested an important role for lysine 131 of CLP in
mediating 5LO binding. In view of the ability of CLP to bind 5LO and
filamentous actin (F-actin), we determined whether CLP could physically
link 5LO to actin filaments. However, no F-actin-CLP·5LO
ternary complex was observed. In contrast, 5LO appeared to compete with
F-actin for the binding of CLP. Moreover, 5LO was found to interfere
with actin polymerization. Our results indicate that the 5LO-CLP and
CLP-F-actin interactions are mutually exclusive and suggest a
modulatory role for 5LO in actin dynamics.
5-Lipoxygenase (5LO)1 is
of central importance in cellular leukotriene (LT) synthesis. This
enzyme converts arachidonic acid released from the membranes by the
cytosolic phospholipase A2 into
5(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and subsequently into the epoxide intermediate LTA4 (1).
LTA4 is further metabolized into LTB4 by the
LTA4 hydrolase or into LTC4 through the action
of the LTC4 synthase. LTC4 is then sequentially degraded into LTD4 and LTE4. Whereas
LTB4 exerts potent stimulatory effects on various leukocyte
functions, including chemotaxis, adhesion, degranulation, and
aggregation, the cysteinyl-LTs (LTC4, LTD4, and
LTE4) are known to contract airway smooth muscle, increase vascular permeability, and promote mucus secretion (2). 5LO and LTs
are, therefore, key components involved in inflammatory disorders,
including arthritis, asthma, and allergic reactions.
Recently, novel modulatory mechanisms determining cellular 5LO activity
were identified. 5LO is phosphorylated by p38 mitogen-activated protein
kinase-activated protein (MAPKAP) kinases prepared from stimulated myeloid cells (3). In addition, Mg2+ increases
5LO activity in vitro (4). Furthermore, a stimulatory Ca2+ binding site has been localized in the N-terminal
domain of 5LO, that may function as a C2 domain in the calcium
regulation of 5LO catalytic activity (5). C2 domains have also been
shown to mediate protein-protein interactions (6).
Additional lines of evidence indicate that cellular 5LO activity and
distribution is regulated by interaction with other proteins. For
example, the subcellular distribution of 5LO differs among cell types
and changes in response to various stimuli. In particular, 5LO
translocates to the nuclear membrane from either the cytosol (for
polymorphonuclear leukocytes) (7) or from inside the nucleus (for
alveolar macrophages) (8). In that perspective, an association of 5LO
with cytoskeletal structures in vivo, which is a reasonable possibility after demonstration of a direct association between 5LO and
actin in vitro (9), could have important implications for
translocation and modulation of cellular 5LO activity.
In our attempt to determine the protein partners of 5LO using the yeast
two-hybrid system, we identified coactosin-like protein (CLP) as a
potential 5LO-interacting protein (10). The CLP nucleotide sequence was
initially found as a sequence flanking a deletion on chromosome 17 characterizing the Smith-Magenis syndrome (11). In a separate paper, we
have characterized CLP as a human filamentous actin (F-actin)-binding
protein.2 Here, we
characterize the CLP-5LO interaction and investigate whether 5LO or the
CLP-5LO tandem is recruited by F-actin.
Cloning, Expression, and Purification of CLP and 5LO--
The
human CLP cDNA was obtained by screening of a human lung cDNA
library using 5LO as a bait (10). The CLP cDNA was amplified by
polymerase chain reaction and cloned in-frame into the
BamHI/XhoI sites of pGEX-5X-1 vector (Amersham
Pharmacia Biotech). The pGEX-5X-1-CLP construct was also subjected to
site-directed mutagenesis, as described in the "site-directed
mutagenesis" section, to generate the CLP mutant K131A.
The pGEX-5X-1-CLP and pGEX-5X-1-CLP K131A constructs as well as the
empty pGEX-5X-1 vector were transformed into the Escherichia coli bacterial strain BL21 (Amersham Pharmacia Biotech) for
expression and purification of the GST-CLP fusion proteins or GST only,
according to the manufacturer's instructions. In some experiments, the
GST-CLP proteins were eluted from the glutathione-Sepharose 4B beads
(Amersham Pharmacia Biotech) with 4 volumes of glutathione (10 mM) in 50 mM Tris-HCl pH 8.0 (elution buffer).
The CLP proteins were then cleaved from the GST moiety with factor Xa
(Amersham Pharmacia Biotech) and purified by anion exchange chromatography.
The E. coli bacterial strain MV1190 was transformed with the
plasmid pT3-5LO and used for expression of 5LO, as described previously (12). The 5LO protein was purified by affinity
chromatography using an ATP-agarose column (12).
Protein Determination--
The protein concentrations were
determined by the method of Bradford (13) using the Bio-Rad dye reagent
with bovine serum albumin (BSA) as standard.
GST Binding Assays--
For binding studies in vitro,
20 µg of the GST-CLP or GST-CLP K131A fusion protein or GST alone,
linked to glutathione-Sepharose 4B beads, was incubated with 0, 0.05, 0.25, and 1 µg of purified 5LO and/or 0, 2, 10, and 20 µg of
F-actin in the presence of BSA (50 µg) in 200 µl of buffer A (2 mM Tris-Cl, 0.2 mM ATP, 0.2 mM CaCl2, and 0.5 mM Mammalian Cell Culture and Transfections--
Human embryonic
kidney 293 (HEK 293) cells were grown in Dulbecco's minimal essential
medium supplemented with 10% fetal bovine serum, 1 mM
sodium pyruvate, 100 units/ml penicillin, and 100 µg/ml streptomycin
in a humidified incubator under 5% CO2 at 37 °C. For
immunoprecipitation experiments, the 5LO cDNA, preceded by a Kozak
consensus sequence (14) and a FLAG epitope
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), was cloned into the pcDNA3.1
mammalian expression vector (Invitrogen), whereas the CLP cDNA was
cloned into the pcDNA3.1/MycHis expression vector (Invitrogen),
which introduced an Myc epitope
(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu) at the C terminus. Plasmid
DNAs were prepared using the Qiagen plasmid DNA purification kit,
precipitated with ethanol, and resuspended in water. The HEK 293 cells
cultured in 100-mm tissue culture dishes were transiently transfected
with 10 µg of each plasmid by the calcium phosphate method. In the
case of transfection with a single plasmid, the total amount of DNA was
adjusted to 20 µg with empty vector.
Immunoprecipitation Experiments--
The HEK 293 cells were
harvested 40 h post-transfection, washed twice with ice-cold
phosphate-buffered saline, and solubilized in 1 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA acid, 0.5% Nonidet P-40) supplemented with
Complete protease inhibitor mix (Roche Molecular Biochemicals). Total
lysates were cleared by centrifugation at 15,000 × g
for 15 min at 4 °C. One-tenth of each cell lysate was kept for
protein determination and analysis by immunoblotting to verify 5LO and
CLP protein expression. Nine-tenths of each cell lysate was incubated
with either anti-Myc monoclonal antibody (9E10) (5 µg/ml; Santa Cruz
Biotechnology), anti-FLAG M2 monoclonal antibody (10 µg/ml; Sigma),
or normal mouse IgG (5 or 10 µg/ml; Santa Cruz Biotechnology) under
continuous rotation for 4 h at 4 °C. GammaBind Plus Sepharose
(Amersham Pharmacia Biotech) was then added, and the incubation was
continued for an additional hour. The immune complexes were washed four
times with 1 ml of lysis buffer and eluted by boiling for 7 min in
Laemmli sample buffer. After centrifugation, the supernatants were
analyzed by immunoblotting, as described below.
Nondenaturing PAGE--
Interaction between 5LO and CLP was
examined by nondenaturing PAGE, as described by Safer et al.
(15). 5LO and CLP were incubated alone or in combination at the
indicated concentrations in buffer A (2 mM Tris, 0.2 mM ATP, 0.2 mM CaCl2, and 0.5 mM
After nondenaturing PAGE of the combination of CLP or CLP K131A and
5LO, the lane was cut out and incubated in SDS sample buffer at room
temperature for 20 min with gentle shaking. The lane was then inserted
on top of a second-dimensional SDS-PAGE gel, and the electrophoresis
was performed in the presence of SDS (0.1%) in the running buffer. The
gels were stained with Coomassie Blue.
Chemical Cross-linking Experiments--
Purified CLP (5 µM) and 5LO (1 µM) proteins were mixed and
incubated for 2 h at room temperature in buffer B (10 mM MOPS, pH 7.0, containing 2 mM
MgCl2 and 50 mM KCl). The reaction mixtures were cross-linked by the addition of 4 mM concentration of
the zero-length cross-linker
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
(Pierce) and 4 mM N-hydroxysulfosuccinimide (sulfo-NHS) (Pierce). After incubation at room temperature, the reactions were quenched by the addition of 20 mM Tris, and
the cross-linked samples were boiled with SDS sample buffer. The
presence of cross-linked CLP·5LO complexes was analyzed on SDS-PAGE
gels and either stained with Coomassie Blue or analyzed by immunoblotting.
F-actin Binding Assay--
For high speed cosedimentation
assays, actin was purified from rabbit skeletal muscle as described by
Rees and Young (16). The GST-CLP fusion, CLP, and 5LO proteins were
purified as described above. After clearing the protein solutions at
100,000 × g for 30 min at 4 °C, the reaction
mixtures were prepared and incubated for 2 h at room temperature
in Beckman centrifuge tubes in the standard polymerization buffer B (10 mM MOPS, pH 7.0, containing 2 mM
MgCl2 and 50 mM KCl). In some experiments, the
reaction mixtures were supplemented with increasing concentrations of
KCl. The samples (100 µl) were then centrifuged at 100,000 × g for 1 h at 4 °C in a Beckman Ultracentrifuge. The
pellet and supernatant of each sample were collected and boiled with
SDS sample buffer. The proteins in the pellet and supernatant fractions
were resolved by SDS-PAGE, and the gels were stained with Coomassie Blue.
Actin Polymerization Assay--
Actin was specifically labeled
at a cysteine residue using acrylodan (Molecular Probes), a
thiol reactive adduct of Prodan (17), as described by Marriott
et al. (18). G-actin-acrylodan was used in a molar ratio of
1:7 with unlabeled G-actin for actin polymerization assays. Neither the
rate nor the extent of polymerization differed significantly between
the native and fluorescent actin (18). The polymerization of G-actin
and G-actin-acrylodan (2 µM) in buffer A in the presence
of 5LO was initiated by the addition of MgCl2 and KCl at
final concentrations of 2 and 50 mM, respectively. In some
experiments, BSA was included or salts were omitted in the assay
mixtures. The progress of the polymerization reaction was monitored for
2 h using a luminescence spectrometer (Aminco-Bowman Series 2).
The excitation wavelength was 400 nm (4-nm bandwidth), and the emission
wavelength was collected through a monochromator at 465 nm (4-nm bandwidth).
Immunoblot Analysis--
Protein suspensions or cell lysates
were fractionated by SDS-PAGE using the Mini-Protean system (Bio-Rad),
transferred to nitrocellulose membranes (Amersham Pharmacia Biotech),
and immunoblotted as described previously (10). A polyclonal anti-human
CLP antiserum2 was raised in rabbits against amino acids
116-130 of human CLP. Monoclonal anti-Myc antibody was from Santa Cruz
Biotechnologies, anti-actin (clone AC-40) antibody was from Sigma, and
rabbit polyclonal 5LO antibody 1551 was used after immunopurification.
Immunoreactive proteins were visualized by using alkaline phosphatase
conjugates and substrates (nitro blue tetrazolium and
5-bromo-4-chloro-3-indolylphosphate).
Yeast Two-hybrid System--
The Gal4 DNA binding
domain vector pGBT9, carrying the TRP1 gene, and the Gal4
DNA-activating domain vector pACT2, carrying the LEU2 gene, were
obtained from CLONTECH. To construct the
vector pGBT9-CLP, the CLP cDNA insert generated by polymerase chain
reaction as a BamHI/XhoI fragment and used for
the pGEX-5X-1-CLP construct, as described above, was cloned in-frame
into the BamHI/SalI restriction sites of pGBT9.
To construct the vector pACT2-5LO, a short
EcoRI/KpnI fragment of the 5LO cDNA from
pT3-5LO (19) was amplified by polymerase chain reaction and cloned
into the EcoRI/KpnI sites of the construct
pAS2-1-5LO (produced by the ligation of the 5LO cDNA from pT3-5LO
into the EcoRI/SalI sites of pAS2-1
(CLONTECH)), and the 5-lipoxygenase cDNA was
cut out as a EcoRI/SalI fragment and then cloned
in-frame into the EcoRI/XhoI sites of the vector pACT2 (CLONTECH). The constructs pACT2- Site-directed Mutagenesis--
Site-directed mutagenesis of the
lysine tandem 130KK131 of CLP, cloned in pGBT9,
was performed by using the QuikChange site-directed mutagenesis kit
(Stratagene) according to the manufacturer's instructions. The desired
mutations were introduced with mutagenic primers, designed with the
help of The Primer Generator (21), that created a
unique SstI, AflII, PstI, or a new
BssHII restriction site in the
130KK131 motif. The conserved
71SKRSK75 motif of CLP was subjected to
site-directed mutagenesis by sequential polymerase chain reaction (22).
The desired mutations were introduced with mutagenic primers that
disabled a unique AvaII restriction site present in the
71SKRSK75 motif. The presence of the mutations
was verified by restriction analysis and confirmed by sequencing the
complete CLP cDNA insert using the DYEnamic ET terminator cycle
sequencing kit (Amersham Pharmacia Biotech) at the KISeq core
facilities (Karolinska Institute, Stockholm, Sweden).
Circular Dichroism Measurements and Analysis--
An AVIV 62DS
circular dichroism spectrometer was used to measure the ellipticity of
samples of purified CLP and CLP K131A at 50 µg/ml in 10 mM sodium phosphate, pH 7.0, using a 1-cm path length cell
thermostatted at 25 °C. For each measurement, four CD spectra were
acquired in the far-UV region from 260 to 200 nm at 0.5-nm intervals
and averaged; the buffer background was then subtracted. Measured
ellipticities in millidegrees were converted into mean residue
ellipticity units and expressed as degrees × cm2 × dmol 5LO and CLP Interact Directly in Vitro--
To study the
interaction between 5LO and CLP in vitro, GST binding
experiments were performed. The GST-CLP fusion protein or GST alone was
expressed in bacteria, coupled to glutathione-Sepharose 4B beads, and
incubated with 0, 0.25, 1.25, and 5.0 µg/ml purified 5LO. After a
30-min incubation, the beads were washed, and the protein complexes
were eluted with glutathione. The presence of 5LO in the eluate was
assayed by SDS-PAGE followed by immunoblot analysis using anti-5LO
antibody. As shown in Fig. 1, no binding of 5LO to the beads coupled to GST alone was detected. However, a
dose-dependent association of 5LO to GST-CLP was observed,
indicating that CLP and 5LO interact directly with each other.
5LO Associates with CLP in Mammalian Cells--
For
coimmunoprecipitation experiments, HEK 293 cells were transfected with
expression plasmids producing a FLAG epitope-tagged 5LO and an Myc
epitope-tagged CLP protein. No CLP (data not shown) or 5LO protein was
found in cells transfected with empty vector only, and no
immunoreactivity to anti-Myc or anti-5LO antibody was observed (Fig.
2A). Transfection of the HEK
293 cells induced the expression of FLAG-tagged 5LO (Fig.
2A) with enzymatic activity similar to that obtained after
transfection with nontagged 5LO (data not shown). Immunoblots also
showed CLP expression in cells transfected with the Myc-tagged CLP
construct; the presence of the Myc tag slightly decreased its
electrophoretic mobility as compared with wild-type CLP. A lysate
fraction of transfected cells was immunoprecipitated by anti-Myc
antibody and subjected to SDS-PAGE. Immunoblotting with the anti-5LO
antibody revealed that 5LO was coimmunoprecipitated with CLP (Fig.
2B). In the reciprocal experiment in which anti-FLAG
antibody was used to immunoprecipitate 5LO, followed by
immunoblotting, CLP coimmunoprecipitated with 5LO (Fig.
2C). No 5LO or CLP was detected in immunoprecipitates prepared with normal mouse IgG (Fig. 2, B and C).
These findings indicate that 5LO forms a complex with CLP in mammalian
cells.
5LO Specifically Interacts with CLP in a
Ca2+-independent Manner--
The 5LO-CLP interaction was
also investigated using nondenaturing PAGE. On this gel, the rate of
migration is determined by the ratio of charge to volume of a protein.
Under the conditions used, purified 5LO migrated as a smeary band with
a low electrophoretic mobility and remained mainly on the top of the
gel (Fig. 3A). CLP had a
greater electrophoretic mobility and migrated as a sharp band in the
middle part of the gel. LTA4 hydrolase, used as a nonbinding control, migrated a little further than CLP, but as a
doublet. When 5LO or CLP was incubated with LTA4 hydrolase, no additional bands or changes in the migration patterns or intensity of the bands was observed. However, when equimolar amounts of CLP and
5LO were mixed, the CLP band disappeared, and the intense 5LO band was
slightly shifted upwards. In the presence of EGTA, the upward shift
remained discernible, although the electrophoretic mobility of free 5LO
was increased.
To confirm that the upward shift observed when CLP was incubated with
5LO is the result of CLP·5LO complex formation, nondenaturing PAGE
was followed by either immunoblotting or SDS-PAGE as a
second-dimensional electrophoresis. Immunoblotting of the nondenaturing
polyacrylamide gels with anti-5LO and anti-CLP antibody indicated the
presence of CLP in the 5LO band (Fig. 3B). As shown in Fig.
3C, analysis of the incubation mixtures by a
second-dimensional SDS-PAGE confirmed the localization of CLP in the
5LO band. The effect of EGTA on the 5LO·CLP complex formation was
further investigated in GST binding experiments. The GST-CLP fusion
protein coupled to glutathione-Sepharose 4B beads were incubated with
increasing amounts of purified 5LO in the presence of calcium (0.2 mM) or in the absence of calcium (with EGTA, 1 mM). As illustrated in Fig. 3D, the presence of EGTA had no influence on 5LO binding to CLP. Taken together, these results indicate an interaction between 5LO and CLP that is
Ca2+-independent.
5LO Binds CLP in a 1:1 Molar Stoichiometry--
The stoichiometry
of the 5LO·CLP complex was determined by titration binding assays on
nondenaturing PAGE. In these assays, a known amount of 5LO was
incubated with increasing amounts of CLP and vice versa, and the ratio
at which unbound CLP appeared or disappeared was determined. When a
fixed amount of CLP was titrated by increasing amounts of 5LO, the
intensity of the CLP band was gradually decreased and disappeared at a
molar ratio of 1:1 or greater (Fig.
4A). When a fixed amount of
5LO was titrated by increasing amounts of CLP, the CLP band became
apparent at a molar ratio greater than 1:1 (Fig. 4B).
In these nondenaturing PAGE experiments, the formation of multiple
equimolar complexes between 5LO and CLP, such as 2:2, 3:3, or higher,
cannot be ruled out. Therefore, the composition of the
CLP·5LO-interacting complex was investigated in chemical
cross-linking experiments. CLP and 5LO were incubated alone or in
combination in the absence or presence of the zero-length cross-linker
EDC and sulfo-NHS, and the samples were analyzed by SDS-PAGE and
Coomassie Blue staining. In the absence of cross-linking agents, CLP
and 5LO migrated as sharp bands at their expected molecular mass (Fig. 4C). No additional band was observed when CLP or 5LO alone
was incubated with EDC/sulfo-NHS for 5 min. However, when CLP was incubated with 5LO in the presence of EDC/sulfo-NHS for 5 min, a new
band of higher molecular mass appeared on the SDS-PAGE gels (Fig.
4C). A time course experiment showed increasing amounts of
this high molecular mass species with time (Fig. 4D).
To ascertain that the newly formed high molecular mass band represents
a covalently linked 5LO·CLP complex, immunoblot analysis using
anti-5LO and anti-CLP antibody was performed. As shown in Fig.
4E, this new band observed on Coomassie Blue-stained gels was immunoreactive with both anti-5LO and anti-CLP antibodies and
migrated at the expected molecular mass for an equimolar 5LO·CLP (1:1) complex. These results indicate that the 5LO·CLP protein complex is composed of 1 molecule of 5LO bound to 1 molecule of CLP.
Role for Lysine 131 of CLP in 5LO Binding--
To identify amino
acid residues of CLP involved in the binding of 5LO, the yeast
two-hybrid system was used. The yeast was cotransformed with 5LO and
various CLP mutant constructs. The entire CLP cDNA insert of at
least one clone per mutated CLP construct was sequenced to confirm the
presence of the intended mutation and the absence of unintended
changes.
All the CLP mutants were able to bind 5LO, except one: the CLP mutant
construct K130A/K131A. This latter CLP mutant interacted with actin but
not with 5LO (Table I). When testing
single-point mutants of CLP, we found that the mutant K130A could still
interact with 5LO, whereas the alanine mutation of lysine 131 prevented 5LO binding. Similar results were obtained when lysine 131 was replaced
by glutamine. Substitution of lysine 131 by arginine maintained the
binding of CLP to 5LO, indicating the requirement of a basic amino acid
residue at position 131.
Based on the observation that a basic amino acid residue is
involved in 5LO binding, another region of CLP containing basic residues was targeted by site-directed mutagenesis: the central 71SKRSK75 motif, in which 3 of the 5 amino
acids are basic residues. Individual substitution of all the residues
of the 71SKRSK75 motif by alanine did not
affect the 5LO binding of CLP.
Analysis of the CLP K131A Mutant Protein in Vitro--
To confirm
the two-hybrid results, the importance of lysine 131 of CLP in 5LO
binding was tested in GST binding experiments and in two-dimensional
gel electrophoresis. For the GST binding assays, the GST-CLP or GST-CLP
K131A fusion protein was expressed in bacteria, coupled to
glutathione-Sepharose 4B beads, and incubated with increasing amounts
of purified 5LO. The dose-dependent association of 5LO with
CLP was markedly reduced by substituting lysine 131 of CLP by an
alanine residue (Fig. 5A).
To exclude any contribution of the GST moiety, the interaction between
purified CLP K131 mutant protein and 5LO was analyzed by
nondenaturing PAGE. When CLP K131A and 5LO were mixed, no
additional bands or other changes in the migration patterns
relative to the individual proteins were observed (Fig.
5B, left panel). In particular, no upward shift
of the 5LO band, suggestive of a protein interaction, was
discernible. Subsequent analysis of the CLP K131A-5LO
incubation mixtures by a second-dimensional SDS-PAGE did not
show colocalization of the two proteins (Fig. 5B,
right panel), thereby confirming the lack of
interaction between the CLP K131A mutant and 5LO. Together, these
observations support an important role for lysine 131 in 5LO binding.
The secondary structure of CLP as well as that of the CLP K131A mutant
was evaluated by circular dichroism spectroscopy, and fractional
composition in terms of 5LO Interferes with the Binding of CLP to F-actin--
A study by
Lepley and Fitzpatrick (9) reports that 5LO interacts with actin
in vitro. In view of the ability of CLP to bind 5LO (this
study) as well as F-actin,2 it was of interest to determine
whether CLP provides a link between F-actin and 5LO.
This issue was first examined in GST binding experiments. The GST-CLP
fusion protein was coupled to glutathione-Sepharose 4B beads and
incubated with various concentrations of F-actin and/or 5LO. Whereas
F-actin and 5LO individually and dose-dependently interacted with CLP, the binding of F-actin to CLP was gradually diminished by increasing concentrations of 5LO (Fig.
6A).
Since the beads could have physically interfered with the formation of
an F-actin-CLP·5LO ternary complex, cosedimentation assays using
purified proteins were performed. In these assays, 5LO was incubated at
increasing concentrations with G-actin in the presence or absence of
CLP, and actin polymerization was induced by the addition of KCl and
MgCl2. After ultracentrifugation, the F-actin-containing
pellet and the supernatant fraction containing the non-pelletizing
actin were analyzed for the presence of 5LO and CLP by SDS-PAGE and
Coomassie Blue staining. As shown in Fig. 6B, inclusion of
CLP in the incubation mixtures did not mediate 5LO binding to F-actin.
Rather, as in the GST binding assays, the absence of an
F-actin-CLP·5LO ternary complex in these experiments unveiled a
mutually exclusive relationship. Binding of CLP to F-actin was observed
in the absence or at low concentrations of 5LO. This CLP-F-actin
association was reduced by equimolar amounts of 5LO (Fig.
6B). In fact, under these conditions, a 5LO·CLP complex, but not a ternary F-actin-CLP·5LO complex, was detected by chemical cross-linking (data not shown).
5LO Inhibits Actin Polymerization--
In cosedimentation assays
performed in the absence of CLP, no significant association of 5LO with
F-actin was found (Fig. 7A).
Instead, 5LO caused an increase in non-pelletizing actin, with a
maximal effect at 1 µM. This effect of 5LO on actin was not influenced by a 5-fold molar excess of BSA relative to 5LO and was
not mimicked by the 5LO buffer only (Fig. 7B).
The observation that 5LO increased the amount of non-pelletizing actin
in cosedimentation assays was further investigated in a
fluorescence-based actin polymerization assay. The polymerization of
acrylodan-labeled G-actin, incubated with increasing concentrations of
5LO, was initiated by the addition of KCl and MgCl2, and
the progress of the polymerization reaction was monitored. As shown in
Fig. 7C, polymerization conditions induced in the control a rapid and gradual increase in fluorescence. The addition of 5LO reduced
the final extent but not the initial rate of actin polymerization; the
degree of inhibition reached about 50% at a 5LO concentration of 1 µM. 5LO buffer alone had no effect on actin
polymerization (data not shown). In a control not subjected to
polymerization conditions, a stable fluorescence signal was observed
(dotted line in Fig. 7C). To rule out the
possibility that the inhibitory effect of 5LO is due to a nonspecific
interaction with other proteins, experiments were performed in the
presence of 2 µM BSA. Although BSA was in an 11-fold
molar excess relative to 5LO, it did not influence the degree of
inhibition; 175 nM 5LO inhibited by about 40% in the
presence and absence of BSA (Fig. 7D), demonstrating that
this effect was specific to the interaction of 5LO with actin.
The cellular activity and localization of 5LO may depend on its
physical interaction with other proteins. To identify interacting proteins, we have used a two-hybrid approach (10). One of the proteins
discovered by this approach, CLP, shows homology to coactosin, an actin
binding protein from Dictyostelium discoideum (24). CLP has
recently been characterized as a human F-actin-binding protein.2 In the present study, we show that 5LO interacts
directly with CLP in vitro and in vivo. In
vitro, the interaction resulted in a heterodimeric complex. The
formation of this complex proved to be independent of Ca2+,
different from the membrane association of 5LO, which is promoted by
Ca2+. We have recently shown that binding of
Ca2+ to the N-terminal domain of 5LO stimulates the
activity of this enzyme (5). Neither
Ca2+-dependent activation nor the membrane
association of 5LO seems to involve CLP. In fact, CLP has no direct
effect on 5LO activity when purified proteins were mixed in
vitro (data not shown).
In a search for amino acid residues involved in the actin binding of
CLP, we found one mutant, CLP K131A, with a strongly reduced capacity
to interact with 5LO. The substitution of lysine 131 by arginine
preserved 5LO binding, indicating the requirement of a basic,
positively charged amino acid residue at position 131. The fact that
the CLP K131A mutant retained its ability to bind actin indicates that
its overall secondary structure was not drastically affected. In
accord, the replacement of lysine 131 by alanine only slightly changed
the circular dichroism spectrum. Lysine 131 thus appears to be part of
the surface interacting with 5LO or to be required in particular for
maintaining the conformation of CLP necessary for 5LO binding.
The regulation of actin polymerization and depolymerization and the
localized assembly of filamentous actin into a network within the
cortex of polymorphonuclear leukocytes are essential for generating the
force necessary for leukocyte locomotion, shape change, phagocytosis,
adhesion, and spreading (25, 26). A connection between 5LO and actin,
as reported previously (9), is therefore of considerable interest. It
raises the possibility that actin is involved in the intracellular
translocation of 5LO, which concomitantly with LT biosynthesis follows
activation of the cells (for review, see Ref. 27). There is evidence
that the activity of 5LO is influenced by the state of actin in
vivo; an up-regulation of
formylmethionylleucylphenylalanine-induced leukotriene generation in
polymorphonuclear leukocytes is observed when the cells were treated
with cytochalasin B (28, 29), which interferes with actin filament
formation. In turn, the catalytic products of 5LO appear to affect the
actin system. For example, monohydroxy acids, including the 5LO
derivate 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid
(5-HETE), are capable of binding cytosolic actin (30). LTB4
induces oscillatory actin polymerization/depolymerization in
polymorphonuclear leukocytes (31, 32), and LTD4 triggers reorganization of the actin network in intestinal epithelial cells (33). Furthermore, leukotrienes have been implicated with the formation of stress fibers in endothelial cells subjected to mechanical stretching (34).
In our experiments, 5LO inhibited the polymerization of actin in
vitro. This inhibitory effect was observed in both cosedimentation and actin polymerization assays. 5LO exerted this effect at
submicromolar concentrations, in accord with the estimated
concentration of monomeric actin (~0.1-0.2 µM) in
equilibrium with polymerized actin (35, 36). These data are compatible
with the possibility that 5LO inhibits actin polymerization by the
sequestration of G-actin. On the other hand, the possibility remains
that 5LO interferes with polymerization by the severing or capping of
actin filaments, as for instance gelsolin does (37). In our actin
polymerization and cosedimentation assays, which provide no evidence
for the incorporation of a putative actin-5LO complex into actin, the binding of 5LO to one or the other end of actin filaments would have
escaped detection. In fact, the finding that inhibition of actin
polymerization by 5LO is not complete and saturates halfway (Fig.
7C), is most easily explained by assuming that 5LO caps the
plus ends of actin filaments, thereby raising the critical concentration of G-actin.
An indirect relationship of 5LO to the actin system is established by
the binding of 5LO to CLP. The question has been addressed whether CLP,
5LO, and actin form a ternary complex. The mutational analysis of CLP
indicated that lysine 131 is important for the binding of 5LO but not
for the binding of actin. On the contrary, lysine 75 is critical for
actin binding but not for the interaction with 5LO.2 Based
on these data, the two binding sites on CLP appear to be distinct.
However, they may be overlapping, or simultaneous binding of 5LO (78 kDa) and actin (42 kDa) to the small CLP molecule (16 kDa) may suffer
from steric hindrance. Our attempts to identify a ternary complex of
5LO, CLP, and filamentous actin were unsuccessful. Rather, data suggest
that 5LO prevents CLP from the interaction with actin. This is relevant
since preliminary experiments suggest that CLP might antagonize the
activity of gelsolin as a capper of the plus ends of actin-filaments
(data not shown), a function reported for coactosin, which interfered
with the actin-capping activity of fragment S1 of severin, a
gelsolin-related protein in Dictyostelium (38). In the
presence of CLP and gelsolin, 5LO may restore the capping activity by
sequestering CLP. Thus, one could speculate that 5LO may inhibit the
polymerization of actin in two ways: by maintaining the activity of
capping proteins in the presence of CLP and by acting itself as an
inhibitor of actin polymerization. Considering the role of 5LO in
vivo, the caveat has to be taken into account that modification of
the proteins involved, such as phosphorylation (3, 39), or the presence of cofactors may turn the inhibitory effect of 5LO into a supporting one.
In conclusion, our results demonstrate that 5LO directly interacts with
CLP. We also show that 5LO inhibits actin polymerization and interferes
with the binding of CLP to F-actin. It may be hypothesized that 5LO,
in addition to its key role in leukotriene synthesis, modulates the
actin dynamics in inflammatory cells, thus representing a novel
regulator of actin function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol, pH 8.0)
supplemented with 2 mM MgCl2 and 50 mM KCl. The effect of EGTA on 5LO·CLP complex formation
was examined in buffer A supplemented with 50 mM KCl. After
a 30-min incubation at room temperature, the beads were washed five
times in incubation buffer, and the GST-CLP fusion proteins or
GST-bound 5LO and/or F-actin complex was eluted with glutathione
elution buffer (10 mM glutathione in 50 mM
Tris-Cl, pH 8.0). The beads were sedimented by centrifugation, and the supernatant was mixed with SDS sample buffer and boiled. The presence of the interacting 5LO and/or actin protein in the supernatant was
assayed by SDS-polyacrylamide gel electrophoresis (PAGE) followed by
immunoblot analysis.
-mercaptoethanol, pH 8.0) for 15 min at room
temperature. LTA4 hydrolase was used as a non-interacting
protein control. The samples were then mixed with native sample buffer,
loaded immediately, and run at 20 mA constant current at 4 °C.
Proteins were then either stained with Coomassie Brilliant Blue or
transferred to nitrocellulose membranes for immunoblot analysis.
-actin
and pACT2-
-actin were obtained by screening of a human lung cDNA
library using human CLP as a bait.2 The constructs
pGBT9-SNF1 and pACT2-SNF4, used as controls, were described previously
(10). The yeast strain PJ69-4A (MATa trp1-901 leu2-3, 112 ura3-52
his3-200 gal4
gal80
LYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) (20) was used to assay protein-protein
interaction, as previously described (10).
1. The fractional composition of the
secondary structure of CLP and CLP K131A in terms of
-helix,
-sheet, and random coil was analyzed using the program K2d
(available on the Internet) (23).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 1.
5LO interacts directly with CLP in
vitro. Twenty micrograms of the GST-CLP fusion
protein or GST alone coupled to glutathione-Sepharose 4B beads were
incubated with 0-1.0 µg of purified 5LO. After elution with 10 mM glutathione and centrifugation, the supernatant was
analyzed and compared with a reference of 5LO (first lane)
by SDS-PAGE and immunoblotting with anti-5LO antibody.
View larger version (40K):
[in a new window]
Fig. 2.
5LO coimmunoprecipitates with CLP.
Epitope-tagged FLAG-5LO and Myc-CLP were expressed in HEK 293 cells.
A, the epitope-tagged Myc-CLP (upper panel) and
FLAG-5LO (lower panel) proteins in the cell lysates (10 µg
of proteins) were detected by immunoblot analysis with anti-Myc and
anti-5LO antibodies, respectively. B, CLP was
immunoprecipitated with anti-Myc antibody (IP anti-Myc) or
normal mouse IgG (last lane). 5LO in the immunoprecipitates
was detected by immunoblot analysis with anti-5LO (IB
anti-5LO). C, 5LO was immunoprecipitated with anti-FLAG
M2 antibody (IP anti-FLAG) or normal mouse IgG (last
lane). Myc-CLP in the immunoprecipitates was detected by
immunoblot analysis with anti-Myc antibody (IB
anti-Myc).
View larger version (29K):
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Fig. 3.
5LO interacts with CLP in a
Ca2+-independent manner. A, nondenaturing
PAGE followed by Coomassie Blue staining. 5LO, CLP, and
LTA4 hydrolase (7.5 µM each) were incubated
alone or in combination in the presence of CaCl2 (0.5 mM) or in absence of Ca2+ (with EGTA 1 mM) (last three lanes). LTA4
hydrolase was used as a noninteracting protein control. B,
nondenaturing PAGE followed by Coomassie Blue staining (CB)
or by immunoblotting using anti-5LO or anti-CLP antibody as indicated.
CLP, 5LO, and the combination of CLP and 5LO (7.5 µM
each) were incubated and subjected to the gels. C,
two-dimensional gel electrophoresis. After nondenaturing PAGE of CLP,
5LO, or a mixture of CLP and 5LO (7.5 µM each), a lane
run in parallel to the third lane was excised, incubated in SDS sample
buffer, and inserted on top of a gel for second-dimensional SDS-PAGE.
Gels were stained with Coomassie Blue. D, GST binding
assays. Twenty micrograms of the GST-CLP fusion protein coupled to
glutathione-Sepharose 4B beads were incubated with 0-1.0 µg of
purified 5LO in the presence of CaCl2 (0.2 mM)
or in absence of Ca2+ (with EGTA 1 mM). After
elution with 10 mM glutathione and centrifugation, the
supernatant was analyzed and compared with a reference of 5LO
(first lane) by SDS-PAGE and immunoblotting with anti-5LO
antibody.
View larger version (26K):
[in a new window]
Fig. 4.
5LO interacts with CLP in a 1:1 molar
stoichiometry. A and B, analysis by
nondenaturing PAGE followed by staining with Coomassie Blue.
A, 5LO (7.5 µM), CLP (7.5 µM),
or incubation mixtures of CLP (7.5 µM) with increasing
concentrations of 5LO (from molar ratio 0.1:1.3). B, CLP (5 µM), 5LO (5 µM), or incubation mixtures of
5LO (5 µM) with increasing concentrations of CLP (from
molar ratio 0.1:2). C and D, CLP (5 µM), 5LO (1 µM), or the combination of CLP
(5 µM) and 5LO (1 µM) was incubated in the
absence or presence of the zero-length cross-linking agents EDC and
sulfo-NHS, as indicated. Gels were stained with Coomassie Blue.
E, CLP (5 µM), 5LO (1 µM), or
the combination of CLP (5 µM) and 5LO (1 µM) was incubated in the presence of EDC/sulfo-NHS and
analyzed by SDS-PAGE followed by either Coomassie Blue (CB)
staining or immunoblotting using anti-5LO or anti-CLP antibody, as
indicated. An additional band of ~32 kDa detected by the anti-CLP
antibody appears to represent a CLP dimer.
-Actin and
-actin were used as positive controls, and
SNF4 was used as a noninteracting protein control.
Lysine 131 of CLP is involved in 5LO binding in the yeast two-hybrid
system
, indicates the absence of yeast
growth at 7 days. In these two-hybrid experiments, actin, a
CLP-interacting protein, and SNF4 served as interacting and
noninteracting controls, respectively. All CLP mutants combined with
actin except for the CLP K75A mutant, indicating that the Gal4 DNA
binding domain moiety of the CLP mutant two-hybrid constructs was
functionally preserved. In addition, all CLP mutants tested in
combination with SNF4 were negative, suggesting that the mutations
introduced in CLP did not induce auto-activation of the HIS3
reporter gene. Similar two-hybrid results were obtained using the Ade2
and LacZ reporter genes (data not shown).
View larger version (31K):
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Fig. 5.
Analysis of the CLP K131A mutant
protein. A, GST binding assays. Twenty micrograms of
the GST-CLP or GST-CLP K131A fusion proteins coupled to
glutathione-Sepharose 4B beads were incubated with 0-1 µg of
purified 5LO. After elution with 10 mM glutathione and
centrifugation, the supernatant was analyzed and compared with a
reference of 5LO (first lane) by SDS-PAGE and immunoblotting
with anti-5LO antibody. B, two-dimensional gel
electrophoresis. Left panel, CLP K131A (5 µM),
5LO (5 µM), or incubation mixtures of CLP K131A and 5LO
(5 µM each) were analyzed by nondenaturing PAGE.
Right panel, a lane run in parallel to the third
lane was excised, incubated in SDS sample buffer, and
inserted on top of a second-dimensional SDS-PAGE. Gels were stained
with Coomassie Blue. C, circular dichroism spectra of CLP
and CLP K131A mutant. Spectra were corrected for buffer background.
Measured ellipticities in millidegrees were converted into mean residue
ellipticity units and expressed as degrees × cm2 × dmol 1.
-helix,
-sheet, and random coil was
estimated. As shown in Fig. 5C, the CD spectra of the CLP
K131A mutant was slightly different from wild-type CLP. Using the
program K2d (23), CLP was predicted to be composed of 9%
-helix,
37%
-sheet, and 54% random coil, whereas the CLP K131A mutant was
found to be composed of 8%
-helix, 44%
-sheet, and 48% random
coil. These results indicate that substitution of lysine 131 by
alanine does not prevent folding of the CLP protein but induces a
subtle change in the secondary structure that might impair the binding
of 5LO.
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Fig. 6.
5LO and F-actin compete rather than cooperate
for the binding of CLP. A, GST binding assays. Twenty
micrograms of the GST-CLP fusion protein were coupled to
glutathione-Sepharose 4B beads and incubated with 0 to 20 µg of
F-actin (left panel) or incubated with 20 µg of F-actin in
the presence of 0 to 20 µg of 5LO (right panel). After
elution with 10 mM glutathione and centrifugation, the
supernatant was analyzed and compared with a reference of actin (first
lane of each panel) by SDS-PAGE and
immunoblotting with anti-actin antibody. B, high speed
cosedimentation experiments. Purified actin and CLP (5 µM) were incubated without or with increasing
concentrations of 5LO at room temperature for 2 h in standard
polymerization buffer (10 mM MOPS, pH 7.0, containing 2 mM MgCl2 and 50 mM KCl). After
centrifugation at 100,000 × g for 1 h, the pellet
(P) fractions, containing F-actin and bound proteins, and
supernatant (S) fractions, containing non-pelletizing actin
and unbound proteins, were analyzed by SDS-PAGE. Gels were stained with
Coomassie Blue.
View larger version (24K):
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Fig. 7.
5LO inhibits actin polymerization.
A and B, high speed cosedimentation experiments.
A, purified G-actin (5 µM) was incubated alone
or with increasing concentrations of 5LO at room temperature for 2 h in standard polymerization buffer. B, purified actin (5 µM), 5LO (1 µM), and BSA (5 µM) were incubated in various combinations at room
temperature for 2 h in standard polymerization buffer. Actin was
also incubated without or with 5LO buffer only under the same
conditions, as indicated. Gels were stained with Coomassie Blue.
C and D, fluorescence-based actin polymerization
assay. Acrylodan-labeled G-actin (2 µM) was polymerized
by the addition of 2 mM MgCl2 and 50 mM KCl. C, polymerization in the absence (5LO
buffer control) or presence of increasing concentrations of 5LO.
Acrylodan-labeled G-actin was also incubated without the addition of
MgCl2 and KCl, i.e. under nonpolymerizing
conditions. D, polymerization in the presence of BSA (2 µM) without or with 5LO (175 nM). The
progress of the polymerization reaction was monitored for 2 h
using a luminescence spectrometer.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Timo Pikkarainen, Alexander Stock, Gerard Marriott, and Patrik Andersson for fruitful discussions and Agneta Nordberg for excellent technical assistance. We are grateful to Philip James for providing the yeast strain PJ69-4A and Jesper Z. Haeggström for providing purified LTA4 hydrolase.
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FOOTNOTES |
---|
* This work was supported by Swedish Medical Research Council Grant 03X-217 and grants from the European Union, the Verum Foundation, the Wallenberg Foundation, and the Deutsche Forschungsgemeinschaft.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.
Recipient of fellowships from the Heart and Stroke Foundation of
Canada and the Medical Research Council of Canada.
¶ To whom correspondence should be addressed. Tel.: 46 8 728 7624; Fax: 46 8 736 0439; E-mail: olof.radmark@mbb.ki.se.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M011205200
2 P. Provost, J. Doucet, A. Stock, G. Gerisch, B. Samuelsson, and O. Rådmark, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: 5LO, 5-lipoxygenase; BSA, bovine serum albumin; CLP, coactosin-like protein; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; F-actin, filamentous actin; G-actin, globular actin; GST, glutathione S-transferase; HEK 293 cells, human embryonic kidney 293 cells; LT, leukotriene; MOPS, 3-(N-morpholino)propanesulfonic acid; NHS, N-hydroxysulfosuccinimide; PAGE, polyacrylamide gel electrophoresis.
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