1 School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2
9JT, UK
2 Cancer Research UK Clinical Centre, St. James' Hospital, University of Leeds,
Leeds, LS9 7TF, UK
* Author for correspondence (e-mail: j.h.walker{at}leeds.ac.uk)
Accepted 4 September 2002
![]() |
Summary |
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Key words: Nucleus, Endothelial, EA.hy.926, Cytosolic phospholipase A2-
![]() |
Introduction |
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cPLA2- belongs to a growing family of phospholipase
A2 enzymes that catalyse the hydrolysis of the sn-2
fatty-acyl bond of phospholipids to liberate free fatty acids
(Dennis, 1997
).
cPLA2-
preferentially liberates arachidonic acid and thus is
considered to be the key enzyme in receptor-mediated eicosanoid production.
This 85 kDa calcium-sensitive protein is subject to complex regulation at the
transcriptional and post-translational level
(Clark et al., 1991
). Previous
studies have shown that cPLA2-
is present in the cytosol of
resting cells and relocates to cellular membranes following stimulation with a
variety of agonists (Glover et al.,
1995
; Peters-Golden et al.,
1996
; Schievella et al.,
1995
; Sierra-Honigmann et al.,
1996
). This translocation process is mediated by its
calcium-dependent lipid binding (CaLB) or C2 domain, which promotes binding to
phospholipids upon elevation of intracellular calcium concentrations
(Gijon et al., 1999
).
Several studies have shown that cPLA2- is also subject to
regulation by phosphorylation. Phosphorylation of cPLA2-
on
Ser505 by p38MAPK enhances its intrinsic activity in platelets
(Kramer et al., 1996
); however
this modification is not essential for catalytic activity. In addition, a
MAPK-activated kinase that may be related to MNK1 has been shown to be
responsible for the concomitant phosphorylation on Ser727
(Hefner et al., 2000
). Other
studies in fibroblasts have implicated the p42/44MAP kinases in the
regulation of cPLA2-
(Mitchell et al., 1999
;
Nemenoff et al., 1993
). More
recently, Ca2+/calmodulin-dependent protein kinase II (CaMK II) was
shown to activate cPLA2-
in myeloblastic leukaemia U937
cells (Muthalif et al.,
2001b
). To date, the studies carried out on endothelial cells have
demonstrated that both the p38MAPK and the p42/44MAPK
members of the MAPK family, as well as protein kinase C (PKC) and an unknown
kinase, are involved in cPLA2-
mediated arachidonic acid
release (Gliki et al., 2001
;
Gudmundsdottir et al., 2001
;
Houliston et al., 2001
;
Sa et al., 1995
;
Wheeler-Jones et al.,
1997
).
The effects of phosphorylation on the subcellular location of
cPLA2- have not been investigated. A previous study on human
umbilical vein endothelial cells (HUVECs) demonstrated that the distribution
of cPLA2-
was dependent on cell density, with subconfluent
cells showing increased nuclear localisation of cPLA2-
compared with confluent cells
(Sierra-Honigmann et al.,
1996
). In addition, close inspection of data obtained from the
recent direct labelling of cPLA2-
with green fluorescent
protein (GFP) reveals some degree of nuclear localisation
(Evans et al., 2001
;
Gijon et al., 1999
;
Hirabayashi et al., 1999
;
Hirabayashi and Shimizu,
2000
). Following on from this, we investigate the subcellular
location of cPLA2-
in the EA.hy.926 endothelial cell line
and examine the effects of cell density, protein phosphorylation and
inhibition of nuclear import and export on nuclear localisation.
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Materials and Methods |
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Cell culture
The EA.hy.926 endothelial cell line, a hybrid of human umbilical vein
endothelial cells (HUVECs) and A549 human lung carcinoma epithelial cells, was
a generous gift from C. J. Edgell (University of North Carolina). Cells were
cultured at 37°C in a humid atmosphere containing 5% CO2 in
air. Cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% foetal bovine serum, penicillin (100 U/ml), streptomycin
(100 µg/ml) and HAT (100 µM hypoxanthine, 0.4 µM aminopterin, 16
µM thymidine).
Immunofluorescence microscopy
The method for immunofluorescence microscopy was adapted from Barwise and
Walker (Barwise and Walker,
1996) and Heggeness et al.
(Heggeness et al., 1977
).
Cells were grown on glass coverslips in six-well dishes overnight. Media was
removed, and the cells were washed three times with pre-warmed (37°C) PBS
and fixed in pre-warmed 10% formalin in neutral buffered saline (approximately
4% formaldehyde, Sigma) for 5 minutes. All subsequent steps were performed at
room temperature. After fixation, the cells were permeabilised with 0.1%
Triton X-100 in PBS for 5 minutes and fixed once again for 5 minutes. The
cells were then washed three times with PBS and incubated in sodium
borohydride solution (1 mg/ml in PBS) for 5 minutes. Following three further
PBS wash steps, the cells were blocked in 5% rabbit serum in PBS for 3 hours.
The cells were then incubated with primary antibody (diluted 1:100 into PBS-5%
serum) overnight followed by the appropriate FITC-conjugated secondary for 3
hours. For antigenic adsorption, the antibody was incubated with its
corresponding blocking peptide (1:5 ratio of mg antibody to mg antigen) for 30
minutes at room temperature prior to being added to the cells. The cells were
then washed eight times with PBS and mounted onto slides in Citifluor mounting
medium (Agar Scientific, Hertfordshire, UK).
Confocal imaging
Confocal fluorescence microscopy was performed using a Leica TCS NT
spectral confocal imaging system coupled to a Leica DM IRBE inverted
microscope. Each confocal section was the average of four scans to obtain
optimal resolution, and the system was used to generate individual sections
that were 0.485 µm thick. All figures shown in this study represent 0.485
µm sections taken through the centre of the nucleus and thus do not include
cytosolic staining above and below the nucleus. Measurements of fluorescence
intensity were performed using the integral quantification module of the Leica
TCS NT software as recommended by the manufacturer
(Brokstad et al., 2001).
Briefly, capture levels were first adjusted so as to avoid saturation and then
kept constant throughout experiments. For each section examined, a line was
drawn through the nucleus in a random orientation. The pixel intensity value
along this line (in arbitrary units) was measured and a mean value was
obtained. Values shown in all figures are a mean value of intensity
(n=90)±s.e.m., taken from cell populations investigated over
three independent experiments.
SDS-PAGE and western blotting
Proteins (20 µg per well) were separated on SDS-polyacrylamide gels
using a discontinuous buffer system
(Laemmli, 1970). For western
blot analysis, proteins were transferred to nitrocellulose
(Towbin et al., 1979
).
Subsequently, the nitrocellulose blots were blocked in 5% non-fat milk in
PBS-0.1% Triton X-100 for 1 hour. Primary antibody incubations (1:1000) were
carried out overnight at room temperature, followed by 1 hour incubations with
the appropriate horseradish-peroxidase-conjugated secondary antibody. For
antigenic adsorption, the antibody was incubated with its corresponding
blocking peptide (1:5 ratio of mg antibody to mg antigen) for 30 minutes at
room temperature prior to being incubated with the nitrocellulose blot.
Immunoreactive bands were visualised using an ECL detection kit (Pierce)
according to the manufacturers instructions. Developed films were photographed
and captured using the FujiFilm Intelligent dark Box II with the Image Reader
Las-1000 package. The intensity of the bands was quantified densitometrically
using the AIDA (Advanced Image Data Analyzer) 2.11 software package in
accordance with the manufacturer's instructions. Average band intensities from
three independent experiments and s.e.m.s were calculated.
Preparation of EA.hy.926 cell nuclei
This method was carried out as described previously
(Compton et al., 1976).
Briefly, cells were grown in flasks to the appropriate level of confluency.
Cells were then washed twice with ice-cold PBS, scraped into ice-cold PBS
(containing 1 mM PMSF and 0.1 mg/ml leupeptin) and pelleted by centrifugation
at 160 g for 10 minutes at 4°C. The cells were then
resuspended in a hypotonic solution (Buffer C: 10 mM Tris-HCl, pH 7.5, 1 mM
MgCl2) and allowed to swell on ice for 10 minutes. Cells were then
lysed mechanically with a Dounce homogeniser. The lysate was layered onto 3 ml
of 1.7 M sucrose in Buffer C and centrifuged for 1 hour at 650
g at 4°C. The supernatant was removed, and the nuclear
pellet was resuspended in 1 ml PBS (containing 1 mM PMSF and 0.1 mg/ml
leupeptin). This suspension of nuclei was then layered onto 3 ml of a 2.0 M
sucrose solution and centrifuged for 1 hour at 3000 g at 4°C. The
resultant nuclear pellet was again resuspended in PBS and layered onto a 2.12
M sucrose solution and centrifuged. The final nuclear pellet obtained after
this procedure was analysed by immunofluorescence microscopy and western
blotting. To obtain nuclei from stimulated cells, the procedure was carried
out using a Buffer C that contained 1 mM CaCl2 instead of
MgCl2. For immunofluorescence microscopy, a small volume of the
final suspension of nuclei obtained was placed onto polylysine-coated
microscope slides in a moist chamber and allowed to adhere for 15 minutes. The
slides were then washed three times in PBS before being subjected to the
immunofluorescence microscopy procedure described above.
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Results |
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|
Using this specific antibody, a comparison of the location of
cPLA2- in confluent and subconfluent EA.hy.926 endothelial
cells revealed that a higher level of nuclear staining was present in
subconfluent cells (Fig. 1B).
Measurements of fluorescence intensity across individual cells illustrated a
distinct elevation of fluorescence staining in the region corresponding to the
nucleus (Fig. 1C), in
particular in the case of the subconfluent cells. The detection of a
non-related nuclear protein, NuMA, revealed that the level of nuclear staining
did not vary with cell density (Fig.
1E), indicating that the changes seen for cPLA2-
were specific and proliferation dependent. Secondary antibody controls gave no
staining (data not shown).
Isolation of purified nuclei from subconfluent and confluent EA.hy.926
cells also confirmed the presence of a higher level of nuclear
cPLA2- in subconfluent cells. Immunofluorescence microscopy
of isolated nuclei demonstrated elevated levels of staining in nuclei obtained
from subconfluent cells compared with those obtained from confluent cells
(Fig. 2A). Analysis of nuclei
isolated from A23187-stimulated cells revealed that this pool of nuclear
cPLA2-
relocates to the nuclear membrane following elevation
of intracellular calcium concentrations
(Fig. 2A). Furthermore, western
blots of identical amounts of nuclear fractions confirmed that subconfluent
cells contained a higher proportion of nuclear cPLA2-
than
confluent cells (Fig. 2B).
Analysis of the distribution of the cytosolic marker, lactate dehydrogenase
(LDH) revealed that the nuclear fractions did not contain this protein
(Fig. 2B). Quantification of
the amounts of nuclear cPLA2-
(Fig. 2C) showed that confluent
cells contained approximately half the amount of nuclear
cPLA2-
that subconfluent cells contain (46.2%±7.4
compared with 83.4%±3.3 of the total amount). It was also noted that in
all experiments the total cellular amount of cPLA2-
did not
show any change, indicating that only the subcellular location and not the
overall expression levels of cPLA2-
are dependent on cell
density.
|
Effect of serum starvation on nuclear localisation
The effect of serum starvation on nuclear localisation of
cPLA2- was studied by immunofluorescence microscopy. The
results showed that the level of nuclear staining decreased following the
removal of serum (Fig. 3A).
Quantification of this reduction (Fig.
3B) showed that the intensity of nuclear staining in cells starved
for 48 hours (34.4±1.5 arbitrary units) was approximately half that
observed in control serum-fed cells (63.9±2.1 arbitrary units).
Interestingly, although removal of serum caused a decrease in nuclear
staining, it appeared to cause a change in morphology and an increase in
staining around the periphery of the nucleus, particularly after the longer
period of 48 hours of starvation.
|
In order to confirm that the presence of nuclear cPLA2-
is dependent on factors present in serum, starved cells were re-fed with
complete serum-containing medium for 24 hours. The results
(Fig. 3C) demonstrated that
cPLA2-
in cells that had been starved for 24 or 48 hours
returns to the nucleus upon the addition of serum. Interestingly, the
morphology of the cells returned to normal; however, the
cPLA2-
-enriched cytosolic speckles that arose during the
starvation period did not disappear completely.
The broad range kinase inhibitor, staurosporine, causes a decrease in
the extent of nuclear localisation
Serum factors regulate cell function via phosphorylation of target proteins
by protein kinases (Karin,
1992). In order to determine whether phosphorylation events were
mediating nuclear localisation of cPLA2-
, the effects of the
broad-range kinase inhibitor, staurosporine, were studied using
immunofluorescence microscopy. This cell-permeable inhibitor is known to
inhibit CaM kinase, myosin light chain kinase, protein kinase A, protein
kinase C and protein kinase G. Previous studies on HUVECs using this inhibitor
have shown that it has no effect on histamine-induced arachidonic acid release
(Gudmundsdottir et al., 2001
).
By contrast, studies on Chinese hamster ovary cells demonstrated that
ATP-mediated phosphorylation and activity of cPLA2-
could be
inhibited by pre-treatment with staurosporine
(Lin et al., 1992
). To date,
however, no studies characterising the effects of staurosporine on the
subcellular location of cPLA2-
have been performed.
The results (Fig. 4A)
indicated that pre-treatment of cells with 1 µM staurosporine for 30
minutes led to a decrease in cPLA2- staining within the
nuclei of both subconfluent and confluent cells. Quantification of the
relative intensities demonstrated that in subconfluent cells, staurosporine
treatment reduces the intensity of nuclear staining from 67.0±1.4
arbitrary units to 46.2±1.1 units
(Fig. 4B). A similar 30%
decrease in nuclear staining of confluent cells was evident (from
35.5±1.4 to 25.7±1.7 arbitrary units).
|
The protein phosphatase inhibitor, okadaic acid, increases nuclear
localisation
The results above indicated that cPLA2- nuclear
localisation could be reduced by inhibiting protein phosphorylation. Following
this, the protein phosphatase inhibitor, okadaic acid, was applied to see if
conditions that promote protein phosphorylation would lead to increased
nuclear localisation of cPLA2-
. In previous studies on
coronary endothelial cells (Kan et al.,
1996
) and macrophages (Gijon
and Leslie, 1999
; Qiu et al.,
1998
), okadaic acid has been shown to cause a slight increase in
arachidonic acid release. However, the effect of okadaic acid on the
subcellular localisation of cPLA2-
has not been studied.
Pre-treatment of confluent monolayers of EA.hy.926 cells with 1 µM okadaic acid for 30 minutes led to small but reproducible increases in the level of nuclear staining (Fig. 5A). Quantification of this increase demonstrated that the level of nuclear staining rises by approximately 35% (Fig. 5B). Studies on the effect of okadaic acid on subconfluent cells were also carried out. However, it was noticed that pre-treatment of subconfluent cells with okadaic acid lead to cell death and necrosis. Under these conditions, many of the cells no longer remained attached to the coverslip and of those that did, all were spherical in appearance (data not shown).
|
Effects of the specific MEK and p38MAPK inhibitors,
PD98059 and SB203580, on the nuclear localisation of
cPLA2-
Previous studies have shown that cPLA2- in platelets and
in HeLa cells is phosphorylated on Ser505 and Ser727
(Borsch-Haubold et al., 1998
).
In platelets, p38MAPK has been shown to be responsible for
phosphorylation on Ser505, and it is believed that a MAPK-activated kinase is
involved in phosphorylation at Ser727
(Hefner et al., 2000
). Studies
on endothelial cells also demonstrate the importance of both p38 and p42/44
MAP kinases in the control of arachidonic acid release
(Gudmundsdottir et al., 2001
;
Wheeler-Jones et al., 1997
).
Consequently, the effects of specific inhibitors of the p42/44MAPK
activator, MEK-1, and p38MAPK were studied.
Treatment of subconfluent EA.hy.926 cells with the MEK-1 inhibitor, PD98059
(20 µM), for 30 minutes resulted in a dramatic decrease in the levels of
nuclear cPLA2- (Fig.
6A). Similarly, the intensity of nuclear staining decreased
following treatment with the p38MAPK inhibitor, SB203580
(Fig. 6B). Quantification of
the relevant intensities of nuclear staining
(Fig. 6C) demonstrated that
treatment of cells with PD98059 and SB203580 decreased
cPLA2-
staining in the nucleus by approximately 39% and 36%,
respectively.
|
The data above showed that treatment of cells with the kinase inhibitors,
staurosporine, PD98059 and SB203580, results in a decrease in the intensity of
nuclear cPLA2- staining. In order to demonstrate this
biochemically, nuclei from control cells and cells treated with the inhibitors
were isolated and analysed by western blotting. The result
(Fig. 6C) confirmed that the
level of cPLA2-
in the nucleus decreases dramatically
following treatment with PD98059, SB203580 or staurosporine. Furthermore,
quantification confirmed that these inhibitors caused a decrease of greater
than 50% in the amount of cPLA2-
present within isolated
nuclei (Fig. 6D).
These results demonstrate that the proliferation-dependent nuclear
localisation of cPLA2- is mediated by phosphorylation
events. In particular, both the p42/44 and the p38 MAP kinases appear to be
critical in controlling the levels of cPLA2-
present within
the nucleus.
Agaricus bisporus lectin blocks re-entry of
cPLA2- into the nucleus
In order to determine whether or not the nuclear import of
cPLA2- is dependent on a functional nuclear localisation
signal (NLS), the edible mushroom (Agaricus bisporus) lectin (ABL) was used as
an inhibitor of NLS-dependent import. This Galß1-3GalNAc
(TF
antigen)-binding lectin has been shown to block NLS-dependent protein uptake
into the nucleus without any apparent cytotoxic effects
(Yu et al., 1999
).
Cells were grown on coverslips overnight and serum starved for 24 and 48
hours, which resulted in a decrease in nuclear staining
(Fig. 7A). Consistent with
previous experiments, the levels of nuclear cPLA2- returned
to normal following the addition of serum-containing media. However,
pre-treatment of cells with ABL (20 µg/ml) for 6 hours prior to the
re-addition of serum inhibited the re-entry of cPLA2-
into
the nucleus (Fig. 7A).
Quantification of the relative levels of nuclear staining revealed that
re-feeding following treatment with ABL did not cause a significant increase
in nuclear cPLA2-
(Fig.
7B). This effect suggests that the import of
cPLA2-
into the nucleus is indeed dependent on an
NLS-mediated mechanism.
|
Leptomycin B blocks nuclear export of cPLA2-
Leptomycin B (LMB), an anti-fungal agent, inhibits nuclear export signal
(NES)-dependent nuclear export by binding to CRM1, a NES receptor in the
nuclear pore complex. Using this inhibitor the subcellular location of several
proteins, including actin (Wada et al.,
1998) and phospholipase C-
1
(Yamaga et al., 1999
), has
been shown to be controlled through regulated nuclear export. Hence LMB was
used to assess whether or not cPLA2-
contains a functional
NES. Cells were grown overnight on coverslips and prior to serum starvation,
cells were treated with LMB (10 ng/ml) for 1 hour. The results
(Fig. 8A) indicated that
treatment with LMB inhibits the serum-starvation-induced export of
cPLA2-
from the nucleus. Quantification of the intensity of
nuclear cPLA2-
staining
(Fig. 8B) showed that no
significant decrease in nuclear staining was observed if cells were
pre-treated with LMB prior to serum starvation.
|
![]() |
Discussion |
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Using both confocal immunofluorescence microscopy and western blot analysis
of isolated nuclei, we have shown that cPLA2- is present in
the nuclei of EA.hy.926 endothelial cells. This finding correlates with
previous data on human umbilical vein endothelial cells (HUVECs) presented by
Sierra-Honigmann et al. (Sierra-Honigmann
et al., 1996
), which showed a higher degree of nuclear
localisation in subconfluent cells compared with confluent cells. We have also
investigated the localisation of cPLA2-
in HUVECs and have
seen high levels of nuclear cPLA2-
in these cells (data not
shown). Furthermore, immunofluorescence studies on HeLa and A549 epithelial
cells, which do not exhibit contact-inhibition, also reveal a high degree of
cPLA2-
nuclear localisation in these cell types (data not
shown).
Several recent studies have demonstrated that GFP-cPLA2-
fusion proteins are also localised in the nuclei of cells in culture. Studies
on MDCK, CHO and PtK2 cells show various levels of nuclear
GFP-cPLA2-
(Evans et
al., 2001
; Hirabayashi et al.,
1999
; Perisic et al.,
1999
), confirming that the nuclear staining observed by
immunofluorescence microscopy is not an antibody or fixation artefact.
Interestingly, in some of these studies, cells were maintained in low levels
of serum, perhaps explaining why the levels of nuclear
GFP-cPLA2-
in these cells were comparatively low. In
addition, we have also noted that such GFP-cPLA2-
fusion
proteins reside in the nuclei of EA.hy.926 cells, HeLa cells and HEK 293 cells
(data not shown).
Many previous immunofluorescence microscopy studies on the localisation of
cPLA2- showed little or no nuclear staining. It is possible
that this may be due to the diverse range of cell types studied, since we have
observed cell-specific variation in the extent of nuclear localisation of
cPLA2-
(data not shown). Alternatively, these differences
could be due to the variety of fixation methods employed. In many cases, in
particular the less recent studies (Kan et
al., 1996
; Peters-Golden et
al., 1996
; Schievella et al.,
1995
), methanol and acetone were used to fix and permeabilise the
cells. This method, however, has been shown to result in poor preservation of
cell morphology and can often destroy protein antigens
(Pastan and Willingham, 1985
),
suggesting a possible explanation for the absence of nuclear staining in these
studies. Paraformaldehyde fixation followed by permeabilisation with a
non-ionic detergent, such as Triton X-100, is also often used in
immunofluorescence studies; however, previous work carried out in this
laboratory indicates that prolonged fixation with paraformaldehyde can inhibit
antibody penetration in to the nucleus (data not shown). Some studies on
cPLA2-
(Glover et al.,
1995
; Liu et al.,
2001
) have used fixation periods of up to 30 minutes; hence it is
possible that the absence of nuclear staining in these cases may be due to
overfixing.
A previous study that did report the presence of nuclear
cPLA2- in HUVECs
(Sierra-Honigmann et al.,
1996
) suggested that the nuclear species of
cPLA2-
had a lower Mr of 70,000. No such
species was observed in the study presented here. Interestingly, several more
recent studies have shown that cPLA2-
is subject to
caspase-mediated cleavage during apoptosis
(Adam-Klages et al., 1998
;
Atsumi et al., 1998
). The
resulting 70-80 kDa fragment has been shown to be catalytically inactive and
present solely within the nucleus (Atsumi
et al., 2000
). Following this, it is probable that the low
molecular weight nuclear species observed previously corresponds to a
proteolytic fragment.
We also show here that the pool of cPLA2- present within
the nucleus is capable of relocating to its phospholipid substrate at the
nuclear membrane in a calcium-dependent manner. This would lead to the
generation of nuclear arachidonic acid and arachidonic acid metabolites.
Nuclear localisation of cPLA2- is dependent on
proliferation
The studies carried out here demonstrate that the presence of
cPLA2- within the nuclei of EA.hy.926 endothelial cells is
dependent on cell density. Thus, the nuclei of subconfluent cells were shown
to contain higher levels of cPLA2-
than those of
contact-inhibited confluent cells. The reduced level of nuclear
cPLA2-
in confluent cells could be mimicked by removing
serum from the growth medium of sub-confluent cells, indicating that a growth
factor within the serum is necessary for cPLA2-
nuclear
localisation. In addition to this, immunofluorescence studies on HeLa cells
demonstrated that these highly proliferating cells contained elevated levels
of nuclear cPLA2-
(data not shown), suggesting a possible
link between nuclear cPLA2-
and proliferation.
p38MAPK and p42/44MAPK mediate the nuclear
localisation of cPLA2-
To date, various studies of many cell types have demonstrated that
cPLA2- activity can be enhanced by exposure to diverse
agonists that promote phosphorylation
(Borsch-Haubold et al., 1999
;
Buschbeck et al., 1999
;
de Carvalho et al., 1996
;
Hernandez et al., 1997
;
Kramer et al., 1993
;
Nemenoff et al., 1993
;
Sa et al., 1995
;
Schalkwijk et al., 1995
). The
effects of phosphorylation on the subcellular location of
cPLA2-
, however, have not been examined. Here, studies using
inhibitors that block activation of p42/44MAPK and
p38MAPK demonstrate that the nuclear localisation of
cPLA2-
is dependent on these kinase activities. Vascular
endothelial growth factor (VEGF), which is essential for endothelial cell
growth and differentiation, is known to activate MAPK signalling cascades,
including those involving the p42/44 kinases. In addition, VEGF-induced
mitogenesis, cyclin D1 synthesis and cyclin-dependent kinase 4 activation were
inhibited by PD98059 (Pedram et al.,
1998
); thus activation of MAP kinases by VEGF is likely to play a
central role in the stimulation of endothelial cell proliferation. Although
the precise signalling events that mediate the biological effects of VEGF
remain unclear, it is possible that phosphorylation and nuclear localisation
of cPLA2-
play a role in VEGF-mediated proliferation.
One of the potential cPLA2- phosphorylation sites,
Ser505, lies within the PXSP motif, which represents a consensus MAPK
phosphorylation site. Phosphorylation on this residue enhances the intrinsic
activity of cPLA2-
, leading to increased levels of
Ca2+-induced arachidonic acid release in several different models
(Abdullah et al., 1995
;
Lin et al., 1992
;
Nemenoff et al., 1993
). Using
specific inhibitors in platelets, p38MAPK has been shown to be
responsible for phosphorylation of cPLA2-
on Ser505, and
this causes a decrease in the level of arachidonic acid release
(Kramer et al., 1996
).
Furthermore, transfected cells expressing S505A, S727 or double S505A S727A
mutant cPLA2-
show significantly decreased levels of
arachidonic acid release (Hefner et al.,
2000
). It is possible, however, that phosphorylation at these
sites is also able to control the subcellular location of
cPLA2-
. Alternatively, phosphorylation on the more recently
identified Ser515 (Muthalif et al.,
2001a
), by either p42/44MAPK or p38MAPK, may
be involved in nuclear localisation. Thus, the activation of cellular kinases
by growth factors together with activation of phosphatases induced upon
contact-inhibition would provide complementary mechanisms of regulating the
nuclear compartmentalisation of cPLA2-
.
cPLA2- contains putative nuclear import and export
signals
The nuclear localisation of proteins that are too large to simply cross the
nuclear envelope by diffusion through nuclear pores is dependent on the
presence of NLSs that target the protein specifically to the nucleus. Analysis
of the amino-acid sequence of cPLA2- reveals potential NLSs
(amino acids 54-60: PDSRKRT and amino acids 269-283: PQKVKRYVESLWKKK) that may
be involved in the targeting of this protein. Similarly, a putative NES can
also be identified (amino acids 552-562: LTFNLPYPLIL). The inhibition of
nuclear uptake and export using ABL and LMB, respectively, suggests that the
nuclear compartmentalisation of cPLA2-
is indeed dependent
on such targeting signals; however, the functionality of those signals
suggested above remains to be investigated. One of the suggested NLSs lies
within the C2 domain. Interestingly, it was observed that a GFP-C2 fusion
protein shows a higher degree of nuclear localisation than a full-length
fusion (Evans et al., 2001
;
Perisic et al., 1999
), which
is in agreement with the C2 domain containing a functional NLS. Furthermore,
the proteolytic fragment of cPLA2-
generated during
apoptosis (amino acids 1-522) contains only the two putative NLSs and not the
NES. This caspase-mediated fragment was found to be exclusively intranuclear
(Atsumi et al., 2000
), further
supporting the functionality of the potential NLSs. In addition, analysis of
the 3D structure of cPLA2-
shows that both the potential
NLSs are placed in exposed positions in the cleft between the catalytic and C2
domains (Fig. 9). Since these
two domains are interconnected by a hinge region that has been shown to be
highly flexible (Dessen et al.,
1999
), it is possible that phosphorylation results in rotation of
the two domains, thereby varying the exposure of the NLSs.
Phosphorylation-mediated regulation of such NLSs is a common phenomenon
(reviewed in Jans and Hubner,
1996
); hence it is feasible that phosphorylation of
cPLA2-
regulates not only its activity but also its cellular
localisation.
|
The role of nuclear cPLA2-
The role of nuclear cPLA2- in the EA.hy.926 endothelial
cell line remains unclear. An increasing body of evidence shows that many key
lipids and the enzymes involved in their metabolism reside within the nucleus
(reviewed in Maraldi et al.,
1999
). The nuclear lipid second messengers generated are thought
to play crucial roles in processes such as gene expression, proliferation,
differentiation and apoptosis. With regards to cPLA2-
metabolites, for example, arachidonic acid activates PPAR
(peroxisome
proliferator-activated receptor-
), a member of the PPAR family of
nuclear receptor transcription factors
(Delerive et al., 2000
). These
receptors bind to PPAR response elements (PPREs) and control transcription of
genes involved in fatty acid metabolism and lipid homeostasis. Prostacyclin,
on the other hand, has recently been shown to promote apoptosis by activating
PPAR
(Hatae et al.,
2001
). In addition, activation of the serum response element
(SRE), a primary nuclear target for many diverse signals, has also been shown
to be dependent on cPLA2-
activity
(Oh et al., 2000
). These
findings imply that cPLA2-
and its downstream products play
significant roles in nuclear functions such as regulation of gene expression.
In particular, the products of cPLA2-
action have been
strongly implicated in proliferation. Increased lysophosphatidylcholine
production, for example, has been shown to lead to proliferation of U937 cells
(Muthalif et al., 2001b
).
Increased arachidonic acid levels also have been associated with proliferation
of A549 cells (Croxtall et al.,
1998
; Croxtall et al.,
1996
). The need for nuclear cPLA2-
in
proliferation is further supported by the high levels of nuclear
cPLA2-
observed in cancerous cells, such as HeLa and A549,
which are continually proliferating.
Future perspectives
The elucidation of the mechanisms regulating cPLA2-
nuclear localisation and the exact role of nuclear cPLA2-
requires extensive further studies. Whether or not cPLA2-
is
associated with any accessory proteins or structures within the nucleus also
remains to be investigated. It is possible that entry into the nucleus simply
provides cPLA2-
with an additional pool of phospholipid
substrate. Previous data have shown that actively growing endothelial cells
are able to liberate more arachidonic acid than growth-arrested cells
(Whatley et al., 1994
). Thus,
while endothelial cells are proliferating, for example at sites of wounds,
they can produce elevated levels of prostacyclin, resulting in decreased
platelet activation. By contrast, the nuclear pool of cPLA2-
may be involved more directly in the process of proliferation and control of
gene expression. Hence, the transport of cPLA2-
into the
nuclei of proliferating cells may be a means of controlling gene expression in
these cells. Interestingly, lipid second messengers such as arachidonic acid
and lysophosphatidylcholine have been shown to stimulate proliferation, and
inhibition of cPLA2-
itself results in decreased
proliferation in a number of cell types. The studies presented here suggest
that the growth-regulated compartmentalisation of cPLA2-
within the nucleus may be a mechanism for regulating the levels of such second
messengers within the nucleus, implicating cPLA2-
as a
pivotal enzyme in controlling proliferation. With regards to angiogenesis in
particular, it can be seen that cPLA2-
may be playing a
crucial role in controlling endothelial cell growth. A further understanding
of the regulation of cPLA2-
and the factors that control its
nuclear localisation may be beneficial in controlling angiogenesis in the
growth of tumours.
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References |
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Abdullah, K., Cromlish, W. A., Payette, P., Laliberte, F., Huang, Z., Street, I. and Kennedy, B. P. (1995). Human cytosolic phospholipase A2 expressed in insect cells is extensively phosphorylated on Ser-505. Biochim. Biophys. Acta 1244,157 -164.[Medline]
Adam-Klages, S., Schwandner, R., Luschen, S., Ussat, S., Kreder,
D. and Kronke, M. (1998). Caspase-mediated inhibition of
human cytosolic phospholipase A2 during apoptosis. J.
Immunol. 161,5687
-5694.
Atsumi, G., Murakami, M., Kojima, K., Hadano, A., Tajima, M. and
Kudo, I. (2000). Distinct roles of two intracellular
phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic
fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced
arachidonate release, whereas that of type VI Ca2+-independent
phospholipase A2 augments spontaneous fatty acid release. J. Biol.
Chem. 275,18248
-18258.
Atsumi, G., Tajima, M., Hadano, A., Nakatani, Y., Murakami, M.
and Kudo, I. (1998). Fas-induced arachidonic acid release is
mediated by Ca2+-independent phospholipase A2 but not cytosolic
phospholipase A2, which undergoes proteolytic inactivation. J.
Biol. Chem. 273,13870
-13877.
Barwise, J. L. and Walker, J. H. (1996).
Annexins II, IV, V and VI relocate in response to rises in intracellular
calcium in human foreskin fibroblasts. J. Cell Sci.
109,247
-255.
Borsch-Haubold, A. G., Bartoli, F., Asselin, J., Dudler, T.,
Kramer, R. M., Apitz-Castro, R., Watson, S. P. and Gelb, M. H.
(1998). Identification of the phosphorylation sites of cytosolic
phospholipase A2 in agonist-stimulated human platelets and HeLa cells.
J. Biol. Chem. 273,4449
-4458.
Borsch-Haubold, A. G., Ghomashchi, F., Pasquet, S., Goedert, M.,
Cohen, P., Gelb, M. H. and Watson, S. P. (1999).
Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by
multiple stress-activated protein kinase pathways. Eur. J.
Biochem. 265,195
-203.
Brokstad, K. A., Kalland, K. H., Russell, W. C. and Matthews, D. A. (2001). Mitochondrial protein p32 can accumulate in the nucleus. Biochem. Biophys. Res. Commun. 281,1161 -1169.[CrossRef][Medline]
Buschbeck, M., Ghomashchi, F., Gelb, M. H., Watson, S. P. and Borsch-Haubold, A. G. (1999). Stress stimuli increase calcium-induced arachidonic acid release through phosphorylation of cytosolic phospholipase A2. Biochem. J. 344,359 -366.[CrossRef][Medline]
Carmeliet, P. (2000). Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389-395.[CrossRef][Medline]
Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N. and Knopf, J. L. (1991). A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell 65,1043 -1051.[Medline]
Compton, J. L., Bellard, M. and Chambon, P. (1976). Biochemical evidence of variability in the DNA repeat length in the chromatin of higher eukaryotes. Proc. Natl. Acad. Sci. USA 73,4382 -4386.[Abstract]
Croxtall, J. D., Choudhury, Q. and Flower, R. J. (1998). Inhibitory effect of peptides derived from the N-terminus of lipocortin 1 on arachidonic acid release and proliferation in the A549 cell line: identification of E-Q-E-Y-V as a crucial component. Br. J. Pharmacol. 123,975 -983.[Abstract]
Croxtall, J. D., Choudhury, Q., Newman, S. and Flower, R. J. (1996). Lipocortin 1 and the control of cPLA2 activity in A549 cells. Glucocorticoids block EGF stimulation of cPLA2 phosphorylation. Biochem. Pharmacol. 52,351 -356.[CrossRef][Medline]
de Carvalho, M. G., McCormack, A. L., Olson, E., Ghomashchi, F.,
Gelb, M. H., Yates, J. R., 3rd and Leslie, C. C. (1996).
Identification of phosphorylation sites of human 85-kDa cytosolic
phospholipase A2 expressed in insect cells and present in human
monocytes. J. Biol. Chem.
271,6987
-6997.
Delerive, P., Furman, C., Teissier, E., Fruchart, J., Duriez, P. and Staels, B. (2000). Oxidized phospholipids activate PPARalpha in a phospholipase A2-dependent manner. FEBS Lett. 471,34 -38.[CrossRef][Medline]
Dennis, E. A. (1997). The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22,1 -2.[CrossRef][Medline]
Dessen, A., Tang, J., Schmidt, H., Stahl, M., Clark, J. D., Seehra, J. and Somers, W. S. (1999). Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism. Cell 97,349 -360.[Medline]
Evans, J. H., Spencer, D. M., Zweifach, A. and Leslie, C. C.
(2001). Intracellular calcium signals regulating cytosolic
phospholipase A2 translocation to internal membranes. J.
Biol. Chem. 276,30150
-30160.
Gijon, M. A. and Leslie, C. C. (1999). Regulation of arachidonic acid release and cytosolic phospholipase A2 activation. J. Leukoc. Biol. 65,330 -336.[Abstract]
Gijon, M. A., Spencer, D. M., Kaiser, A. L. and Leslie, C.
C. (1999). Role of phosphorylation sites and the C2 domain in
regulation of cytosolic phospholipase A2. J. Cell
Biol. 145,1219
-1232.
Gliki, G., Abu-Ghazaleh, R., Jezequel, S., Wheeler-Jones, C. and Zachary, I. (2001). Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-delta and by mobilization of intracellular Ca2+. Biochem. J. 353,503 -512.[CrossRef][Medline]
Glover, S., de Carvalho, M. S., Bayburt, T., Jonas, M., Chi, E.,
Leslie, C. C. and Gelb, M. H. (1995). Translocation of the
85-kDa phospholipase A2 from cytosol to the nuclear envelope in rat
basophilic leukemia cells stimulated with calcium ionophore or IgE/antigen.
J. Biol. Chem. 270,15359
-15367.
Gudmundsdottir, I. J., Halldorsson, H., Magnusdottir, K. and Thorgeirsson, G. (2001). Involvement of MAP kinases in the control of cPLA(2) and arachidonic acid release in endothelial cells. Atherosclerosis 156,81 -90.[CrossRef][Medline]
Hatae, T., Wada, M., Yokoyama, C., Shimonishi, M. and Tanabe, T. (2001). Prostacyclin-dependent apoptosis mediated by PPAR {delta}. J. Biol. Chem. 10, 10.
Hefner, Y., Borsch-Haubold, A. G., Murakami, M., Wilde, J. I.,
Pasquet, S., Schieltz, D., Ghomashchi, F., Yates, J. R., 3rd, Armstrong, C.
G., Paterson, A. et al. (2000). Serine 727 phosphorylation
and activation of cytosolic phospholipase A2 by MNK1-related
protein kinases. J. Biol. Chem.
275,37542
-37551.
Heggeness, M. H., Wang, K. and Singer, S. J. (1977). Intracellular distributions of mechanochemical proteins in cultured fibroblasts. Proc. Natl. Acad. Sci. USA 74,3883 -3887.[Abstract]
Hernandez, M., Bayon, Y., Sanchez Crespo, M. and Nieto, M. L. (1997). Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1. Biochem. J. 328,263 -269.[Medline]
Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T., Iino, M.,
Itoh, H. and Shimizu, T. (1999). Critical duration of
intracellular Ca2+ response required for continuous translocation
and activation of cytosolic phospholipase A2. J. Biol.
Chem. 274,5163
-5169.
Hirabayashi, T. and Shimizu, T. (2000). Localization and regulation of cytosolic phospholipase A(2). Biochim. Biophys. Acta 1488,124 -138.[Medline]
Houliston, R. A., Pearson, J. D. and Wheeler-Jones, C. P.
(2001). Agonist-specific cross talk between ERKs and p38(mapk)
regulates PGI(2) synthesis in endothelium. Am. J. Physiol. Cell.
Physiol. 281,C1266
-C1276.
Jans, D. A. and Hubner, S. (1996). Regulation
of protein transport to the nucleus: central role of phosphorylation.
Physiol. Rev. 76,651
-685.
Kan, H., Ruan, Y. and Malik, K. U. (1996). Involvement of mitogen-activated protein kinase and translocation of cytosolic phospholipase A2 to the nuclear envelope in acetylcholine-induced prostacyclin synthesis in rabbit coronary endothelial cells. Mol. Pharmacol. 50,1139 -1147.[Abstract]
Karin, M. (1992). Signal transduction from cell
surface to nucleus in development and disease. FASEB
J. 6,2581
-2590.
Kramer, R. M., Roberts, E. F., Manetta, J. V., Hyslop, P. A. and
Jakubowski, J. A. (1993). Thrombin-induced phosphorylation
and activation of Ca(2+)-sensitive cytosolic phospholipase A2 in
human platelets. J. Biol. Chem.
268,26796
-26804.
Kramer, R. M., Roberts, E. F., Um, S. L., Borsch-Haubold, A. G.,
Watson, S. P., Fisher, M. J. and Jakubowski, J. A. (1996).
p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase
A2 (cPLA2) in thrombin-stimulated platelets. Evidence
that proline-directed phosphorylation is not required for mobilization of
arachidonic acid by cPLA2. J. Biol. Chem.
271,27723
-27729.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Lin, L. L., Lin, A. Y. and Knopf, J. L. (1992). Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. USA 89,6147 -6151.[Abstract]
Liu, J., Takano, T., Papillon, J., Khadir, A. and Cybulsky, A. V. (2001). Cytosolic phospholipase A2-alpha associates with plasma membrane, endoplasmic reticulum and nuclear membrane in glomerular epithelial cells. Biochem. J. 353, 79-90.[CrossRef][Medline]
Maraldi, N. M., Zini, N., Santi, S. and Manzoli, F. A. (1999). Topology of inositol lipid signal transduction in the nucleus. J. Cell Physiol. 181,203 -217.[CrossRef][Medline]
Mitchell, R. A., Metz, C. N., Peng, T. and Bucala, R.
(1999). Sustained mitogen-activated protein kinase (MAPK) and
cytoplasmic phospholipase A2 activation by macrophage migration
inhibitory factor (MIF). Regulatory role in cell proliferation and
glucocorticoid action. J. Biol. Chem.
274,18100
-18106.
Muthalif, M. M., Hefner, Y., Canaan, S., Harper, J., Zho, H.,
Parmentier, J. H., Aebersold, R., Gelb, M. H. and Malik, K. U.
(2001a). Functional interaction of calcium/calmodulin-dependent
protein kinase II and cytosolic phospholipase A2. J.
Biol. Chem. 276,39653
-39660.
Muthalif, M. M., Ljuca, F., Roaten, J. B., Pentapaty, N., Uddin,
M. R. and Malik, K. U. (2001b).
Ca2+/calmodulin-dependent protein kinase II and cytosolic
phospholipase A2 contribute to mitogenic signaling in myeloblastic
leukemia U-937 cells. J. Pharmacol. Exp. Ther.
298,272
-278.
Nemenoff, R. A., Winitz, S., Qian, N. X., van Putten, V.,
Johnson, G. L. and Heasley, L. E. (1993). Phosphorylation and
activation of a high molecular weight form of phospholipase A2 by
p42 microtubule-associated protein 2 kinase and protein kinase C.
J. Biol. Chem. 268,1960
-1964.
Oh, J., Rhee, H. J., Kim, S., Kim, S. B., You, H., Kim, J. H. and Na, D. S. (2000). Annexin-I inhibits PMA-induced c-fos SRE activation by suppressing cytosolic phospholipase A2 signal. FEBS Lett. 477,244 -248.[CrossRef][Medline]
Pastan, I. and Willingham, M. C. (1985). Endocytosis. Plenum Press.
Pedram, A., Razandi, M. and Levin, E. R.
(1998). Extracellular signal-regulated protein kinase/Jun kinase
cross-talk underlies vascular endothelial cell growth factor-induced
endothelial cell proliferation. J. Biol. Chem.
273,26722
-26728.
Perisic, O., Paterson, H. F., Mosedale, G., Lara-Gonzalez, S.
and Williams, R. L. (1999). Mapping the phospholipid-binding
surface and translocation determinants of the C2 domain from cytosolic
phospholipase A2. J. Biol. Chem.
274,14979
-14987.
Peters-Golden, M., Song, K., Marshall, T. and Brock, T. (1996). Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipid hydrolysis. Biochem. J. 318,797 -803.[Medline]
Qiu, Z. H., Gijon, M. A., de Carvalho, M. S., Spencer, D. M. and
Leslie, C. C. (1998). The role of calcium and phosphorylation
of cytosolic phospholipase A2 in regulating arachidonic acid
release in macrophages. J. Biol. Chem.
273,8203
-8211.
Sa, G., Murugesan, G., Jaye, M., Ivashchenko, Y. and Fox, P.
L. (1995). Activation of cytosolic phospholipase
A2 by basic fibroblast growth factor via a p42 mitogen-activated
protein kinase-dependent phosphorylation pathway in endothelial cells.
J. Biol. Chem. 270,2360
-2366.
Schalkwijk, C. G., Spaargaren, M., Defize, L. H., Verkleij, A. J., van den Bosch, H. and Boonstra, J. (1995). Epidermal growth factor (EGF) induces serine phosphorylation-dependent activation and calcium-dependent translocation of the cytosolic phospholipase A2. Eur. J. Biochem. 231,593 -601.[Abstract]
Schievella, A. R., Regier, M. K., Smith, W. L. and Lin, L.
L. (1995). Calcium-mediated translocation of cytosolic
phospholipase A2 to the nuclear envelope and endoplasmic reticulum.
J. Biol. Chem. 270,30749
-30754.
Sierra-Honigmann, M. R., Bradley, J. R. and Pober, J. S. (1996). "Cytosolic" phospholipase A2 is in the nucleus of sub-confluent endothelial cells but confined to the cytoplasm of confluent endothelial cells and redistributes to the nuclear envelope and cell junctions upon histamine stimulation. Lab. Invest. 74,684 -695.[Medline]
Towbin, H., Staehelin, T. and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350 -4354.[Abstract]
Vane, J. R., Anggard, E. E. and Botting, R. M. (1990). Regulatory functions of the vascular endothelium. N Engl. J. Med. 323,27 -36.[Medline]
Wada, A., Fukuda, M., Mishima, M. and Nishida, E.
(1998). Nuclear export of action: a novel mechanism regulating
the subcellular localization of a major cytoskeletal protein. EMBO
J. 17,1635
-1641.
Whatley, E., Satoh, K., Zimmerman, A., Mcintyre, T. M. and Prescott, S. M. (1994). Proliferation-dependent changes in release of arachidonic acid from endothelial cells. J. Clin. Invest. 94,1889 -1990.[Medline]
Wheeler-Jones, C., Abu-Ghazaleh, R., Cospedal, R., Houliston, R. A., Martin, J. and Zachary, I. (1997). Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A2 in endothelial cells via p42/p44 mitogen-activated protein kinase. FEBS Lett. 420, 28-32.[CrossRef][Medline]
Yamaga, M., Fujii, M., Kamata, H., Hirata, H. and Yagisawa,
H. (1999). Phospholipase C-deltal contains a functional
nuclear export signal sequence. J. Biol. Chem.
274,28537
-28541.
Yu, L. G., Fernig, D. G., White, M. R., Spiller, D. G.,
Appleton, P., Evans, R. C., Grierson, I., Smith, J. A., Davies, H.,
Gerasimenko, O. V. et al. (1999). Edible mushroom
(Agaricus bisporus) lectin, which reversibly inhibits epithelial cell
proliferation, blocks nuclear localization sequence-dependent nuclear protein
import. J. Biol. Chem.
274,4890
-4899.