Stimulation-dependent recruitment of cytosolic phospholipase A2-ato EA.hy.926 endothelial cell membranes leads to calcium-independent association Seema Grewal, Jennifer Smith, Sreenivasa
Trang 1Stimulation-dependent recruitment of cytosolic phospholipase A2-a
to EA.hy.926 endothelial cell membranes leads
to calcium-independent association
Seema Grewal, Jennifer Smith, Sreenivasan Ponnambalam and John Walker
School of Biochemistry and Molecular Biology, University of Leeds, UK
Cytosolic phospholipase A2-a (cPLA2-a) is a
calcium-activated enzyme involved in agonist-induced arachidonic
acid release In endothelial cells, free arachidonic acid is
predominantly converted into prostacyclin, a potent
vaso-dilator and inhibitor of platelet activation As the
rate-lim-iting step in prostacyclin production is the generation of free
arachidonic acid by cPLA2-a, this enzyme has become an
attractive pharmacological target and the focus of many
studies Following stimulation with calcium-mobilizing
agonists, cPLA2-a translocates to intracellular phospholipid
membranes via its C2 domain In this study, the
calcium-induced association of cPLA2-a with EA.hy.926 endothelial
cell membranes was investigated Subcellular fractionation
and immunofluorescence studies showed that following
stimulation with histamine, thrombin or the calcium
ionophore A23187, cPLA2-a relocated to intracellular
membranes Treatment of A23187-stimulated cells with
EGTA or BAPTA-AM demonstrated that a substantial
pool of cPLA2-a remained associated with membrane
frac-tions in a calcium-independent manner Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-a Calcium-independent membrane association of cPLA2-a was not due to hydrophobic or cytoskeletal inter-actions Finally, the recombinant C2 domain of cPLA2-a exhibited calcium-independent membrane binding to mem-branes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-a Such regulatory associations could enable the cell to dis-criminate between the varying levels of cytosolic calcium induced by different stimuli
Keywords: endothelium; cPLA2-a; arachidonic acid; calcium; C2 domain
Cytosolic phospholipase A2-a (cPLA2-a) belongs to a
growing family of phospholipase A2enzymes that catalyse
the hydrolysis of the sn-2 fatty-acyl bond of phospholipids
to liberate free fatty acids [1] In the endothelium, cPLA2-a
plays a pivotal role in releasing free arachidonic acid from
membrane phospholipids This arachidonic acid is the
precursor for prostacyclin, a member of the eicosanoid
family of inflammatory mediators, which acts as a potent
vasodilator and inhibitor of platelet aggregation [2] As the
rate-limiting step in the production of prostacyclin is the
generation of arachidonic acid by cPLA2-a, it can be seen
that cPLA2-a plays a crucial role in several endothelial
functions such as haemostasis, angiogenesis, control of
vascular tone and prevention of thrombosis formation
Consequently, cPLA2-a has become an attractive target for
the development of novel pharmacological therapeutics
against various pathological conditions [3,4] To date, however, the exact mechanisms involved in the control of this important enzyme remain unclear
cPLA2-a is an 85 kDa, calcium-sensitive protein and is subject to regulation at both the transcriptional and post-translational level [5] Early studies on the cloning, expres-sion and purification of cPLA2-a show that purified recombinant cPLA2-a binds to natural membranes in the presence of physiological concentrations of calcium [6] More recently, studies on cultured mammalian cells have shown that cPLA2-a is present in the cytosol of resting cells and relocates to intracellular membranes following stimu-lation with a variety of agonists that cause an increase in cytosolic calcium levels [7–10] In accordance with this, studies on platelets and endothelial cells have shown that this membrane relocation is consistent with an increased PLA2activity in membrane fractions [11,12] Several studies have also shown that cPLA2-a activity in endothelial cells is regulated by phosphorylation [13–15], however the role of these modifications in the translocation and membrane association of cPLA2-a is unclear
The translocation of cPLA2-a to membrane phospho-lipids has been shown to be mediated by its calcium-dependent lipid binding or C2 domain, which promotes binding to phospholipids upon elevation of intracellular calcium concentrations [16] C2 domains are remark-able modules present in over 100 proteins including the
Correspondence to J Walker, School of Biochemistry and Molecular
Biology, University of Leeds, Leeds LS2 9JT, UK.
Fax: + 44 1133433167, Tel.: + 44 1133433119,
E-mail: j.h.walker@leeds.ac.uk.
Abbreviations: cPLA 2 -a, cytosolic phospholipase A2 alpha.
Note: A departmental web site is available at http://www.bmb.
leeds.ac.uk
(Received 3 September 2003, revised 27 October 2003,
accepted 30 October 2003)
Trang 2GTPase-activating protein, phospholipase C and the
syn-aptic vesicle protein, synaptotagmin [17] The Ca2+-binding
properties of these domains allow them to act as
electro-static switches that bind phospholipid membranes in the
presence of calcium without requiring large conformational
changes [18] Based on this, the binding of cPLA2-a to
membranes is believed to be reversible, with translocation
occurring only in the presence of calcium In agreement with
this, binding of cPLA2-a to synthetic liposomes was shown
to be fully reversible, and the addition of an excess of the
calcium chelator, EGTA, abolished binding [19] Recently,
however, in Chinese hamster ovary cells transfected with
GFP-cPLA2-a, there is evidence for a translocation of
cPLA2-a to membranes that persists even after calcium
levels have returned to resting levels [20]
Here, using biochemical subfractionation and
immuno-fluorescence localization studies, we investigated the
relo-cation of cPLA2-a to membranes in EA.hy.926 endothelial
cells Our results demonstrate an association of cPLA2-a
with endothelial cell membranes that is not consistent with a
simple reversible calcium-dependent interaction of cPLA2-a
with phospholipids These results imply that some
mechan-ism, other than simple C2-dependent association with lipids,
must be involved in the regulation of cPLA2-a
Experimental procedures
Materials
Tissue culture media, enzymes and antibiotics were
pur-chased from Gibco BRL (Paisley, Scotland) Goat
poly-clonal antibodies to cPLA2-a were obtained from Santa
Cruz Biotechnology Inc (CA, USA) Secondary fluorescein
isothiocyanate-conjugated secondary antibodies were from
Sigma and anti-goat horseradish peroxidise-conjugated Igs
were from Pierce (Cheshire, UK) All other standard
reagents and chemicals were from Sigma (Poole, Dorset,
UK) or BDH (Poole, Dorset, UK)
Cell culture
The EA.hy.926 cell line, a hybrid of human umbilical vein
endothelial cells (HUVEC) and A549 human lung
carci-noma epithelial cells [21], was a generous gift from C J
Edgell (University of North Carolina, USA) Cells were
cultured on plasticware at 37°C in a humid atmosphere
containing 5% (v/v) CO2 in air Cells were grown in
Dulbecco’s modified Eagles medium supplemented with
10% fetal bovine serum, penicillin (100 UÆmL)1),
strepto-mycin (100 lgÆmL)1) and HAT (100 lM hypoxanthine,
0.4 lMaminopterin, 16 lMthymidine)
Subcellular fractionation
This method was carried out as described previously [22]
and cells were grown to confluence Medium was removed
and the cells were washed twice with prewarmed NaCl/Pi
(Dulbecco A, Oxoid Ltd, Hampshire, UK) For
stimula-tions, the cells were then incubated for the appropriate time
at 37°C with 5 lM A23187 in Hepes/Tyrode’s buffer
containing 1 mMCaCl2 Cells were then washed twice with
NaCl/P, scraped into ice-cold Buffer A (100 mM KCl,
10 mM Pipes, 1 mM NaN3, 1 mM phenylmethanesulfonyl fluoride, 1 mMsodium orthovanadate, 50 mMbenzamidine, 0.1 mgÆmL)1leupeptin) and lysed either by freeze-thawing
or by homogenization with a Dounce homogeniser Veri-fication of lysis was performed using Trypan Blue staining (Sigma) according to the manufacturer’s instructions The cell lysate was centrifuged at 200 000 g for 10 min at 4°C and the resultant soluble cytosolic fraction (C1) was removed The insoluble pellet was washed twice in Buffer
A (generating cytosol washes C2 and C3) and finally solubilized in Buffer B [Buffer A containing 1% (v/v) Triton X-100] Insoluble material was removed by centrifugation and the Triton-soluble membrane fraction (M) was collec-ted Preparations were carried out at various free calcium concentrations by adding the appropriate amounts of CaCl2 and EGTA to Buffer A as determined by theMETLIG pro-gram [23] Equivalent amounts of the subcellular fractions were analysed by SDS/PAGE and Western blotting Preparation of cell cytoskeletons
This method was carried out as described previously [22] Cells were grown to confluence, washed three times with NaCl/Piand then collected by scraping into ice-cold Buffer
B Cytoskeleton fractions were isolated by centrifugation at
200 000 g for 2 h at 4°C The supernatant (representing cytosol and membrane proteins) was removed and the insoluble pellet (representing the cytoskeletal fraction) was solubilized in Laemmli sample buffer Various free calcium concentrations were maintained by adding the appropriate amounts of CaCl2and EGTA to Buffer B, as determined by theMETLIGprogram Equivalent amounts of the fractions were analysed by SDS/PAGE and Western blotting Temperature-induced phase separation of Triton X-114 The separation procedure was carried out as described in
a previous report [24] EA.hy.926 cell membrane fractions were prepared as described above Membrane fractions were resuspended in 1% (w/v) Triton X-114, 150 mMNaCl,
10 mM Tris, 2 lM CaCl2, pH 7.4 Samples were then incubated at 30°C for 10 min and centrifuged at 3000 g for 3 min The aqueous upper phase (representing hydro-philic proteins) was separated from the detergent-rich lower phase (representing hydrophobic proteins) and equivalent amounts of the two were analysed by Western blotting Immunofluorescence microscopy
The method for immunofluorescence microscopy was adapted from previous reported methods [25,26] Cells were grown on glass coverslips overnight Media was removed and the cells were washed three times with prewarmed (to
37°C) NaCl/Piand fixed in prewarmed 10% (v/v) formalin
in neutral buffered saline [approximately 4% (v/v) formal-dehyde, Sigma] for 5 min All subsequent steps were performed at room temperature After fixation, the cells were permeabilized with 0.1% (v/v) Triton X-100 in NaCl/
Pifor 5 min and fixed once again for 5 min The cells were then washed three times with NaCl/Pi and incubated in freshly prepared sodium borohydride solution (1 mgÆmL)1
in NaCl/P) for 5 min to reduce autofluorescence Following
Trang 3three further NaCl/Piwash steps, the cells were blocked in
5% (v/v) rabbit serum in NaCl/Pifor 3 h The cells were
then incubated with primary antibody [diluted 1 : 100 into
NaCl/Pi, 5% (v/v) serum] overnight followed by fluorescein
isothiocyanate-conjugated secondary antibody for 3 h, with
eight NaCl/Pi washes performed in between incubations
Sodium azide (1 mM) was included in all incubations to
prevent bacterial growth The cells were then washed eight
times with NaCl/Pi and mounted onto slides in Citifluor
mounting medium (Agar Scientific, Hertfordshire, UK)
Confocal imaging
Confocal fluorescence microscopy was performed using a
Leica TCS SP 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 The system was used to generate individual
sections that were 0.485 lm thick All figures shown in this
study represent 0.485 lm sections taken through the
nucleus
SDS/PAGE and Western blotting
Proteins (20 lg per well) were separated on
SDS/polyacryl-amide gels using a discontinuous buffer system [27] For
Western blot analysis, proteins were transferred to
nitrocel-lulose [28] Subsequently, the nitrocelnitrocel-lulose blots were
blocked in 5% (w/v) nonfat milk in NaCl/Pi, 0.1% (v/v)
Triton X-100 for 1 h Primary antibody incubations
(1 : 1000) were carried out overnight at room temperature,
followed by 1 h incubations with the appropriate
horse-radish peroxidase-conjugated secondary antibody For
antigenic adsorption, the antibody was incubated with its
corresponding blocking peptide (1 : 5 ratio of lg antibody
to lg antigen) for 30 mins at room temperature prior to
being incubated with the nitrocellulose blot
Immunoreac-tive bands were visualized using an enhanced
chemilumines-cence detection kit (Pierce) according to the manufacturer’s instructions Following this, the 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 general, the average
independent experiments were calculated
C2 domain binding assays The binding assays were based on those described previ-ously [29] Membrane fractions from nonstimulated and A23187-stimulated cells were prepared as described above Membrane fractions (corresponding to approximately
100 lg of total protein) were incubated with 0.1 lg purified C2 domain for 30 min at 30°C The sample was then centrifuged at 200 000 g for 10 min at 4°C to sediment the membranes Any unbound C2 domain in the supernatant was removed whilst any bound material was solubilized
in Buffer B Insoluble material was removed by centri-fugation and the soluble membrane fraction was collected Samples were analysed by SDS/PAGE and Western blotting
Results
CPLA2-a relocates to intracellular membranes following
an elevation in cytosolic calcium concentration The subcellular location of cPLA2-a in EA.hy.926 endo-thelial cells was investigated by immunofluorescence micro-scopy using an antibody that specifically recognizes the a-isoform of cPLA2(Fig 1A, lane 1) Antigenic adsorption
of this antibody with the appropriate blocking peptide abolished detection of cPLA2-a by both Western blotting (Fig 1A, lane 2) and immunofluorescence microscopy (data not shown) Using this specific antibody, a comparison of
Fig 1 Relocation of cPLA 2 -a to intracellular
membranes following A23187-stimulation (A)
cPLA 2 -a was detected by Western blotting of
EA.hy.926 lysates (20 lg protein) using a goat
polyclonal antibody (i) Also shown are
con-trol lanes corresponding to antigen-adsorbed
antibody (ii), and horseradish peroxidase
conjugated anti-(goat IgG) controls (iii) (B)
Cells were grown on coverslips and incubated
with buffer alone (i) or stimulated with 5 l M
A23187 (ii), 10 l M histamine (iii) or 1 UÆmL)1
thrombin (iv) in the presence of 1 m M
extra-cellular calcium for 1 min Cells were then
fixed and permeabilized, and cPLA 2 -a was
detected using immunofluorescence
micros-copy Scale bar, 10 lm.
Trang 4the location of cPLA2-a in resting and stimulated
EA.hy.926 cells was carried out In nonstimulated cells,
cPLA2-a was present throughout the cytosol and the
nucleus (Fig 1B, panel i) Following elevation of the
cytosolic calcium concentration in response to the
physio-logical stimulus histamine or thrombin, or the calcium
ionophore, A23187, a specific relocation of cPLA2-a to
intracellular membranes resembling the endoplasmic
reti-culum and nuclear envelope was evident (Fig 1B, panels
ii–iv) Both secondary antibody and peptide-adsorbed
antibody controls gave no staining (data not shown)
confirming that the staining observed corresponded
specifi-cally to cPLA2-a Measurement of intracellular calcium
concentrations using Fura-2-AM demonstrated that
expo-sure to either 10 lMhistamine, 1 UÆmL)1thrombin or 5 lM
A23187 in the presence of 1 mMextracellular calcium led to
an increase in cytosolic calcium concentration from a resting
value of 100 nMto approximately 1–2 lM(data not shown)
These values were consistent with those obtained from other
studies on endothelial cells [30,31] Based on these findings,
future experiments were performed primarily with A23187,
to avoid the complication of agonist-specific signalling
events
To investigate the calcium-dependency of relocation
further, fractionations were performed under the resting
and elevated calcium levels observed in endothelial cells
Firstly, cytosol and membrane fractions were obtained from
resting cells that were lysed in the presence of various free
calcium concentrations Analysis of the resultant samples
indicated that, with increasing concentrations of free
calcium, an increasing amount of cPLA2-a was found
associated with membranes (Fig 2A) In the complete
absence of calcium, no cPLA2-a was present in the
membrane fraction In contrast, all of the endogenous
cPLA2-a was found to be membrane-bound at calcium
concentrations of 800 nMor above (Fig 2)
Subfractionation experiments were also performed in the
presence of either 100 nM free calcium for resting cells or
2 lM free calcium following A23187 stimulation These
calcium concentrations were representative of the
intracel-lular cytosolic calcium concentration in the absence and
presence of stimulation, respectively (as described above)
The results of these fractionation studies (Fig 3A) indicated
that under resting calcium levels of 100 nM, cPLA2-a was
predominantly cytosolic In contrast, when cells were
stimulated with 5 lMA23187 for 10 min and fractionated
in the presence of 2 lMcalcium, most of the cPLA2-a was
membrane-bound The cytosolic location of lactate
dehy-drogenase confirmed that cells were lysed sufficiently
Quantification of the relative levels of cPLA2-a in the
fractions showed that in resting cells 79.9% ± 6.9 of the
total amount was present in the cytosol with only
19.1% ± 6.0 present in the membrane fraction In contrast,
following stimulation only 28.5% ± 1.9 remained in the
cytosolic fraction whereas 71.5% ± 1.9 was found to be
membrane-associated (Fig 3B)
The association of cPLA2-a with membranes is
EGTA-resistant and time-dependent
To further characterize the binding of cPLA2-a to
endo-thelial cell membranes, the effects of the calcium chelator
EGTA on membrane association were studied Cells were stimulated and fractionated as above, and membrane fractions were washed with 5 mM EGTA The results (Fig 4) show that, as demonstrated above, only approxi-mately 30% of the total amount of cPLA2-a remained cytosolic following stimulation with A23187 and homo-genization in the presence of 2 lM Ca2+
however, only 14.8% ± 2.6 of the total protein could be eluted from the membrane pellet by washing with EGTA, with greater than half the amount of total cPLA2-a (52.5% ± 4.65) remaining tightly associated with the membrane in a manner that resisted extraction with EGTA Similar results were seen following treatment of cells with
10 lMhistamine or 1 UÆmL)1thrombin with minimal loss
of cPLA2-a from stimulated membrane fractions following washing with EGTA (Fig 4C)
The effects of calcium chelation on the subcellular location of cPLA2-a were examined using immunofluores-cence microscopy Cells were stimulated with 5 lMA23187 for various time periods To test the effects of reducing cytosolic calcium levels, cells were stimulated in the same way then the extracellular and intracellular calcium chela-tors, EGTA and BAPTA-AM respectively, were added to the cells
3 After a 1 min stimulation period followed by calcium chelation, a cytosolic staining pattern resembling
Fig 2 Calcium dependencyof membrane binding (A) Cells were grown to confluence in flasks, and scraped into Buffer A containing the free calcium levels indicated The cells were homogenized and fract-ionated into cytosol and membrane Fractions were separated by SDS/ PAGE and Western blotted, and cPLA 2 -a was detected (B) The rel-ative amount of cPLA 2 -a in each fraction was quantified and expressed
as a percentage of the total amount of cPLA 2 -a The amounts in the respective cytosol and membrane fractions were plotted against the corresponding calcium concentration The data is representative of results obtained from three independent experiments.
Trang 5that of a nonstimulated cell was evident (Fig 5A) In
contrast, cells stimulated for 10 min showed a high
proportion of membrane-relocated cPLA2-a that was
resistant to the calcium chelation Consistent with this,
subfractionation of cells directly into EGTA following
A23187-stimulation showed that increased stimulation time
led to an increase in the EGTA-resistant pool of
membrane-bound cPLA2-a (Fig 5B) Under these conditions, more
than 20% of the total cPLA2-a pool was found to be
EGTA-resistant when directly solubilized in a Triton/
EGTA buffer following a 10 min stimulation period
(Fig 5C)
EGTA-resistant membrane binding is not dependent
on a change in hydrophobicity or the cytoskeletal
association of cPLA2-a
It was possible that the binding of calcium to the C2 domain
results in a change in the overall hydrophobicity of the
protein, allowing it to partially insert itself into the lipid
bilayer To address this question, temperature-induced
phase separation of Triton X-114 was performed A
solution of Triton X-114 is homogenous at temperatures
below 20°C Above this temperature, the solution separates into an aqueous phase and a detergent phase Previous studies have shown that integral membrane proteins and proteins with exposed hydrophobic regions partition into the detergent phase [21] Analysis of cPLA2-a in membrane fractions prepared from resting and stimulated cells showed that no change in hydrophobicity occurred, and all the protein was exclusively in the aqueous phase of a Triton X-114 solution (Fig 6A)
The possibility that the observed EGTA-resistant binding of cPLA2-a to membranes was due to a cytoskeletal interaction was investigated Cytoskeleton fractions were isolated by direct solubilization and sedi-mentation from nonstimulated and A23187-stimulated cells The results from these studies showed that there was
no association of cPLA2-a with the cytoskeletal pellet in either resting cells isolated in EGTA or 100 nM calcium,
or in A23187-stimulated cells isolated in 2 lM calcium (Fig 6B)
Fig 3 Calcium-induced relocation of cPLA 2 -a (A) Resting EA.hy.926
cells scraped into 100 n M free calcium buffer, or A23187-stimulated
cells (10 min at 37 °C) scraped into 2 l M free calcium buffer were
homogenized and subfractionated into cytosolic (C) and membrane
(M) fractions, including intermediate wash steps (C2, C3 and M2).
Samples, including total lysates (T), were separated by SDS/PAGE
and Western blotted, and cPLA 2 -a was detected The distribution of
the cytosolic marker, lactate dehydrogenase (LDH) in resting cells was
also determined (B) Quantification of the amount of cPLA 2 -a present
in each of the indicated fractions, expressed as a percentage of the total
amount of cPLA 2 -a (± SEM, n ¼ 3).
Fig 4 EGTA-resistant binding of cPLA 2 -a to EA.hy.926 cell mem-branes (A) Cells were stimulated with 5 l M A23187 for 10 min in the presence of 1 m M extracellular calcium Cells were then scraped into lysis buffer containing 2 l M free calcium, homogenized and subfract-ionated Following removal of the cytosolic fraction, the remaining pellet containing membrane proteins was washed twice in lysis buffer containing 5 m M EGTA The remaining pellet was solubilized in EGTA/Triton X-100 to give the membrane fraction The samples were then immunoblotted to detect cPLA 2 -a (B) Quantification of the amount of cPLA 2 -a present in each of the indicated fractions, expressed as a percentage of the total amount of cPLA 2 -a (± SEM,
n ¼ 3) (C) Subcellular fractionation was also carried out following
10 min stimulation with 1 UÆmL)1thrombin and 10 l M histamine, as described above The amount of cPLA 2 -a in the samples was detected
by Western blotting.
Trang 6The C2 domain of cPLA2-a demonstrates
calcium-independent binding to membranes
To determine whether the C2 domain alone was able to
confer EGTA-resistant membrane binding, in vitro binding
studies of purified recombinant C2 domain to EA.hy.926 cell
membranes were performed The results of these studies
(Fig 7A) show that the calcium dependency of binding of
purified C2 domain to membranes prepared from
nonstim-ulated cells is identical to that of the binding of the
endogenous protein To determine whether this binding
was reversible, membrane fractions containing the bound C2
domain were washed with EGTA As expected, the C2
domain could be removed from nonstimulated membrane
fractions by washing with 5 mM EGTA, and was found
exclusively in the EGTA wash fraction (Fig 7B) To examine
whether any changes occurred in the membrane following
stimulation, studies were also performed using membrane
fractions isolated from A23187-stimulated cells (in the
pres-ence of 2 lMfree calcium) Using this approach, remarkable
differences in the binding properties of the C2 domain
were observed (Fig 7B) In contrast to the data shown
above, only a small proportion of the C2 was able to bind to
the membranes, resulting in a large pool that remained in the
soluble fraction Furthermore, these studies showed that
the protein that did bind could not be removed from the
membrane by EGTA washing, hence was found tightly
associated with the EGTA-resistant membrane fraction
Discussion
To date, the association of cPLA2-a with cellular
mem-branes has been attributed to the calcium-dependent
binding of its C2 domain to membrane phospholipids This
domain promotes the reversible binding of proteins to
phospholipids in the presence of calcium The results shown here, however, demonstrate a novel mode of binding of cPLA2-a to EA.hy.926 cell membranes in a manner that resists extraction with the calcium chelator EGTA Using subcellular fractionation experiments, it was observed that endogenous cPLA2-a binds to EA.hy.926 cell membranes in a calcium-dependent manner At con-centrations below 200 nMthe protein was largely cytosolic, whereas it was completely membrane-associated at physio-logically elevated calcium concentrations of 800 nM and above In resting endothelial cells, the basal levels of arachidonic acid release and prostacyclin production [32] imply that a pool of cPLA2-a is constitutively membrane-associated and catalytically active Not surprisingly there-fore a small proportion of cPLA2-a was found to be associated with a nonstimulated membrane fraction The exact role and nature of this constitutively membrane-associated cPLA2-a requires further investigation
Most interestingly, more than 50% of the total cellular pool of cPLA2-a relocated to membranes following stimu-lation with A23187 and remained associated with a membrane fraction even after extraction with EGTA Similar results were also seen using 10 lM histamine or
1 UÆmL)1thrombin (Fig 4) Immunofluorescence micros-copy also confirmed that membrane-relocated cPLA2-a remained associated with membranes even in the presence
of intracellular and extracellular calcium chelators Fur-thermore, the amount of cPLA2-a present in the EGTA-resistant membrane fraction was seen to increase with stimulation time, suggesting that prolonged activation leads
to membrane association that resists extraction by the removal of calcium These findings are consistent with those published by Hirabayashi and coworkers [20] which suggested that stimulation periods of less than 2 min caused only partial activation and reversible relocation of cPLA-a
Fig 5 Effects of EGTA and BAPTA-AM on the relocation of cPLA 2 -a Cells were stimulated with 5 l M A23187 in the presence of 1 m M
extracellular calcium for the times indicated (A) Cells were fixed and permeabilized, and cPLA 2 -a was detected by fluorescence microscopy For EGTA/BAPTA-AM treatment, cells were stimulated and processed as above However, following stimulation, cells were washed with 5 m M
EGTA/5 m M BAPTA-AM for 5 mins directly prior to fixation Scale bar, 5 lm (B) Cells were treated as indicated, scraped into a 5 m M EGTA buffer, homogenized and subfractionated into cytosolic and membrane fractions and cPLA 2 -a was detected by immunoblotting (C) Quantification
of the amount of cPLA 2 -a present in each of the indicated fractions, expressed as a percentage of the total amount of cPLA 2 -a (± SEM, n ¼ 3).
Trang 7in CHO cells, whereas longer stimulations caused binding
that persisted even after reduction of cytosolic levels of
calcium to resting values This may be a mechanism for
allowing the cell to discriminate appropriate signals from
small transient fluctuations in intracellular calcium
concen-trations Hence, once the calcium transient exceeds a critical
level, a tight-binding state of cPLA2-a could lead to a
continuous membrane localization and arachidonic acid
production for prolonged periods, even after the calcium
levels return to their resting value
A recently published study also demonstrates a prolonged
ionophore-stimulated, perinuclear membrane association of
wild type cPLA2-a for several minutes after the return of
intracellular calcium to unstimulated levels [33] This
phenomenon was seen to be dependent on the
phosphory-lation of S505, which enhanced the hydrophobic interaction
of catalytic domain residues to membrane phospholipids
In support of this, Evans and colleagues [34] demonstrated
that full-length cPLA-a dissociated more slowly from
membranes than the C2-domain alone, also indicating that the catalytic domain may be involved in prolonged mem-brane binding In the present study, however, no change in the overall hydrophobicity of cPLA2-a in ionophore-treated cells was observed by the Triton X-114 phase separation method (Fig 6) However it may be possible that this method is insufficiently sensitive to detect subtle changes in hydrophobicity
Analysis of the calcium dependency of binding of the purified C2 domain of cPLA2-a to EA.hy.926 membranes demonstrated that it exhibited calcium-dependent mem-brane binding properties identical to that of the endogenous full-length protein Interestingly, it was observed that recombinant C2 domain could bind to nonstimulated membranes in a reversible manner, whereas binding to stimulated membranes was EGTA-resistant Most import-antly, it was noticed that only partial binding of the C2 domain to stimulated membranes occurred This raises the possibility that under stimulated conditions, the binding site
Fig 7 In vitro binding of pure re-folded C2 domain to to EA.hy.926 cell membranes (A) Cells were grown to confluence in flasks, scraped into EGTA buffer and fractionated into cytosol and membrane fractions Membrane fractions were incubated with 0.1 lg pure C2 domain at
30 °C for 30 min in the presence of the indicated levels of free calcium Following centrifugation at 200 000 g any unbound protein (soluble) was collected and the pellet (membrane) was solubilized (B) Cell membranes were prepared from resting (in EGTA) and stimulated (5 l M A23187 for 10 min, in 2 l M calcium) cells Membranes were incubated with 0.1 lg pure C2 domain at 30 °C for 30 mins in the presence of 2 l M free calcium Following centrifugation at 200 000 g any unbound protein (soluble) was collected and the membrane pellet was washed in 5 m M EGTA Following a further centrifugation step, the EGTA-elutable fraction was collected (EGTA wash) and the EGTA-resistant cell pellet (membrane) was solubilized in Triton X-100 Fractions were analysed by Western blotting using a mouse anti-(cPLA 2 -a) mAb
Fig 6 Temperature-induced phase separation of Triton X-114 and
extraction of EA.hy.926 cytoskeletons (A) Cytosol and membrane
fractions from nonstimulated cells and A23187-stimulated cells were
prepared in the presence of 100 n M and 2 l M free calcium levels,
respectively The membrane fractions were resuspended in 1% (v/v)
Triton X-114 and temperature-induced phase separation was
per-formed The aqueous and detergent phases were separated and made
up to equal volumes Samples were immunoblotted for cPLA 2 -a (B)
Triton X-100 soluble (S, representing cytosol and membranes) and
insoluble (P, representing cytoskeleton) fractions were prepared from
nonstimulated cells (in EGTA or 100 n M free calcium levels) and cells
stimulated with A23187 for 10 mins (in 2 l M free calcium) Equivalent
amounts of the fractions were immunoblotted for cPLA 2 -a.
Trang 8for cPLA2-a may be partially blocked or saturated by
endogenous cPLA2-a These findings imply that, following
stimulation, the membrane fraction undergoes a change
that allows the anomalous binding of cPLA2-a It is possible
that this may be due to a change in the protein or lipid
composition, implying that some other protein or lipid
interaction is involved in the EGTA-resistant binding of
cPLA2-a to membranes Previous studies have shown that
cPLA2-a is able to bind to ceramide, cholesterol and
phosphatidylinositol 4,5-bisphosphate [35–37] thus it is
possible that such interactions mediate the
calcium-inde-pendent membrane associations observed here This, and
the possible involvement of binding proteins, is further
supported by the observation that cPLA2-a relocates to
specific cellular membranes indicating that a specific
mech-anism for targeting is present In particular there is no
relocation of cPLA2-a to the plasma membrane whereas C2
domains in other proteins (e.g protein kinase C-c [38]),
result in these proteins moving exclusively from the cytosol
to the plasma membrane Overexpression of the C2 domain
of cPLA2-a alone [8] or GFP–C2–cPLA2-a fusion proteins
[34,39] demonstrate that this truncated protein exhibits the
same relocation patterns as the full-length protein,
indica-ting that the targeindica-ting information or mechanism lies within
this region of the protein C2 domains are also known to
mediate protein–protein interactions hence the presence of a
C2 domain in cPLA2-a further supports the possibility that
an accessory protein is involved in the regulation of
cPLA2-a Previous far Western studies identified the
inter-mediate filament protein vimentin as an adaptor protein
that interacts with the C2 domain of cPLA2-a in a
calcium-dependent manner [40] Whether vimentin plays a role in the
calcium-independent association of cPLA2-a with
phos-pholipids remains to be investigated Furthermore, a
grow-ing body of evidence supports the functional couplgrow-ing of
cPLA2-a to its downstream cyclo-oxygenase enzymes,
COX-1 and COX-2 [41,42] It is possible that these enzymes,
which show similar subcellular localization [43,44], may
specifically interact with cPLA2-a and act as accessory
proteins The identification and characterization of these
and/or other binding partners or adapter proteins would
give further insight into the novel mechanism of cPLA2-a
regulation identified here
In conclusion, it has been demonstrated here that cPLA2
-a reloc-ates to cellul-ar membr-anes following elev-ations in
cytosolic free calcium concentration; however, it is able to
remain tightly associated with the membrane in a
calcium-independent manner Prolonged association with
mem-branes, despite a return of cytosolic calcium to resting levels,
could be of physiological significance in prolonging
arachi-donate production in response to cell stimulation These
results indicate that this novel binding is not due simply to
the calcium-dependent lipid binding capacity of the C2
domain, and that some other binding partner or accessory
protein may be involved in the regulation of cPLA2-a
Acknowledgements
This work was funded by the British Heart Foundation and the
BBSRC We thank Dr C J Edgell for the gift of the EA.hy.926 cells,
Dr R Williams for purified C2 domain and Dr E E Morrison for
assistance with confocal imaging.
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