Department of Pharmacology and Centers for Connective Tissue Diseases and Vascular Biology, College of Medicine, The University of Tennessee Center for Health Sciences, Memphis, TN 38163, USA
* Author for correspondence (e-mail: kmalik{at}utmem.edu)
Accepted 29 October 2002
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Summary |
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Key words: Calmodulin, Ca2+Calmodulin-dependent protein kinase II, ein kinase II, Phosphorylation, Dephosphorylation
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Introduction |
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cPLA2 is activated by low levels of calcium (Ca2+)
(Kramer et al., 1991;
Gronich et al., 1990
;
Clark et al., 1990
;
Wijkander and Sundler, 1991
),
which is not directly involved in its catalytic activity but is believed to be
required for its binding to membranes
(Wijkander and Sundler, 1992
;
Nalefski et al., 1994
).
cPLA2 translocates from cytosol to membranes in the presence of
submicromolar Ca2+ concentrations
(Clark et al., 1991
;
Channon and Leslie, 1990
;
Yoshihara and Watanabe, 1990
).
The increase in intracellular Ca2+ triggered by Ca2+
ionophores or agonists such as norepinephrine (NE), angiotensin II,
bradykinin, epidermal growth factor, IgE-antigen, histamine and thrombin
translocates the enzyme from the cytosol to the nuclear envelope or promotes
its association with cell membrane fraction
(Muthalif et al., 1996
;
Muthalif et al., 1998
;
Freeman et al., 1998
;
Schievella et al., 1995
;
Kast et al., 1993
;
Schalkwijk et al., 1995
;
Glover et al., 1995
;
Sierra-Honigmann et al., 1996
;
McNicol and Shibou, 1998
).
Recently, it has been shown that short duration intracellular Ca2+
[Ca2+]i transients translocate cPLA2 to the
Golgi, whereas long [Ca2+]i transients promote its
translocation to Golgi, endoplasmic reticulum and perinuclear membrane
(Evans et al., 2001
). These
observations, and the demonstration that AA-metabolizing enzymes also localize
to the nuclear envelope, suggest that cPLA2 releases AA for
prostanoid production from the membrane phospholipids of the nuclear envelope
and adjacent endoplasmic reticulum (Woods
et al., 1993
; Regier et al.,
1995
; Coffey et al.,
1997
; Serhan et al.,
1996
).
The mechanism by which Ca2+ influx promotes translocation of
cPLA2 is not known. Ca2+ produces several of its
cellular actions, including activation of Ca2+-calmodulin
(CaM)-dependent protein kinase II (CaMKII) and cPLA2, by binding to
CaM (Dupont and Goldbeter,
1998). These observations raise the possibility that CaMKII might
mediate cPLA2 translocation to the nuclear envelope. To test this
hypothesis, we have investigated the effect of inhibitors of CaM and CaMKII on
cPLA2 translocation and its phosphorylation in response to NE and
ionomycin. The present study demonstrates that phosphorylation of
cPLA2 by CaMKII in response to NE mediates its translocation by a
mechanism independent of its catalytic activity and that Ca2+ alone
is not sufficient for cPLA2 translocation to the nuclear envelope
in rabbit vascular smooth-muscle cells (VSMCs).
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Materials and Methods |
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Confocal microscopy and quantitation of confocal images
Cells were viewed by confocal fluorescence microscopy (BioRad MRC-1000,
Laser Scanning Confocal Imaging system using an argon-krypton lamp with a
100x objective lens) with anti-cPLA2 monoclonal antibody as
described (Muthalif et al.,
1996). The original Texas Red confocal images, obtained in red,
were converted to black and white images in Adobe® Photoshop® 7.0.
This made it easier to quantify the density of cPLA2 accumulation
around the nuclear envelope. A fixed number of pixels were quantified in
several cells using NIH image 1.62 and the values were averaged for each
experiment and grouped for different batches of cells for statistical
analysis. Then, the images were converted into pseudocolour in the RGB mode of
Adobe Photoshop 7.0 to enhance the visual appearance of cPLA2
distribution around the nuclear envelope.
Measurement of cytosolic Ca2+ levels
VSMCs were loaded with fura-2 (5 µM for 30 minutes at 37°C) and the
level of cytosolic Ca2+ was determined as described
(Cornwell and Lincoln, 1989).
The effects of NE (10 µM) and ionomycin (1 µM) were determined in the
presence of inhibitors of CaM (10 µM W-7, 1 µM CLMD and 10 µM
E6-B), CaMKII (10 µM KN-93) or their vehicle.
Treatment of cells with phosphatases and phosphatase inhibitors
Cells grown to sub-confluence on slides and arrested for growth were
permeabilized with ß-escin (Sigma, St Louis, MO) as described
(Kobayashi et al., 1989). They
were washed to remove ß-escin, incubated with 0.5 Unit ml-1
phosphatase (alkaline phosphatase, pH 8.0, or potato acid phosphatase, pH 4.8;
Calbiochem, La Jolla, CA) for 15 minutes at 37°C in the presence and
absence of serine/threonine phosphatase inhibitor (1 µM okadaic acid;
Biomol, Plymouth Meeting, PA), allowed to reseal for 1 hour at 37°C, and
then exposed to NE (10 µM), ionomycin (1 µM) or their vehicle for 10
minutes. Cells were washed and processed for confocal microscopy.
Permeabilization and resealing were verified by uptake of Texas-Red-conjugated
bovine serum albumin (Molecular Probes, Eugene, OR). Earlier studies from our
laboratory have shown that VSMCs transiently permeabilized with ß-escin
and resealed maintain the same responsiveness to NE as nonpermeabilized cells
(Nebigil and Malik, 1993
).
Cell viability was also determined with trypan blue exclusion. More than 95%
of the cells treated with phosphatases were viable.
cPLA2 assay
cPLA2 activity was determined from the hydrolysis of substrate
L-[1-14C]arachidonyl phosphatidylcholine (40-100 µM, 200 Ci
mmol-1; American Radiolabeled Chemicals, St Louis, MO) using 25
µg protein from cell lysates as described
(Muthalif et al., 1996).
Phosphorylation and immunoprecipitation of cPLA2
Cells were grown on 100 mm tissue culture dishes to sub-confluence and
arrested for growth. Phosphorylation and immunoprecipitation was performed as
described (Akiba et al., 1995).
Briefly, cells were labelled with 300 µCi ml-1
32P-orthophosphoric acid (Amersham Pharmacia Biotech, Piscataway,
NJ) for 4 hours in phosphate-free Dulbecco's modified Eagle's medium (DMEM)
along with inhibitors and treated with NE (10 µM) or ionomycin (1 µM)
for 10 minutes. The cells were lysed in HEPES buffer containing protease and
phosphatase inhibitors (350 mM sucrose, 1 mM EGTA, 100 µg ml-1
aprotinin and 20 µg ml-1 soybean trypsin inhibitor) and
cPLA2 was immunoprecipitated using anti-cPLA2 monoclonal
antibodies (Genetics Institute, Cambridge, MA). 32P-labelled
cPLA2 immunoprecipitate was subjected to 10% SDS-PAGE. The gel was
dried and the radioactivity was detected by autoradiography.
Immunoblotting
To analyse CaMKII activity, samples (20 µg of protein) were
resolved on 10% SDS-polyacrylamide gels and then transferred to a
nitrocellulose membrane. After blocking with 2% milk and 2% BSA in TTBS for
1-2 hours, the membrane was incubated overnight with
anti-phospho-CaMKII
polyclonal antibody (Santa Cruz, San Diego, CA) at
1:1000 dilution in 20 mM Tris, pH 7.6, 137 mM NaCl and 0.05% Tween (TTBS
buffer) containing 5% BSA, followed by incubation with
anti-goat-IgG-horseradish-peroxidase antibody (1:20,000 dilution in TTBS) for
1 hour at 25°C. The immunoreactive protein was detected using the Amersham
ECL Plus system. CaMKII protein levels were detected using anti-CaMKII goat
polyclonal antibody (Santa Cruz, San Diego, CA).
In vitro phosphorylation of cPLA2 by CaMKII and its
loading in VSMCs
A recent study from our laboratory has shown that CaMKII phosphorylates
recombinant cPLA2 or that immunoprecipitated from VSMC in the
presence but not absence of Ca2+ and CaM
(Muthalif et al., 2001). To
prepare phosphorylated cPLA2 for introduction into VSMCs grown on
slides, 3 µg of recombinant cPLA2 (Genetics Institute,
Cambridge, MA) was incubated for 4 hours with 60 ng of purified rat brain
CaMKII (Calbiochem, La Jolla, CA) and 0.4 µg of CaM (Calbiochem, La Jolla,
CA) at 30°C in kinase buffer containing 20 mM MOPS (pH 7.2), 25 mM
ß-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM
CaCl2, 75 mM MgCl2, and 500 µM ATP in a total volume
of 30 µl. To prepare dephosphorylated cPLA2, 3 µg of
phosphorylated cPLA2 was incubated with 0.5 Unit ml-1
alkaline phosphatase at 37°C for 15 minutes in a total volume of 30 µl
with 20 mM MOPS (pH 8.0). For unphosphorylated cPLA2, 3 µg of
recombinant cPLA2 in 30 µl of 20 mM MOPS (pH 7.2) was used.
Aliquots (0.5 µl) of cPLA2 solutions were used to load VSMC
reversibly permeabilized with ß-escin as described
(Nebigil and Malik, 1993
). The
cells were washed to remove ß-escin, incubated with 0.5 µl of
cPLA2 solution (unphosphorylated, phosphorylated or
dephosphorylated) in the presence of 10 mM
ethyleneglycol-bis(ß-aminoethyl)-N,N,N',N'-tetraacetic
acid (EGTA) and 1 µM
1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic
acid (BAPTA, both from Calbiochem, La Jolla, CA) for 10 minutes, and then
allowed to reseal for 1 hour at 37°C. Permeabilization and resealing were
verified, and the cells were processed for confocal microscopy to determine
the localization of cPLA2 as described above.
The phosphorylation of cPLA2 by CaMKII and its dephosphorylation by alkaline phosphatase were confirmed by gel-shift analysis of cPLA2 protein. A sample (1 µg) of cPLA2 protein was resolved on 20-cm 10% SDS-polyacrylamide gels (1% bisacrylamide, pH 8.3) and then transferred to a nitrocellulose membrane. Immunoblot analysis with anti-cPLA2 polyclonal antibody at 1:2000 dilution was carried out as described above.
In vitro labelling of phosphorylated, unphosphorylated, and
dephosphorylated cPLA2 with fluorescent dye Alexa 488 and its
loading in VSMCs
Phosphorylated, unphosphorylated and dephosphorylated recombinant
cPLA2 were tagged with the fluorescent dye Alexa 488 according to
the manufacturer's protocol (Molecular Probes, Eugene, OR). Briefly, the
recombinant cPLA2 was labelled with the reactive dye and the
unincorporated dye was separated from that incorporated into the protein by
passing through a column packed with the purification resin provided by
Molecular Probes. The labelled cPLA2 protein was quantified, and
0.5 µg was loaded into VSMCs reversibly permeabilized with ß-escin (20
µM) in the presence of 10 mM EGTA and 1 µM BAPTA; its localization in
the cells was viewed by confocal microscopy as described above.
Analysis of data
The density of immunostaining of cPLA2 and its catalytic
activity are expressed as mean ± s.e.m. Data were analysed by one-way
analysis of variance (ANOVA); the Newman-Keuls multiple-range test was used to
determine the difference between multiple groups. The unpaired Student's
t-test was used to determine the difference between two groups. A
value of P<0.05 was considered to be statistically
significant.
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Results |
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|
Effect of inhibitors of CaM and CaMKII on NE-induced cPLA2
translocation to the nuclear envelope in subconfluent cells
Ca2+ is known to produce several of its biological actions by
interacting with CaM, including AA release for prostacyclin synthesis in VSMCs
(Nebigil and Malik, 1993;
Muthalif et al., 1996
). To
investigate the possible contribution of CaM to NE-induced translocation of
cPLA2, the effects of three structurally distinct CaM inhibitors
were examined: CLMD (Gietzen et al.,
1982
), E6-B (Hu et
al., 1992
) and W-7 (Tanaka et
al., 1983
). All three inhibitors blocked NE-induced
cPLA2 translocation to the nuclear envelope from the cytoplasm and
nucleus (Fig. 2). W-5, a
structural analogue of W-7 that does not inhibit CaM activity
(Hidaka et al., 1981
) in
concentrations similar to that of W-7, failed to alter NE-induced
cPLA2 translocation (data not shown).
|
Because CaM activates CaMKII in VSMCs
(Muthalif et al., 2001), we
studied the effect of the CaMKII inhibitor KN-93 and that of an inactive
structural analogue, KN-92 (Fan et al.,
1997
), on NE-induced cPLA2 translocation and
CaMKII
activity. KN-93 (Fig.
3) but not KN-92 (data not shown) inhibited NE-induced
cPLA2 translocation from the cytoplasm and nucleus to the nuclear
envelope. The CaMKII
subtype has been shown to be involved in the
activation of cPLA2 in VSMCs
(Muthalif et al., 1996
). To
determine the possible involvement of CaMKII
in the NE-induced
cPLA2 translocation, the effect of CaMKII
antisense, sense
and scrambled oligonucleotides were tested. Treatment of cells with
CaMKII
antisense (Fig.
3), but not sense or scrambled, oligonucleotides (data not shown)
inhibited NE-induced cPLA2 translocation from the nucleus and
cytoplasm to the nuclear envelope. The concentration of CaMKII
antisense used in these experiments has been previously shown in our
laboratory selectively to deplete CaMKII protein levels in rabbit VSMCs
(Muthalif et al., 1996
). In
sub-confluent VSMCs, ionomycin also caused cPLA2 translocation from
the nucleus and cytoplasm to the nuclear envelope, which was blocked by
inhibitors of CaM (W-7) and CaMKII (KN-93;
Fig. 4) but not by their
corresponding inactive structural analogues (W-5 and KN-92) at similar
concentrations (data not shown). The effect of other CaM inhibitors (CLMD,
E6-B) on ionomycin-induced cPLA2 translocation was not
tested. In confluent VSMCs, the inhibitors of CaM and CaMKII (data not shown)
also blocked VSMC translocation of cPLA2 from the cytoplasm to the
nuclear envelope elicited by NE or ionomycin.
|
|
Effect of inhibitors of CaM, CaMKII and their structural analogues on
CaMKII activity in VSMCs
To determine the selectivity of inhibitors of CaM and CaMKII, the effects
of W-5 and KN-92 (the inactive structural analogues of W-7 and KN-93) on
CaMKII activity was determined. The inhibitors of CaM (W-7) and CaMKII (KN-93)
but not their corresponding structural analogues (W-5 and KN-92) attenuated an
NE- or ionomycin-induced increase in CaMKII activity, as detected from
immunoblot analysis using phospho-CaMKII monoclonal antibodies
(Fig. 5). KN-93 at the 10 µM
concentration used in this study did not alter the activity of NE-induced
increase in phosphorylation of protein kinase C
, another
serine/threonine kinase. Moreover, KN-93 was also effective at concentration
as low as 1 µM at inhibiting NE-induced increase in CaMKII activity and
cPLA2 translocation to the nuclear envelope (data not shown). These
studies and those presented below were performed only on sub-confluent
cells.
|
Effect of CaM and CaMKII inhibitors on NE- and ionomycin-induced
increase in cytosolic Ca2+ and cPLA2 phosphorylation in
VSMCs
To determine whether Ca2+ is required to activate CaMKII to
phosphorylate cPLA2 or directly for cPLA2 translocation
to the nuclear envelope, the effects of NE and ionomycin on cytosolic
Ca2+ and cPLA2 phosphorylation were investigated in the
presence of inhibitors of CaM (W-7) and CaMKII (KN-93). Both NE and ionomycin
increased the level of cytosolic Ca2+, which was not altered by W-7
and KN-93 (Fig. 6A). The CaM
inhibitors E6-B and CLMD also failed to interfere with the NE-or
ionomycin-induced rise in cytosolic Ca2+ (data not shown). NE and
ionomycin increased cPLA2 phosphorylation, which was inhibited in
the absence of extracellular Ca2+ and by W-7 and KN-93, whereas W-5
and KN-92 had no effect on cPLA2 phosphorylation
(Fig. 6B-F).
|
Effect of phosphatases and phosphatase inhibitors on NE-induced
cPLA2 translocation in VSMCs
To assess the contribution of phosphorylation to NE-induced
cPLA2 translocation to the nuclear envelope, alkaline phosphatase
(0.5 Unit ml-1) was introduced to VSMCs by reversibly
permeabilizing them with ß-escin in the presence or absence of the
phosphatase inhibitor okadaic acid (1 µM). Earlier studies from our
laboratory have shown that VSMCs transiently permeabilized and resealed
maintained the same responsiveness to NE as nonpermeabilized cells
(Nebigil and Malik, 1993).
Okadaic acid alone did not cause cPLA2 translocation to the nuclear
envelope. Treatment with alkaline phosphatase in the absence, but not the
presence, of okadaic acid blocked NE-induced cPLA2 translocation
(Fig. 7A,B), suggesting that
Ca2+-induced phosphorylation of cPLA2 requires its
translocation to the nuclear envelope. Similar results were obtained with
potato acid phosphatase (data not shown).
|
NE and okadaic acid increased cPLA2 activity about fourfold over their vehicle controls. The activity of cPLA2 in response to NE was further enhanced in the presence of okadaic acid. Alkaline phosphatase reduced cPLA2 activity in both the absence and presence of NE; the reduction was minimized in the presence of okadaic acid. However, NE in the presence of okadaic acid and alkaline phosphatase caused the same order of increase in cPLA2 activity as NE alone (Fig. 7C).
NE-induced cPLA2 phosphorylation was also increased in the presence of okadaic acid. Alkaline phosphatase inhibited NE-induced cPLA2 phosphorylation, which was restored in the presence of okadaic acid (Fig. 7D,E). Alkaline phosphatase also inhibited ionomycin-induced phosphorylation of cPLA2, which was restored in the presence of okadaic acid (data not shown). Treatment of cells with alkaline phosphatase also caused dephosphorylation of CaMKII, which was prevented by okadaic acid (data not shown). Therefore, it appears that phosphorylation caused by an increase in cytosolic Ca2+ in response to NE and ionomycin is required both for cPLA2 activation and for its translocation to the nuclear envelope in VSMCs.
Effect of inhibitors of cPLA2 activity on NE-induced
cPLA2 translocation in VSMCs
To determine whether both phosphorylation and catalytic activity of
cPLA2 are required for NE-induced translocation to the nuclear
envelope, VSMCs were treated with two different inhibitors of cPLA2
catalytic activity AACOCF3 (10 µM) and MAFP (10 µM)
and the effects of NE on cPLA2 translocation, activity and
phosphorylation were examined. Neither inhibitor prevented NE from causing
cPLA2 translocation to the nuclear envelope
(Fig. 8A,B); the inhibitors
attenuated cPLA2 activity (Fig.
8C) but failed to alter NE-induced cPLA2
phosphorylation (Fig. 8D,E).
These observations indicate that the phosphorylation, but not the catalytic
activity of cPLA2, is essential for its translocation to the
nuclear envelope from the cytosol and nucleus in VSMCs.
|
Distribution of exogenous phosphorylated, unphosphorylated, and
dephosphorylated cPLA2 in VSMCs
Phosphorylation of recombinant cPLA2 by CaMKII and its
dephosphorylation by alkaline phosphatase was confirmed by its gel shift
(Fig. 9A). In VSMCs loaded with
cPLA2 after reversible permeabilization with ß-escin in the
presence of EGTA and BAPTA, the phosphorylated, but not unphosphorylated or
dephosphorylated, form of cPLA2, as detected by immunofluorescence
(Fig. 9B) or by conjugation to
Alexa 488 (Fig. 9C), was found
to accumulate around the nucleus.
|
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Discussion |
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In sub-confluent cells, cPLA2 (as detected by immunostaining)
was distributed in both the cytosol and the nucleus. However, in confluent
VSMCs, cPLA2 was localized primarily to the cytosol. Exposure to NE
or ionomycin of both sub-confluent and confluent cells caused accumulation of
cPLA2 in the perinuclear region and the nuclear envelope. Similar
distribution of cPLA2 in sub-confluent and confluent endothelial
cells, and its translocation to the perinuclear region and the nuclear
envelope in response to histamine have been reported
(Sierra-Honigmann et al.,
1996). Ca2+ has been shown to be essential for the
binding of cPLA2 to phospholipid vesicles or membranes
(Clark et al., 1991
;
Wijkander and Sundler, 1992
;
Nalefski et al., 1994
;
Channon and Leslie, 1990
;
Yoshihara and Watanabe, 1990
).
The enzyme contains an N-terminal Ca2+-dependent
phospholipid-binding domain (CaLB) that is believed to bind Ca2+
and to promote the attachment of cPLA2 to membranes
(Nalefski and Falke, 1998
;
Zhang et al., 1996
;
Xu et al., 1998
); deletion of
this but not the C-terminal domain prevents its membrane binding
(Nalefski et al., 1994
). In
our study, in the absence of extracellular Ca2+, NE and ionomycin
failed to cause translocation of cPLA2 to the nuclear envelope in
both sub-confluent and confluent VSMCs. Although NE causes release of
intracellular Ca2+ in the VSMCs via activation of
-1
adrenergic receptors (Nebigil and Malik,
1993
), this appears to be insufficient to cause redistribution and
activation of cPLA2 (Muthalif
et al., 1996
). Our findings that, first, three structurally
distinct inhibitors of the Ca2+-binding protein CaM (CLMD,
E6-B and W-7) blocked NE- or ionomycin-induced cPLA2
accumulation around the nuclear envelope in both sub-confluent and confluent
cells and, second, that W-5 (a structural analogue of W-7 that does not
inhibit CaM activity at similar concentrations) failed to alter NE- or
ionomycin-induced cPLA2 translocation suggest an involvement of CaM
in cPLA2 translocation to the nuclear envelope in VSMCs.
CaM is known to produce several of its cellular actions by activating
CaMKII (Soderling et al.,
1990). CaMKII has been reported to cause activation of
cPLA2 and to be translocated to the nucleus in response to NE in
VSMCs (Muthalif et al., 1996
).
Our demonstration that CaMKII inhibitor KN-93 and CaMKII antisense, which
decrease CaMKII phosphorylation and activity
(Muthalif et al., 1998
), but
not KN-92 (an inactive structural analogue of KN-93), blocked translocation of
cPLA2 to the nuclear envelope in both sub-confluent and confluent
cells suggests that CaMKII mediates cPLA2 translocation. Inhibitors
of CaM and CaMKII did not alter the NE-induced rise in cytosolic
Ca2+ levels. Moreover, the effect of the Ca2+ ionophore
ionomycin to cause cPLA2 translocation, but not to increase
cytosolic Ca2+ levels, was also blocked by inhibitors of CaM (W-7)
and CaMKII (KN-93). These observations strongly suggest that CaM-activated
CaMKII mediates the effect of increased cytosolic Ca2+ to promote
cPLA2 translocation to the nuclear envelope in VSMCs. Because NE
and ionomycin caused cPLA2 phosphorylation and inhibitors of CaM
(W-7) and CaMKII (KN-93) but not their respective structural analogues (W-5
and KN-92) blocked cPLA2 phosphorylation and increase its catalytic
activity in response to NE and ionomycin, it would appear that phosphorylation
and activation of cPLA2 is required for its translocation to the
nuclear envelope. However, our demonstration that AACOCF3 and MAFP
inhibited cPLA2 activity but not its phosphorylation in response NE
in VSMCs suggests that cPLA2 phosphorylation, but not its activity,
is an essential component of the mechanism for its translocation to the
nuclear envelope.
That cPLA2 phosphorylation by CaMKII mediates its translocation induced by NE and ionomycin is supported by our observation that this was prevented by treatment of VSMCs with alkaline or potato acid phosphatase, which inhibited the phosphorylation of cPLA2 and CaMKII. Moreover, the inhibitory effect of phosphatases on NE- and ionomycin-induced cPLA2 phosphorylation and translocation was attenuated in the presence of the phosphatase inhibitor okadaic acid. Although okadaic acid alone increased the extent of cPLA2 phosphorylation and its catalytic activity, this was less than that caused by NE or ionomycin and was insufficient to cause cPLA2 translocation to the nuclear envelope. Therefore, it appears that phosphorylation of cPLA2 by Ca2+CaM-dependent CaMKII in response to NE and ionomycin is responsible for its translocation to the nuclear envelope. The effect of phosphatases to prevent cPLA2 translocation and phosphorylation in response to NE or ionomycin was not due to their cytotoxic effects, because they did not cause any morphological change or entry of Texas-Red-conjugated BSA or trypan blue into VSMCs.
Direct evidence that phosphorylation of cPLA2 by CaMKII promotes its accumulation around the nuclear envelope was provided by our demonstration that recombinant cPLA2 phosphorylated by purified CaMKII in vitro (but not cPLA2 that was unphosphorylated or dephosphorylated by treatment with alkaline phosphatase) was localized around the nuclear envelope in VSMCs that had been reversibly permeabilized by ß-escin. Similarly, cPLA2 cross-linked with Alexa 488 and phosphorylated by CaMKII, but not unphosphorylated or dephosphorylated cPLA2, was found to be localized around the nuclear envelope in reversibly permeabilized cells in the absence of extracellular Ca2+ and the presence of EGTA and the intracellular Ca2+ chelator BAPTA. These findings also support the view that cPLA2 phosphorylation by CaMKII mediates its translocation from the nucleus and cytosol to the nuclear envelope in the rabbit VSMCs. The accumulation of phosphorylated but not dephosphorylated cPLA2 to the nuclear envelope in the absence of Ca2+ suggests that dephosphorylation and not lack of Ca2+ promotes the dissociation of cPLA2 from the nuclear envelope in the rabbit VSMCs.
The site of phosphorylation of cPLA2 by CaMKII that is involved
in its translocation and association with components of the nuclear envelope
and/or endoplasmic reticulum is not known. Recently, we have reported that
CaMKII phosphorylates cPLA2 at Ser515 in response to NE
in VSMCs (Muthalif et al.,
2001). Therefore, it is possible that phosphorylation of
cPLA2 at this site might be responsible for its translocation to
the nuclear envelope by NE. However, further studies on the translocation of
cPLA2 mutated at this site (S515A) are required to address this
issue. cPLA2 is also known to be phosphorylated on
Ser505 and/or on Ser727 by mitogen-activated-protein
kinases (MAPK) ERK1/2 and p38 MAPK and by MAPK-activated protein kinase 1
(MNK-1) or a related kinase (Lin et al.,
1993
; Borsch-Haubold et al.,
1998
; Gijon and Leslie,
1999
; Gijon et al.,
2000
; Hefner et al.,
2000
). In embryonic chick heart cells,
ß-2-adrenergic-receptor-stimulated cPLA2 translocation to the
perinuclear region and AA release have been reported to be mediated by ERK1/2
and p38 MAPK activation (Magne et al.,
2001
). However, in CHO cells the Ca2+ ionophore A23187
caused translocation of cPLA2 that was mutated at the site (S505A)
phosphorylated by these kinases
(Schievella et al., 1995
).
Because, in VMSCs, ERK1/2 is phosphorylated by AA metabolites generated by
CaMKII-stimulated cPLA2, and the inhibitor of cPLA2
activity MAFP [which attenuates ERK1/2 phosphorylation
(Muthalif et al., 1998
)] did
not alter cPLA2 translocation to the nuclear envelope, it is
unlikely that ERK1/2 phosphorylation is required for cPLA2
translocation to the nuclear envelope in response to NE in VSMCs. p38 MAPKs,
which also cause phosphorylation and/or activation of cPLA2 in some
cell systems (Kramer et al.,
1995
; Waterman et al.,
1996
; Kramer et al.,
1996
; Borsch-Haubold et al.,
1998
; Hiller and Sundler,
1999
), do not mediate NE-induced activation or translocation of
cPLA2 to the nuclear envelope in VSMCs
(Fatima et al., 2001
).
The mechanism by which cPLA2 binds to the perinuclear membranes
is not known. In MDCK cells, depletion of ATP has been shown to result in
Ca2+-induced translocation of cPLA2 to the perinculear
region (Scheridan et al.,
2001). It is not known whether the translocation of
cPLA2 produced by neurohumoral agents is due to decreased ATP
levels in VSMCs. In HEK 293 cells stably transfected with cPLA2,
the Ca2+ ionophore A23187 promotes co-localization of
cPLA2 with the intermediate filament protein vimentin around the
perinuclear region by an interaction between C2 domain of cPLA2 and
the head domain of vimentin (Nakatani et
al., 2000
). Therefore, it is possible that phosphorylated
cPLA2 introduced into permeabilized VSMCs, which was translocated
to the nuclear envelope in the presence of EGTA and BAPTA, might bind with
vimentin upon translocation to the nuclear envelope.
Although phosphorylation of cPLA2 results in its activation, it
has been reported to be insufficient for its maximal activation and AA release
(Shalkwijk et al., 1995; Qiu et al.,
1998; Gijon and Leslie,
1999
). Moreover, it has been shown that, for continuous membrane
localization and full activation of cPLA2, a critical time period
of increased cytosolic Ca2+ level is required
(Hirabayashi et al., 1999
). It
has been reported that mutation of the Ca2+-binding residues in the
C2 domain leads to a higher Ca2+ requirement for cPLA2
binding to its substrates and its activity
(Bittova et al., 1999
).
However, our demonstration that the inhibitors of cPLA2 AACOCF3 and
MAFP, which attenuated NE-induced cPLA2 activation and AA release
in VSMCs (Muthalif et al.,
1996
; LaBelle and Polyak,
1998
), failed to block the phosphorylation and translocation of
cPLA2 to the nuclear envelope suggests that the release of AA in
response to NE is independent of cPLA2 translocation to the nuclear
envelope. These observations also raise the possibility that NE might release
AA from sites other than the nuclear envelope in VSMCs. The significance of
cPLA2 translocation to the nuclear envelope during inhibition of
its activity in response to NE is not known. One might speculate that the
translocation of cPLA2 to the nuclear envelope in response to NE in
VSMCs could be a part of an inactivating rather than an activating mechanism
of cPLA2 (i.e. transient removal of cPLA2 away from its
site(s) of action).
In conclusion, the present study provides an evidence that cPLA2 translocation from the cytosol to the nuclear envelope in response to the adrenergic transmitter NE is mediated via phosphorylation of cPLA2 by Ca2+CaM-dependent CaMKII, and that Ca2+ does not directly cause cPLA2 translocation to the nuclear envelope. Moreover, cPLA2 translocation to the nuclear envelope is independent of its catalytic activity.
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