School of Biochemistry and Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK
* Author for correspondence (e-mail: j.walker{at}leeds.ac.uk)
Accepted 27 February 2003
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Summary |
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Key words: Golgi, Phospholipase A2, Cyclooxygenase, Arachidonic acid, Calcium, Ca2+
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Introduction |
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cPLA2-a 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). Since
cPLA2-a preferentially liberates arachidonic acid, it is considered
the key PLA2 enzyme involved in receptor-mediated PG production.
This 85 kDa, Ca2+-sensitive protein is subject to complex
regulation at the transcriptional and post-translational level (reviewed by
Clark et al., 1991
). Previous
studies have shown that cPLA2-a is present in the cytosol of
resting cells and relocates to intracellular membranes following stimulation
with a variety of agonists (Evans et al.,
2001
; Glover et al.,
1995
; Hirabayashi et al.,
1999
; Peters-Golden et al.,
1996
; Schievella et al.,
1995
; Sierra-Honigmann et al.,
1996
). This translocation process is mediated by its
Ca2+-dependent lipid-binding (CaLB) or C2 domain, which promotes
binding to phospholipids upon elevation of intracellular Ca2+
concentrations (Gijon et al.,
1999
).
Downstream of cPLA2-a, COX converts arachidonic acid into
PGH2. COX exists as two isoforms encoded by distinct genes. COX-1
is a constitutively expressed housekeeping gene, whereas COX-2 is an inducible
gene expressed at higher levels in response to inflammatory and mitogenic
stimuli (Smith and Langenbach,
2001). Despite similarity in structure and catalytic properties,
COX-1 and COX-2 have been shown to use different intracellular pools of
arachidonic acid and are believed to have distinct cellular functions
(Reddy and Herschman, 1997
).
In addition, accumulating evidence suggests that both the COX isoenzymes are
functionally coupled to cPLA2-a to control the immediate and
delayed phases of PG synthesis (Croxtall et
al., 1996
).
Airway epithelial cells play a crucial role in the modulation of the
inflammatory response by controlling the level of PGs that they produce. Human
lung epithelial cells release increased levels of PGs in response to various
stimuli, including cytokines such as interleukin-1ß (IL-1ß),
interferon-g (IFN-) and tumour necrosis factor-
(TNF-
),
or Ca2+-mobilizing agents such as bradykinin and Ca2+
ionophores (Choudhury et al.,
2000
; Mitchell et al.,
1999
; Saunders et al.,
1999
). Such release has been shown to be dependent on elevation of
cytosolic Ca2+ levels and activation of cPLA2-
(Tokumoto et al., 1994
).
Surprisingly, the intracellular membrane to which cPLA2-
relocates following elevation of cytosolic Ca2+ in airway
epithelial cells has not been investigated previously. In addition, although
the expression levels and activities of the COX-1 and COX-2 enzymes have been
studied extensively in epithelial cells, their subcellular locations have not
been determined. In this study, we show that cPLA2-
relocates to the Golgi apparatus of human A549 airway epithelial cells in a
Ca2+-dependent manner, where its distribution overlaps that of the
COX-1 isoform.
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Materials and Methods |
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Cell culture
A549 human lung carcinoma epithelial cells (ATCC, Rockville, MD) were
cultured at 37°C in a humid atmosphere containing 5% CO2 in
air. Cells were grown in DMEM supplemented with 10% foetal bovine serum,
penicillin (100 U/ml), streptomycin (100 µg/ml) and 2 mM L-glutamine.
Immunofluorescence microscopy
The method for immunofluorescence microscopy was adapted from earlier
protocols (Barwise and Walker,
1996; Heggeness et al.,
1977
). Cells were grown on glass coverslips in 6-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 permeabilized 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
antibodies (diluted 1:100 into PBS-5% serum) overnight. Cells were washed
eight times with PBS then incubated with AlexaFluor488- and 594-conjugated
secondary antibodies, or rhodamine-conjugated WGA or ConA (10 µg/ml) for 3
hours. The cells were then washed eight times with PBS and mounted onto slides
in Citifluor mounting medium (Agar Scientific, Stansted, UK).
A23187 and brefeldin A (BFA) treatment
Cells were grown on coverslips in 6-well dishes overnight. Prior to
stimulation, media was removed and the cells were washed three times with
pre-warmed (37°C) PBS. For A23187 stimulations, PBS was removed and 5
µM A23187 in HEPES/Tyrode's buffer containing 1 mM Ca2+ was
added. The cells were then incubated at 37°C for 1 minute, after which
time the buffer was removed and fixative was added immediately. For BFA
treatment, cells were stimulated as above and then BFA was added to the cells
at a final concentration of 10 µg/ml. Cells were incubated at 37°C for
30 minutes then fixed immediately.
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. 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. To capture double-labelled
samples, sequential scanning of each fluorescence channel was performed
(according to the manufacturers guidelines) to avoid cross-contamination of
fluorescence signals. To quantify the degree of cPLA2-
overlap with Golgi markers and the COX isoforms, the number of
cPLA2-
+ structures was calculated by visual
inspection of 0.485 µm thick sections from four individual cells. For each
marker, the number of cPLA2-
-positive structures that were
labelled by the specific marker were counted and expressed as a percentage of
the total number of cPLA2-
-positive structures.
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:mg antigen) for 30 minutes at room
temperature prior to being incubated with the nitrocellulose blot.
Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL)
detection kit (Pierce) according to the manufacturer's instructions. Developed
films were photographed and captured using the FujiFilm Intelligent dark Box
II with the Image Reader Las-1000 package.
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Results |
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Using this specific antibody, a comparison of the location of
cPLA2- in resting and Ca2+ ionophore-treated A549
cells was carried out (Fig.
1B). In nonstimulated cells, cPLA2-
was present
in the cytosol and in the nucleus. Following elevation of the cytosolic
Ca2+ concentration using the Ca2+ ionophore A23187, a
specific relocation of cPLA2-
to a compact juxtanuclear
reticulum was evident (Fig.
1B). Secondary antibody and peptide-adsorbed antibody controls
gave no staining (data not shown), confirming that the staining observed
corresponded to specific staining of cPLA2-
. Identical
staining patterns, corresponding to juxtanuclear relocation, were also seen
following stimulation with the physiological stimulus histamine (data not
shown).
A strong possibility was that the juxtanuclear staining pattern observed in
ionophore-treated cells corresponded to the Golgi apparatus. To test this
possibility, ionophore-treated cells were counterstained with
rhodamine-conjugated WGA, a lectin that selectively labels
N-acetyl-ß-D-glucosaminyl residues, which are found on proteins
in the Golgi apparatus as well in the plasma membrane and the nuclear envelope
(Virtanen et al., 1980). The
results (Fig. 2A) revealed
co-localization of cPLA2-
with WGA-positive perinuclear
structures corresponding to the Golgi complex. No overlap was seen at the
plasma membrane or nuclear membrane. Counterstaining cells with ConA, a lectin
that can be used as a marker for the endoplasmic reticulum (ER)
(Virtanen et al., 1980
),
revealed very little overlap (Fig.
2B), indicating that cPLA2-
was not relocating
to the ER.
|
cPLA2- co-localizes with trans-Golgi stack and
trans-Golgi network (TGN) markers
In order to determine the precise subcompartment of the Golgi apparatus to
which cPLA2- was relocating, cells were labelled with
antibodies specific for various Golgi subcompartments. First, in order to
determine whether cPLA2-
was localized to the ER-Golgi
intermediate compartment (ERGIC), ionophore-treated cells were selectively
labelled with antibodies against the 53 kDa ERGIC-resident protein ERGIC53
(Schweizer et al., 1988
;
Schweizer et al., 1990
).
Double labelling of cells with this antibody and an
anti-cPLA2-
antibody
(Fig. 3A) demonstrated that,
although the juxtanuclear staining pattern given by each of these antibodies
was similar, only a small amount of overlap (indicated by yellow in the merged
image) was evident. Thus, cPLA2-
does not appear to
relocalize to the ERGIC upon ionophore stimulation.
|
In order to label specifically the cis-Golgi network and the cis-cisternae
of the Golgi apparatus, cells were labelled with an antibody against
ß-COP, a subunit of the Golgi-associated coatamer protein complex COPI
(Wieland and Harter, 1999).
Again, this antibody gave a staining pattern similar to that observed for
cPLA2-
(Fig.
3B). However, only partial overlap of the two staining patterns
was observed and close inspection revealed that cPLA2-
and
ß-COP were associated with neighbouring structures that were not
superimposed (Fig. 3B,
merge).
The distribution of cPLA2- was also compared with the
distribution of proteins of the trans-Golgi cisternae and the TGN. First,
cells were specifically labelled with antibodies against GalT, an enzyme found
in both the trans-Golgi cisternae and the TGN
(Nilsson et al., 1993
;
Roth et al., 1985
). The
results (Fig. 4A) demonstrated
that a high degree of overlap was evident, indicating that
cPLA2-
is localized predominantly to the trans side of the
Golgi complex. To investigate this further, cells were counterstained with an
antibody against TGN46, a TGN-resident protein
(Prescott et al., 1997
).
Again, a high degree of overlap between the two antibodies was observed
(Fig. 4B), indicating that
cPLA2-
locates to both the trans-Golgi stack and the TGN
subcompartments following ionophore stimulation.
|
Quantification of the degree of overlap between cPLA2-
and the various Golgi and ER markers was performed as described in the
Materials and Methods. The results (Fig.
5) confirm that cPLA2-
overlaps predominantly
with markers of the Golgi apparatus, whereas little overlap is seen with the
ER marker (approximately 72% overlap with WGA compared with only 23% with
ConA). In particular, cPLA2-
showed approximate 70% and 60%
overlap with the trans-Golgi markers, GalT and TGN46, respectively. By
contrast, markers of the cis-Golgi (ERGIC53, ß-COP) showed only 20-25%
overlap.
|
Ca2+-induced Golgi localization of
cPLA2- can be disrupted with BFA
To test whether cPLA2- was relocating to the lipid
bilayer membranes of the trans-Golgi stack and the TGN, and was not just in
close proximity to these membranes, cells were treated with BFA, a potent
fungal metabolite that is known to inhibit trafficking through the secretory
pathway (reviewed by Klausner et al.,
1992
). Previous studies using BFA have shown that it blocks
protein transport into the Golgi complex, resulting in the redistribution of
Golgi-stack proteins and TGN-resident proteins to the ER and endosomal
systems, respectively (Lippincott-Schwartz
et al., 1990
;
Lippincott-Schwartz et al.,
1991
; Wood et al.,
1991
).
Cells were treated with BFA (10 µg/ml) for 30 minutes following
stimulation, and the location of cPLA2- was analysed by
immunofluorescence microscopy. The results indicate that BFA treatment caused
a redistribution of cPLA2-
from the Golgi to dispersed
cytosolic structures (Fig.
6).
|
cPLA2- co-localizes with COX-1 but not with
COX-2
The data above demonstrated that, in A549 lung epithelial cells,
cPLA2- relocates primarily to the Golgi complex and the TGN.
In order to determine whether COX enzymes showed a similar localization, their
distribution was analysed by confocal microscopy. Cells were double labelled
with anti-cPLA2-
antibodies and specific monoclonal
antibodies raised against either COX-1 or COX-2. The results
(Fig. 7) demonstrated that the
two COX isoforms were present at distinct subcellular locations in A549 lung
epithelial cells. COX-1 was constitutively localized at the Golgi complex and
in cytoplasmic structures in both control and ionophore-stimulated cells,
whereas COX-2 was always present at the nuclear membrane and in cytosolic
speckles. Prior to stimulation, no co-localization of cPLA2-
with either of the COX isoenzymes was observed (data not shown).
Interestingly, however, following stimulation with A23187, a specific
co-localization of cPLA2-
with COX-1 but not with COX-2 was
evident in regions of the Golgi apparatus
(Fig. 7A and 7B, merged
images). Quantification of the degree of overlap revealed that 63±14%
of the cPLA2-
+ structures overlapped COX-1
immunoreactivity, whereas only a 3±5% overlap was evident for COX-2
(Fig. 7C).
|
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Discussion |
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Further investigation of the Golgi localization using markers for specific
Golgi subcompartments (Figs 3,
4) revealed that
cPLA2- was localized primarily at the trans-Golgi cisternae
of the Golgi complex and at the TGN. Only a small amount of
cPLA2-
was found associated with the ERGIC or the cis face
of the Golgi stack. In addition, disruption of the Golgi apparatus using BFA
caused redistribution of cPLA2-
to cytosolic structures, as
expected for a Golgi-associated protein
(Fig. 6). This finding is
consistent with previous work on Madin-Darby canine kidney epithelial cells,
which demonstrated that the Golgi localization of over-expressed
GFP-cPLA2-
could be disrupted with BFA
(Evans et al., 2001
).
Previous immunofluorescence studies on non-epithelial cells have shown that
cPLA2- relocates predominantly to the ER, the nuclear
envelope or the perinuclear region (Gijon
and Leslie, 1999
; Glover et
al., 1995
; Hirabayashi et al.,
1999
; Peters-Golden et al.,
1996
; Schievella et al.,
1995
). However, here we found very little relocation of
cPLA2-
to the ER of A549 cells. Thus, it appears that Golgi
localization of cPLA2-
may be specific for epithelial cells,
suggesting that the precise relocation of cPLA2-
to
intracellular membranes is dependent on cell type.
Co-localization of cPLA2- with COX-1
Following our observations on the cell-specific relocation of
cPLA2- to the Golgi apparatus, we investigated the
subcellular location of the COX enzymes that lie downstream of
cPLA2-
in PG synthesis. Our results revealed that, in A549
cells, the COX-1 and COX-2 isoforms have distinct subcellular locations. COX-1
was constitutively localized to the Golgi apparatus and small cytoplasmic
structures, whereas COX-2 was detected mainly on ER-like punctate structures
and the nuclear membrane. Previous studies on bovine aortic endothelial cells
have also shown distinct intracellular locations for these two proteins, with
COX-1 localized at the nuclear membrane and ER, and COX-2 present in cytosolic
vesicular structures (Liou et al.,
2000
). It is feasible that such distinct spatial organization of
the COX isoforms may, in part, provide an explanation for their observed
differences in function. Although the two COX enzymes show similar enzymatic
properties in vitro, recent evidence has shown that their role in the
regulation of PG synthesis is clearly segregated (reviewed in
Smith and Langenbach, 2001
).
COX-1 has been shown to be pivotal in the immediate phase of PG synthesis,
which is elicited by Ca2+-mobilizing agonists, whereas COX-2 is
involved in the delayed response, which lasts for several hours following
activation by proinflammatory stimuli. Thus, it is possible that the distinct
subcellular location of the two isoenzymes is crucial for mediating specific
responses that depend on the length and magnitude of the stimulus.
Most interestingly, our results here revealed a specific co-localization of
a pool of COX-1 with cPLA2- at the Golgi complex. By
contrast, no co-localization with COX-2 was evident. Whether this is due to
direct interaction of the COX-1 isoform with cPLA2-
remains
to be investigated. If this is the case, the constitutive localization of
COX-1 at the Golgi complex could be involved in recruiting
cPLA2-
specifically to this site following stimulation.
Alternatively, both proteins could favour localization in phospholipid
membranes of a certain composition, which are present only in specific
subdomains of the Golgi apparatus.
The co-localization of cPLA2- with the COX-1 isoform
presented here is a novel finding. Previous studies have shown that COX-1 but
not COX-2 co-localizes with prostacyclin synthase in phorbol ester-induced
endothelial cells (Liou et al.,
2000
), further suggesting that enzymes involved in eicosanoid
production might be complexed or in very close proximity to one another. Such
an association could ensure the immediate synthesis of PGs following an
increase in intracellular Ca2+. It must be noted, however, that the
studies carried out here were performed on cells that displayed basal,
uninduced levels of COX-2 expression
(Croxtall et al., 1996
).
Following exposure to mitogenic or inflammatory stimuli, cells exhibit
increased levels of COX-2 expression
(Smith and Langenbach, 2001
)
and it is possible that this induced COX-2 shows an alternative intracellular
localization. Furthermore, whether or not any of this induced COX-2
co-localizes with cPLA2-
under these conditions remains to
be investigated.
Potential roles for cPLA2- in the Golgi
complex
Previous reports have implicated PLA2 activity in the
maintenance of correct Golgi structure and function. These include studies on
Golgi formation (Choukroun et al.,
2000), tubulation (de
Figueiredo et al., 1999
) and membrane remodelling
(Schmidt et al., 1999
). In
addition, several studies using PLA2 inhibitors have highlighted
the importance of PLA2 activity in trafficking between the ER and
the Golgi (de Figueiredo et al.,
2000
; Drecktrah and Brown,
1999
; Slomiany et al.,
1992
), endocytosis (Mayorga et
al., 1993
), exocytosis
(Slomiany et al., 1998
) and
the intracellular trafficking of secretory proteins
(Choukroun et al., 2000
;
Tagaya et al., 1993
). Since
most of the inhibitors used in such studies were broad-range inhibitors, the
precise species of PLA2 involved in these processes is unclear.
Nevertheless, studies related directly to cytosolic PLA2-
do
confirm that this member of the PLA2 family is involved in
trafficking events and maintenance of Golgi structure and function
(Choukroun et al., 2000
;
Slomiany et al., 1992
).
The exact role of cPLA2 in Golgi structure and function is
unclear. It is evident that PLA2s have several features that could
be potentially very useful in controlling the membrane fusion processes that
mediate intracellular trafficking. PLA2 activity generates two
bioactive compounds, lysophospholipid and arachidonic acid. It has been shown
previously that lysophospholipid is able to stimulate secretion
(Murakami et al., 1991) and
arachidonic acid is known to promote membrane bilayers to form a HII phase
(Burger and Verkleij, 1990
), a
lipid arrangement that may be imperative during membrane fusion events.
Alternatively, the PLA2-dependent changes in trafficking observed
might be due to a direct effect of the enzyme on the lipid composition of the
Golgi membrane. It is known that the asymmetry of the lipid bilayer is
necessary to maintain the fundamental properties of the various intracellular
compartments within the cell (Verkade and
Simons, 1997
). Following this, it is possible that hydrolysis of
membrane phospholipids by PLA2 could modify the composition of the
two leaflets, thereby playing a crucial role in controlling the structure and
trafficking of Golgi membranes. Furthermore, whether the cell-specific Golgi
localization of cPLA2-
in A549 cells is of particular
importance in maintaining the polarity of these epithelial cells remains to be
investigated. Epithelial cell polarity is derived from the selective
distribution of their proteins and lipids into distinct plasma membrane
domains. Previous studies have implied that cPLA2 activity has
effects on the distribution of only a subset of proteins
(Choukroun et al., 2000
); thus,
it is possible that cPLA2-
may be playing a role in
selective membrane trafficking in these cells.
In conclusion, the studies presented here demonstrate that
cPLA2- relocates in a Ca2+-dependent manner to
the Golgi apparatus of human A549 lung epithelial cells where it co-localizes
with COX-1. The functional relevance of this co-localization and the
mechanisms that underlie the specific targeting to this region remain to be
investigated. Nevertheless, these data, together with recent studies on the
role of PLA2 activity in maintaining Golgi structure and function,
suggest that the Golgi location of cPLA2-
might play an
important role in membrane trafficking events.
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
Burger, K. N. and Verkleij, A. J. (1990). Membrane fusion. Experientia 46,631 -644.[Medline]
Choudhury, Q. G., McKay, D. T., Flower, R. J. and Croxtall, J.
D. (2000). Investigation into the involvement of
phospholipases A(2) and MAP kinases in modulation of AA release and cell
growth in A549 cells. Br. J. Pharmacol.
131,255
-265.
Choukroun, G. J., Marshansky, V., Gustafson, C. E., McKee, M.,
Hajjar, R. J., Rosenzweig, A., Brown, D. and Bonventre, J. V.
(2000). Cytosolic phospholipase A(2) regulates golgi structure
and modulates intracellular trafficking of membrane proteins. J.
Clin. Invest. 106,983
-993.
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]
Croxtall, J. D., Newman, S. P., Choudhury, Q. and Flower, R. J. (1996). The concerted regulation of cPLA2, COX2, and lipocortin 1 expression by IL-1ß in A549 cells. Biochem. Biophys. Res. Commun. 220,491 -495.[CrossRef][Medline]
de Figueiredo, P., Polizotto, R. S., Drecktrah, D. and Brown, W.
J. (1999). Membrane tubule-mediated reassembly and
maintenance of the Golgi complex is disrupted by phospholipase A2 antagonists.
Mol. Biol. Cell 10,1763
-1782.
de Figueiredo, P., Drecktrah, D., Polizotto, R. S., Cole, N. B., Lippincott- Schwartz, J. and Brown, W. J. (2000). Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum. Traffic 1, 504-511.[CrossRef][Medline]
Dennis, E. A. (1997). The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem. Sci. 22,1 -2.[CrossRef][Medline]
Drecktrah, D. and Brown, W. J. (1999).
Phospholipase A(2) antagonists inhibit nocodazole-induced Golgi ministack
formation: evidence of an ER intermediate and constitutive cycling.
Mol. Biol. Cell 10,4021
-4032.
Earnest, D. L., Hixson, L. J. and Alberts, D. S. (1992). Piroxicam and other cyclooxygenase inhibitors: potential for cancer chemoprevention. J. Cell. Biochem. Suppl. 16I,156 -166.
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. Leukocyte 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.
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
[published erratum appears in J. Biol. Chem. (1995). 270,
20870]. J. Biol. Chem.
270,15359
-15367.
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]
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.
Klausner, R. D., Donaldson, J. G. and Lippincott-Schwartz, J. (1992). Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116,1071 -1080.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Liou, J. Y., Shyue, S. K., Tsai, M. J., Chung, C. L., Chu, K. Y.
and Wu, K. K. (2000). Colocalization of prostacyclin
synthase with prostaglandin H synthase-1 (PGHS-1) but not phorbol
ester-induced PGHS-2 in cultured endothelial cells. J. Biol.
Chem. 275,15314
-15320.
Lippincott-Schwartz, J., Donaldson, J. G., Schweizer, A., Berger, E. G., Hauri, H. P., Yuan, L. C. and Klausner, R. D. (1990). Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway. Cell 60,821 -836.[Medline]
Lippincott-Schwartz, J., Yuan, L., Tipper, C., Amherdt, M., Orci, L. and Klausner, R. D. (1991). Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic. Cell 67,601 -616.[Medline]
Mayorga, L. S., Colombo, M. I., Lennartz, M., Brown, E. J., Rahman, K. H., Weiss, R., Lennon, P. J. and Stahl, P. D. (1993). Inhibition of endosome fusion by phospholipase A2 (PLA2) inhibitors points to a role for PLA2 in endocytosis. Proc. Natl. Acad. Sci. USA 90,10255 -10259.[Abstract]
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.
Murakami, M., Kudo, I., Fujimori, Y., Suga, H. and Inoue, K. (1991). Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells. Biochem. Biophys. Res. Commun. 181,714 -721.[Medline]
Nilsson, T., Pypaert, M., Hoe, M. H., Slusarewicz, P., Berger, E. G. and Warren, G. (1993). Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells. J. Cell Biol. 120,5 -13.[Abstract]
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]
Prescott, A. R., Lucocq, J. M., James, J., Lister, J. M. and Ponnambalam, S. (1997). Distinct compartmentalization of TGN46 and ß 1,4-galactosyltransferase in HeLa cells. Eur. J. Cell Biol. 72,238 -246.[Medline]
Reddy, S. T. and Herschman, H. R. (1997).
Prostaglandin synthase-1 and prostaglandin synthase-2 are coupled to distinct
phospholipases for the generation of prostaglandin D2 in activated mast cells.
J. Biol. Chem. 272,3231
-3237.
Roth, J., Taatjes, D. J., Lucocq, J. M., Weinstein, J. and Paulson, J. C. (1985). Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation. Cell 43,287 -295.[Medline]
Saunders, M. A., Belvisi, M. G., Cirino, G., Barnes, P. J.,
Warner, T. D. and Mitchell, J. A. (1999). Mechanisms of
prostaglandin E2 release by intact cells expressing cyclooxygenase-2: evidence
for a `two-component' model. J. Pharmacol. Exp. Ther.
288,1101
-1106.
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.
Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A. V., Witke, W., Huttner, W. B. and Soling, H. D. (1999). Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature 401,133 -141.[CrossRef][Medline]
Schweizer, A., Fransen, J. A., Bachi, T., Ginsel, L. and Hauri, H. P. (1988). Identification, by a monoclonal antibody, of a 53-kD protein associated with a tubulo-vesicular compartment at the cis-side of the Golgi apparatus. J. Cell Biol. 107,1643 -1653.[Abstract]
Schweizer, A., Fransen, J. A., Matter, K., Kreis, T. E., Ginsel, L. and Hauri, H. P. (1990). Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. Eur. J. Cell Biol. 53,185 -196.[Medline]
Sierra-Honigmann, M. R., Bradley, J. R. and Pober, J. S. (1996). `Cytosolic' phospholipase A2 is in the nucleus of subconfluent 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]
Slomiany, A., Grzelinska, E., Kasinathan, C., Yamaki, K., Palecz, D. and Slomiany, B. L. (1992). Function of intracellular phospholipase A2 in vectorial transport of apoproteins from ER to Golgi. Int. J. Biochem. 24,1397 -1406.[Medline]
Slomiany, A., Nowak, P., Piotrowski, E. and Slomiany, B. L. (1998). Effect of ethanol on intracellular vesicular transport from Golgi to the apical cell membrane: role of phosphatidylinositol 3-kinase and phospholipase A2 in Golgi transport vesicles association and fusion with the apical membrane. Alcohol Clin. Exp. Res. 22,167 -175.[Medline]
Smith, W. L. and Langenbach, R. (2001). Why
there are two cyclooxygenase isozymes. J. Clin.
Invest. 107,1491
-1495.
Tagaya, M., Henomatsu, N., Yoshimori, T., Yamamoto, A., Tashiro, Y. and Fukui, T. (1993). Correlation between phospholipase A2 activity and intra-Golgi protein transport reconstituted in a cell-free system. FEBS Lett. 324,201 -204.[CrossRef][Medline]
Tokumoto, H., Croxtall, J. D. and Flower, R. J.
(1994). Differential role of extra- and intracellular calcium in
bradykinin and interleukin 1 stimulation of arachidonic acid release
from A549 cells. Biochim. Biophys. Acta
1211,301
-309.[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]
Towler, M. C., Prescott, A. R., James, J., Lucocq, J. M. and Ponnambalam, S. (2000). The manganese cation disrupts membrane dynamics along the secretory pathway. Exp. Cell Res. 259,167 -179.[CrossRef][Medline]
Vane, J. R. and Botting, R. M. (1995). New insights into the mode of action of anti-inflammatory drugs. Inflamm. Res. 44,1 -10.[Medline]
Verkade, P. and Simons, K. (1997). Robert Feulgen Lecture 1997. Lipid microdomains and membrane trafficking in mammalian cells. Histochem. Cell Biol. 108,211 -220.[CrossRef][Medline]
Virtanen, I., Ekblom, P. and Laurila, P. (1980). Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells. J. Cell Biol. 85,429 -434.[Abstract]
Wieland, F. and Harter, C. (1999). Mechanisms of vesicle formation: insights from the COP system. Curr. Opin. Cell Biol. 11,440 -446.[CrossRef][Medline]
Wood, S. A., Park, J. E. and Brown, W. J. (1991). Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes. Cell 67,591 -600.[Medline]