From the Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel
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ABSTRACT |
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The activation of cellular phospholipase D (PLD)
is implicated in vesicular trafficking and signal transduction. Two
mammalian PLD forms, designated PLD1 and PLD2, have been cloned, but
their cellular localization and function are not fully understood.
Here, we report that in HaCaT human keratinocytes, as well as other cell lines, PLD activity is highly enriched in low density, Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin-1. Similar to other PLDs, the PLD activity in these
membrane domains is stimulated by phosphatidylinositol 4,5-bisphosphate and is inhibited by neomycin. Immunoblot analysis indicated that caveolin-rich membrane domains do not contain the PLD1 isoform. Stable
transfection of mouse PLD2 in Chinese hamster ovary cells greatly
increased PLD activity in these domains compared with PLD activity in
control Chinese hamster ovary cells transfected with vector alone. PLD
activity is enriched in low density Triton-insoluble membrane domains
also in U937 promonocytes, even though these cells do not express
caveolin-1. In U937 cells, also, PLD1 is largely excluded from low
density Triton-insoluble membrane domains. Expression of recombinant
caveolin-1 in v-Src-transformed NIH-3T3 cells resulted in up-regulation
of PLD activity in the caveolin-containing membrane domains. The
caveolin scaffolding peptide (caveolin-182-101) modulated the caveolar PLD activity, causing stimulation at
concentration of 1-10 µM and inhibition at
concentrations >10 µM. We conclude that a PLD activity,
which is likely to represent PLD2, is enriched in low density
Triton-insoluble membrane domains. The effects of caveolin-1 expression
and of the caveolin scaffolding peptide suggest that in cells that
express caveolin-1, PLD may be targeted to caveolae. The possible
functions of PLD in the dynamics of caveolae and related domains and in
signal transduction processes are discussed.
The basal activity of phospholipase D
(PLD)1 in mammalian cells is
very low, yet the enzyme can be activated in a variety of cell types,
rapidly and dramatically, by a wide range of stimuli (hormones,
neurotransmitters, growth factors, cytokines, etc.) (1-4). Recently,
multiple forms of eukaryotic PLDs have been molecularly cloned. These
include three plant enzymes (5, 6), two mammalian PLDs (PLD1 (7) and
PLD2 (8, 9)), and a yeast PLD (10-12). These PLD genes all belong to
an extended gene family that also includes bacterial PLDs, as well as
certain non-PLD phosphatidyltransferases (13-15). Additional forms of
PLD that do not belong to the PLD/phosphatidyltranferase family may
exist (16, 17). Although the activation of PLD isoforms is likely to be
involved in signal transduction and membrane traffic events, their
precise cellular localization and function(s) are still poorly defined.
There is growing evidence for the existence in biological membranes of
microdomains that are laterally segregated in the plane of the bilayer
and that are enriched in sphingolipids and in cholesterol. Such
microdomains have been variously termed detergent-insoluble glycosphingolipid-rich complexes (DIGs), glycosphingolipid-enriched membranes, and sphingolipid-cholesterol "rafts" (18). DIGs are related, in their lipid composition and their resistance to detergent solubilization, to specific morphologically and biochemically well
defined cellular structures called caveolae (19). Caveolae are
non-clathrin-coated plasma membrane invaginations, 50-100 nm in size,
that have a characteristic striated coat structure (20). A major
caveolar coat protein is caveolin, a 21-kDa integral membrane protein
(21). Caveolin forms high molecular weight homo-oligomers (22) and acts
as a scaffolding protein for various other proteins (23). The lipid
composition of caveolae and DIGs facilitates their isolation as low
density, Triton-insoluble (LDTI) membrane particles on discontinuous
sucrose density gradients (24, 25). Analyses of a large variety of cell
types have indicated that DIGs are present in most, if not all,
eukaryotic cells. In contrast, caveolae are found mostly (although not
exclusively) in epithelial cells and generally are absent from
hematopoietic cells (21). Caveolae have been implicated in transport
processes such as endocytosis and transcytosis (Ref. 26 and citations therein), as well as cholesterol efflux (27). Both caveolae and DIGs
are thought to play an important role in cellular signal transduction
(28, 29).
It was recently reported that low density Triton X-100-insoluble
membrane domains are enriched in polyphosphoinositides, especially in phosphatidylinositol 4,5-bisphosphate (PIP2) (30-34).
PIP2 is a required cofactor for PLD activity in
vitro (35-37), and PLD activation depends on ongoing
PIP2 synthesis in permeabilized cells (38) and intact cells
(39). These observations prompted us to examine the presence of PLD
activity in DIGs and caveolae. We now report that a PLD activity
is enriched in these membrane domains. We further show that this
enzyme is not PLD1, and that recombinant PLD2 is preferentially
targeted to LDTI membranes in stably transfected CHO cells. Finally, we
demonstrate that the PLD activity in LDTI membranes is markedly
up-regulated in cells that overexpress recombinant caveolin-1 and is
modulated in vitro by the caveolin scaffolding domain
peptide (caveolin-182-101). Parts of this work have been
published in abstract form (40).
Cell Culture--
HaCaT, a human keratinocyte cell line (41),
was kindly provided by Dr. Norbert Fusenig
(Deutcheskrebsforschungszentrum, Heidelberg, Germany). HaCaT cells were
cultured at 37 °C in a humidified atmosphere containing 5%
CO2 in Dulbecco's modified Eagle's medium supplemented
with 10% (v/v) fetal calf serum and antibiotics
(penicillin/streptomycin). U937 cells were grown according to Pertile
et al. (38). v-Src-transformed NIH 3T3 (v-Src-3T3) cells and
COS-7 cells were grown in the same media and conditions as HaCaT cells.
v-Src-3T3 cells were transfected with a full-length human caveolin-1
cDNA subcloned into a pJB20 vector using EcoRI sites,
which was kindly provided by Dr. A. Taraboulos (Hebrew University of
Jerusalem). Ten µg of DNA were transfected per 100-mm, 70% confluent
dish using LipofectAMINE (Life Technologies, Inc.) according to the
manufacturer's instructions. Neomycin-resistant cells were selected
for at least 3 weeks in medium containing 400 µg/ml G418
(Calbiochem), and the stable transfectants were pooled and used as a
population. CHO cells were grown in Alfa+ medium supplemented with 10%
fetal calf serum and antibiotics (penicillin/streptomycin). cDNA
encoding mouse PLD2 (kindly provided by Dr. Michael Frohman) was
subcloned into a plasmid vector for stable transfection (pEGFPN3,
CLONTECH) using NheI and SmaI
restriction sites. Four µg of DNA were transfected per 35-mm, 70%
confluent dish using LipofectAMINE (Life Technologies, Inc.) according
to the manufacturer's instructions. Cells were selected with 600 µg/ml G418 in the culture medium and used after 2 months.
Isolation of Low Density Triton X-100-insoluble Membrane
Domains--
Low density Triton X-100-insoluble membrane domains were
purified from cultured cells essentially as described (25). Briefly, cell monolayers (two confluent 150-mm dishes; ~3 × 107 cells) were scraped in 1 ml of ice-cold lysis buffer
containing 25 mM MES, pH 6.5, 0.15 M NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1% Triton X-100. After homogenization, cell
extracts were adjusted to 40% sucrose by addition of 1 ml of the above
buffer (minus Triton X-100) containing 80% sucrose and placed at the
bottom of an 12-ml ultracentrifuge tube. A discontinuous gradient was
formed above the lysate by adding 4 ml each of 30 and 5% sucrose
solutions, and the tubes were centrifuged at 190,000 × g (39,000 rpm) for 16-20 h in an SW-41 rotor at 4 °C.
Fractions (0.9 ml) were collected beginning at the top of the gradient.
The pellet was resuspended in 0.9 ml of MES-NaCl buffer. The protein
content of each fraction was determined according to a modified Lowry
procedure using a commercially available kit (Bio-Rad). In experiments
in which the distribution of total PLD activity was determined directly
in aliquots of gradient fractions (Figs. 1 and 2), a Triton X-100
concentration of 0.5% was maintained along the entire gradient to
ensure an equal detergent concentration and minimize surface-dilution
effects on PLD activity. LDTI membrane domains of U937 cells were
prepared as above with some minor modifications as described in Ref.
42. For characterization of PLD activity (Tables I and II), the sucrose
gradient fractions containing membrane domains were diluted 2-fold in
25 mM MES, 0.15 M NaCl buffer, and the
membranes were precipitated by centrifugation at 30,000 × g for 1 h. The membrane pellet was resuspended in MES-NaCl buffer and utilized in PLD assays or immunoblot analysis for
caveolin-1 and PLD1.
Assay of Phospholipase D in Vitro--
The activity of PLD was
determined with 1-acyl-2-(6-N-(7-nitrobenzo-2-oxa-1,3
diazol-4-yl)amino)caproyl-phosphatidylcholine (C6-NBD-phosphatidylcholine) (Avanti Polar Lipids) as
substrate, measuring the production of
C6-NBD-phosphatidylpropanol. The assay was carried out
essentially as described before (43), with minor modifications.
Briefly, the reaction mixtures (120 µl) contained 50 mM
Na-HEPES, pH 7.2, 0.3 mM
C6-NBD-phosphatidylcholine, 150 mM 1-propanol,
and 2-5 mol % PIP2 (Boehringer Mannheim). The amount of
protein present in the assay varied when PLD was determined directly in
aliquots of gradient fractions. In the PLD characterization experiments
(Table I) protein concentration was 13 µg/ml. Reactions were carried
out for 60 min at 37 °C. Termination and separation of the products
by thin layer chromatography were carried out as described previously
(43). Results are expressed in terms of
C6-NBD-phosphatidylpropanol fluorescence units produced.
Assay of Phospholipase D in Intact Cells--
PLD activity was
assayed in intact [3H]oleic acid-prelabeled cells by
measuring production of the specific PLD product
[3H]phosphatidylethanol according to a previously
published procedure (44).
Generation of Antibodies to Human PLD1--
Rabbit antibodies
were raised to a peptide sequence (CARMPWHDIASAVHGK) that corresponds
to residues 675-688 of human PLD1, to which a Cys-Ala N-terminal
extension was added to facilitate cross-linking to keyhole limpet
hemocyanin. Rabbits were immunized against the keyhole limpet
hemocyanin peptide antigen by injection of 500 µg of the conjugate in
complete Freund's adjuvant, and antibody production was boosted by
four additional injections of the antigen in incomplete Freund's
adjuvant at 3-week intervals. This antiserum, designated anti-PLD1
(3074), interacts with a ~120-kDa protein band in a number of human
cell types, including HeLa, U937, and HT-29 cell lines, as well as
human platelets. The interaction of anti-PLD1 (3074) with this band was
competitively blocked by the peptide antigen, indicating that it binds
to the same epitope on the ~120-kDa protein band. In addition, the
3074 antiserum cross-reacted with recombinant human PLD1 expressed in
COS-7 cells, indicating that it recognizes authentic human PLD1 (data
not shown).
Immunoblot Analysis of Caveolin-1 and PLD--
Aliquots taken
from each of the sucrose density gradient fractions were separated by
SDS-PAGE. Proteins were transferred to nitrocellulose membranes and
blocked by incubation for 1 h with 5% skim milk (w/v) in
phosphate-buffered saline containing 0.1% Triton X-100. Immunoblot
analysis was carried out with a monoclonal antibody to caveolin-1
(clone 2297, Transduction Laboratories) or anti-PLD1 (3074), both
utilized in a dilution of 1:1000 in the blocking buffer. Another
anti-PLD1 serum (Upstate Biotechnology Inc.) was utilized according to
the manufacturer's recommendations. The blots were then washed
extensively and incubated with horseradish peroxidase-linked goat
anti-rabbit or goat anti-mouse IgG. Bands were visualized by enhanced
chemiluminescence using a commercially available kit (Amersham
Pharmacia Biotech).
To obtain LDTI membrane domains, Triton X-100-based lysates were
prepared from HaCaT human keratinocytes. Lysates were fractionated on a
discontinuous sucrose density gradient. The fractions were analyzed for
protein concentration, caveolin-1 immunoreactivity, and PLD activity.
The distribution of protein along the gradient was highly skewed, with
greater than 90% of the total protein present in the high density
fractions, starting at fraction 9 (Fig.
1A, open circles). These
fractions contain 40% sucrose and represent the combined cytosolic and
Triton-soluble proteins. The last fraction represents the pellet and
contains Triton-insoluble protein complexes, probably comprising
nuclear and cytoskeletal proteins. In contrast, immunoblot analysis
with a specific antibody to caveolin-1 indicated that low density
fractions 5 and 6 contained nearly all of the cellular caveolin-1, a
specific coat protein of caveolae (Fig. 1A, inset). These
data confirm previous results obtained in other cell types and indicate
that LDTI membrane domains include caveolae. Samples of each gradient
fraction were tested for PLD activity utilizing a fluorescent
phospholipid, C6-NBD-phosphatidylcholine, as a substrate in
an in vitro assay system that includes PIP2 as
the sole exogenously added cofactor (36, 43). Significant levels of PLD
activity (evinced by production of
C6-NBD-phosphatidylpropanol) were observed in most gradient
fractions (Fig. 1A, shaded columns). However, there was a
peak of PLD activity associated with the caveolin-rich membranes in
fractions 4-7. Under these assay conditions, the activity present in
these domains comprised 16% of the total cellular PLD activity. The
highest specific PLD activity was present in fraction 5 (Fig.
1B). The specific activity of PLD in fraction 5 was 7.7-fold
higher than the activity detected in high density gradient fractions
(fractions 10-13). To confirm the generality of these findings, we
examined the distribution of PLD activity in NIH 3T3 mouse fibroblasts
(Fig. 2). Overall, the pattern of PLD
activity distribution in this cell line was very similar to that
observed in HaCaT cells. The peak of PLD activity associated with DIGs
and caveolae in LDTI fractions comprised 16.1% of the total activity
distributed along the gradient (Fig. 2A), and the peak
specific PLD activity was 5-fold higher that the activity present in
the high density fractions (Fig. 2B). Qualitatively similar
results were obtained in other cell lines, including COS-7 cells and
U937 promonocytes (see below), MCF-7 human breast adenocarcinoma cells,
and HT-29 human colon
adenocarcinoma.2 These data
indicate that the presence of significant PLD activity in low density,
Triton-insoluble and caveolin-rich membrane domains is a general
phenomenon.
INTRODUCTION
Top
Abstract
Introduction
References
MATERIALS AND METHODS
RESULTS
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Fig. 1.
Localization of phospholipase D activity in
low density sucrose gradient fractions containing caveolin-1 prepared
from HaCaT human keratinocytes. Triton X-100 lysates were prepared
from HaCaT cells and fractionated by flotation in a discontinuous
sucrose density gradient, and the fractions were analyzed for protein
concentration, PLD activity, and caveolin-1 immunoreactivity as
described under "Materials and Methods." A, distribution
of total protein concentration, total PLD activity, and caveolin-1
immunoreactivity (inset). B, specific PLD
activity.
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Fig. 2.
Localization of phospholipase D activity in
low density sucrose gradient fractions containing caveolin-1 prepared
from NIH 3T3 mouse fibroblasts. Triton X-100 lysates were prepared
from NIH 3T3 cells and fractionated by flotation in a discontinuous
sucrose density gradient, and the fractions were analyzed for protein
concentration, PLD activity, and caveolin-1 immunoreactivity as
described under "Materials and Methods." A, distribution
of total protein concentration, total PLD activity, and caveolin-1
immunoreactivity (inset). B, specific PLD
activity.
Multiple PLD activities exhibiting different biochemical and
enzymological properties have been reported (see Ref. 45 for review). A
preliminary characterization of the PLD in LDTI membrane domains was
carried out in order to assess its relationship to previously reported
activities. The activity of PLD in the domains was linear with time of
incubation for up to 90 min and was maximal at pH 7.5-8.0. Although
substantial enzymatic activity could be observed in the absence of any
added lipid or protein cofactor, the addition of PIP2
stimulated enzyme activity 5-17-fold, depending on other assay
conditions (Table I). Maximal activation
of PLD was observed at PIP2 concentrations 1 mol %.
Sodium oleate, added at a concentration previously determined to be
optimally effective in stimulation of brain membrane PLD (60 mol %),
abolished enzyme activity, indicating that this is not the
oleate-activated PLD. The divalent cation chelators EGTA and EDTA did
not inhibit PLD activity; rather, in the absence of PIP2,
enzyme activity was increased, and addition of Ca2+ reduced
it to the levels observed in the absence of any divalent cation
chelator (Table I). These data indicate that PLD in LDTI domains is not
stimulated by divalent cations such as Ca2+ and
Mg2+, but is similar to other PLD activities in its
sensitivity to the lipid cofactor PIP2. The PLD activity in
LDTI membrane domains was high despite the absence of exogenously added
protein cofactors such as the small G proteins RhoA and
ADP-ribosylation factor, known to activate human PLD1 (7, 46). Because
guanosine 5'-3-O-(thio)triphosphate was not routinely
included in the assay mixture, it is unlikely that endogenous RhoA or
ADP-ribosylation factor was involved in supporting the high PLD
activity in LDTI membrane domains under our in vitro assay
conditions. Furthermore, addition of guanosine 5'-3-O-(thio)triphosphate (in the presence of MgATP) had no
effect on PLD activity in either the absence or presence of
PIP2 (Table I).
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The possibility that PLD is anchored to LDTI membrane domains via its cofactor PIP2 was examined by testing the effect of neomycin, a ligand of PIP2 that previously has been shown to inhibit PLD activity (36, 47). Neomycin was shown also to release PLD from rat brain membranes, presumably by competitively displacing the enzyme from its PIP2 binding site in the membrane (37). The PLD activity in isolated LDTI membranes was 73-80% inhibited by neomycin when present in the assay mixture (Table II). Pretreatment of the LDTI membranes with 5 mM neomycin, followed by wash and PLD assay, revealed that the aminoglycoside failed to release the enzyme from the membranes, as the activity in the neomycin-treated membranes was virtually identical to that in the control membranes. The PLD activity in the dialyzed supernatant was undetectable.
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Two distinct mammalian PLD genes have been sequenced recently,
designated PLD1 and PLD2 (7-9). PLD1 encodes a 120-kDa enzyme, the
activity of which is greatly stimulated by ADP-ribosylation factor and
RhoA and by the regulatory domain of protein kinase C- (7, 46, 48).
In contrast, the activity of PLD2 is high in the absence of these
protein factors and it is sensitive to neither of them (8, 9). Both PLD
forms exhibit a nearly absolute requirement for PIP2 (7,
8). We have raised polyclonal antibodies directed against a highly
conserved peptide sequence found within the human PLD1 gene (residues
675-688; see under "Materials and Methods"). Immunoblot analysis
of samples derived from sucrose density gradient fractions prepared
from HaCaT cells revealed a ~120-kDa, PLD1-immunoreactive band in
high density fractions but not in the low density fractions containing
caveolin (Fig. 3A). When equal
amounts of protein from caveolin-rich LDTI membrane domains (prepared
from fractions 5-6) and non-caveolin-containing membranes (prepared
from fractions 10-12) were analyzed by an antiserum directed to a
different epitope on the PLD1 molecule (residues 908-918), an
identical ~120-kDa protein band was seen in non-caveolin-containing
membranes, but not in caveolin-rich LDTI membrane domains (Fig.
3B). The distribution of PLD activity, PLD1, and caveolin-1
was examined also in COS-7 cells, in which a pronounced peak of PLD
activity is present in LDTI membranes (Fig.
4A). In these cells, again, a
clear dissociation between the distribution of caveolin-1 and that of
PLD1 was observed (Fig. 4B). These data clearly indicate
that the PLD found in caveolin-rich LDTI membrane domains is not PLD1.
Rather, PLD1 may very well contribute to the activity measured in the
high density fractions.
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The biochemical properties of the PLD found in caveolin-rich LDTI membrane domains, mainly its high constitutive activity in vitro and its stimulation by PIP2, are reminiscent of those reported for the mammalian PLD2 gene product (8). Unfortunately, antibodies that can recognize endogenous PLD2 (as opposed to the recombinant enzyme) are not yet available. An attempt was made to test the hypothesis that the PLD found in LDTI membrane domains is PLD2 by expression of a HA-tagged mouse PLD2 gene in COS-7 cells and examination of the distribution of PLD activity and PLD2 immunoreactivity. Transient expression of recombinant PLD2 resulted in a dramatic increase of COS-7 cellular PLD activity and immunoreactive HA-tagged PLD2, which was evident in LDTI membrane domains (~30%) as well as in non-caveolin-containing fractions (~70%).3 However, similar results were obtained upon transient expression of human HA-tagged PLD1 in COS-7 cells.3 Thus, the distribution of recombinant HA-tagged PLD1 and endogenous PLD1 (cf. Figs. 3 and 4) were found to be distinctly different. This suggests that the distribution of recombinant PLDs transiently expressed in COS-7 cells may not reflect the localization of endogenous PLDs.
The inconclusive results obtained using transient transfection in COS-7 cells prompted us to examine the distribution of non-tagged mouse PLD2 in stably transfected CHO cells. CHO cells normally exhibit very low PLD activity, either in vivo or in vitro, and are thus suitable for functional studies designed to examine the regulation and function of stably expressed PLD2. PLD activity was initially tested in the intact [3H]oleic acid-prelabeled cells, measuring production of [3H]phosphatidylethanol (expressed as a percentage of total lipid-incorporated radioactivity). As shown in Fig. 5A, basal PLD activity in the vector-control cells was low, whereas the phorbol ester PMA stimulated PLD activity markedly and dose-dependently. In PLD2-transfected cells basal activity was virtually undetectable, whereas the maximal PMA-stimulated PLD response was elevated significantly (albeit not dramatically) in comparison with the control cells, indicating that the expressed mouse PLD2 was functional. The distribution of PLD activity in sucrose density fractions in vitro was examined next (Fig. 5B). Control cells had low in vitro PLD activity in most gradient fractions (Fig. 5B, open columns). In contrast, PLD activity in the gradient from PLD2-transfected cells was greatly elevated, most dramatically in fractions 5 and 6. These results clearly indicate that PLD2 is preferentially targeted to LDTI membrane domains in CHO-PLD2 cells and strongly suggest that the endogenous PLD found in these domains in cell lines such as HaCaT, NIH 3T3, U937, and COS-7 is PLD2.
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The preparations of LDTI membrane domains from various cells most likely contain sphingolipid- and cholesterol-rich microdomains (variously termed rafts, glycosphingolipid-enriched membranes, or DIGs (18)), as well as caveolae proper. To determine whether PLD is specifically localized to caveolae, the distribution of PLD activity and PLD immunoreactivity was examined in U937 human promonocytic cells. Like other hematopoietic cells (42, 49), U937 cells do not express caveolin and lack morphologically identifiable caveolae.4 As shown in Fig. 6A, there is a marked enrichment of PLD activity in LDTI membrane fractions of U937 cells, with a specific activity in fraction 6 that is 13-fold higher than that measured in the total cell lysate. The PLD activity present in the LDTI membranes represent 64% of the total cellular activity (Fig. 6B). Immunoblot analysis shows that PLD1 is localized almost exclusively in high density fractions (Fig. 6C). These data show the PLD activity is localized in LDTI membranes also in the absence of caveolin.
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The fact that PLD activity is enriched in LDTI membrane domains in the absence of caveolin does not exclude the possibility that the same activity may be targeted to caveolae proper in cells that do express this protein. To examine the possible interaction between caveolin and PLD, we tested the effect of expression of caveolin-1 on the distribution of PLD activity. It has been demonstrated that caveolin-1 expression is down-regulated in oncogenically transformed cells (50). Thus, in order to have a low baseline expression of endogenous caveolin we elected to use v-Src-transformed NIH 3T3 cells in these experiments. Expression of recombinant caveolin-1 dramatically elevated immunoreactive caveolin levels in the low density fractions of the sucrose gradient (Fig. 7, top). Analysis of PLD activity in the fractions revealed that overexpression of caveolin markedly elevated PLD activity in low density fractions 4-6 but had little effect on PLD activity in the other fractions (Fig. 7, bottom). At present, it is impossible to determine whether the additional recombinantly expressed caveolin-1 stimulated the activity of PLD or caused an increase in the number of PLD molecules present in the fractions. The latter effect could be due either to increased expression of PLD or to a caveolin-mediated increase in its targeting to caveolae.
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To further examine the relationship between caveolin-1 and PLD, we tested the effect of a caveolin-derived peptide (caveolin-182-101) on the PLD activity in LDTI membranes. This peptide corresponds to the caveolin scaffolding domain, which is thought to mediate the interaction of caveolin with other proteins (23). As shown in Fig. 8, the caveolin scaffolding domain peptide had a dose-dependent modulatory effect on PLD activity. At low concentrations (1-10 µM), it stimulated the enzymatic activity ca. 40%. However, higher concentrations caused a progressive decrease in PLD activity, resulting in a nearly complete inhibition at a concentration of 50 µM. These data strongly suggest that caveolin-1 may interact with PLD via the same caveolin scaffolding domain with which it binds to other signaling proteins.
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DISCUSSION |
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The evidence provided herein indicates that low density Triton-insoluble membrane domains are highly enriched in a phospholipase D activity, which has been provisionally identified as PLD2 and which is up-regulated by overexpression of caveolin-1. In the present study, we have utilized a simple and facile approach for the preparation of LDTI membrane domains, based on two of their distinct physical properties: insolubility in nonionic detergents (such as Triton X-100) and low density. The fraction thus isolated typically contains less than 10% of the total cell protein and therefore may be expected to be highly pure. However, these domains include at least two types of structures: (i) caveolae (characterized by a caveolin-based coat structure), and (ii) DIGs (also known as glycosphingolipid-enriched membranes and cholesterol/sphingolipid rafts). The latter structures share with caveolae their distinct lipid composition but are devoid of caveolin, and they have been found in cells that do not express this protein (42, 49, 51-53).
Our results show that PLD is targeted to caveolin-free LDTI membrane
domains (isolated from U937 cells), indicating that caveolin is not a
necessary requirement for targeting PLD to these domains. However,
these results do not exclude the possibility that in cells that do
express caveolin, some or all of the cellular PLD complement will be
localized in caveolae. In fact, we demonstrate that the activity of PLD
in LDTI membrane domains is increased upon expression of recombinant
caveolin-1, as well as in multidrug resistance cells2 that
endogenously express high level of caveolin-1 (54). These data suggest
that caveolin could either modulate or target PLD activity to these
domains, implying a direct interaction between these two proteins. This
conclusion is strongly supported by the modulatory effect of the
caveolin scaffolding domain peptide (caveolin-182-101) on
the caveolar PLD activity. Curiously, the peptide exhibits a
stimulatory action on PLD activity at low concentrations ( 10 µM), and a strong inhibitory effect at higher
concentrations (10 µM). The molecular basis for this
bimodal effect is unclear. A possible explanation is that caveolin-1
has a constitutive stimulatory effect on PLD activity that is mimicked
by the the caveolin scaffolding peptide. The peptide-induced
stimulation is not extensive, possibly because the PLD is already in a
caveolin-activated state, albeit not a fully active one. The inhibitory
effect of caveolin-182-101 at higher concentrations could
be due to physical displacement of PLD from the caveolae and hence its
dissociation from its membrane bound substrate. These possibilities are
currently being explored in our laboratory. Together, the effect of
caveolin-1 expression on PLD and the action of the caveolin scaffolding
peptide strongly suggest that PLD will be localized in caveolae proper
in cells that do have these structures.
What is the identity of the PLD found in LDTI/caveolae? The present work unequivocally excludes PLD1 and the oleate-activated PLD as candidates. The high constitutive activity of PLD in LDTI membranes in vitro suggests that it may be PLD2 because, unlike PLD1, PLD2 exhibits high enzymatic activity in the absence of G protein activators (8). The distribution of recombinant PLD2 in stably transfected CHO cells provides strong support to the identification of the PLD found in LDTI membranes as PLD2. Notably, immunofluorescence analysis of the distribution of recombinant PLD2 in serum-treated rat embryo fibroblasts (REF-52) revealed its localization in submembraneous vesicles near the plasma membrane (8), consistent with a localization in caveolae. However, only the availability of sensitive anti-PLD2 antibodies could unequivocally confirm the identification of the PLD found in LDTI membranes as PLD2.
Like that of PLD2 (and PLD1), the PLD activity in LDTI membranes is stimulated by PIP2. Caveolae and DIGs are enriched in this minor but biologically important phospholipid (30-34). This fact, which served as the starting point for the present investigation, raises two questions: first, if these domains are highly enriched in PIP2, how is exogenous PIP2 able to further stimulate PLD activity? Second, does PIP2 serve as a membrane anchor for PLD in DIGs and caveolae? Studies have demonstrated that preparation of DIGs and caveolae as LDTI membranes results in loss of PIP2 from these domains (32, 34). It is therefore reasonable to assume that a similar loss has also occurred in our study, leaving the PLD unsaturated with PIP2. A corollary of this assumption is that PLD is not anchored to DIGs and caveolae via PIP2. Indeed, our experiments with neomycin (Table II) show that it is incapable of releasing the PLD from LDTI membranes, indicating that it is most likely anchored to the membranes in PIP2-independent manner, unlike the PLD in rat brain membranes (37). This conclusion is fully consistent with our caveolin scaffolding domain data, which suggest, instead, another anchoring mechanism involving direct interaction with caveolin.
The localization of PLD2 in LDTI membrane domains raises a number of
questions regarding its regulation and its possible function(s). As
compared with the regulation of PLD1, little is known about PLD2
regulation other than the fact that its activity is stimulated by
PIP2 (8, 9). It was also shown that PLD2 activity is inhibited by one or more cytosolic factors (8), which have recently
been reported to be - and
-synucleins (55). The present findings
may help identify additional factors, either protein or lipid, that
regulate PLD2 and that reside in LDTI membranes or are recruited to
these domains upon cell activation.
The activation of PLD1 is believed to take part in ADP-ribosylation
factor-dependent intracellular vesicle trafficking, where PLD-produced phosphatidic acid has been suggested to promote the recruitment of coat proteins onto the budding vesicle (56). Caveolar
PLD may be speculated to have an analogous function in caveolae
dynamics. Alternatively, the caveolar PLD may participate in
caveolae-resident receptor signaling. LDTI membrane domains in general,
and caveolae in particular, have been implicated in cell-surface
receptor-mediated signal transduction. PLD is activated by growth
factors acting via receptor tyrosine kinases, such as epidermal growth
factor (57, 58) and platelet-derived growth factor (58-61).
Platelet-derived growth factor stimulates the phosphorylation on
tyrosine residues of a number of proteins that are recruited onto the
platelet-derived growth factor receptor (62). Many of these proteins,
including the platelet-derived growth factor receptor itself, are
mobilized into caveolae upon stimulation (63). Similarly, epidermal
growth factor-induced recruitment of Raf-1 seems to occur in caveolae
(64). It remains to be seen whether the activation of PLD in DIGs and
caveolae by growth factors and other extracellular signal
molecules participates in signaling cascades launched from these
intriguing lipid platforms.
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ACKNOWLEDGEMENTS |
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We are grateful to Yona Eli and Tova Harel for excellent technical assistance and to all members of our laboratory for many helpful discussions. We thank Dr. Norbert Fusenig for providing the HaCaT cells, Prof. I. Cabantchik for providing the HT-29 cells, Dr. A. Taraboulos for providing caveolin-1 cDNA, and Drs. Michael Frohman and Andrew Morris for providing the PLD1 and PLD2 plasmids.
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FOOTNOTES |
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* This work was supported in part by grants from the United States-Israel Binational Science Foundation (Jerusalem, Israel), the Israel Science Foundation, the Minerva Foundation (Munich, Germany), and the Forchheimer Center for Molecular Genetics (Rehovot, Israel).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The first two authors contributed equally to this work.
§ Recipient of a FEBS Long-term Fellowship.
¶ Recipient of a grant-in-aid from the Israel Ministry of Absorption.
Incumbent of the Harold L. Korda Professorial Chair in
Biology. To whom correspondence should be addressed. Tel.:
972-8-9342773; Fax: 972-8-9344116; E-mail:
lhliscov{at}weizmann.weizmann.ac.il.
The abbreviations used are: PLD, phospholipase D; C6-NBD, (6-N-(7-nitrobenzo-2-oxa-1,3 diazol-4-yl)amino)caproyl; DIG, detergent-insoluble glycosphingolipid-rich complex; LDTI, low density Triton X-100-insoluble; PIP2, phosphatidylinositol 4,5-bisphosphate; CHO, Chinese hamster ovary; MES, 4-morpholineethanesulfonic acid.
2 G. Fiucci, M. Czarny, and Y. Lavie, unpublished observations.
3 Y. Lavie and M. Czarny, unpublished observations.
4 M. Czarny, unpublished results.
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REFERENCES |
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