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J Biol Chem, Vol. 274, Issue 40, 28652-28659, October 1, 1999
From the Laboratoire de Biochimie des Transports Cellulaires, CNRS,
Unité Mixte de Recherche 8619, Bâtiment 432, Université Paris XI, 91 405 Orsay Cedex, France
Colonic epithelial HT29-cl19A cells are polarized
and secrete proteins among which The protein machinery underlying protein transport from the
endoplasmic reticulum to the cell surface has been extensively studied
(for a review, see Ref. 1). However, interest in the regulatory
mechanisms that control the different steps of the intracellular
traffic and more particularly the role of the lipids in this processes
is rapidly growing at the moment.
The possible involvement of different signal transduction pathways in
the control of intracellular protein transit has recently been
investigated; the first evidence indicating that anterograde transport
along the secretory pathway is regulated by protein phosphorylation was
provided by the use of the phosphatase inhibitor okadaic acid
(2-5).
The involvement of protein kinase A
(PKA)1 in regulating membrane
traffic was suggested when it had been found that the regulatory RII
subunit of PKA was partially associated with Golgi membranes (6-8).
Pimplikar and Simons (9) and Hansen and Casanova (10), respectively,
showed that PKA activators increase the apical transport of influenza
hemagglutinin and the secretion of the endogenous glycoprotein gp80 in
Madin-Darby canine kidney cells. More recently, Jilling and Kirk (11)
showed that, in cultured colonic epithelial cells, cAMP stimulated
constitutive membrane traffic from the TGN to the apical cell surface.
Muniz et al. (12) demonstrated that, in NRK cells, the
inhibition of PKA by the PKA inhibitor H89 decreased protein transit
from the ER to the Golgi and blocked the exit of the vesicular
stomatitis virus G glycoprotein from the Golgi. In lacrimal glands
Robin et al. (13) showed that the PKA inhibitor H89 affected
the intracellular transit of regulated secretory proteins.
As far as a regulatory effect of protein kinase C (PKC) on
intracellular traffic is concerned, few data are available. Membrane trafficking between the ER and the Golgi apparatus of NRK cells was
found to be regulated by a diacylglycerol/phorbol ester-binding protein
(14), whereas in Madin-Darby canine kidney cells Pimplikar and Simons
(9) and Cardone et al. (15), respectively, described activation of apical transport of influenza hemagglutinin and stimulation of transcytosis of polymeric IgA receptor, by phorbol esters. In cell-free systems, PKC has also been shown to play a role in
the formation of vesicles from the TGN (16-21).
Finally, enzymes that modify membrane lipids may also regulate
constitutive membrane traffic. Evidence for the participation of
phosphoinositides and phosphatidylinositol kinases in the regulation of
membrane traffic has been reported (for a review, see Ref. 22). Lipases
are also implicated in these phenomena; as long ago as 1993, Stutchfield and Cockcroft (23) showed that phospholipase D (PLD)
activity was correlated with secretion in HL60 cells. More recently,
PLD and phosphatidic acid (PA) produced by the hydrolysis of
phosphatidylcholine (PC) by PLD, were shown to be required for the
formation of Golgi secretory vesicles (24-26) and for the regulation
of transport from the endoplasmic reticulum to the Golgi complex
(27).
To study the effect of the signal transduction pathways on the
intracellular transit of proteins more precisely, the human colonic
cell line HT29-cl19A appeared to be a good model system. These
epithelial cells, like the HT29-cl16E, emerged from the parental HT29
cells after the induction of differentiation by treatment with butyrate
(28) and are stably differentiated. HT29-cl19A cells have mostly been
used to study chloride secretion and cystic fibrosis transmembrane
conductance regulator expression, whereas HT29-cl16E cells were
selected to study mucin secretion (29-31). HT29-cl19A cells are unable
to secrete mucins but synthesize and secrete different proteins and
peptides including The aim of this study was to investigate in intact cells, which
normally secrete proteins, the different pathways controlling protein
transit and to determine the nature of the second messenger(s) connecting the signaling events, evoked by the binding of
receptor-directed agonists, to protein secretion.
In this work we quantified the amounts of We can conclude that in HT29-cl19A cells, an increase of PA level,
either directly by PLD action or by a two-step mechanism involving the
action of both PLC and DAG kinase is correlated to an enhancement in
the rate of apical protein transit.
Materials--
Tissue culture media were from Life Technologies,
Inc., Cergy Pontoise, France. [3H]Leucine (126-184
Ci/mmol, 4.66-6.81 TBq/mmol to 5 mCi/ml, 185 MBq/ml) was obtained from
Amersham Pharmacia Biotech, Les Ulis, France.
[3H]Myristic acid (49 Ci/mmol, 1.81 TBq/mmol - 1 mCi/ml,
37 MBq/ml) was purchased from NEN Life Science Products, Le Blanc
Mesnil, France. myo-2-[3H]Inositol was
obtained from Amersham Pharmacia Biotech (17.6 Ci/mmol, 0.65 TBq/mmol
to 1 mCi/ml, 37 MBq/ml). Carbachol, forskolin, dibutyryl cAMP, PMA,
PDBu, vasoactive intestinal peptide, propranolol, and the calcium
ionophore A23187 came from Sigma, Saint Quentin Fallavier, France.
Bisindolylmaleimide I (BIM), diacylglycerol kinase inhibitor II
(R59949), and tricyclodecan-9-ylxanthate (D609) were purchased from
Calbiochem, France Biochem, Meudon, France. All solvents were from
Prolabo, Fontenay sous Bois, France.
Cell Culture--
HT29-cl19A cells (kindly donated by Dr.
C. L. Laboisse) were a cloned line derived from the parental HT29
cells after treatment by butyrate (28). The cells were grown in Falcon
culture flasks (25 cm2) in a humidified atmosphere of 95%
air, 5% CO2, at 37 °C. The cells were fed every day
with Dulbecco's modified Eagle's medium (DMEM; 4.5g/liter glucose)
supplemented with 10% heat-inactivated fetal bovine serum. Cells were
subcultured after trypsin treatment every 7 days when they had reached
about 90% confluence. The passage number of the cells used in this
study varied between 16 and 30.
For all experiments, cells were seeded (about 0.25 × 106 cells/filter) onto Falcon cell culture inserts (10.5-mm
membrane diameter, 0.4-µm pore size, 1.0 × 108
pores/cm2). Cells were used between 14 and 20 days
following seeding, i.e. at least 1 week after they had
become confluent.
Metabolic Labeling and Measurement of Protein Secretion--
For
metabolic labeling, cells were incubated for 30 min in leucine-free
medium supplemented with 1% BSA. They were then pulsed for 10 min with
[3H]leucine (38.5 Ci/mmol) in 1% BSA leucine-free
medium; [3H]leucine was added to the basolateral side (65 µCi/380 µl volume). Cells were washed three times with 1 mM leucine in PBS to stop the labeling, and 1 ml of 1% BSA
in DMEM was placed in both the apical and the basolateral compartments.
Secretagogues, when used, were added to the basolateral medium and the
cells were incubated for 3-6 h at 37 °C. Medium samples (duplicate
aliquots of 80 µl) were collected at various time periods,
centrifuged at 2,000 × g to remove any detached cells,
and kept at 4 °C. Cells were lysed for 15 min in 1 ml of 0.5 N NaOH. The media samples and duplicate aliquots (80 µl)
of cell lysates were subjected to two cycles of 20% trichloroacetic
acid - 0.1% phosphotungstic acid precipitation, and the insoluble
radioactivity was counted in a 1212 Rackbeta liquid scintillation
counter (Amersham Pharmacia Biotech, Saint-Quentin en Yvelines, France).
When secretagogues were solubilized in Me2SO, the final
concentration of this solvent in the incubation medium was below
0.2%.
Protein secretion was expressed as the percentage of
3H-labeled proteins released into either the apical or
basolateral medium (i.e. 100 × total amount of
3H-labeled proteins released in one medium/total amount of
3H-labeled proteins in the two media and in the cells). The
secretion stimulation factor represents the ratio of protein secretion
in the presence of a secretagogue to control protein secretion.
SDS-PAGE Analysis and Fluorography of Secreted Proteins--
For
these experiments, proteins were radiolabeled for 30 min with
[3H]leucine (47.5 Ci/mmol) and then incubated with 300 µl of DMEM in both compartments for 6 h. BSA was absent from all
the culture media. The radiolabeled proteins secreted in the incubation
media were denaturated by adding 5× denaturating sample buffer to each sample and heating to 100 °C. Samples were separated by SDS-PAGE on
10% acrylamide gels. After fixation and staining with Coomassie Blue,
the gels were soaked in Amplify (Amersham Pharmacia Biotech), dried,
and exposed to Amersham Hyperfilms-MP for 1 to 5 days. Films were then
quantified by laser scanning densitometry with a Personal Densitometer
SI and ImageQuaNT software (Molecular Dynamics, Evry, France).
Immunoblotting--
Polypeptides separated by SDS-PAGE on 10%
acrylamide gels were electroblotted onto nitrocellulose
(Immobilon-NC, 0.45-µm pore, Millipore) or polyvinylidene difluoride
membranes (Hybond-P, 0.45-µm pore, Amersham Pharmacia Biotech). The
secreted protein, Immunoprecipitation--
Secretion media were obtained as
described for fluorography experiments. Cells were lysed in 300 µl of
0.1% triton X-100 in DMEM in the presence of protease inhibitors
(antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl
fluoride, benzamidine). Aliquots of identical volumes of media and
lysates were then incubated at 4 °C with polyclonal antibody against
ELISA--
Secretion media were obtained as described for
fluorography experiments, and cells were lysed as described for
immunoprecipitation experiments. The quantification of
Lipid Analysis--
For the measurement of PA and
phosphatidylethanol (PEt) production, HT29-cl19A cells grown for 14-20
days on cell culture inserts were washed in serum-free DMEM and then
labeled for 2 days with [3H]myristic acid in DMEM
containing 1 mg/ml lipid-free BSA. [3H]Myristic acid (49 Ci/mmol) was added to the basolateral side (2 µCi/1 ml of medium).
Before beginning the experiments, the cells were washed with PBS and
incubated in 1 ml of DMEM in the presence of the drugs to be tested. At
the end of the experiment, the cells were collected by cutting the
filters from the filter cups and the cellular lipids were extracted by
vortex-mixing in 1.8 ml of methanol/chloroform/HCl (100:50:1) at
4 °C. 30 min later 0.5 ml of water, 0.6 ml of chloroform, and 0.6 ml
of 2 M KCl were successively added to produce an aqueous
and an organic phase. The phases were separated by centrifugation at
1000 × g for 10 min; the aqueous phase was discarded,
and the organic phase containing the lipids was evaporated. The dried
samples were dissolved in chloroform/methanol (19:1), loaded on TLC
silica plates (Whatman LK6D), and resolved by developing with the
organic phase of the solvent system prepared by mixing isooctane/acetic
acid/ethyl acetate/water (2:3:13:10). The radiolabeled lipids were
detected and quantified with a Tracemaster 20 automatic TLC-linear
analyzer (Berthold, EGG, Evry, France).
When PLD activity was determined through the transphosphatidylation
reaction, experiments were performed as described above except that the
cells were preincubated 15 min in the presence of 3% ethanol prior to
the addition of the drugs to be tested.
The identity of PA, PEt, and PC were confirmed by comparison of their
RF values with PA and PEt standards revealed
after iodine sublimation, and [3H]choline-labeled PC radiodetected.
The amounts of PA and PEt formed were expressed as percentages of the
amount of PC detected.
Measurement of Inositol Phosphates Production--
For the
measurement of inositol phosphates (IPs) production, HT29-cl19A cells
grown for 14-20 days on cell culture inserts were washed in serum-free
DMEM and then labeled for 2 days with [3H]inositol in
DMEM containing 1 mg/ml lipid-free BSA. [3H]Inositol
(17.6 Ci/mmol) was added to the basolateral side (5 µCi/1 ml of
medium). Before beginning the experiments, the cells were washed with
PBS and incubated in 1 ml of DMEM, supplemented with 10 mM
LiCl, in the presence of the drugs to be tested. The reaction was
stopped by addition of 1 ml of cold 7% trichloroacetic acid and the
cells were scrapped from the inserts. Cell homogenates were centrifuged
for 10 min at 15,000 × g and 4 °C. The
IPs-containing supernatants were treated (4 × 6 ml) with
diethylether to remove trichloroacetic acid, neutralized with Tris
base, and then applied to anion-exchange columns containing about
1 g of Dowex 1 (AG 1-X8, 200-400-mesh, formate form, Bio-Rad).
Columns were washed with 10 ml of water to remove the excess of
myo-inositol; glycerophosphoinositols were eluted with 10 ml
of 60 mM ammonium formate, 5 mM disodic tetraborate. Total IPs were eluted with 12 ml of 1 M
ammonium formate, 0.1 M formic acid. The radioactivity of
aliquots of all the eluates was determined.
Characterization of Protein Secretion in HT29-cl19A Cells--
In
order to understand the regulation of protein secretion in these
filter-grown cells, we first analyzed the unstimulated secretion of
metabolically pulse-labeled proteins by different methods.
When we followed kinetics of secretion of leucine-labeled,
trichloroacetic acid-precipitated proteins in apical medium, we observed a lag time of about 1 h before the beginning of protein release; this may correspond to the minimal duration of intracellular transit. The rate of protein release then increased linearly for the
next 3 h (data not shown). The amount of radiolabeled proteins released in the apical medium after 3 and 6 h of incubation is shown in Table I. In the basolateral
medium, the secretion was lower (Table I).
The [3H]leucine-labeled proteins released by HT29-cl19A
cells were analyzed by SDS-PAGE and fluorography. Fig.
1a shows the fluorographic
patterns of the proteins secreted in the two media. In the apical
medium from unstimulated cells (lane 4), a 55-kDa polypeptide was observed. When the film was overexposed, minor polypeptides were detected (lane 4'); the 55-kDa
polypeptide was found to represent about 95% of the secreted
radiolabeled proteins. In the basolateral medium from unstimulated
cells (lane 5), the 55-kDa polypeptide was also
present, but its amount was lower than in the apical medium. Minor
polypeptides with molecular weights different of those of the
polypeptides found in the apical medium were seen when the film was
overexposed (lane 5'). The nature of the proteins
secreted and the relative proportion of the 55-kDa band are not
identical in the two media, indicating a polarized secretion.
Jilling and Kirk (11) identified the 55-kDa polypeptide as
Finally, we quantified by ELISA the amount of
Effect of Phorbol Esters on Protein Secretion--
PKA activators
have been described to selectively stimulate apical protein secretion
in HT29-cl19A cells (11), but no data were available concerning the
possible involvement of PKC in the regulation of protein transit in
these polarized cells. We thus tested the hypothesis that phorbol
esters, potent PKC activators, might also stimulate apical protein secretion.
We investigated the dose-dependent effect (3 nM
to 3 µM) of PDBu, one phorbol ester, on radiolabeled
protein secretion and showed that it was able to stimulate the protein
release in both compartments but with different efficiencies. In the
apical medium, a maximal effect on protein release was observed for
PDBu concentrations
The time course of apical radiolabeled protein secretion stimulated by
PDBu was followed; the lag time was shortened to about 50 min, and the
rate of protein release was increased compared with unstimulated cells
(data not shown). The effect of PDBu on radiolabeled protein secretion
into both media was quantified, and the results are given in Table I.
Under the same experimental conditions as those used for PDBu, PMA,
another phorbol ester, used at a concentration of 1 µM,
yielded identical results (data not shown).
The fluorographic pattern of the proteins secreted into the apical
medium after PDBu stimulation (Fig. 1a, lane
1 compared with lane 4) showed that
the intensity of the 55-kDa band was increased. When the film was
overexposed, the minor polypeptides were always detected (data not
shown); their relative amounts toward the 55-kDa band were found to be
identical to that of the control.
The fluorographic patterns of the immunoprecipitates (Fig.
1c) confirmed these results. After PDBu stimulation, the
intensity of the 55-kDa band was increased and the amount of
The results obtained by the two kinds of quantification
(trichloroacetic acid precipitation assay and ELISA) are coherent. After 6 h of incubation, phorbol esters enhanced the apical
secretion about 2-fold and the basolateral one only 1.3-fold (Table I). Consequently, for subsequent experiments, the trichloroacetic acid
precipitation assay was mainly used because it was easier than ELISA to
perform routinely.
We verified that the stimulation of secretion by PDBu was neither a
consequence of lysis of the cells nor the result of a PDBu-induced
elevation of the rate of protein synthesis (data not shown).
To evaluate the requirement of a phosphorylating activity of PKC in the
pathway enhanced by phorbol esters, we tested the effect of a
competitive inhibitor for the ATP-binding site of PKC, BIM (33). The
dose-dependent effect of BIM (0.15-10 µM) on
the apical secretion of radiolabeled proteins enhanced by PDBu was
investigated. Under our experimental conditions, the maximal effect of
the drug was obtained for concentrations > 2.5 µM
and a plateau was reached; for these concentrations BIM was found to
decrease the PDBu stimulation factor of about 60%. These results indicate that a part of the transit regulated by phorbol esters is
dependent on the phosphorylating activity of PKC.
Implication of PLD and PA in Protein Secretion--
Recent data
obtained from experiments performed with cell-free systems have
pinpointed the role of PLD in vesiculation (24, 25). Since this enzyme
can be activated by PKC (34, 35), we wondered whether, in HT29-cl19A
cells, the PDBu-activated PKC could regulate the PLD activity and
consequently the secretory process.
In the presence of primary alcohols, PLD catalyzes a
transphosphatidylation reaction; the production of PA is inhibited, and a phosphatidyl alcohol product is formed instead. We tested the effect
of different alcohols on protein secretion. The unstimulated secretion
in the apical medium was reduced by ethanol in a
dose-dependent way (Fig.
2a). After 3 h of
incubation in the presence of 3% ethanol, the secretion rate decreased
by 36%. The effect of 0.8% butanol-1 (another primary alcohol) was
close to that of 3% ethanol, whereas 0.8% butanol-3 (a tertiary
alcohol, which is not a substrate for the transphosphatidylation
reaction) was unable to reduce secretion (data not shown). It is also
noteworthy that ethanol had no effect on basolateral protein secretion
(data not shown).
To investigate the relationship between secretion and PLD activity, we
measured the activity of this enzyme under different experimental
conditions (Fig. 2, b and c). In control cells, a small amount of PEt was detected, indicating a basal activity of PLD.
PDBu enhanced the amount of PEt formed by about 9-fold, and BIM reduced
the effect of PDBu by about 65%. This effect of BIM is in good
correlation with its 60% inhibitory effect on protein secretion
enhanced by PDBu.
Since very recent data have suggested a role for PA, the physiological
product of PC hydrolysis by PLD, in vesiculation (26, 27), we wondered
whether this phospholipid would be able to regulate protein secretion.
We thus determined the amounts of PA formed under different
experimental conditions and showed that they were accurately correlated
with the percentages of secretion (Fig.
3); in the presence of 3% ethanol the
amount of PA decreased by 35% and the secretion rate by 36%, whereas
in the presence of PDBu the amount of PA increased by 2.45-fold and the
secretion rate by 2.2-fold.
Regulation of Protein Secretion by Carbachol--
Since phorbol
esters stimulate protein transit partly via PKC activation, we wondered
whether receptor-directed agonists, known to lead to PKC activation,
would also be able to regulate this transit. HT29-cl19A cells possess
muscarinic M3 receptors (36); therefore, we tested the
effect of carbachol, an agonist of this receptor type, on protein
secretion. The dose-dependent effect of this agonist was
investigated; carbachol (concentrations in the range of 30-100
µM) induced a maximal increase of protein secretion in
the apical medium but was without effect on the basolateral secretion.
The stimulation factors obtained for a carbachol concentration of 100 µM were 1.45 ± 0.04 and 1.32 ± 0.03 after 3 and 6 h of incubation, respectively (n > 10).
This carbachol concentration was used for further experiments.
The fluorographic pattern of the proteins secreted in the apical medium
shows a small increase in the intensity of the 55-kDa band (Fig.
1a, lane 2 compared with
lane 4).
The activation of muscarinic receptors can be coupled, via a G protein,
to the hydrolysis of phosphatidylinositol bisphosphate (PIP2) by phosphatidylinositol-phospholipase C (PI-PLC),
which produces DAG and inositol trisphosphate (IP3).
IP3 production then leads to Ca2+ mobilization,
and DAG activates PKC. In order to examine whether this
Ca2+ pathway is implicated in the secretory response to
carbachol, we first quantified the amount of total IPs. We found that
10 min, 30 min, and 2 h after the stimulation by carbachol the
amount of IPs was increased by about 3-, 7-, and 16-fold respectively. This result is in agreement with previous data indicating that, in
HT29-cl19A cells, carbachol stimulates increases in cellular IP3 and cytosolic Ca2+ (37). Nevertheless, the
Ca2+ pathway may not be implicated in the regulation of
protein secretion, since 1 µM ionomycin (11) and 10 µM of the ionophore A23187 (data not shown) were unable
to enhance protein release.
Since DAG is the physiological activator of most PKCs, we tested the
effect of the PKC inhibitor, BIM, on the secretory response triggered
by carbachol. 5 µM BIM was unable to inhibit the
enhancement of protein release induced by carbachol (data not shown).
These results suggest that PKC is not implicated in the regulation of protein transit when the cells are stimulated by carbachol.
Finally, as about 40% of the PLD activation triggered by PDBu was
independent of the PKC activity, we wondered whether carbachol could
activate PLD by a PKC-independent mechanism. Fig. 2c shows that carbachol was unable to trigger the activation of PLD since no PEt
was formed in these conditions.
These results are not in agreement with the commonly admitted statement
that PLD activation involves the phosphoinositide cycle and PKC
(35).
Implication of PA in Protein Secretion Enhanced by
Carbachol--
Since Ca2+ release, PKC, and PLD
activations could not explain protein release stimulated by carbachol,
we investigated the possible implication of another signaling pathway.
DAG produced by PIP2 hydrolysis can be converted into PA by
means of a DAG kinase. To enhance the amount of PA, we used
propranolol, an inhibitor of the phosphatidic acid
phosphohydrolase which hydrolyzes PA into DAG (38), and tested
the effect of this drug on protein secretion. 300 µM
propranolol increased the secretion obtained with carbachol; the
stimulation factor obtained after 6 h of incubation was
1.55 ± 0.06 (n = 3).
In order to check whether the amount of PA was increased by carbachol
treatment, we quantified the amounts of PA in different experimental
conditions. The chromatograms presented in Fig.
4a show that the amount of PA
formed after stimulation with carbachol was increased. Carbachol
increased the amount of PA by about 1.7- and 1.4-fold in control cells
or in cells pretreated with 300 µM propranolol,
respectively (Fig. 4, b and c). It is noteworthy that this increase in the amount of PA is less important than after a
PLD stimulation triggered by PDBu (2.45-fold increase) but is well
correlated with a lower effect of carbachol on protein secretion
(stimulation factor 1.45 with carbachol and 2.2 with PDBu). When cells
were pretreated with 10 µM DAG kinase inhibitor R59949
(39) prior to a stimulation by carbachol, the increase of PA was
reduced by about 70% (Fig. 4b). These results confirmed the
hypothesis of a production of PA, via the activation of both PLC and
DAG kinase, which can explain the regulatory effect of carbachol on
protein transit.
Nevertheless, DAG, and consequently PA, can also be produced by the
carbachol-triggered hydrolysis of PC by a phosphatidylcholine phospholipase C (PC-PLC) (40, 41). Since PC is the main phospholipid labeled with [3H]myristic acid (data not shown), we
wondered whether labeled PA might have also been produced by PC
hydrolysis. To investigate the possible implication of the PC-PLC in
the formation of PA, we tested the effect of a preincubation of the
cells with 200 µM D609, a competitive inhibitor of this
enzyme (42). Fig. 4c shows that this inhibitor reduced by
about 70% the amount of PA formed after incubation with carbachol in
the presence of propranolol. This effect was not due to an inhibition
of PI-PLC, since we verified that D609 did not reduce the production of
IPs stimulated by carbachol (data not shown).
Independence of the Pathways Regulating Protein
Secretion--
Since our results showed that the secretory responses
to carbachol and PDBu occurs via different pathways, we wondered
whether they were independent and additive. Fig.
5a shows that the effects of
100 µM carbachol and 0.1 or 1 µM PDBu were
additive (the PDBu concentration of 0.1 µM was tested,
since 80% of the total
PKA activators had also been shown to stimulate apical transport in
HT29-cl19A cells (11). We thus asked the question of whether the
secretory responses to secretagogues that increase the intracellular
cAMP level are independent of, and consequently additive with, the
secretory responses studied in this work. We first compared, under our
experimental conditions, the effect of vasoactive intestinal peptide, a
physiological stimulator of the HT29-cl19A cells, dibutyryl cAMP, a
permeant cAMP analogue, as well as forskolin, a direct activator of
adenylyl cyclase. The stimulation factors were close to 1.4 for the
three secretagogues (data not shown) and in the fluorographic pattern
of the proteins secreted in the apical medium after forskolin
stimulation, the intensity of the 55-kDa band was slightly increased
(Fig. 1a, lane 3 compared with
lane 4). We also found that forskolin acted by a
completely different mechanism from that of carbachol or phorbol
esters, as it did not increase the production of PA (data not shown).
When 100 µM carbachol and 10 µM forskolin
were added together, the secretion stimulation factor was increased and
corresponded to a complete additivity of the effects of the two
secretagogues (Fig. 5b). This result confirms that those two
regulatory pathways are fully independent.
Regulation of Protein Transit at a TGN or Post-TGN Step of the
Secretory Pathway--
We finally investigated whether the different
modulators of protein secretion act at earlier or later steps of the
transit. To answer this question, we used the "20 °C block"
(43). HT29-cl19A cells were pulse-labeled at 37 °C and then
incubated for 3 h at 20 °C. At this temperature, the
radiolabeled proteins should be entrapped within the TGN. The different
drugs to be tested, PDBu, carbachol, and ethanol, were then added to
the basolateral medium, and the incubation was continued at 37 °C
for 3 h. A typical experiment showing the kinetics of radiolabeled
protein secretion in the apical medium is shown in Fig.
6. After a lag time of about 45 min,
secretion started and exhibited linear kinetics. The stimulation factors calculated after 3 h were 1.88 ± 0.20, 1.31 ± 0.12, and 0.55 ± 0.02 for PDBu, carbachol, and ethanol,
respectively. They are in the same range as the stimulation factors
calculated when the drugs were added just after the pulse.
Although these results do not allow us to exclude a role of the
secretagogues at the ER-to-Golgi step, they clearly indicate that a TGN
(or post-TGN) step in protein transit is regulated in HT29-cl19A cells.
In this work we present new data concerning the regulation of
apical protein transit by several different signaling pathways. Colonic
epithelial HT29-cl19A cells, 5-10 days after they have reached
confluence, exhibit morphological cell polarity and functional differentiation (28). They secrete in a constitutive way
We showed that phorbol esters reduce the duration of transit and
enhance the rate of apical protein transit by 2.2-fold. This increase
in protein transit is inhibited by BIM, a PKC inhibitor. Since BIM
interacts specifically with the catalytic domain of PKC, we conclude
that PKC activity is needed for the regulation of protein transit in
HT29-cl19A cells. The requirement of the phosphorylating activity of a
Golgi-associated PKC in the vesicle formation and release was described
by Westerman et al. (21) and Simon et al. (19);
our experiments in intact cells are in good agreement with these
findings from cell-free systems.
The hypothesis that PKC could play a role in vesicular transport
through the activation of PLD, which has been found to be enriched in
Golgi membranes (44, 45) is attractive. The possible implication of PLD
in the regulation of protein transit in HT29-cl19A was thus
investigated. Ethanol was found to reduce unstimulated secretion in a
dose-dependent way; moreover, in unstimulated cells incubated in the presence of ethanol, a small amount of PEt was detected, indicating a low basal PLD activity. The suggestion that a
basal activity of this enzyme may mediate constitutive secretion was
recently put forward by Bi et al. (27). To confirm the
involvement of PLD and PA in protein transit, we determined the amounts
of PA (or PEt) in different experimental conditions and observed a
close correlation between these results and our data concerning
secretion. (i) PDBu increased the amount of PA by 2.45-fold, whereas it
stimulated secretion by 2.2-fold; (ii) ethanol decreased the amount of
PA by 35%, whereas it lowered secretion by 36%; (iii) BIM inhibited
the PDBu-stimulated PLD activity (65% inhibition) to the same degree
as it reduced protein release triggered by PDBu (60% inhibition).
Nevertheless, about 40% of both PLD activation and protein secretion
triggered by phorbol esters bypasses the PKC step, and at present we do
not have explanation for this pathway.
Taken together our results strongly suggest that, in intact cells,
phorbol esters regulate protein transit via PLD-catalyzed PA
production. They are in good agreement with the recent findings, obtained from experiments performed with a cell-free system, that PA
increases vesicle budding from the TGN (26).
In this work, phorbol esters were shown to activate PKCs and PLD with a
resulting increase in protein transit. However, although these drugs
are useful pharmacological tools, they are not physiological modulators
of the regulatory pathways. We took advantage of the fact that
HT29-cl19A cells possess muscarinic M3 receptors (36) to
study the effect of carbachol, a cholinergic receptor-directed agonist,
on protein transit. Activation of these receptors results in the
triggering of a well established signal transduction pathway; the
activation of a PI-PLC generates IP3, which controls
cytosolic Ca2+ levels, and DAG, which is an important
regulator for PKC.
Carbachol was less efficient than phorbol esters in increasing protein
secretion (stimulation factor 1.45 compared with 2.2 obtained with
PDBu). Nevertheless, the results obtained with carbachol were rather
unexpected. Although carbachol, via PI-PLC activation, was able to
produce IP3 (and consequently DAG), (i) Ca2+
was not found to be involved in the control of protein transit; (ii)
DAG was not found to activate PLD via a PKC activation as it was the
case for PDBu (perhaps the PKC isoform(s) activated by DAG is not
implicated in PLD stimulation). Another mechanism explaining the
secretory effect of carbachol had to be searched. A product derived
from DAG may be involved. As DAG can be phosphorylated into PA by a DAG
kinase, we analyzed the PA content of the cells and showed that the
level of this phospholipid was increased by about 1.7-fold on carbachol
stimulation. Thus, carbachol, on the one hand, enhanced the PA level in
the cells and, on the other hand, increased protein secretion. These
two results are well correlated and strengthen the conclusion that PA
plays a role in the regulation of protein transit.
DAG (and consequently PA) was also reported to be produced by direct
hydrolysis of PC by a PC-PLC; cholinergic and bradykinin receptors have
been shown to activate this enzyme (40, 41, 46). In HT29-cl19A cells,
we found that D609, a specific inhibitor of this enzyme (42),
partly inhibited the production of PA triggered by carbachol. The two
phospholipases may play a role in the physiological regulation of
protein transit, as McKenzie et al. (41) showed that, in
fibroblasts treated with carbachol, PI-PLC activation lead to a
transient increase in DAG level, whereas PC-PLC activation would
produce a sustained level of DAG. Since secretion is a phenomenon that
lasts several hours, a sustained PC-PLC-dependent
production of PA is not unlikely in our system.
Finally we show that PA production is not the sole mechanism
controlling secretion, since we demonstrated that cAMP controls protein
transit by a PA-independent pathway.
Our results allow us to describe a part of the regulation of protein
secretion in HT29-cl19A cells according to the scheme presented in Fig.
7; the constitutive secretion depends on
a basal PLD activity. A phorbol ester-triggered enhancement of the rate of protein transit is well correlated to a classical PLD-catalyzed PA
production. Nevertheless, when the cells are stimulated via their
cholinergic receptors, the increase in the rate of protein transit,
also in good correlation with an enhancement of the amount of PA,
implicates a non-classical pathway involving the activation of PLCs and
DAG kinase.
We thank Dr. Blandine Geny, Dr. Hervé
Le Stunff, and Dr. Philippe Mauduit for helpful discussions and Dr.
Gillian Barratt for critical reading of the manuscript.
*
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 abbreviations used are:
PKA, protein kinase
A;
PKC, protein kinase C;
ER, endoplasmic reticulum;
PLD, phospholipase
D;
PLC, phospholipase C;
TGN, trans-Golgi network;
DAG, diacylglycerol;
ELISA, enzyme-linked immunosorbent assay;
BSA, bovine serum albumin;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel
electrophoresis;
DMEM, Dulbecco's modified Eagle's medium;
PA, phosphatidic acid;
PEt, phosphatidylethanol;
PC, phosphatidylcholine;
IP3, inositol trisphosphate;
IP, inositol phosphate;
PIP2, phosphatidylinositol bisphosphate;
PDBu, phorbol
12,13-dibutyrate;
BIM, bisindolylmaleimide I;
PMA, phorbol
12-myristate 13-acetate.
Relationship between Phosphatidic Acid Level and Regulation of
Protein Transit in Colonic Epithelial Cell Line HT29-cl19A*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin
represents about 95%. Secretion occurs via a constitutive pathway, so
that the rates of secretion directly reflect the rates of protein
transit. In this paper we have demonstrated that: 1) in resting cells
phospholipase D (PLD) is implicated in the control of apical protein
transit; 2) phorbol esters stimulate apical protein transit
(stimulation factor 2.2), which is correlated with a PLD-catalyzed
production of phosphatidic acid (PA) (2.45-fold increase); 3) the
stimulation of cholinergic receptors by carbachol results in an
increase (stimulation factor 1.45) of apical protein transit which is
independent of protein kinase C and PLD activities, but related to PA
formation (1.7-fold increase) via phospholipase(s) C and diacylglycerol
kinase activation; 4) an elevation of the cAMP level enhances apical
protein transit by a PA-independent mechanism; 5) a trans-Golgi network
or post-trans-Golgi network step of the transit is the target for the
regulatory events. In conclusion, we have shown that PA can be produced
by two independent signaling pathways; whatever the pathway followed, a
close relationship between the amount of PA and the level of secretion
was observed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin (11). These cells have
been used for studies of ion secretion, and were shown to respond to
the cholinergic agonist carbachol, to the PKC activators PDBu and PMA
(32) to vasoactive intestinal peptide and to forskolin (31). Finally,
these cells become polarized after confluence, so when they are grown
on filters, basolateral and apical secretions can be analyzed independently.
1-antitrypsin,
the major protein secreted. We showed that protein transit can be regulated at a TGN or post-TGN step of the pathway and confirmed that
agents that increase the cAMP level enhanced apical protein transit. We
demonstrated that other mechanisms also controlled secretion: (i)
phorbol esters, which we showed to activate PLD, enhanced the PA amount
and the apical transit; (ii) carbachol, which we showed to activate PLC
and produce an elevation of the PA level (but without activating PLD),
also increased apical transit, but to a lower extent.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin, was detected with a
polyclonal peroxidase-labeled antibody against this protein (ICN
Pharmaceuticals, Orsay, France; dilution 1:5000) and revealed with the
ECL kit (Amersham Pharmacia Biotech).
1-antitrypsin (Sigma; dilution 1:250) for 2.5 h
followed by incubation with immobilized protein A (Amersham Pharmacia
Biotech) for 30 min. Immunoprecipitates were washed twice with DMEM and
solubilized in sample buffer for SDS-PAGE. In some experiments,
immunoprecipitations were performed in the absence of antibody in order
to determine the aspecific binding of radiolabeled proteins on protein
A-Sepharose.
1-antitrypsin in the media was performed using a
competition ELISA. Human 1-antitrypsin (ICN) was used to
standardize this assay, in the concentration range 3 × 10
10 to 107 M. The samples
containing the antigen were incubated, first for 2 h with the
anti-1-antitrypsin peroxidase-labeled polyclonal antibody (ICN; dilution 1:10000) and then for 2 h on a titration plate coated with 1-antitrypsin. The peroxidase activity
of the antigen-antibody complex bound to the plate was determined using 3,3',5,5'-tetramethyl benzidine as a substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of quantifications of protein secretion by trichloroacetic
acid assay and ELISA
1-antitrypsin was quantified by ELISA after
6 h of incubation. Values are means ± S.E.,
n > 10 for trichloroacetic acid assay and
n = 4 for ELISA. The total 1-antitrypsin
(secreted + non-secreted) was estimated as 250 ± 50 pmol/insert (n = 2).
View larger version (60K):
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Fig. 1.
Analysis of secreted proteins by
fluorography, immunoblotting, and immunoprecipitation. a,
the cells were labeled with [3H]leucine for 30 min and
the secretagogues were added just after. The media obtained after
6 h of incubation were analyzed by fluorography. Lanes 1-4', proteins secreted in the apical medium in the absence
of secretagogue (lanes 4 and 4'), or
after addition of 100 µM carbachol (lane 2), 10 µM forskolin (lane 3), 1 µM PDBu (lane 1).
The same volumes of the different media were loaded on the gel.
Lanes 5 and 5', proteins secreted in
the basolateral medium in the absence of secretagogue. Lanes 4' and 5' correspond to overexposed views of
media shown in lanes 4 and 5,
respectively. The results are representative of three independent
experiments. b, apical medium (lane 1), basolateral medium (lane 2), and
cell lysate (lane 3) obtained after 6 h of
incubation of unstimulated cells were analyzed by immunoblotting with
an antibody against 1-antitrypsin. The results are
typical of one experiment among three. c, the cells were
labeled with [3H]leucine for 30 min and the secretagogues
were added just after. The media obtained after 6 h of incubation
and the cell lysate were immunoprecipitated with an antibody against
1-antitrypsin as described under "Experimental
Procedures" and the immunoprecipitates were analyzed by fluorography:
apical medium (lane 1), basolateral medium
(lane 2), and cell lysate (lane 4) of unstimulated cells. Lanes 3 and
5 represent the aspecific bindings to protein A-Sepharose of
the apical medium and the cell lysate of unstimulated cells,
respectively. Lanes 6-8, apical medium
(lane 6), basolateral medium (lane 7), and cell lysate (lane 8) of cells
stimulated with 1 µM PDBu. The results are typical of one
experiment among two.
1-antitrypsin. The immunoblots presented in Fig.
1b confirm the presence of this protein in the two media and
the cell lysate. We thus analyzed the secretion of
1-antitrypsin by immunoprecipitation. Fig. 1c
(lanes 1-5) shows the result of the fluorography
of the immunoprecipitates obtained from the medium and from the cell lysate. The intensity of the band of radiolabeled
1-antitrypsin from the apical medium was higher than
that from the basolateral medium.
1-antitrypsin secreted in both the apical and the
basolateral medium (Table I). We also found a higher amount of
1-antitrypsin in the apical medium than in the
basolateral one.
1 µM; the 1 µM
concentration was used for further experiments.
1-Antitrypsin was quantified by ELISA after PDBu
stimulation. The results, given in Table I, indicate that about 80% of the 1-antitrypsin is secreted from the cells after
6 h of stimulation with PDBu.
1-antitrypsin in the cell lysate greatly reduced.
View larger version (31K):
[in a new window]
Fig. 2.
Effect of ethanol on protein secretion and
PLD activity measurement. a, effect of ethanol on apical
protein release. The cells were pulse-labeled with
[3H]leucine and ethanol (concentrations ranging between
1% and 3%) was added immediately after the pulse. The stimulation
factors were calculated after 3 h of incubation. Values are
means ± S.E., n = 3. b, PLD activity
measurements. The cells were labeled with [3H]myristic
acid for 48 h, washed, and pretreated with 3% ethanol for 15 min.
1 µM PDBu was then added or not, and the incubations were
continued for 45 min; lipids were extracted and analyzed by TLC. Two
typical radiochromatograms are shown. c, after a 48-h
labeling with [3H]myristic acid, the cells were
pretreated with 3% ethanol for 15 min. 1 µM PDBu (in the
absence or presence of 5 µM BIM) or 100 µM
carbachol (carba) were then added, and the incubations were
continued for 45 min. The PEt amounts were determined from the TLC
radiochromatograms. Values are means ± S.E., n = 5.
View larger version (36K):
[in a new window]
Fig. 3.
Comparative effects of ethanol and PDBu on
protein secretion and PA amount. For secretion measurements, the
cells were pulse-labeled with [3H]leucine; 3% ethanol, 1 µM PDBu (or nothing) was added immediately after, and the
radiolabeled proteins were quantified in the apical medium after 3 h of incubation. Values are means ± S.E., n = 3. For PA amount determinations, the cells were labeled with
[3H]myristic acid for 48 h, washed, and treated with
either 3% ethanol or 1 µM PDBu (or nothing) for 45 min;
lipids were extracted, and the PA amounts determined from the TLC
radiochromatograms. Values are means ± S.E., n 7.
View larger version (35K):
[in a new window]
Fig. 4.
Effect of carbachol on apical protein
secretion and on PA formation. a, the cells were labeled
with [3H]myristic acid for 48 h and treated for 45 min with 100 µM carbachol (carba). Lipids were
extracted and analyzed by TLC. Two typical radiochromatograms are
shown. b, after a 48-h labeling with
[3H]myristic acid, the cells were pretreated (or not) for
2 h with 10 µM R59949. They were then stimulated (or
not) for 45 min with 100 µM carbachol. The PA amounts
were determined from the TLC radiochromatograms. Values are means ± S.E., n 4. c, after a 48-h labeling
with [3H]myristic acid, the cells were pretreated for 30 min with 300 µM propranolol (pro) in the
presence (or absence) of 200 µM D609. They were then
stimulated (or not) for 45 min with 100 µM carbachol. The
PA amounts were determined from the TLC radiochromatograms. Values are
means ± S.E., n = 4. Paired Student's
t test gave p values < 0.03.
1-antitrypsin being released
after 6 h of incubation with 1 µM PDBu, an
additional increase in secretion was difficult to detect).
View larger version (27K):
[in a new window]
Fig. 5.
Additive effects of secretagogues on apical
protein secretion. The cells were pulse-labeled with
[3H]leucine. a, 100 µM carbachol
(carba) and 0.1 or 1 µM PDBu were added alone
or together (as indicated in the graphic) immediately after the pulse.
The stimulation factors were calculated after 6 h of
incubation. Values are means ± S.E., n = 3. b, 100 µM carbachol and 10 µM
forskolin (forsko) were added alone or together immediately
after the pulse. The stimulation factors were calculated after 6 h
of incubation. Values are means ± S.E., n 3.
View larger version (18K):
[in a new window]
Fig. 6.
Effect of 20 °C block on apical protein
secretion. The cells were pulse-labeled with
[3H]leucine at 37 °C and chased for 3 h at
20 °C. The temperature was then increased to 37 °C and 1 µM PDBu, 100 µM carbachol
(carba), 3% ethanol, or nothing (control) were
added. The kinetics of protein secretion in the apical medium were
followed. Values are results from one of three typical
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-antitrypsin, which represents about 95% of the
apically secreted proteins.
View larger version (27K):
[in a new window]
Fig. 7.
Proposed pathways for PA formation. PA
can be produced by two reactions: phosphorylation of DAG by a DAG
kinase and hydrolysis of PC by PLD. DAG is produced either from
PIP2 by a PI-PLC or from PC by a PC-PLC. These two enzymes
are regulated by muscarinic receptors. PLD is activated by phorbol
esters, partly via a PKC isoform that is not activated by muscarinic
receptors.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correpondence should be addressed.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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