From INSERM, U-338 Biologie de la Communication Cellulaire, 5 rue Blaise Pascal, 67084 Strasbourg Cedex, France
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ABSTRACT |
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Besides having a role in signal transduction,
heterotrimeric G proteins may be involved in membrane trafficking
events. In chromaffin cells, Go is associated with
secretory organelles, and its activation inhibits the
ATP-dependent priming of exocytosis. By using permeabilized
cells, we previously described that the control exerted by the
granule-bound Go on exocytosis may be related to effects on
the cortical actin network through a sequence possibly involving Rho.
To provide further insight into the function of Rho in exocytosis, we
focus here on its intracellular localization in chromaffin cells. By
immunoreplica analysis, immunoprecipitation, and confocal
immunofluorescence, we found that RhoA is specifically associated with
the membrane of secretory chromaffin granules. Parallel subcellular
fractionation experiments revealed the occurrence of a
mastoparan-stimulated phosphatidylinositol 4-kinase activity in
purified chromaffin granule membranes. This stimulatory effect of
mastoparan was mimicked by GAP-43, an activator of the
granule-associated Go, and specifically inhibited by
antibodies against Go. In addition, Clostridium
botulinum C3 exoenzyme completely blocked the activation of
phosphatidylinositol 4-kinase by mastoparan. We propose that the
control exerted by Go on peripheral actin and exocytosis is related to the activation of a downstream RhoA-dependent
phosphatidylinositol 4-kinase associated with the membrane of secretory
granules.
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INTRODUCTION |
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Studies on diverse secretory cell types have established a crucial role for GTP-binding proteins in the regulation of calcium-dependent exocytosis. Trimeric G proteins have been found associated with the membrane of various secretory granules (1-3), suggesting their participation in the exocytotic reaction. Accordingly, the involvement of a plasma membrane-bound Gi3 protein in the late stages of exocytosis in mast cells has been demonstrated (4). Direct control of exocytosis by Gi and Go proteins has also been described in insulin-secreting cells (5) and in chromaffin cells (3, 6-9). Besides heterotrimeric G proteins, a subset of small GTPases appears to control the exocytotic reaction. Rab3 and the ADP-ribosylation factor 6 which are specifically localized on secretory vesicles (10, 11) have been proposed to serve as a regulator of the exocytotic fusion in chromaffin cells (12, 13), anterior pituitary cells (14), and melanotrophs (15). In addition, recent investigations led to the idea that Rho may be a component of the molecular machinery underlying regulated secretion (16-20).
Rho belongs to the GTPase family consisting of Rho, Rac, and Cdc 42 proteins. This family has been implicated in a number of cellular functions requiring the reorganization of actin-based structures (21-23). In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis since actin forms a cortical barrier that must be reorganized to enable docking and/or fusion of the secretory granules with the plasma membrane (24-26). Rho together with a trimeric G protein regulates the changes in the actin cytoskeleton observed in activated mast cells (17, 27). In chromaffin cells, we recently described that the secretory granule-associated Go controls the priming of exocytosis by modifying the cortical actin network through a sequence of events that eventually involves Rho (20). Thus, Rho seems to be an integral component of the signaling pathway leading to the cytoskeletal redistribution necessary for secretion, although the mechanism by which Rho relates to the actin organization remains to be elucidated.
To identify the partners of Rho signaling in calcium-evoked secretion,
we examine here the intracellular distribution of Rho in chromaffin
cells. By immunoreplica analysis and confocal immunofluorescence, we
demonstrate that RhoA is a specific component of the membrane of
secretory chromaffin granules. Furthermore, our data reveal that the
phosphatidylinositol
(PtdIns)1 4-kinase present on
chromaffin granules (28, 29) can be stimulated by specific activators
of Go through a mechanism sensitive to Clostridium
botulinum C3 ADP-ribosyltransferase. We propose that the
granule-bound Go controls the peripheral actin cytoskeleton and exocytosis through an effector pathway involving the sequential participation of RhoA and PtdIns 4-kinase both located on the membrane
of secretory chromaffin granules.
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MATERIALS AND METHODS |
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Culture of Chromaffin Cells-- Chromaffin cells were isolated from fresh bovine adrenal glands by retrograde perfusion with collagenase and purified on self-generating Percoll gradients (30). Cells were suspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and containing cytosine arabinoside (10 µM), fluorodeoxyuridine (10 µM), streptomycin (50 µg/ml), and penicillin (50 units/ml). Cells were cultured as monolayers on either 24 multiple 16-mm Costar plates (Cambridge, MA) at a density of 2.5 × 105 cells/well or fibronectin-coated glass coverslips at a density of 2 × 105 cells or 100-mm Costar plates at a density of 5 × 106 cells/plate. Experiments were performed 3-7 days after plating.
Subcellular Fractionation of Cultured Chromaffin Cells-- Cultured chromaffin cells (50 × 106 cells) were washed with Locke's solution, rapidly frozen and thawed, and collected in 3.7 ml of buffer A containing 20 mM HEPES, pH 7.5, 1 mM EDTA, 1 mM DTT, 20 µg/ml pepstatin, 20 µg/ml aprotinin, 20 µg/ml trypsin inhibitor, 20 µg/ml RNase, 0.1 mM phenylmethylsulfonyl fluoride (total homogenate). Following 5 min incubation on ice, 2.5 ml of the homogenate was centrifuged for 45 min at 100,000 × g. The supernatant was saved (cytosol) and the pellet homogenized in 500 µl of buffer A (membrane-bound fraction).
Subcellular Fractionation of Bovine Adrenal Medulla--
Plasma
and chromaffin granule membranes were purified from bovine adrenal
medulla as described previously (3). Briefly, adrenal medullary glands
were homogenized in 0.32 M sucrose (10 mM
Tris-HCl, pH 7.4) and then centrifuged at 800 × g for
15 min. After centrifugation at 20,000 × g for 20 min,
the pellet was resuspended in 0.32 M sucrose (10 mM Tris-HCl, pH 7.4), layered on a continuous sucrose
density gradient (1-2.2 M sucrose, 10 mM
Tris-HCl, pH 7.4), and centrifuged for 1 h at 100,000 × g. Twelve 1-ml fractions were collected from top to bottom
and analyzed for protein by the Bradford procedure. The distribution of
dopamine--hydroxylase (D
H) and Na+/K+
ATPase was estimated as described previously (31, 32). Plasma membranes
were purified from fractions 2 and 3, which contained the highest
Na+/K+ ATPase activity. Chromaffin granule
membranes were recovered from fractions 11 and 12, which contained the
highest D
H activity. Fractions were diluted 10 times in TED buffer
(20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT), and membranes were collected by centrifugation for
30 min at 100,000 × g. Membranes were suspended in TED
buffer and stored at
20 °C.
ADP-ribosylation Assay with Recombinant C3 Enzyme-- ADP-ribosylation with C. botulinum exoenzyme C3 ADP-ribosyltransferase (5 µg/ml) was performed with 15 µg of proteins in 20 mM HEPES, pH 7.5, 10 mM thymidine, 1 mM DTT, 1 mM EDTA, 5 mM MgCl2, 1 mM ATP, 100 µM GTP, 0.5 µCi of [32P]NAD (0.15 µM) (30 Ci/mmol, 2 mCi/ml, NEN Life Science Products). The reaction was carried out for 30 min at 37 °C in a final volume of 120 µl. Proteins were then precipitated with 10% trichloroacetic acid, centrifuged (15 min at 12,000 rpm), and dissolved in sample buffer for SDS-polyacrylamide gel separation. Labeled proteins were analyzed by autoradiography with a Bio-Imaging Analyzer FUJIX BAS1000 (Fuji, Tokyo, Japan).
Phosphatidylinositol Kinase Assay--
PtdIns kinase activity
was detected as described by Husebye and Flatmark (28). Chromaffin
granule membrane proteins (50 µg) were preincubated for 10 min in the
presence or absence of the indicated concentrations of mastoparan in
assay buffer containing 30 mM HEPES, pH 7.0, 0.1 mM EGTA, 5 mM MgCl2 in a final
volume of 300 µl. The reaction was started by addition of 30 µl of
the ATP solution containing 2.5 µCi of [-32P]ATP
(3000 Ci/mmol, Amersham Pharmacia Biotech) and 5 mM MgATP. After 2 min, the reaction was stopped by addition of 800 µl of chloroform, methanol, 12 M HCl (20/40/1, v/v/v). The
chloroform phase was extracted, evaporated under vacuum, and then
redissolved in 20 µl of chloroform/methanol (2/1, v/v). Phospholipids
were separated on one-dimensional TLC oxalate-coated silica gel plates in chloroform, methanol, 4 M NH4OH (9/7/2,
v/v/v). Labeled phospholipids were visualized with
32P-imaging plates using the Bio-Imaging Analyzer FUJIX
BAS1000. Labeled phospholipids were compared with standard lipids
stained with iodine vapor.
Deacylation and HPLC Analysis--
For definitive identification
of the phosphatidylinositol monophosphates detected on the TLC plates,
the radioactive spots were excised, deacylated (33), and subjected to
HPLC analysis using a 10-µm Partisil SAX ion-exchange column
(Interchim, Montluçon, France). After sample injection, the
column was equilibrated with water for 5 min, and
32P-labeled glycerophosphatidylinositol monophosphates were
separated using a 60-min linear gradient of 0-0.18 M
ammonium phosphate, pH 3.8 (33). A mixture of AMP and ADP was applied
with the deacylated samples, and their 260-nm absorbance served to
standardize elution times that were similar from one run to the next.
Radioactivity was detected by assaying 200-µl eluate fractions.
32P-Labeled glycerophosphatidylinositol 4-phosphate
(gPtdIns-4-P) standard was prepared by deacylation of
[32P]PtdIns-4-P obtained by lipid extraction and TLC
separation of 32P-labeled quiescent CHO cells as described
(33). The 32P-labeled glycerophosphatidylinositol
3-phosphate (gPtdIns-3-P) standard was prepared by deacylation of
[32P]PtdIns-3-P produced in vitro by
phosphatidylinositol 3-kinase (p85) immunoprecipitated from CHO cells
and incubated in the presence of PtdIns and [-32P]ATP
(33).
GTPase Activity--
The GTPase activity of purified chromaffin
granule membranes was estimated according to the procedure previously
described (3, 6). Briefly, granule membrane proteins (10 µg) were incubated for 10 min at 30 °C in 100 µl of 50 mM
HEPES, pH 8.0, 1 mM EDTA, 1 mM DTT, 100 mM NaCl, and 1 mM MgCl2 in the
presence of the indicated concentrations of mastoparan. The GTPase
reaction was started by the addition of GTP at a final concentration of 0.8 µM with 1 nCi [-32P]GTP (NEN Life
Science Products, 30 Ci/mmol) and stopped by the addition of 250 µl
of cold 5% (w/v) Norit in phosphate buffer. After centrifugation, the
32Pi in the supernatant was determined by
32P Cerenkov counting. Non-enzymatic hydrolysis of
[
-32P]GTP during the assay was subtracted from all
data. Assays were performed in triplicate.
Immunocytochemistry and Confocal Laser Scanning Microscopy-- Chromaffin cells grown on fibronectin-coated glass coverslips were washed with Locke's solution and subsequently fixed for 15 min in 4% paraformaldehyde in 0.12 M sodium/potassium phosphate, pH 7.0, and for a further 10 min in fixative containing 0.1% Triton X-100. Following several rinses with phosphate-buffered saline (PBS), cells were pretreated with 3% bovine serum albumin, 10% normal goat serum in PBS to reduce nonspecific staining. Cells were incubated for 2 h at 37 °C with the primary antibodies in PBS containing 3% bovine serum albumin in a moist chamber. Cells were then washed with PBS and subsequently incubated for 1 h at 37 °C with the respective secondary antibodies diluted to 1:200 in PBS containing 3% bovine serum albumin. Finally, coverslips were extensively washed with PBS, rinsed with water, and mounted in Moviol 4-88 (Hoechst).
Immunofluorescence staining was monitored with a Zeiss laser scanning microscope (LSM 410 invert) equipped with a planapo oil (63 ×) immersion lens (numerical aperture =1.4). DTAF emission was excited using the argon laser 488-nm line, whereas TRITC was excited using the helium/neon laser 543-nm line. The emission signals were filtered with a Zeiss 515-565-nm filter (DTAF emission) or with a long pass 595-nm filter (TRITC signal). Nonspecific fluorescence was assessed by incubating cells with the secondary fluorescent antibodies and measuring the average intensity value for each fluorochrome. This value was subtracted from all images.Immunoprecipitation and Immunoblotting--
Chromaffin granule
membrane proteins (250 µg) were subjected to ADP-ribosylation with
recombinant C3 transferase. Granule membranes were subsequently
immunoprecipitated with 30 µl of anti-DH antiserum and 100 µl of
ImmunoPure Immobilized protein A (Pierce) (10). The beads were
collected by centrifugation, and washed and bound proteins were
analyzed by SDS-gel electrophoresis and autoradiography.
Antibodies--
The rabbit polyclonal anti-DH antiserum used
for chromaffin granule membrane immunoprecipitation was prepared in our
laboratory and its specificity demonstrated (24). Rabbit polyclonal
anti-bovine chromogranin A (CGA) antibodies (34) were used at 1:5000
dilution. Mouse monoclonal antibodies against RhoA and rabbit
polyclonal antibodies against RhoB (Santa Cruz Biotechnology, INC) were
used at 1:10 dilution for immunocytochemical experiments and at 1:100 for immunodetection on nitrocellulose sheets. Donkey anti-rabbit IgGs
conjugated to dichlorotriazinyl aminofluorescein (DTAF) were used at
1:200 dilution (Chemicon International Inc.). Goat anti-mouse IgG
conjugated to tetramethylrhodamine (TRITC) were used at 1:200 dilution
(Chemicon International Inc.). Affinity purified antibodies against the
COOH-terminal G
o peptide (ANNLRGCGLY) or
G
i peptide (KNNLKDCGLF) were prepared as described
previously (6), and their specificity against non-denaturated G
protein was demonstrated. Mouse monoclonal antibodies against the p85
subunit of phosphatidylinositol 3-kinase were purchased from Upstate
Biotechnology (Lake Placid, NY).
Peptides, Proteins, and Toxins-- Mastoparan was obtained from Sigma. Cytosolic and membrane-bound forms of GAP-43 were purified from bovine brain according to a previously published procedure (8). C. botulinum exoenzyme C3 ADP-ribosyltransferase (C3 transferase) prepared and purified as described (35) was a generous gift of Dr. M. R. Popoff (Institut Pasteur, Paris).
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RESULTS |
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Subcellular Localization of C3-catalyzed ADP-ribosylated Proteins in Chromaffin Cells-- To investigate the intracellular distribution of Rho in chromaffin cells, we made use of the specific ADP-ribosylation reaction with C. botulinum C3 ADP-ribosyltransferase, in which [32P]ADP-ribose is incorporated on residue asparagine 41 of Rho (36, 37). Plasma membranes, chromaffin granule membranes, and cytosolic proteins were prepared from bovine adrenal medulla by subcellular fractionation on sucrose density gradients, and the presence of substrates for C3 transferase was analyzed by gel electrophoresis and autoradiography. We found that C3 transferase consistently labeled a 21-kDa protein present in chromaffin granule membranes and to a smaller extent in the cytosol (Fig. 1A). In contrast, the plasma membrane contained virtually no detectable ADP-ribosylated product when incubated with C3 transferase (Fig. 1A). Control experiments performed in the absence of C3 transferase confirmed that the granule-associated 21-kDa protein was specifically labeled in a toxin-dependent fashion (Fig. 1A).
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RhoA Is a Specific Component of the Secretory Chromaffin Granule Membrane-- Since Rho and Rac are potential substrates of C3 transferase (37), we characterized further the granule-associated 21-kDa protein by immunodetection on nitrocellulose sheets. Fig. 2 illustrates a Western blot analysis of adrenal medullary plasma membranes, chromaffin granule membranes, and cytosolic proteins using anti-RhoA and anti-RhoB antibodies. RhoA was detected in the granule membrane fraction, displaying an apparent molecular mass of 21 kDa (Fig. 2A). We also observed the presence of a substantial amount of RhoA in the cytosolic fraction but not in the plasma membrane (Fig. 2A), in agreement with the results obtained by C3-induced ADP-ribosylation. In contrast, no specific immunosignal for RhoB was detectable among the three subcellular fractions (Fig. 2B). To evaluate the portion of RhoA present in the cytosol, cultured chromaffin cells were collected, and the content of RhoA was estimated in three fractions defined as the total homogenate, the cytosol, and the membrane-bound compartment. By C3-catalyzed ADP-ribosylation and immunoreplica analysis using the anti-RhoA antibodies, we found that the cytoplasmic pool represented approximately 15-20% of the total RhoA present in chromaffin cells (Table I).
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Effect of Mastoparan and GAP-43 on the Secretory Granule-associated
Phosphatidylinositol 4-Kinase Activity--
We previously reported
that the secretory granule-associated Go protein regulates
the peripheral cytoskeleton in chromaffin cells by a mechanism
involving the small GTP-binding protein Rho (20). Since
phosphoinositide kinases are potential effectors for Rho in mediating
cytoskeletal rearrangements (21-23), we investigated the possible
functional relationship between the granule-bound form of
Go, RhoA, and the PtdIns 4-kinase activity described in chromaffin granule membranes (28, 29). Mastoparan, a selective activator of Gi and Go proteins (39), was used
in our previous studies of permeabilized chromaffin cells to stimulate
the granule-associated Go and its downstream effectors (3,
6, 20). Hence, we first examined the effect of mastoparan on PtdIns
kinase activity in purified chromaffin granule membranes. Fig.
4 shows the PtdIns kinase activity
detected in plasma and chromaffin granule membranes using endogenous
PtdIns as substrate. PtdIns monophosphate (PtdInsP) was the only
32P-labeled derivative of PtdIns found in lipid extracts of
both plasma and granule membranes labeled with
[-32P]ATP (Fig. 4A). Note that the PtdIns
kinase activity was approximately 10 times more important in the
granule membrane than in the plasma membrane. A quantitative analysis
of PtdIns labeling in granule membranes was performed in the presence
of either quercetin, an inhibitor of PtdIns 3- and 4-kinases (40), or
LY294002, a specific inhibitor of PtdIns 3-kinase (41). As illustrated
in Fig. 4B, only quercetin inhibited the formation of
radiolabeled PtdInsP, in line with the presence of PtdIns 4-kinase
among the components of the chromaffin granule membrane (28, 29).
Mastoparan strongly stimulated the formation of radiolabeled PtdInsP in
purified chromaffin granule membranes (Fig. 4B). Moreover,
the mastoparan-stimulated PtdIns kinase activity was not significantly
affected by LY294002 but largely inhibited by quercetin (Fig.
4B). To identify further the PtdIns kinase activated by
mastoparan, radiolabeled PtdInsP was extracted from the thin layer
plate, deacylated, and analyzed by anion-exchange HPLC. Fig.
4C shows that deacylated PtdIns-3-P and PtdIns-4-P are
readily separated by this method. The deacylated PtdInsP recovered from
granule membranes incubated in the presence of mastoparan eluted as one
single peak that comigrated with the 4-isomer (Fig. 4C). The
3-isomer was not detectable in either control or mastoparan-stimulated
samples (Fig. 4C), thereby confirming the identity of the
mastoparan-activated lipid kinase in secretory granule membranes as
PtdIns 4-kinase.
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Mastoparan-induced Activation of the Granule-associated
Phosphatidylinositol 4-Kinase Requires Go--
Mastoparan
has been reported to activate Gi and Go
proteins by interacting with the carboxyl terminus of the subunit
(42). To assess whether mastoparan enhanced the granule-associated
PtdIns 4-kinase activity by stimulating the granule-bound
Go, we attempted to antagonize the effect of mastoparan
with affinity purified antibodies prepared against the carboxyl
terminus of G
o and G
i. Chromaffin granule
membranes were incubated overnight with either anti-G
o
or anti-G
i antibodies and then exposed to 20 µM mastoparan. Fig. 7 shows
that the presence of the anti-G
i antibodies did not
significantly affect the mastoparan-stimulated PtdIns kinase activity.
In contrast, the stimulating effect of mastoparan on PtdInsP formation
was completely abolished by the anti-G
o antibodies (Fig.
7), in agreement with the idea that mastoparan enhanced the
granule-associated PtdIns 4-kinase activity by stimulating the
granule-bound Go. It is noteworthy that the
anti-G
o antibodies reversed at the same concentration
the effects of mastoparan on secretory granule-associated PtdIns
4-kinase, peripheral actin organization (20), and
ATP-dependent catecholamine secretion (3), implying a
possible link between these three events in the exocytotic pathway.
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Effect of C. botulinum C3 Transferase on Mastoparan-induced
Activation of the Granule-associated Phosphatidylinositol
4-Kinase--
To probe the possible role of Rho in the
mastoparan-induced activation of PtdIns 4-kinase, we used the C3
ADP-ribosyltransferase to specifically inactivate the
granule-associated Rho protein. Purified chromaffin granule membranes
were incubated for 10 min with increasing concentrations of C3 toxin in
the presence or in the absence of 20 µM mastoparan. The
reaction was then started by adding [-32P]ATP, and the
formation of radiolabeled PtdInsP was analyzed. Treatment with C3
transferase did not modify the basal level of PtdIns kinase activity
(Fig. 8). However, C3 transferase induced a dose-dependent inhibition of the mastoparan-induced
PtdIns kinase activity (Fig. 8). At 5 µg/ml, C3 toxin completely
abolished the mastoparan-dependent formation of PtdInsP
(Fig. 8), indicating that the inactivation of Rho prevented mastoparan
from activating PtdIns 4-kinase in chromaffin granule membranes. These
results reinforce the idea that the secretory granule-associated
Go controls exocytosis by stabilizing the actin
cytoskeleton (20) through a pathway involving RhoA and the
granule-associated PtdIns 4-kinase.
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DISCUSSION |
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Exocytotic fusion of secretory granules with the plasma membrane
requires calcium and ATP. By using permeabilized cell models, it has
been possible to establish that exocytosis consists of sequential
ATP-dependent and ATP-independent phases of release (7, 43,
44). The ATP-independent phase has been attributed to the fusion of a
small number of docked granules (45, 46), whereas the
ATP-dependent component has been assumed to include docking
of granules and priming of the exocytotic machinery (43). We previously
investigated the function(s) of trimeric G proteins in chromaffin cell
exocytosis by using selective modulators of G proteins (3, 6-9,
20). Our findings indicate that two trimeric G proteins act in series
in the exocytotic pathway as follows: a secretory granule-associated
Go controls the ATP-dependent priming reaction,
whereas a plasma membrane-bound Gi3 is involved in the late
ATP-independent fusion step (3, 6-9). More recently, immunocytochemical and functional studies performed on permeabilized chromaffin cells indicated that the control exerted by the
granule-associated Go on the priming of exocytosis is
related to effects on the subplasmalemmal actin cytoskeleton (20). Our
results suggested that activated Go stabilized the actin
network through a sequence of events possibly implicating the small
GTPase Rho (20). In line with this hypothesis, Rho-dependent pathways have been previously described to be
under the control of trimeric G proteins in diverse cell types,
including fibroblasts (47), leukocytes (48), myocytes (49), and mast cells (27).
To investigate the intracellular localization of Rho in secretory cells, we used here the exoenzyme C3 from C. botulinum which specifically ADP-ribosylates the protein at an asparagine residue in the effector domain (36, 37). We found that a 21-kDa substrate of C3 toxin is specifically associated with the membrane of purified secretory chromaffin granules. Accordingly, the presence of C3 substrates in granule-enriched fractions has been previously described in neutrophils (50) and anterior pituitary cells (51). In the present study, we demonstrate by confocal immunocytochemistry the colocalization of RhoA with the chromaffin granule marker chromogranin A, further confirming the specific association of RhoA with the membrane of secretory organelles. This granular distribution fits well with the idea that RhoA may act as a downstream effector for the granule-bound Go controlling the subplasmalemmal actin network in chromaffin cells (20).
Although the best established function of Rho relates to the cytoskeletal organization, the pathways involved in Rho-induced actin regulation remain to be identified. To date, several candidates have been proposed to serve as signaling elements mediating the Rho-dependent cytoskeletal features. For instance, protein kinase N (52, 53) and Rho kinase (54, 55) have been identified as possible downstream effectors of Rho in actin stress fiber formation. In permeabilized fibroblasts, Mackay et al. (56) demonstrated that moesin and its close relatives, ezrin and radixin, can reconstitute stress fiber assembly and cortical actin polymerization in response to Rho activation. Several reports also indicate that Rho controls the activity of various phosphoinositide kinases (21-23), thereby regulating the synthesis of PtdIns 4,5-bisphosphate (PtdIns-4,5-P2), a known regulator of actin polymerization (57).
In chromaffin cells, the ATP dependence of secretion has been suggested
to reside, at least in part, in maintaining cellular phosphoinositides
at a defined level (58). The involvement of phosphoinositides in
exocytosis is further supported by the recent identification of three
proteins required for the priming of exocytosis, as PtdIns transfer
protein (59), PtdIns 5-kinase (60), and PtdIns 4-kinase (29). Both
PtdIns transfer protein and PtdIns 5-kinase are cytosolic proteins. In
contrast, PtdIns 4-kinase has been identified among the components of
the chromaffin granule membrane (28, 29). These observations led us to
investigate whether Rho-dependent activation of PtdIns
4-kinase might be part of the molecular machinery linking the
granule-bound Go to the regulation of the cortical actin
network. By using purified chromaffin granule membranes, we show here
that activation of Go stimulates the granule-associated
PtdIns 4-kinase activity. Moreover, incubation of granule membranes
with C. botulinum C3 transferase completely abolished the
Go-stimulated PtdIns kinase activity, indicating the
participation of Rho in the sequence of events coupling Go to the granule-bound PtdIns 4-kinase. These results correlate well with
the idea that Rho-dependent synthesis of PtdIns-4P may be
the link integrating the granule-bound Go with the
regulation of cortical actin network.
Although PtdIns 4-kinase has been identified as a component of the priming reaction (29), its exact function in the exocytotic machinery remains to be determined. PtdIns 4-kinase produces PtdIns-4P by specifically phosphorylating the D-4 position of the inositol ring. PtdIns-4P can be subsequently phosphorylated by PtdIns 5-kinase to generate PtdIns-4,5-P2. Indeed, cytosolic PtdIns 5-kinase has been described as an essential factor for the ATP-dependent priming of chromaffin cell secretion (60). PtdIns-4,5-P2 is an important regulator of the cytoskeleton organization in many cell types (57). In chromaffin cells, PtdIns-4,5-P2 decreases the actin filament severing activities of gelsolin and scinderin (57, 61), two proteins that have been found associated with the cortical ring of actin filaments (30, 61). Moreover, recombinant scinderin facilitates exocytosis in permeabilized chromaffin cells, an effect that can be blocked by PtdIns-4,5-P2 (62). Thus, by decreasing the actin-severing activity of scinderin and/or gelsolin, Go/Rho-dependent PtdIns 4-kinase may well contribute to the stabilization of peripheral actin filaments that exclude secretory granules from the subplasmalemmal zone in resting chromaffin cells. Alternatively, PtdIns 4,5-P2 may be required to maintain or regulate the interactions between actin filaments and the plasma membrane in stimulated cells. For instance, it has been recently demonstrated that Rho-dependent synthesis of PtdIns 4,5-P2 stimulates the association of ezrin, radixin, and moesin proteins with their membrane-binding partner CD44 and thereby contributes to the cross-linking of actin filaments with plasma membranes (56, 63). The occurrence of ezrin, radixin, and moesin proteins in neuroendocrine cells is currently unknown. However, it can be predicted that termination of exocytosis and/or endocytosis requires the rapid re-assembly of cortical actin filaments. In view of its granular location, the Go/Rho-dependent PtdIns 4-kinase represents an attractive candidate capable of maintaining or stimulating the formation of actin filaments specifically at the site of fusion between granule and plasma membranes.
To conclude, the present data indicate that stimulation of the secretory granule-associated Go results in the Rho-dependent activation of PtdIns 4-kinase, a pathway that may contribute to the local increase of PtdIns-4,5-P2 on the granule membrane. Further investigations are now required to correlate the intracellular fluctuations of PtdIns-4,5-P2 with the activation/inactivation cycle of the granule-bound Go. It is tempting to propose that Go/Rho-dependent activation of PtdIns 4-kinase represents an important basis for the actin cytoskeletal reorganization underlying membrane trafficking at the site of exocytosis.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. Michel R. Popoff for the generous gift of the C. botulinum exoenzyme C3 ADP-ribosyltransferase and Dr. Chris J. Vlahos (Lilly) for kindly providing us with LY294002. We thank Dr. Claude Leray for helping with the HPLC analysis; Danièle Thiersé for culturing chromaffin cells; and Dr. Nancy Grant for revising the manuscript.
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FOOTNOTES |
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* This work was supported by Association de la Recherche sur le Cancer ARC Grant 9101 and the Ligue Nationale Contre le Cancer.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.
To whom correspondence should be addressed. Tel.: 33 3 88 45 67 13; Fax: 33 3 88 60 08 06; E-mail: bader{at}neurochem.u-strasbg.fr.
1
The abbreviations used are: PtdIns,
phosphatidylinositol; gPtdIns-4-P, glycerophosphatidylinositol
4-phosphate; HPLC, high pressure liquid chromatography; PBS,
phosphate-buffered saline; DTT, dithiothreitol; CHO, Chinese hamster
ovary; DH, dopamine-
-hydroxylase; PtdInsP, phosphatidylinositol
monophosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; DTAF, dichlorotriazinyl aminofluorescein; TRITC, tetramethylrhodamine isothiocyanate; CGA, chromogranin A.
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REFERENCES |
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