1 Dipartimento di Fisiologia Generale ed Ambientale, University of Bari, 70126
Bari, Italy
2 Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
3 Freie Universität Berlin, Institut für Pharmakologie, Berlin,
Germany
* Author for correspondence (e-mail: g.valenti{at}biologia.uniba.it)
Accepted 7 January 2003
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Summary |
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Key words: Aquaporin 2, RhoA, Rho-GDI, PKA, Actin cytoskeleton
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Introduction |
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G proteins are involved in the regulation of vesicle redistribution between
intracellular compartments of the exocytic and endocytic pathways
(Nuoffer and Balch, 1994), and
subunits of Gi and Go proteins have been found associated with membranes in
different cell lines. Heterotrimeric G proteins from the Gi family are
required for cAMP-triggered trafficking of AQP2
(Valenti et al., 1998
).
Furthermore, monomeric GTP-binding proteins from the Rab family were found
in a kidney preparation enriched in AQP2-containing vesicles, and are likely
involved in AQP2 intracellular membrane traffic
(Liebenhoff and Rosenthal,
1995).
Depolymerization of cortical F-actin has been considered an important
prerequisite for facilitation of exocytosis. Complex biological processes such
as vesicle trafficking require precise control of actin cytoskeleton
organization, which involves activation of the small GTP-binding protein Rho
(Ren et al., 1999;
Ridley et al., 1993
). Rho is a
member of the Ras superfamily of small GTP-binding proteins. Proteins from the
Rho family (Cdc42, Rac1 and Rho) are involved in the regulation of actin
polymerization. In particular, Cdc42 activates Rac1 and Rho proteins in
serum-starved Swiss 3T3, and induces the formation of actin-based structures
known as filopodia. The activation of Rac1 results in the formation of
lamellipodia. In addition, Rho regulates the organization of stress fibers and
the assembly of focal adhesion complexes
(Nobes and Hall, 1995
).
GTP-binding proteins from the Rho family cycle between an active GTP-bound
state and an inactive GDP-bound form. The exchange of hydrolyzed GDP for GTP
results in a conformational change, unmasking structural domains by which they
bind to the effectors. The activity of monomeric G proteins is controlled by
different types of factors: GDP/GTP exchange protein (GEP) stimulates the
interconversion between the GDP form and GTP form; and GTPase activating
protein (GAP) binds to the GTP-form and stimulates the intrinsic GTPase
activity of monomeric G-proteins. GDP dissociation inhibitor (GDI) inhibits
GDP dissociation, prevents GTP hydrolysis and maintains the Rho family members
in a soluble form (Sasaki and Takai,
1998
; Van Aelst and
D'Souza-Schorey, 1997
). The switch process for Rho family proteins
presents an additional layer of regulation.
GTP-binding proteins from the Rho family are maintained associated with
membrane fraction (active form) interacting with specific target molecules
involved in the regulation of actin cytoskeleton. Translocation of Rho
proteins from the membrane fraction to a soluble compartment correlates with
their inactivation and with subsequent interaction with Rho-GDI. Recently, it
was shown that RhoA is phosphorylated by PKA at Ser-188, and that this
phosphorylation increases the interaction with GDP dissociation inhibitor
(GDI), inhibiting the binding with downstream targets independently of the
GTP/GDP state (Dong et al.,
1998; Forget et al.,
2002
). This suggests that the cAMP signal mediates down-regulation
of RhoA. Moreover, the cytosolic complex of the Rho GDP-bound form and Rho-GDI
is not activated by Rho-GEP, suggesting the existence of another regulatory
factor.
In renal collecting duct cells, we have recently demonstrated that
inhibition of RhoA GTPase with Clostridium difficile toxin B and with
Clostridium botulinum C3 toxin caused actin depolymerization and
translocation of AQP2 to the plasma membrane in the absence of hormonal
regulation (Klussmann et al.,
2001; Tamma et al.,
2001
). The present work was undertaken to verify whether Rho
inhibition is a physiological step in the signal transduction cascade
activated by vasopressin and leading to AQP2 fusion with the apical membrane.
To answer this question, we used a biochemical assay for the quantitation of
Rho activity. Using this assay, we analyzed the contribution of soluble
factors such as Rho-GDI to Rho activation/inactivation cycle in the signal
transduction cascade initiated by elevation of cAMP and leading to AQP2
targeting to the plasma membrane. We also attempted to delineate other
components of the Rho signaling pathway, notably the role of cAMP-dependent
protein kinase A (PKA) phosphorylation of RhoA on a serine residue. We
demonstrate that this event represents an additional layer of regulation of
Rho activity in renal principal cells. The data reported here reveal several
intriguing aspects of the regulatory role of Rho on AQP2 trafficking.
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Materials and Methods |
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Cell culture
CD8 cells were established by stably transfecting the RC.SV3 rabbit
cortical collecting duct cells with cDNA encoding rat AQP2
(Valenti et al., 1996). CD8
cells were grown at 37°C as described in a hormonally defined medium
containing 5% newborn calf serum (Valenti
et al., 1996
). Confluent monolayers were used at days 3-5 after
plating.
Expression and purification of GST-TRBD
The coding sequence for the Rhotekin Rho-binding domain (TRBD) cloned into
the pGEX-2T vector (kindly provided by A. Schwartz, La Jolla, CA) was
amplified in E. coli. E. coli expressing the fusion protein were
inoculated in 1 liter of LB medium with ampicillin using a fresh bacterial
preculture. Incubation was continued at 37°C until OD reached 0.5-0.6.
Protein expression was then induced with 1 mM of
isopropyl-b-D-thiogalactopyranoside (IPTG) for 30 minutes. The bacteria were
collected by centrifugation at 5000 g and resuspended in PBS
with protease inhibitors (3.2 mg/ml trypsin inhibitor, 1.4 mg/ml aprotinin,
0.5 mM benzamidine, 0.5 mM PMSF) before sonication. The bacteria were lysated
for 30 minutes in PBS with 1% Triton X-100. The lysate was clarified by
centrifugation at 17,000 g at 4°C for 10 minutes and the
supernatant obtained was incubated with 450 µl of glutathione beads for 30
minutes at 4°C. The beads were washed three times at 4°C with PBS in
the presence of protease inhibitors and used for affinity precipitation of
cellular GTP-Rho.
Affinity precipitation of cellular GTP-Rho
Rho activity was evaluated in renal CD8 cells in three different
experimental conditions: at rest, after elevation of cAMP levels with
forskolin treatment and in Toxin-B-treated cells. Stimulation with forskolin
was performed with 104 M forskolin for 15 minutes at
37°C. To evaluate the effect of toxin B on Rho activity, confluent
monolayers were incubated in a cell culture medium containing toxin B (200
ng/ml) for 2 hours at 37°C. Cells were washed with ice-cold buffer
containing 150 mM NaCl, 10 mM Tris-buffered pH 7.4 (TBS) and then lysed in
ice-cold RIPA buffer containing 50 mM Tris-HCl pH 7.2, 1% Triton X-100, 0.5%
sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl2, 3.2
µg/µl trypsin inhibitor, 1.4 µg/µl aprotinin, 0.5 mM benzamidine,
0.5 mM PMSF. The cell lysate was clarified by centrifugation at 13,000
g for 5 minutes at 4°C and incubated with GST-RBD beads
(20-30 µg) for 30 minutes at 4°C. The beads were washed three times
with a buffer containing 50 mM Tris-buffered pH 7.2, 1% Triton, 150 mM NaCl,
10 mM MgCl2, 3.2 µg/µl trypsin inhibitor, 1.4 µg/µl
aprotinin, 0.5 mM benzamidine, 0.5 mM PMSF. GTP-Rho was eluted by boiling the
precipitate in Laemmli buffer for 10 minutes in the presence of 40 mM DTT.
Bound Rho proteins were detected by western blotting using a monoclonal
antibody against RhoA. The densitometric analysis was performed using Scion
Image Software for Windows. Statistical analysis was performed by one-way
ANOVA and Tukey's multiple comparison test.
Membrane preparation
For the preparation of the fraction enriched in the plasma membranes (low
speed pellet, LS) or in intracellular vesicles (high speed pellet, HS) control
or forskolin-stimulated cells (104 M for 15 minutes at
37°C) were homogenized with a glass/Teflon homogenizer in ice-cold buffer
containing 250 mM sucrose and 10 mM Tris pH 7.5. Cell suspensions were
centrifuged at 700 g for 10 minutes at 4°C. The
supernatant was centrifuged at 17,000 g for 45 minutes at
4°C. The resulting pellet (LS) enriched in plasma membrane was recovered
in PBS and stored at 20°C. The supernatant was spun at 200,000
g in a Beckman Rotor 50 2Ti for 60 minutes at 4°C. The
final pellet (HS) enriched in intracellular vesicles was recovered in PBS and
stored at 20°C until used for immunoblotting studies.
Co-immunoprecipitation
CD8 cells were washed with PBS and homogenized with a glass Teflon
homogenizer in lysis buffer containing 20 mM Tris-HCl, 1% Igepal, 1 mM EDTA, 1
mM EGTA, 1 mM dithiothreitol, 0.5% deoxycholate, 0.1% SDS, 1.5 mM
MgCl2, 0.15 M NaCl, pH 8.0. The homogenates were centrifuged at
17,000 g, 30 min at 4°C and the supernatants were
incubated overnight with 2 µg of anti-RhoA antibodies. Protein A-conjugated
agarose beads (Sigma) were added and incubated for 2 h. The beads were washed
three times with 1 ml of lysis buffer. Bound proteins were eluted with Laemmli
sample buffer without DTT (95°C, 5 min) and subjected to Western blot
analysis using Rho-GDI antibodies (Santa Cruz Biotechnology). Alternatively,
the homogenates from control and forskolin-stimulated cells were incubated
overnight with 30 µl agarose conjugated monoclonal anti-phosphoserine
antibodies. Immunocomplexes, after washing, were eluted in Laemmli buffer,
resolved in a 13% polyacrylamide gel and subjected to Western blot analysis
for RhoA immunodetection.
Phosphorylation of RhoA in intact cells
Metabolic labeling of confluent CD8 cells with [32P]
orthophosphoric acid (NEN, Life Science Products, Italy, s.r.l.) was performed
as previously described (Valenti et al.,
2000). Briefly, confluent monolayers of CD8 cells, grown on 20 mm
cell culture Petri dishes, were metabolically labeled with 250 µCi/ml of
[32P] orthophosphoric acid (NEN) in 0.5 ml of phosphate-free DMEM
for 2 hours at 37°C in a 5% CO2 atmosphere. CD8 cells were left
untreated or stimulated with forskolin 104 M for 15 minutes
at 37°C in PBS, with or without a 30 minute pretreatment with 30 µM
H89. Cells were washed three times with PBS and quickly lysed in ice-cold
immunoprecipitation buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM KCl, 5
mM EDTA, 2% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM sodium
pyrophosphate, 1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A. The
lysate was clarified by centrifugation at 17,000 g for 10
minutes at 4°C and subjected to immunoprecipitation with 10 µl
agarose-conjugated RhoA (Sigma, Immunochemicals). Immunocomplexes were washed
three times in immunoprecipitation buffer, mixed with 30 µl of Laemmli
buffer, heated at 95°C for 10 minutes and resolved in a 13%
SDS-polyacrylamide gel. Gels were stained, dried and exposed to Kodak X-Omat
AR film Lightning Plus intensifying screen at 80°C. To verify that
identical amounts of RhoA were immunoprecipitated from each sample, gels were
probed with anti-RhoA antibodies by western blotting.
SDS-polyacrylamide gel electrophoresis and immunoblot analysis
Proteins were resolved in a 13% polyacrylamide gel. Proteins were
transferred onto Immobilon-P (Millipore) by standard procedures. Blots were
incubated with primary antibody as reported in the figure legends and then for
45 minutes with a horseradish peroxidase-conjugated secondary antibody (1:3000
Sigma) and visualized using the ECL-Plus detection system (Amersham Life
Science). Alternatively, blots were incubated with goat anti-mouse IgG
alkaline-phosphatase-conjugated antibodies (1:5000 Sigma) and revealed for
alkaline-phosphatase using 0.56 mM 5-bromo-4-chloro-3-indolyl phosphate, 0.48
mM nitro blue tetrazolium in 10 mM Tris-HCl, pH 9.5 (Jansen,
Pharmaceutica).
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Results |
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The activities of the Rho family are regulated by several proteins that
modulate their GTP/GDP state. The cycling between the two nucleotide-bound
states is accompanied by cycling between the cytosolic fraction and the
membranes (Mackay and Hall,
1998; Takaishi et al.,
1995
). To investigate whether Rho inhibition resulted in a
decrease in membrane-associated Rho, cell fractionation of CD8 cells was
performed from control and forskolin-stimulated cells. Cells were homogenized
and the plasma membrane enriched fraction (LS) as well as the intracellular
vesicles enriched fraction (HS) were obtained by differential centrifugation.
Western blotting analysis of separated membrane fractions from control cells
revealed that the majority of immunodetectable RhoA was associated with LS
(Fig. 2A and densitometric
analysis in Fig. 2B). After
stimulation with forskolin, a significant decrease (36.3±0.13,
n=4) in immunoreactive RhoA was observed in LS. This result indicates
that the signal transduction pathway activated by forskolin and leading to
AQP2 translocation involves inhibition of Rho GTPase with a concomitant
dissociation from a membrane compartment.
|
Rho proteins cycle between active, GTP-bound and inactive GDP-bound states
(Mackay and Hall, 1998).
Rho-GDI appears to stabilize the inactive, GDP-bound form of the protein,
forming a cytosolic complex with Rho-GDP
(Bourmeyster and Vignais,
1996
). CD8 cells were tested for the endogenous expression of
Rho-GDI. Equal amounts (30 µg/lane) of proteins enriched in plasma membrane
(LS) or in intracellular vesicles (HS) isolated from control or
forskolin-stimulated CD8 cells were separated by gel electrophoresis and
immunoblotted with specific anti-Rho-GDI antibody
(Fig. 3A). Relative to HS,
Rho-GDI appeared enriched in LS. After forskolin stimulation, the intensity of
the band decreased in both LS and HS fractions
(Fig. 3A and densitometry in
Fig. 3B). This finding suggests
that upon elevation of cAMP, Rho-GDI dissociates from the membranes. It has
been reported that the GDP form of Rho is preferentially bound to Rho-GDI in a
soluble complex (Chuang et al.,
1993
; Hart et al.,
1992
; Leonard et al.,
1992
; Sasaki et al.,
1993
). To investigate whether forskolin stimulation caused an
increase in the molecular interaction between RhoA and RhoGDI, generating a
soluble complex, co-immunoprecipitation experiments were performed in control
and forskolin-stimulated CD8 cells. As shown in
Fig. 4, the amount of
RhoA-RhoGDI complex increased by 1.83±0.17-fold in forskolin-stimulated
cells compared with control cells (Fig.
4A, I.P. RhoA and densitometry in
Fig. 4B). This data confirmed
that elevation of intracellular cAMP caused an increase in the molecular
interaction between RhoA and RhoGDI.
|
|
It has been shown that RhoA phosphorylation at Ser188 by PKA stabilizes and
increases the binding with Rho-GDI (Forget
et al., 2002). Moreover, it has been suggested that following
phosphorylation of RhoA by PKA, GTP-bound Rho can be uncoupled from its
putative effector independently of its binding to GTP or GDP
(Lang et al., 1996
). To
investigate whether RhoA phosphorylation occurs during forskolin stimulation,
CD8 cells were metabolically labeled with [32P] orthophosphate, and
either left untreated or stimulated with forskolin 104 M
followed by RhoA immunoprecipitation. Equal amounts of immunoprecipitated RhoA
were loaded on the gels and subjected to autoradiography. Forskolin
stimulation caused a nearly 2.5-fold increase in RhoA phosphorylation, which
was abolished in H89-pretreated cells, indicating that PKA is the kinase
responsible for this effect (Fig.
5A and relative densitometric analysis). To confirm whether RhoA
phosphorylation occurred at a serine residue, serine-phosphorylated proteins
were immunoprecipitated from control and forskolin-stimulated cells, and RhoA
was detected with specific anti-RhoA antibodies (see Materials and Methods for
details) (Fig. 5B). Relative to
the control, the amount of serine-phosphorylated RhoA immunoprecipitated from
forskolin-stimulated cells was about twofold, demonstrating that the increase
in phosphorylated RhoA involves a serine residue.
|
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Discussion |
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Finally, the experiments reported here also include a novel finding,
suggesting for the first time that the signal transduction pathway used by
vasopressin causes PKA-dependent phosphorylation of RhoA, which might be an
alternative pathway for terminating RhoA signaling in renal cells. We
demonstrated that forskolin stimulation was associated with an increase in Rho
phosphorylation on a serine residue, a protein modification known to stabilize
the inactive form of RhoA and to increase its interaction with Rho-GDI
(Lang et al., 1996). In fact,
it has been reported that RhoA is phosphorylated by PKA at Ser188 and this
phosphorylation increases the interaction with Rho-GDI, inhibiting the binding
of downstream targets independently of the GTP/GDP state, indicating that the
cAMP signal mediates downregulation of RhoA
(Dong et al., 1998
). Following
phosphorylation of RhoA by PKA, GTP-bound Rho can be uncoupled from its
putative effector independently of its binding to GTP or GDP. The emerging
picture (Fig. 6) suggests that
hormonal stimulation raises the level of intracellular cAMP and results in the
activation of PKA which then phosphorylates AQP2 and RhoA, possibly at Ser188.
Rho phosphorylation causes a decrease in the binding to its putative
effectors, the Rho kinases (Dong et al.,
1998
). The attenuation of Rho activity would favor the
depolymerization of F-actin, facilitating AQP2 insertion into the plasma
membrane. Indeed, stimulation of CD8 cells with forskolin induces a
depolymerization of F-actin-containing cytoskeletal structures
(Valenti et al., 2000
).
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Taken together, these data demonstrate that RhoA inhibition through Rho phosphorylation and interaction with Rho-GDI is a key event for cytoskeletal dynamics controlling cAMP-induced AQP2 translocation.
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Acknowledgments |
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