First published online 25 February 2003
doi: 10.1242/jcs.00355
cAMP-induced AQP2 translocation is associated with RhoA inhibition through RhoA phosphorylation and interaction with RhoGDI
Grazia Tamma1,
Enno Klussmann2,
Giuseppe Procino1,
Maria Svelto1,
Walter Rosenthal2,3 and
Giovanna Valenti1,*
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
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Fig. 1. Affinity precipitation of cellular GTP-Rho by pull-down assay. (A) CD8
cells were left untreated (control), stimulated with forskolin
(10–4 M, 15 minutes) or incubated with C. difficile
toxin B (200 ng/ml, 2 hours), which inhibits all proteins from the Rho family.
The lysates from each condition were incubated with 20-30 µg of GST-RBD
(Rho binding domain of rhotekin) conjugated with glutathione-Sepharose 4B
beads. GTP-Rho was precipitated by centrifugation and subjected to western
blotting analysis with anti-RhoA antibodies. Results are representative of
three independent experiments with similar results. (B) Densitometric profile
(mean values±s.e., n=3) of the GTP-RhoA band relative to
control. *P<0.001.
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Fig. 2. Detection of RhoA in membrane fractions from CD8 cells. (A) Fractions
enriched in plasma membrane (low speed pellet, LS) or in intracellular
vesicles (high speed pellet, HS) were prepared from CD8 cells in basal
condition and after forskolin stimulation. Equal amount of protein (30
µg/lane) were subjected to western blotting analysis (see above) with
anti-RhoA-specific antibody (1:500) and revealed with
alkaline-phosphatase-conjugated anti-mouse IgG (1:5000). (B) Densitometric
analysis (mean values±s.e., n=4) of the RhoA 22 kDa band.
*P<0.001.
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Fig. 3. (A) Detection of Rho-GDI in membrane fractions from CD8 cells. Membrane
fractions enriched in plasma membrane (LS) or in intracellular vesicles (HS)
were prepared from control and forskolin-stimulated CD8 cells. Equal amounts
of protein (30 µg/lane) were separated by gel electrophoresis and
immunoblotted with anti-Rho-GDI antibody (1:500). Results are representative
of three independent experiments. (B) Densitometric profile (mean
values±s.e., n=3) of the 29 kDa band.
*P<0.001.
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Fig. 4. Co-immunoprecipitation of RhoA and RhoGDI. (A) RhoA antibody was incubated
with cell lysate from control and forskolin-stimulated CD8 cells.
Immunocomplexes were precipitated with Protein-A-conjugated agarose. Proteins
were eluted using Laemmli buffer and analyzed by immunoblot for detection of
RhoGDI. This result is representative of three independent experiments
demonstrating that forskolin stimulation is associated with the formation of a
soluble RhoA-Rho-GDI complex. (B) Densitometric profile (mean
values±s.e., n=3) of the 29 kDa band.
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Fig. 5. Forskolin stimulation of CD8 cells results in RhoA phosphorylation at a
serine residue. (A) For each experimental condition, confluent monolayers of
CD8 cells were metabolically labeled with [32P] orthophosphate (250
µCi/ml), for 3 hours. Cells were lysed and RhoA was immunoprecipitated with
10 µl agarose-conjugated RhoA and detected by autoradiography. Experimental
conditions included: control condition, stimulation with forskolin
(10–4 M for 15 minutes at 37°C); incubation with H89 (30
µM for 30 minutes at 37°C) followed by stimulation with forskolin.
Equal amounts of RhoA were precipitated in each condition (I.P. RhoA).
Forskolin stimulation increased the phosphorylated state of RhoA nearly
2.5-fold and pretreatment with H89 abolished the forskolin effect. A
densitometric profile (mean values±s.e., n=3) of bands
corresponding to phosphorylated RhoA is shown on the right. (B) Cell lysate
from basal and forskolin-stimulated CD8 cells incubated with 30 µl
monoclonal agarose conjugated anti-phosphoserine. Immunocomplexes were mixed
with 20 µl of Laemmli buffer and blotted with anti-RhoA antibodies.
Forskolin treatment resulted in a nearly two-fold increase in
serine-phosphorylated RhoA. A densitometric profile (mean values±s.e.,
n=3) of bands corresponding to serine phosphorylated RhoA is shown on
the right.
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Fig. 6. Model depicting the role of RhoA in regulating actin cytoskeleton
organization in vasopressin-stimulated renal cells. Vasopressin raises
intracellular cAMP with consequent activation of PKA, which phosphorylates
AQP2 and RhoA. Rho phosphorylation causes a decrease in the binding to its
putative effectors, the Rho kinases. The attenuation of Rho activity results
in depolymerization of F-actin, facilitating AQP2 insertion into the plasma
membrane.
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© The Company of Biologists Ltd 2003
