Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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We characterized the
role of guanine nucleotide dissociation inhibitor (GDI) in
RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth
muscle. Endogenous contents (~2-4 µM) of RhoA and RhoGDI were
near stoichiometric, whereas a supraphysiological GDI concentration was
required to relax Ca2+ sensitization of force by GTP and
guanosine 5'-O-(3-thiotriphosphate) (GTPS). GDI also
inhibited Ca2+ sensitization by GTP · G14V RhoA, by
-adrenergic and muscarinic agonists, and extracted RhoA from
membranes. GTP
S translocated Rho-kinase to a Triton
X-114-extractable membrane fraction. GTP · G14V RhoA complexed
with GDI also induced Ca2+ sensitization, probably through
in vivo dissociation of GTP · RhoA from the complex, because it
was reversed by addition of excess GDI. GDI did not inhibit
Ca2+ sensitization by phorbol ester. Constitutively active
Cdc42 and Rac1 inhibited Ca2+ sensitization by
GTP · G14V RhoA. We conclude that 1) the most likely
in vivo function of GDI is to prevent perpetual "recycling" of
GDP · RhoA to GTP · RhoA; 2) nucleotide
exchange (GTP for GDP) on complexed GDP · RhoA/GDI can precede
translocation of RhoA to the membrane; 3) activation of
Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and
Rac1 can inhibit Ca2+ sensitization by activated
GTP · RhoA.
calcium sensitization; Cdc42; Rac; RhoGDI; Y-27632; smooth muscle
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INTRODUCTION |
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THE SMALL GTPase
RhoA modulates, through its effector(s), the Ca2+
sensitivity of smooth muscle contraction and nonmuscle cell motility, mediating these, and other, important physiological mechanisms (24, 28, 37, 41, 57, 60; reviewed in Refs. 26, 35,
51, 55, 63). Smooth muscle and
nonmuscle myosin II ATPases are physiologically activated by actin upon
phosphorylation of the serine 19 residue of their regulatory light
chain (RLC) by a Ca2+-calmodulin-activated myosin light
chain kinase (MLCK) and are inactivated by dephosphorylation by a
Ca2+-independent myosin light chain phosphatase (MLCP;
reviewed in Refs. 20 and 54). Therefore, inhibition of
MLCP can increase phosphorylation of the RLC of smooth and nonmuscle
myosin II and, consequently, force and motility, without a necessary
increase in intracellular Ca2+ concentration
([Ca2+]i) (55, 56; reviewed in Refs.
20 and 54). Such inhibition of MLCP can be effected by
activation of certain G protein-coupled receptors and, in permeabilized
preparations, by guanosine 5'-O-(3-thiotriphosphate) (GTPS) (30, 53) through activation of RhoA (Ref.
16 and references therein) and its effector, Rho-kinase
(and some other serine/threonine kinases; see
DISCUSSION). Rho-kinase phosphorylates the
regulatory subunit of the trimeric MLCP and inhibits its catalytic activity, causing an increase in RLC phosphorylation and contraction (11, 27, 28; reviewed in Refs. 20 and 55).
Guanine nucleotide dissociation inhibitor (RhoGDI; henceforth referred
to as GDI) is an important component of signaling through Rho subfamily
proteins. Known functions of GDI include 1) inhibition of
both guanine nucleotide dissociation from and hydrolysis by Rho
proteins; 2) complexation with Rho proteins that are
hydrophobic, as the result of their prenylated (geranylgeranylated)
COOH termini, thereby maintaining a large fraction of these proteins as
soluble cytosolic and inactive Rho (Rac, Cdc42)/GDI complexes; and
3) extraction of Rho protein from cell membranes. GDI can
complex with both the GTP- and GDP-bound forms of the Rho subfamily
proteins (RhoA, Cdc42, Rac1) (18, 21, 42, 46) and
interacts with them through their highly conserved GDI binding surfaces
(36). When activated, either indirectly, by agonists
acting on surface membrane receptors, or more directly, by GTPS, in
permeabilized smooth muscle, RhoA dissociates from its cytosolic
complex with GDI and translocates to the surface membrane (13,
14). Activation of RhoA by upstream trimeric G proteins
(G
13) is mediated by guanine nucleotide exchange factors
(GEF) such as p115 RhoGEF (19), a member of the GEF family
containing Dbl and pleckstrin homology domains (reviewed in Ref.
8). The cytosolic RhoA/GDI complex contains GDP as the
bound nucleotide, whereas activation of most Ras family, including Rho
subfamily, proteins requires exchange to the GTP-bound form (reviewed
in Ref. 3). It has not been previously determined whether,
in cells, nucleotide exchange (replacement of GDP by GTP) precedes
translocation and occurs in the cytosol or follows dissociation of Rho
from GDI and occurs at the cell membrane. Similarly, although
considerable information is available about the properties of GDI in
solution (reviewed in Ref. 43), little is known about
details of its physiological mechanism of action and function in cells.
We have used highly purified recombinant RhoA/GDI complexes containing
either GTPS, GTP, or GDP to determine whether it is sufficient to
exchange nucleotide on the complex to enable it to Ca2+
sensitize smooth muscle. We also have determined the exogenous concentrations of GDI required for inhibiting Ca2+
sensitization by GTP or its nonhydrolyzable analog, GTP
S, and compared it with endogenous cellular RhoA and GDI contents. We present
an experimental model indicating that prior formation of the
GTP · RhoA/GDI complex is sufficient for subsequent
translocation of GTP · Rho to the membrane and activation of
Ca2+ sensitization. We further suggest that the
physiological regulatory role of GDI is to prevent the recycling of
non-GDI-complexed GDP · RhoA (and possibly other Rho family
members) generated by GTP hydrolysis to active GTP · RhoA,
rather than to extract GTP-bound RhoA from the membrane, although such
extraction is possible at unphysiologically high GDI-to-RhoA
stoichiometry. We also found that the time course of the response to
GDI indicates close temporal coupling of RhoA to the downstream
mediators of Ca2+ sensitization.
A preliminary report of some of these findings has been published (45a).
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METHODS |
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Smooth muscle preparations.
For measurement of isometric tension, small strips (200 µm wide, 3 mm
long) of rabbit portal vein and ileum longitudinal smooth muscle were
dissected, and isometric tension was measured as published previously
(29, 31). Muscle strips were permeabilized by incubation with -escin (75 µM) for 35 min at 24°C, conditions that retain agonist-induced Ca2+-sensitization pathways. Force was
expressed as a percentage of the maximal Ca2+-induced
contraction obtained in permeabilized tissue at the end of the
experiment. Agonists were generally added to the strips at supramaximal
concentrations to minimize diffusional delays.
Statistical analysis. Results are expressed as means ± SE obtained from n experiments. Statistical analysis was performed using paired or unpaired Student's t-test where appropriate.
Western blot protocols. Tissue homogenates and recombinant Rho protein/GDI complex standards were submitted to SDS-PAGE, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline containing 0.05% Tween 20 (PBST) for 1 h and then incubated with primary antibody for 1 h at 37°C. The following dilutions of primary antibodies were used: anti-RhoA (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:3,000, anti-GDI (Santa Cruz) at 1:5,000, anti-Rac1 (Santa Cruz) at 1:500, anti-Rho-kinase (Transduction Laboratories, Lexington, KY) at 1:500, anti-Ras (Transduction Laboratories) at 1:500, and anti-PAK (Santa Cruz) at 1:2,000. The blots were washed in PBST, incubated in secondary antibody for 1 h at 37°C, and detected with enhanced chemiluminescence (ECL; Amersham). The protein signals were quantitated by densitometry using a Bio-Rad GS-670 imaging densitometer.
Quantitation of Rho proteins and GDI in rabbit smooth muscle.
Rabbit portal vein and the longitudinal smooth muscle layer of ileum
were carefully dissected, and excess connective tissue was removed. The
tissue samples were weighed and homogenized in buffer containing 1%
sodium dodecyl sulfate (SDS) and 10 mM dithiothreitol (DTT). The
homogenized tissues were centrifuged at 800 g for 5 min at
4°C to sediment nonsolubilized material. Protein concentrations were
measured with a modified Lowry protein assay (Bio-Rad) with bovine
serum albumin (BSA) standards. A serial dilution of each homogenate was
used for quantitation, over the linear range, on Western blots
utilizing as standards known quantities of purified recombinant Rho
proteins or GDI that were transferred on the same PVDF membranes.
Comparisons of RhoA, GDI, and Rho-kinase contents in intact and
-escin-permeabilized muscles were done on size-matched strips of muscle.
Coimmunoprecipitation of RhoA with GDI from smooth muscle cytosol. The longitudinal layer of ileum smooth muscle was homogenized in ice-cold homogenization buffer [25 mM Tris · HCl, pH 7.4, 5 mM MgCl2, 150 mM NaCl, and protease inhibitor cocktail (P-8340; Sigma, St. Louis, MO)] and centrifuged at 200,000 g for 20 min at 4°C. The supernatant was collected as the cytosolic fraction, and the protein concentration was determined. Cytosolic proteins (100 µg) were precleared with protein A-agarose (Santa Cruz) and then incubated with 2 µg of an anti-GDI antibody that recognizes the NH2 terminus of GDI (A-20 anti-GDI; Santa Cruz) for 2 h at room temperature. Protein A-agarose was added and incubated for an additional 2 h at room temperature. The immunoprecipitate was collected by centrifugation at 2,500 g for 5 min at 4°C and washed twice with homogenization buffer. The supernatant and immunoprecipitate were made up in Laemmli sample buffer, and a RhoA Western blot was performed to determine whether the RhoA coimmunoprecipitated with GDI.
Determination of the subcellular distribution of RhoA and
Rho-kinase.
A minimum of 10 pooled rabbit portal vein strips per point (dimensions
given in Smooth muscle preparation) were used for
determining the subcellular distribution of RhoA and Rho-kinase.
Tissues were permeabilized with -toxin as previously described
(15), relaxed in Ca2+-free solution, and
stimulated with 50 µM GTP
S for 1 or 20 min; they were then
homogenized in ice-cold NaCl buffer (10 mM Tris · HCl, pH 7.5, 5 mM MgCl2, 2 mM EDTA, and 100 mM NaCl), with the following
protease inhibitors and reducing agent: 1 mM 4-(2-aminoethyl) benzonesulfonyl fluoride, 20 µg/ml leupeptin, 20 µg/ml aprotinin, and 1 mM DTT. The homogenate was centrifuged at 800 g, and
the supernatant was centrifuged at 100,000 g for 30 min at
4°C (Optima TLX ultracentrifuge, TLA 120.1 rotor; Beckman
Instruments); this supernatant was collected as the cytosolic fraction
and the 100,000 g pellet as the membrane fraction. RhoA and
Rho-kinase were detected by Western blotting. As a control, 90% of
total tissue lactic dehydrogenase (a cytosolic marker) was in the
cytosolic fraction.
Phase separation by Triton X-114.
Because Rho-kinase in membrane fractions might be associated with
cytoskeletal proteins that may cosediment with membrane lipids, we
analyzed the distribution of Rho-kinase in membrane fractions using
Triton X-114. This zwitterionic detergent separates proteins containing
hydrophobic domains from hydrophilic proteins (5). For
this purpose, -toxin-permeabilized portal vein tissues were
incubated with or without 50 µM GTP
S in pCa 6.5 solution (20 min)
and then homogenized in a buffer containing 50 mM Tris · HCl,
pH 7.5, 5 mM MgCl2, 2 mM EDTA, 1 mM DTT, 150 mM NaCl, and protease inhibitors. The membrane fraction (100,000 g
pellet, as described above) was resuspended in the same buffer
containing 2% Triton X-114 (Sigma) and kept on ice for 30 min with
occasional mixing. The samples were then warmed to 37°C for 5 min,
and the micelles formed were pelleted by centrifugation at 37°C (5 min, 800 g). The upper fraction containing hydrosoluble
proteins and the pellet containing the hydrophobic proteins were
separated, mixed with 2× sample buffer, run on SDS-PAGE, and
transferred to PVDF membranes. Membranes were immunoblotted for RhoA,
Rho-kinase, and GDI and detected by chemiluminescence.
Extraction of RhoA by GDI from smooth muscle membranes.
Rabbit bladder was dissected from connective tissue and homogenized in
25 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl2, 100 µM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 2 µM
pepstatin A with a polytron and centrifuged for 5 min at 800 g to pellet unbroken cells and cellular debris. For
nucleotide exchange, the supernatant was removed and incubated at room
temperature for 45 min with 10 mM EDTA and 500 µM GTPS. The
membrane fraction was pelleted at 240,000 g for 1 h and
resuspended in 25 mM Tris, pH 8.0, 100 mM NaCl, 5 mM MgCl2,
10 mM EDTA, and 100 mM GTP
S, and a Bio-Rad protein assay was
performed. Mg2+ was added to a concentration of 13 mM, and
aliquots of 100 µg of membrane protein were incubated with increasing
concentrations of recombinant His6 GDI in a final volume of
60 µl for 10 min at room temperature (46). Quantitative
RhoA Western blots using known quantities of purified recombinant
RhoA/GDI complex as a standard were performed to quantitate the RhoA in
the assay. The membranes were pelleted by ultracentrifugation, washed,
and resuspended in 2× Laemmli sample buffer. Western blots were used
to determine the amount of RhoA, Ras, and Rho-kinase in the membrane fractions.
Expression and purification of recombinant His6 GDI.
An amino-terminal His6 affinity tag was added by ligating
the GDI cDNA into a modified pET22B vector with a replaced polylinker from vector pFastBac-HTa (Life Technologies), the resultant plasmid was
used to transform Escherichia coli strain BL21(DE3), and
His6 GDI was expressed and purified as previously described
(52). The purified GDI was concentrated to 30-40
mg/ml with a MICROSEP microconcentrator (Pall Filtron). Protein
concentrations were determined using the Bradford protein assay
(Bio-Rad) with BSA standards. Because imidazole interfered with force
measurements in -escin-permeabilized smooth muscle, the GDI was
buffer-exchanged into PIPES buffer (30 mM
1,4-piperazinediethanesulfonic acid, pH 7.1, 5 mM magnesium
methanesulfonate, and 165 mM potassium methanesulfonate) using a
Sephadex G-25 superfine column (Pharmacia Biotech) and then
reconcentrated to 30-40 mg/ml. This second concentrator filtrate
was used as a buffer control for force measurement in
-escin-permeabilized smooth muscle.
Expression and purification of recombinant His6 G14V RhoA/FLAG-GDI complex. Construction of human Rho protein and GDI expression plasmids for yeast were previously described (46, 47). Coexpression of His6-prenylated G14V RhoA and FLAG-GDI in Saccharomyces cerevisiae and subsequent His6 G14V RhoA/FLAG-GDI complex purification from the yeast cytosolic fraction were previously described in detail (46). Coexpression yields milligram quantities of pure complex with stoichiometrically bound nucleotide and circumvents the low yield and handling difficulties of singly expressed prenylated RhoA. The purified complex was GDP-to-GTP nucleotide-exchanged using a standard nucleotide exchange protocol (46) and then buffer-exchanged into PIPES buffer using a Sephadex G-50 centrifuge column (45). HPLC analysis as previously described (46) revealed that 50-70% of the complex bound GTP, with the remainder binding GDP.
As a control for testing heterodimer stability after GDP-to-GTP nucleotide exchange and buffer exchange into PIPES buffer, 500 µg of GTP-bound complex were run on a Superdex-75 16/60 gel-filtration column (Pharmacia Biotech) equilibrated with PIPES buffer and eluted in this same buffer at 0.4 ml/min. The protein eluted at the same time as a GDP · RhoA/GDI complex control, and both eluted 8 min earlier than control His6 GDI. A silver-stained SDS-PAGE gel of the GTP · RhoA/GDI elution peak revealed equimolar amounts of RhoA and GDI. Therefore, the heterodimer remained complexed after nucleotide exchange to GTP and buffer exchange into PIPES buffer (data not shown). His6 RhoA/FLAG-GDI was similarly expressed and purified and then used as a standard for the quantitation of RhoA and GDI in rabbit portal vein and ileum.Purification of posttranslationally modified His6
G14V RhoA, His6 G12V Cdc42, and His6 Q61L Rac1
from yeast membranes.
SY1 yeast was transformed with
YEpPGAL-His6 G14V
RhoA-tPMA1. The yeast were cultured with galactose
induction of Rho protein expression and lysed, and the cytosol and
membranes were fractionated as previously described (46).
The membrane fractions from an 18 L culture were resuspended in Tris
buffer (25 mM Tris base, pH 8.0, 100 mM NaCl, and 5 mM
MgCl2) with 50 µM GTP. The membrane was solubilized with
1.2% CHAPS {3-[(3-chloramidopropyl)
dimethylammonio]-1-propanesulfonate} with a detergent-to-protein
ratio of 5:1 for 1 h at 4°C. The solubilized membrane fraction
was then ultracentrifuged at 240,000 g for 1 h at 4°C
to sediment nonsolubilized membrane fraction components. The
supernatant was diluted with Tris buffer to reduce the CHAPS concentration to 0.3%, and 50 µM GTP was added. The sample was incubated with 5 ml of pre-equilibrated TALON (Clontech Laboratories, Palo Alto, CA) metal chelate affinity resin for 45 min at room temperature. The resin was packed into a chromatography column and
washed extensively, first with resuspension buffer with 0.3% CHAPS and
then with Tris buffer with 5 mM imidazole. The bound protein was eluted
with Tris buffer with 50 µM GTP, 100 mM imidazole, and 0.1% CHAPS
and then concentrated in a 15-ml Amicon (10 MWCO) centrifuge
concentrator to <1 ml. The purified constitutively active
GTP · Rho proteins were buffer-exchanged into PIPES buffer and
reconcentrated, and the GTP · Rho protein concentration was quantitated by determination of bound nucleotide. The protein was used
for determining its effects on force developed by the -escin-permeabilized rabbit smooth muscle.
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RESULTS |
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Quantitation of RhoA and GDI in rabbit ileum and portal vein smooth muscle. To assess the concentrations of physiologically relevant, endogenous proteins involved in Ca2+ sensitization, we determined the amounts of RhoA and GDI in intact portal vein and ileum longitudinal smooth muscle by quantitative Western blotting using serial dilution of known concentrations of purified recombinant Rho and GDI proteins to generate a standard curve. The quantity of RhoA and GDI in the tissues, interpolated from the standard curves, were 151 ± 17 and 147 ± 12 pg/µg total proteins for RhoA and 390 ± 140 and 254 ± 64 pg/ml total proteins for GDI in portal vein and ileum, respectively (n = 3). Equivalent cellular concentrations were calculated (1.8 and 1.7 µM for RhoA and 4.2 and 2.7 µM for GDI in portal vein and ileum, respectively) on the basis of the assumption that 80% of tissue weight is water and that RhoA and GDI molecular weights are 21.4 and 23.2 kDa, respectively.
We also determined the amount of endogenous GDI, RhoA, Rho-kinase, and p21-activated kinase (PAK) lost from theTranslocation of Rho-kinase to the membrane fraction and Triton
X-114 partition after stimulation with GTPS in
-toxin-permeabilized rabbit portal vein smooth muscle.
After stimulation with GTP
S, Rho-kinase translocated from the
cytosolic to membrane fractions (Fig.
1A) with a significant amount
in the hydrophobic Triton X-114-soluble fraction (Fig. 1B).
As a control, RhoA was immunoblotted on the same PVDF membrane used for
detecting Rho-kinase. As reported previously (14), the
majority of RhoA in unstimulated smooth muscles is cytosolic and
decreased within 1 min after stimulation with GTP
S, while it
increased in the hydrophobic Triton X-114-extractable membrane fraction
(Fig. 1B, pellet). GDI, as also found previously, was recovered in the cytosolic fraction, and its distribution was not
affected by the presence of nucleotide. This result also indicated that
membrane fractions did not contain detectable amounts of cytosoluble
proteins.
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GDI extracts RhoA, but not Rho-kinase or Ras, from membrane
fraction and complexes all detectable cytosolic RhoA.
The concentration of RhoA in GTPS-stimulated rabbit bladder membrane
preparations used for the GDI extraction assay was 70-80 nM. After
the membranes were incubated in buffer containing submicromolar Mg2+ with excess GTP
S to nucleotide exchange the RhoA,
addition of recombinant GDI extracted RhoA from the membranes in a
concentration-dependent manner. GDI (0.50 µM) extracted approximately
half of the RhoA in the membrane fraction, and increasing GDI
concentration above 5 µM caused no significant further extraction.
Thus extraction of GTP
S · RhoA from the membrane fraction
required at least an ~10-fold molar excess of GDI. A small amount of
RhoA could not be extracted from the membranes by even 50 µM GDI. GDI
did not extract Rho-kinase or Ras from the membranes (n = 3; Fig. 2A). All the
detectable cytosolic RhoA was coimmunoprecipitated with GDI from rabbit
smooth muscle cytosol fraction with a rabbit polyclonal IgG antibody
that recognizes the NH2 terminus of GDI (amino acids 2-21) as shown in Fig. 2B.
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Inhibition by GDI and Y-27632 of Ca2+
sensitization induced by constitutively active recombinant prenylated
GTP · G14V RhoA.
The constitutively active mutant GTP · G14V RhoA expressed in
yeast caused significant contraction of -escin-permeabilized rabbit
portal vein strips at constant free [Ca2+], as was
previously seen with GTP · G14V RhoA expressed in a baculovirus/Sf9 system (16).
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GDI inhibits phenylephrine- and carbachol-induced, but not phorbol
ester-induced, Ca2+ sensitization of
rabbit portal vein and ileum smooth muscle.
Phenylephrine (PE; 100 µM) plus GTP (10 µM) induced
Ca2+ sensitization of force (17.4 ± 4.8%,
n = 4) in -escin-permeabilized rabbit portal vein
smooth muscle strips, and GDI (100 µM) relaxed the steady state of
PE- plus GTP-induced contraction (at constant Ca2+) by
76 ± 11.2% (n = 4; Fig.
5). In paired control strips, PE plus GTP
caused a similar magnitude of contraction (20 ± 5.2%, n = 4) that was not significantly relaxed by the
filtrate from the last concentration step of the GDI purification
(6.3 ± 5% relaxation, n = 4; Fig. 5). The
half-time of GDI-induced relaxation in these experiments was 1.80 ± 0.16 min.
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Ca2+ sensitization of force by the
recombinant GTP · G14V RhoA/GDI complex.
His6 G14V RhoA and FLAG-GDI were coexpressed in yeast and
purified as a complex from yeast cytosol (46). Over 90%
of complex purified from this expression system is GDP bound
(46). Therefore, the protein was nucleotide-exchanged to
the GTP-bound form by a 6-h incubation at room temperature in the
presence of 7.5 mM GTP and submicromolar Mg2+, resulting in
50-70% of complex being GTP bound (46).
Concentrations of 1, 3, and 10 µM of the GTP-bound complex of G14V
RhoA and GDI caused 16.5 ± 4.6% (n = 3),
25.6 ± 2.7% (n = 3), and 19.0 ± 1.3% (n = 5) of the maximal Ca2+-induced
contraction in -escin-permeabilized rabbit portal vein, respectively. The control, 10 µM GDP-bound G14V RhoA/GDI complex, caused only 8.9 ± 4.3% (n = 3) of the maximal
Ca2+-induced contraction under identical conditions.
Similarly to the contraction induced by GTP · G14V RhoA alone,
the 3 µM GTP · G14V RhoA/GDI complex-induced contraction was
completely relaxed (Fig. 6) by the
Rho-kinase inhibitor Y-27632 (3 µM), from 25.6 ± 2.7%
(n = 3) to 3.0 ± 1.0% (n = 3, P < 0.01). Contractions induced by 3 µM
GTP · G14V RhoA/GDI complex were also completely relaxed (Fig.
6), from 22.2 ± 6.7% (n = 3) to
2.2 ± 2.6% (n = 3, P < 0.01), by excess
free GDI (20 µM).
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Comparison of inhibition by GDI of GTP- and GTPS-induced
Ca2+ sensitization.
To determine whether GDI-induced inhibition of the Rho/Rho-kinase
pathway was affected by hydrolysis of GTP bound to RhoA, we compared
the activity of GDI in the presence of a nonhydrolyzable nucleotide
analog (GTP
S) with that in the presence of (hydrolyzable) GTP. The
concentrations of GTP and GTP
S were chosen to yield equal levels of
Ca2+-sensitized force, 39% of maximal pCa 4.5-induced
force. The results (Fig. 7) suggest that
GDI was, albeit modestly, more effective in inhibiting the effects of
GTP than those of GTP
S. The concentrations of GDI that resulted in
50% relaxation of 10 µM GTP- and 0.1 µM GTP
S-induced
Ca2+-sensitization were 0.56 ± 0.20 and 0.89 ± 0.11 µM, respectively (n = 4). The half-times of 10 µM GDI-induced relaxation of GTP (10 µM)- and GTP
S (0.1 µM)-induced force (Fig. 7B) were 3.21 ± 0.31 and
4.15 ± 0.35 min, respectively (n = 5).
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Inability of GTP · G14V RhoA to reconstitute
GTPS-induced Ca2+ sensitization of
force in GTP
S-downregulated rabbit portal vein.
Prolonged incubation of
-toxin-permeabilized smooth muscle with
GTP
S causes, by a yet unknown molecular mechanism, loss of the
Ca2+-sensitizing response to GTP
S (downregulation) but
not that to PDBu (15). To determine whether this could be
due to sequestration of endogenous RhoA, we attempted to rescue the
downregulation with constitutively active, recombinant RhoA. However,
GTP · G14V RhoA (1 µM) caused no significant contraction
(1.2 ± 1.2%, n = 5) in downregulated smooth
muscle, and it did not recover the response to GTP
S (Fig.
8), indicating that
downregulation was not simply due to sequestration or loss of function
of endogenous RhoA. In time-matched control strips, 1 µM
GTP · G14V RhoA caused 12.8 ± 2.2% (n = 5) of the maximal Ca2+-induced contraction, which was not
significantly different from the contraction induced by
GTP · G14V RhoA in freshly
-escin-permeabilized tissue (Fig.
8C). Addition of the phosphatase inhibitor microcystin (MC)
produced a large, rapid increase in force, indicative of normal
catalytic phosphatase activity in GTP
S-downregulated muscles.
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Inhibition of GTP · G14V RhoA-induced
Ca2+ sensitization by GTP · G12V
Cdc42 and GTP · Q61L Rac1.
To explore other G protein-coupled mechanisms that may cross talk with
RhoA/Rho-kinase-mediated Ca2+ sensitization, we determined
the effect of two other Rho subfamily GTPases, Rac and Cdc42. The
constitutively active mutant GTP · G12V Cdc42 (10 µM)
significantly inhibited subsequent GTP · G14V RhoA-induced
contraction from 24% (n = 3, 1 µM) to 3.5% of the maximal Ca2+-induced contraction (n = 3, P < 0.01; Fig. 9) but,
by itself, had no significant effect on force (3.1% relaxation in
control with filtrate vs. 0.47% relaxation with Cdc42,
n = 3).
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DISCUSSION |
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RhoA-coupled mechanisms are activated when GDP is replaced by GTP in the RhoA/GDI complex and GTP · RhoA is translocated to the plasma membrane (reviewed in Refs. 9, 43, and 55). Our measurements of endogenous RhoA and its complex with GDI show a close-to-stoichiometric relationship (~2-4 µM each) between endogenous RhoA and GDI, particularly when considering that some GDI is associated with other Rho subfamily proteins, Cdc42 and Rac1; thus there is not a large pool of excess GDI available in vivo for removing and sequestering membrane-bound GTP · RhoA by mass action.
Of the endogenous RhoA/GDI complex, only a small fraction (~30%) was
lost from tissues permeabilized with -escin, which allows transmembrane permeation of up to 130-kDa proteins (22),
including recombinant GTP · RhoA/GDI (Fig. 6). The limited loss
of the small RhoA molecule (62) and the endogenous,
compact heterodimer with GDI (36) suggests that the
mobility of endogenous RhoA/GDI may be limited by its association in a
much larger and/or anchored complex with other protein(s). This
possibility is also supported by the fact that, although purified
RhoA/GDI is readily ADP-ribosylated by the clostridial exoenzyme C3
(46), the unpurified endogenous complex is not (6,
14, 46). The slow leak of GDI from smooth muscle (present study)
contrasts with its reportedly complete loss from mast cells within 5 min of permeabilization with streptolysin-O (44), possibly
due to differences between Rac/GDI (mast cells) and RhoA/GDI (smooth
muscle), respectively, different methods of permeabilization, and/or
that a fraction of poorly diffusible GDI remaining in mast cells was
not measured (44).
GDI relaxed the contractions induced by recombinant GTP · G14V RhoA and agonists (Figs. 4 and 5), consistent with its ability to negatively regulate the Rho/Rho-kinase pathway (38; reviewed in Ref. 43). The relaxant effect of Y-27632 (Fig. 4, A and C), a selective Rho-kinase inhibitor (12, 60), further confirmed that the GDI-inhibitable effects of G14V RhoA and agonists were mediated by Rho-kinase. GDI also extracted RhoA from smooth muscle, as from other, membranes (33).
Inhibition of Ca2+ sensitization fully activated by GTP or
GTPS required considerably more GDI than the endogenous
concentration. The extraction of RhoA by GDI is not catalytic but
requires one-to-one binding. Therefore, the physiological significance
of the recombinant GDI required for extracting RhoA has to be assessed
in light of the total ratio of GDI to RhoA molecules. The volume of
permeabilized smooth muscle used in this study was ~0.036 µl, and,
assuming an upper limit of 4 µM endogenous RhoA, 60% of it
translocated by GTP
S to the membrane is equivalent to 2.4 µM (or
0.086 pmol). The bath volume containing the 500 nM GDI (or 0.06 nmol)
required for 50% relaxation of Ca2+-sensitized force (Fig.
7) was 120 µl, providing a nearly 1,000-fold molar excess of GDI over
RhoA. In contrast, the concentration of endogenous free GDI available
in an intact cell is approximately the same as the amount of RhoA that
dissociated from the complex; this GDI would be insufficient for
removing, on a mole basis, active GTP · RhoA from the membrane
and inactivating the Rho/Rho-kinase pathway.
Activated RhoA (GTP · RhoA) associates with the cell membrane (14); therefore, its removal by GDI presumably represents competition between membrane lipids and GDI for the geranylgeranylated COOH terminus of RhoA and another, positively charged surface that interacts with GDI (21, 36). Hence, although the in vitro affinities of GDI for, respectively, GDP- and GTP-bound Rho family proteins (affinity constant ~30 nM for Cdc42H) (42) are similar, our present results in vivo, in conjunction with studies with liposomes (46), suggest that in cells GDI competes more effectively for GDP · RhoA than for GTP · RhoA. In the absence of GDI, the higher concentration of GTP than GDP in cells would cause GDP · RhoA to recycle to active GTP · RhoA, and Rho signaling would be perpetually "on" through regeneration of GTP from GDP and ATP. Therefore, we conclude that the most likely physiological role of GDI is not to terminate the activity of GTP · RhoA but to sequester GDP · RhoA after GTP hydrolysis.
The relatively rapid time course of GDI-induced relaxation of Ca2+-sensitized force (Fig. 7B) suggests that the activity of the downstream kinase(s) that Ca2+ sensitizes by inhibiting smooth muscle myosin phosphatase (28) is closely temporally coupled to the presence of GTP · RhoA. This implies that activation of the effector kinase by RhoA is transient and that active phosphatase(s) in smooth muscle dephosphorylate the inhibitory site on the regulatory subunit of myosin phosphatase. The major effector kinase first identified as inhibiting MLCP is Rho-kinase (28), but other serine/threonine kinase(s) can also phosphorylate the inhibitory site of smooth muscle myosin phosphatase in vitro (11, 59). Because Rho-activated kinases also bind Rho (23, 34, 35, 39), negative regulation by GDI may also occur through removal of RhoA from its effector kinase(s) as well as from the plasma membrane.
We interpret the Ca2+-sensitizing effect of the GTP · G14V RhoA/GDI complex to result from its in vivo dissociation and the subsequent translocation of GTP · RhoA to the cell membrane, because GTP · RhoA, but not GDP · RhoA, spontaneously translocates from its complex with GDI to liposomes in vitro (46). [Should physiological translocation also require a membrane protein (4), then the results with liposomes (46) suggest that the protein is required for nucleotide exchange.] The Ca2+-sensitizing effect of GTP · G14V RhoA/GDI complex is not due to contamination by free GTP · G14V RhoA because the purified complex contains little or no detectable free RhoA (Ref. 46, Fig. 4, lane 9; see METHODS) and because similar concentrations of the complex and GTP · G14V RhoA caused similar forces (compare Fig. 4 with Fig. 6). It is also unlikely that the undissociated complex is active, like the undissociated Rac1/GDI that activates NADPH oxidase (1, 7), because excess GDI inhibited Ca2+ sensitization induced by the GTP · G14V RhoA/GDI.
A significant portion of Rho-kinase (Fig. 1A) and nearly all
of the RhoA (present study and Ref. 14) translocated by
GTPS to the membrane fraction was Triton X-114 soluble (Fig.
1B). This finding indicates that activation of Rho-kinase
not only translocates it to the membrane fraction, as also suggested by
a recent immunofluorescence study (58), but also exposes
its hydrophobic surface, consistent with an unfolding mechanism of
Rho-kinase activation occurring at the cell membrane.
In addition to Rho-kinase (see above), other
Ca2+-independent kinases can also phosphorylate the
inhibitory site of the regulatory subunit of myosin phosphatase
(Thr-695; Refs. 11 and 59). Furthermore, both Rho-kinase
(32) and another Rho effector, protein kinase N (PKN)
(17), can phosphorylate CPI17, a potent inhibitor, when
phosphorylated, of myosin phosphatase (10). Regardless of
the downstream effector(s), the inhibitory effect of GDI on agonist-
and GTPS-induced Ca2+ sensitization suggests that the
most relevant kinases are RhoA effectors that converge to inhibit
myosin II phosphatase.
Phorbol ester-induced Ca2+ sensitization mediated by protein kinase C(s) was not inhibited by GDI (present study, Fig. 5). This not only confirms but extends the conclusion, based on the use of a Rho-kinase inhibitor (12) and the effects of downregulation of, respectively, G protein-coupled and phorbol ester-induced Ca2+ sensitization (15, 25, 61), that the Rho/Rho-kinase pathway is independent of protein kinase C(s) stimulated by phorbol esters, although both converge on inhibiting myosin phosphatase. Our present results obtained with GDI rule out the involvement of RhoA in phorbol ester-induced Ca2+ sensitization through not only Rho-kinase but also other Rho effectors, such as PKN.
Downregulation of G protein-coupled Ca2+ sensitization
(15) did not decrease Rho-kinase content, and it could not
be rescued by recombinant GTP · G14V RhoA. PDBu, as well as MC,
an inhibitor of the catalytic subunit of myosin phosphatase, retain
their Ca2+-sensitizing effect even when that of GTPS is
downregulated (15); therefore, the present results suggest
that downregulation occurs downstream of RhoA through a
yet-to-be-identified negative regulatory mechanism(s).
The two other constitutively active Rho-subfamily small GTPases, GTP · G12V Cdc42 (Fig. 9) and GTP · Q61L Rac1, significantly inhibited G14V RhoA-induced Ca2+ sensitization of force. Negative regulation of Rho activity by constitutively active Rac1 and Cdc42 in NIH/3T3 cells has been ascribed to inhibition of RhoA activation: nucleotide exchange from GDP · RhoA to active GTP · RhoA (48); GTP · RhoA was estimated as the fraction of RhoA immunoprecipitated by a peptide containing the Rho-binding domain of rhotekin that interacts with GTP · RhoA but not with (inactive) GDP · RhoA (49). However, it is highly unlikely that such inhibition of activating nucleotide exchange (GTP for GDP) accounts for the inhibition of Ca2+ sensitization induced by constitutively active GTP · G14V RhoA (Fig. 9). It is possible that Rac1 and Cdc42 also interfere with the binding of GTP · RhoA to rhotekin and other effectors, including Rho-kinase. Other inhibitory mechanisms could involve PAK, a serine/threonine protein kinase effector of both Cdc42 and Rac1 (2) that can phosphorylate and inhibit MLCK (50). However, inhibition of MLCK by PAK should result in relaxation of the pCa 6.3-induced contraction independently of RhoA/Rho-kinase activity, and this was not the case. In any case, the effects of Cdc42 and Rac1 found in the present study suggest the existence of previously undescribed mechanism(s) through which these two GTPases that do not cause significant Ca2+ sensitization can negatively regulate the Rho-kinase pathway, unlike the apoptosis RhoA pathway, which is not affected by either Rac1 or Cdc42 (40).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Akiko Yoshimura of Welfide Corporation for a generous gift of Y-27632. The human GDI cDNA was a gift from Dr. G. Bokoch, and the RhoA cDNA was a gift from Dr. A. Hall. We thank Barbara Nordin and Ann Folsom for preparation of the manuscript.
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FOOTNOTES |
---|
* M. C. Gong, I. Gorenne, and P. Read contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-48807.
Present address of M. C. Gong: Department of Physiology, University of Kentucky School of Medicine, Lexington, KY 40536-0084.
Address for reprint requests and other correspondence: A. P. Somlyo, PO Box 800736, Charlottesville, VA 22908 (E-mail: aps2n{at}virginia.edu).
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.
Received 9 December 2000; accepted in final form 20 February 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abo, A,
Webb MR,
Grogan A,
and
Segal AW.
Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane.
Biochem J
298:
585-591,
1994[ISI][Medline].
2.
Bagrodia, S,
and
Cerione RA.
PAK to the future.
Trends Cell Biol
9:
350-355,
1999[ISI][Medline].
3.
Bishop, AL,
and
Hall A.
Rho GTPases and their effector proteins.
Biochem J
348:
241-255,
2000[ISI][Medline].
4.
Bokoch, GM,
Bohl BP,
and
Chuang T-H.
Guanine nucleotide exchange regulates membrane translocation of Rac/Rho GTP-binding proteins.
J Biol Chem
269:
31674-31679,
1994
5.
Bordier, C.
Phase separation of integral membrane proteins in Triton X-114 solution.
J Biol Chem
256:
1604-1607,
1981
6.
Bourmeyster, N,
Boquet P,
and
Vignais PV.
Role of bound GDP in the stability of the RhoA-rhoGDI complex purified from neutrophil cytosol.
Biochem Biophys Res Commun
205:
174-179,
1994[ISI][Medline].
7.
Bromberg, Y,
Shani E,
Joseph G,
Gorzalczany Y,
Sperling O,
and
Pick E.
The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages.
J Biol Chem
269:
7055-7058,
1994
8.
Cerione, RA,
and
Zheng Y.
The Dbl family of oncogenes.
Curr Opin Cell Biol
8:
216-222,
1996[ISI][Medline].
9.
Cherfils, J,
and
Chardin P.
GEFs: structural basis for their activation of small GTP-binding proteins.
Trends Biochem Sci
24:
306-311,
1999[ISI][Medline].
10.
Eto, M,
Senba S,
Morita F,
and
Yazawa M.
Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle.
FEBS Lett
410:
356-360,
1997[ISI][Medline].
11.
Feng, J,
Ito M,
Ichikawa K,
Isaka N,
Nishikawa M,
Hartshorne DJ,
and
Nakano T.
Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase.
J Biol Chem
274:
37385-37390,
1999
12.
Fu, X,
Gong MC,
Jia T,
Somlyo AV,
and
Somlyo AP.
The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPS-, and phorbol ester-induced Ca2+-sensitization of smooth muscle.
FEBS Lett
440:
183-187,
1998[ISI][Medline].
13.
Fujihara, H,
Walker LA,
Gong MC,
Lemichez E,
Boquet P,
Somlyo AV,
and
Somlyo AP.
Inhibition of RhoA translocation and calcium sensitization by in vivo ADP-ribosylation with the chimeric toxin DC3B.
Mol Biol Cell
8:
2437-2447,
1997
14.
Gong, MC,
Fujihara H,
Somlyo AV,
and
Somlyo AP.
Translocation of rhoA associated with Ca2+-sensitization of smooth muscle.
J Biol Chem
272:
10704-10709,
1997
15.
Gong, MC,
Fujihara H,
Walker LA,
Somlyo AV,
and
Somlyo AP.
Downregulation of G-protein-mediated Ca2+-sensitization in smooth muscle.
Mol Biol Cell
8:
279-286,
1997[Abstract].
16.
Gong, MC,
Iizuka K,
Nixon G,
Browne JP,
Hall A,
Eccleston JF,
Sugai M,
Kobayashi S,
Somlyo AV,
and
Somlyo AP.
Role of guanine nucleotide-binding proteins-ras-family or trimeric or both in Ca2+ sensitization of smooth muscle.
Proc Natl Acad Sci USA
93:
1340-1345,
1996
17.
Hamaguchi, T,
Ito M,
Feng J,
Seko T,
Koyama M,
Machida H,
Taskase K,
Amano M,
Kaibuchi K,
Hartshorne DJ,
and
Nakano T.
Phosphorylation of CPI-17, an inhibitor of myosin phosphatase, by protein kinase N.
Biochem Biophys Res Commun
274:
825-830,
2000[ISI][Medline].
18.
Hancock, JF,
and
Hall A.
A novel role for RhoGDI as an inhibitor of GAP proteins.
EMBO J
12:
1915-1921,
1993[Abstract].
19.
Hart, MJ,
Jiang X,
Kozasa T,
Roscoe W,
Singer WD,
Gilman AG,
Sternweis PC,
and
Bollag G.
Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by G13.
Science
280:
2112-2114,
1998
20.
Hartshorne, DJ,
Ito M,
and
Erdödi F.
Myosin light chain phosphatase.
J Muscle Res Cell Motil
19:
325-341,
1998[ISI][Medline].
21.
Hoffman, GR,
Nassar N,
and
Cerione RA.
Structure of the Rho family GTP-binding protein Cdc42 in complex with multifunctional regulator RhoGDI.
Cell
100:
345-356,
2000[ISI][Medline].
22.
Iizuka, K,
Ikebe M,
Somlyo AV,
and
Somlyo AP.
Introduction of high molecular weight (IgG) proteins into receptor coupled, permeabilized smooth muscle.
Cell Calcium
16:
431-445,
1994[ISI][Medline].
23.
Ishizaki, T,
Maekawa M,
Fujisawa K,
Okawa K,
Iwamatsu A,
Fujita A,
Watanabe N,
Saito Y,
Kakizuka A,
Moril N,
and
Narumiya S.
The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.
EMBO J
15:
1885-1893,
1996[Abstract].
24.
Itoh, K,
Yoshioka K,
Akedo H,
Uehata M,
Ishizaki T,
and
Narumiya S.
An essential part for Rho-associated kinase in the transcellular invasion of tumor cells.
Nat Med
5:
221-225,
1999[ISI][Medline].
25.
Jensen, PE,
Gong MC,
Somlyo AV,
and
Somlyo AP.
Separate upstream and convergent downstream pathways of G-protein and phorbol ester-mediated Ca2+-sensitization of myosin light chain phosphorylation in smooth muscle.
Biochem J
318:
469-475,
1996[ISI][Medline].
26.
Kaibuchi, K,
Kuroda S,
and
Amano M.
Regulation of cytoskeleton and cell adhesion by the rho family GTPases in mammalian cells.
Annu Rev Biochem
68:
459-486,
1999[ISI][Medline].
27.
Kawano, Y,
Fukata Y,
Oshiro N,
Amazo M,
Nakamura T,
Ito M,
Matsumura F,
Inagaki M,
and
Kaibuchi K.
Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.
J Cell Biol
147:
1023-1037,
1999
28.
Kimura, K,
Ito M,
Amano M,
Chihara K,
Fukata Y,
Nakafuku M,
Yamamori B,
Feng J,
Nakano T,
Okawa K,
Iwamatsu A,
and
Kaibuchi K.
Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase).
Science
273:
245-248,
1996[Abstract].
29.
Kitazawa, T,
Kobayashi S,
Horiuti K,
Somlyo AV,
and
Somlyo AP.
Receptor coupled, permeabilized smooth muscle: role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+.
J Biol Chem
264:
5339-5342,
1989
30.
Kitazawa, T,
Masuo M,
and
Somlyo AP.
G-protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle.
Proc Natl Acad Sci USA
88:
9307-9310,
1991[Abstract].
31.
Kobayashi, S,
Kitazawa T,
Somlyo AV,
and
Somlyo AP.
Cytosolic heparin inhibits muscarinic and -adrenergic Ca2+ release in smooth muscle: physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling.
J Biol Chem
264:
17997-18004,
1989
32.
Koyama, M,
Ito M,
Feng J,
Seko T,
Shiraki K,
Takase K,
Hartshorne DJ,
and
Nakano T.
Phosphorylation of CPI-17, an inhibitory phosphoprotein of smooth muscle myosin phosphatase, by Rho-kinase.
FEBS Lett
475:
197-200,
2000[ISI][Medline].
33.
Leonard, DA,
and
Cerione RA.
Solubilization of Cdc42Hs from membranes by Rho-GDP dissociation inhibitor.
Methods Enzymol
256:
98-105,
1995[ISI][Medline].
34.
Leung, T,
Manser E,
Tan L,
and
Lim L.
A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes.
J Biol Chem
270:
29051-29054,
1995
35.
Lim, L,
Manser E,
Leung T,
and
Hall C.
Regulation of phosphorylation pathways by p21 GTPases. The p21 ras-related rho subfamily and its role in phosphorylation signalling pathways.
Eur J Biochem
242:
171-185,
1996[Abstract].
36.
Longenecker, K,
Read P,
Derewenda U,
Dauter Z,
Liu X,
Garrard S,
Walker L,
Somlyo AV,
Nakamoto RK,
Somlyo AP,
and
Derewenda ZS.
How RhoGDI binds Rho.
Acta Crystallogr D Biol Crystallogr
55:
1503-1515,
1999[ISI][Medline].
37.
Lucius, C,
Steusloff A,
Troschka M,
Hofmann F,
Aktories K,
and
Pfitzer G.
Clostridium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins.
J Physiol (Lond)
506:
83-93,
1998
38.
Mariot, P,
O'Sullivan AJ,
Brown AM,
and
Tatham PER
Rho guanine nucleotide dissociation inhibitor protein (RhoGDI) inhibits exocytosis in mast cells.
EMBO J
15:
6476-6482,
1996[Abstract].
39.
Matsui, T,
Amano M,
Yamamoto T,
Chihara K,
Nakafuku M,
Ito M,
Nakano T,
Okawa K,
Iwamatsu A,
and
Kaibuchi K.
Rho-associated kinase, a novel serine/threonine kinase, as a putative target for the small GTP binding protein Rho.
EMBO J
15:
2208-2216,
1996[Abstract].
40.
Moorman, JP,
Luu D,
Wickham J,
Bobak DA,
and
Hahn CS.
A balance of signaling by Rho family small GTPases RhoA, Rac1 and Cdc42 coordinates cytoskeletal morphology but not cell survival.
Oncogene
18:
47-57,
1999[ISI][Medline].
41.
Nobes, CD,
and
Hall A.
Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia.
Cell
81:
53-62,
1995[ISI][Medline].
42.
Nomanbhoy, TK,
and
Cerione RA.
Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy.
J Biol Chem
271:
10004-10009,
1996
43.
Olofsson, B.
Rho guanine dissociation inhibitors: Pivotal molecules in cellular signalling.
Cell Signal
11:
545-554,
1999[ISI][Medline].
44.
O'Sullivan, AJ,
Brown AM,
Freeman HNM,
and
Gomperts BD.
Purification and identification of FOAD-II, a cytosolic protein that regulates secretion in streptolysin-O permeabilized mast cells, as a Rac/RhoGDI complex.
Mol Biol Cell
7:
397-408,
1996[Abstract].
45.
Penefsky, HS.
A centrifuged-column procedure for the measurement of ligand binding by beef heart F1.
Methods Enzymol
56:
527-530,
1979[Medline].
45a.
Read, PW,
Liu X,
Gong MC,
DiPierro CG,
Somlyo AV,
Somlyo AP,
and
Nakamoto RK.
Mammalian G-protein signal transduction pathways activated by human GTP-RHOA/RhoGDI complex purified from S. cerevisiae (Abstract).
Mol Biol Cell
10:
417a,
1999[ISI].
46.
Read, PW,
Liu X,
Longenecker K,
DiPierro CG,
Walker LA,
Somlyo AV,
Somlyo AP,
and
Nakamoto RK.
Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes.
Protein Sci
9:
1-11,
2000[Abstract].
47.
Read, PW,
and
Nakamoto RK.
Expression and purification of Rho/RhoGDI complexes.
Methods Enzymol
325:
15-25,
2000[ISI][Medline].
48.
Reid, T,
Furuyashiki T,
Ishizaki T,
Watanabe G,
Watanabe N,
Fujisawa K,
Morii N,
Madaule P,
and
Narumiya S.
Rhotekin, a new putative target for Rho bearing homology to a serine/threonine kinase, PKN, and Rhophilin in the Rho-binding domain.
J Biol Chem
271:
13556-13560,
1996
49.
Sander, EE,
Ten Klooster JP,
Delft SV,
Van Der Kammen RA,
and
Collard JG.
Rac downregulates Rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior.
J Cell Biol
147:
1009-1021,
1999
50.
Sanders, LC,
Matsumura F,
Bokock GM,
and
De Lanerolle P.
Inhibition of myosin light chain kinase by P21-activated kinase.
Science
283:
2083-2085,
1999
51.
Seasholtz, TM,
Majumdar M,
and
Brown JH.
Rho as a mediator of G protein-coupled receptor signaling.
Mol Pharmacol
55:
949-956,
1999
52.
Sheffield, PJ,
Garrard SM,
and
Derewenda ZS.
Overcoming expression and purification problems of RhoGDI using a family of "parallel" expression vectors.
Protein Expr Purif
15:
34-39,
1999[ISI][Medline].
53.
Somlyo, AP,
Kitazawa T,
Himpens B,
Matthijs G,
Horiuti K,
Kobayashi S,
Goldman YE,
and
Somlyo AV.
Modulation of Ca2+-sensitivity and of the time course of contraction in smooth muscle: a major role of protein phosphatases?
Adv Prot Phosphatases
5:
181-195,
1989.
54.
Somlyo, AP,
and
Somlyo AV.
Signal transduction and regulation in smooth muscle.
Nature
372:
231-236,
1994[ISI][Medline].
55.
Somlyo, AP,
and
Somlyo AV.
Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II.
J Physiol (Lond)
522:
177-185,
2000
56.
Somlyo, AP,
Wu X,
Walker L,
and
Somlyo AV.
Pharmacomechanical coupling: the role of calcium, G-proteins, kinases and phosphatases.
Rev Physiol Biochem Pharmacol
134:
203-236,
1998.
57.
Somlyo, AV,
Bradshaw D,
Ramos S,
Murphy C,
Myers CE,
and
Somlyo AP.
Inhibition of Rho-kinase retards human prostatic cancer cell migration.
Biochem Biophys Res Commun
269:
652-659,
2000[ISI][Medline].
58.
Taggart, MJ,
Lee YH,
and
Morgan KG.
Cellular redistribution of PKC, rhoA, and ROK
following smooth muscle agonist stimulation.
Exp Cell Res
251:
92-101,
1999[ISI][Medline].
59.
Trinkle-Mulcahy, L,
Ichikawa K,
Hartshorne DJ,
Siegman MJ,
and
Butler TM.
Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in alpha-toxin-permeabilized smooth muscle.
J Biol Chem
270:
18191-18194,
1995
60.
Uehata, M,
Ishizuki T,
Satoh H,
Ono T,
Kawahara T,
Morishita T,
Tamakawa H,
Yamagami K,
Inui J,
Maekawa M,
and
Narumiya S.
Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension.
Nature
389:
990-994,
1997[ISI][Medline].
61.
Walker, LA,
Gailly P,
Jensen PE,
Somlyo AV,
and
Somlyo AP.
The unimportance of being (protein kinase C) epsilon.
FASEB J
12:
813-821,
1998
62.
Wei, Y,
Zhang Y,
Derewenda U,
Liu X,
Minor W,
Nakamoto RK,
Somlyo AV,
Somlyo AP,
and
Derewenda ZS.
Crystal structure of RhoA-GDP and its functional implications.
Nat Struct Biol
4:
699-703,
1997[ISI][Medline].
63.
Zohn, IM,
Campbell SL,
Khosravi-Far R,
Rossman KL,
and
Der CJ.
Rho family proteins and Ras transformation: the RHOad less traveled gets congested.
Oncogene
17:
1415-1438,
1998[ISI][Medline].