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J. Biol. Chem., Vol. 278, Issue 31, 28523-28527, August 1, 2003
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From the
Institut für Toxikologie,
Medizinische Hochschule D-30625 Hannover, Germany, the
||Institut für experimentelle und klinische
Pharmakologie und Toxikologie der Universität Freiburg, D-79098 Freiburg,
Germany, and the ¶Department of Cell
Pharmacology, Nagoya University Graduate School of Medicine, Nagoya 466-8550,
Japan
Received for publication, February 24, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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S-induced release of Rho from GDI-1. Thus, ADP-ribosylation leads to
entrapment of Rho in the GDI-1 complex. The increased stability of the GDI
complex prevented binding of Rho to membrane-associated players of the GTPase
cycle such as the activating guanine nucleotide exchange factors and effector
proteins. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In the GTP-bound form, Rho translocates to plasma membranes where it
interacts with effectors to transduce the signal downstream. In the inactive
GDP-bound form, Rho is complexed by the guanine nucleotide dissociation
inhibitor (GDI),1
thereby localizing it to the cytosolic fraction. Three isoforms of GDI have
been described: Rho-GDI-1 that interacts with Rho, Rac, and Cdc42 and the
D4-GDI or Ly-GDI that is selectively expressed in hematopoetic cell lines
(3–5).
Rho-GDI and the murine form Rho-GDI3 possess a hydrophobic N-terminal
elongation to be associated with membranous compartments
(6,
7). GDI seems to
function with RhoG but not with Rac. It is poorly understood how Rho is
released from the GDI complex. Phospholipids, especially PIP2, are
reported to liberate Rho from the GDI complex
(8,
9). Recently, moesin, a member
of the ERM (ezrin, radixin, moesin) family,
that binds to CD44 or intercellular adhesion molecules (ICAMs) has been
reported to bind GDI thereby releasing monomeric Rho
(10–13).
Because Clostridium botulinum exoenzyme C3 turns off the Rho-dependent signal pathway by selective ADP-ribosylation of RhoA, -B, and -C but not of other low molecular mass GTPases, C3 has been classified as an indispensable tool in cell biology. The disadvantage of poor cell accessibility because of failure of receptor binding and translocation domain has been overcome by different approaches: 1) microinjection of C3 (14); 2) electroporation of cells in the presence of C3 (15); 3) permeabilization by digitonin (16) or streptolysin O (17); 4) intracellular expression of C3 (18–20); 5) using chimeric toxins that recruit the cell entry machinery of other toxins, C3 is fused to enzyme-deficient diphtheria toxin or C. botulinum C2 toxin (21–23). The latter possibility, a chimera with the binary C. botulinum C2 toxin, was applied in this study.
In intact cells, ADP-ribosylation of Rho is accompanied by disaggregation of actin filaments leading to the notion that ADP-ribosylation turns Rho inactive. It has been reported that the typical feature of the disrupted actin filament system caused by C3-catalyzed ADP-ribosylation is reversed by the cytotoxic necrotizing factor (CNF) (22, 24) that is known to transform Rho constitutively active by deamidation of glutamine 63 (25, 26). Because deamidation merely blocks GTP hydrolysis, ADP-ribosylated Rho is able to interact functionally with its effectors. Thus, inhibition of Rho-effector coupling seems not to be the mode of how ADP-ribosylation inactivates Rho functions.
Therefore, we studied the influence of ADP-ribosylation on the Rho-GDI interaction and on the subcellular distribution of Rho and report here that ADP-ribosylation prevents membrane binding and traps Rho in the GDI complex thereby blocking Rho cycling between cytosol and membranes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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S were
obtained from Roche Molecular Biochemicals. The antibody against RhoA and
GDI-1 were purchased from Santa Cruz Biotechnology, Inc. Recombinant Proteins—RhoA was expressed in Sf9 insect cells using a baculovirus expression system (Pharmingen). Sf9 cells (1 x 106 cells/ml) were cultured at 25 °C in TNM-FH insect medium containing 10% fetal calf serum, 100 units/ml penicillin G, and 100 mg/ml streptomycin. Infected with pAcGHLT containing RhoA baculovirus transfer vectors (multiplicity of infection of 5–10) was performed at 25 °C for 48 h. Thereafter, the cells were lysed by sonication in buffer A (50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 1% (v/v) Triton X-100, 10 µM phenylmethylsulfonyl fluoride, and 10 mM Tris-HCl, pH 7.5) and centrifuged at 20,000 x g for 1 h. GST-RhoA from the supernatant was precipitated with glutathione-Sepharose beads at 4 °C for 30 min followed by intensive washing with buffer A to remove unbound proteins. RhoA was released from the parent GST fusion proteins by incubation with thrombin (Pharmingen, 10 units) at 4 °C overnight in buffer (150 mM NaCl, 5 mM MgCl2, 2.5 mM CaCl2, 1 mM dithiothreitol, and 50 mM Tris-HCl, pH 8.0). The beads were removed by centrifugation, and thrombin was precipitated using p-aminobenzamidine beads. The homogeneity of the recombinant RhoA was proven by SDS-PAGE.
GDI-1 was purified as glutathione S-transferase fusion proteins from Escherichia coli followed by thrombin cleavage (100 µg/ml for 30 min at 22 °C). Thrombin was removed by precipitation with benzamidine-Sepharose beads (Amersham Biosciences). The chimeric C2IN-C3 toxin (exoenzyme C3 is fused to the catalytically deficient C2I toxin) (23) and C. botulinum C2 toxin were purified as described (27). In this study C3 is synonymous with C2IN-C3.
Toxin Treatment—NIH3T3 fibroblasts were grown in Dulbecco's medium supplemented with 10% fetal calf serum, 4 mM glutamine, 100 µg/ml penicillin, and 100 µM streptomycin. Fibroblasts on 10-cm dishes were treated with C2IN-C3 fusion toxin or C2 toxin (C2I plus C2II) (each 1 µg/ml) for 3 h. The medium was removed and the cells were washed with 5 ml of ice-cold phosphate-buffered saline and scraped in 300 µl/dish lysis buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 40 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 80 µg/ml benzamidine, 1 mM dithiothreitol, 1 mM EDTA). The cells were disrupted mechanically by sonication (five times on ice), followed by centrifugation for 10 min at 2000 x g to remove the nuclear fraction. The supernatant was used as cell lysate.
Lysates from NIH3T3 cells were centrifuged at 100,000 x g for 1 h to prepare cytosolic and total particulate fractions. The high speed pellet, which consists of the heavy and the light membrane fractions, was washed and resuspended in lysis buffer (28). Cytosolic and membranous fractions were brought to the same concentration of protein measured by the method of Bradford (51).
ADP-ribosylation—RhoA (50 µg/ml) was subjected to
ADP-ribosylation by C3 exoenzyme (1 µg/ml) and 10 µM
[32P]NAD for 30 min at 37 °C. Nucleotide exchange reaction:
isoprenylated RhoA (200 µg/ml) dissolved in lysis buffer was incubated in
the presence of 100 µM GTPS or 100 µM GDP
for 1 h on ice.
Immunoblot Analysis—SDS-polyacrylamide gel electrophoresis was performed on 12.5% acrylamide gels. The gels were analyzed by PhosphorImager SI from Molecular Dynamics. Proteins were separated on 12.5% acrylamide gels and transferred onto nitrocellulose for 2 h at 250 mA. The membranes were blocked for 1 h with 5% (w/v) nonfat dried milk. Blots were incubated for 2 h with anti-RhoA monoclonal antibody (Santa Cruz), anti-GDI-1 polyclonal antibody (both diluted 1:2.000) and anti-PLD1 (1:500) in phosphate-buffered saline containing 0.05% Tween and then for 45 min with a horseradish peroxidase-conjugated secondary antibody. The proteins were visualized by enhanced chemiluminescence. Western blotting of purified recombinant proteins confirmed that the antibodies were specific (results not shown).
Binding of RhoA to Membranes—Recombinant RhoA from
Sf9 cells was ADP-ribosylated as described above followed by
pre-binding of GDP or GTPS. RhoA (2 µg) was incubated with NIH3T3
membranes (50 µg) on ice for 30 min. The reaction mixture was layered onto
200 µl of sucrose (20% (w/v) sucrose supplemented with 0.1 mg/ml bovine
serum albumin and 0.02% (w/v) sodium azide) using microcentrifugation tips and
centrifuged for 1 h at 30,000 x g at 4 °C. The supernatant
was precipitated and resolved in 30 µl of Laemmli sample buffer. The pellet
was dissolved in the same volume of sample buffer. Supernatants and pellets
were analyzed for RhoA content by immunoblot.
Separation of RhoA from the GDI Complex—Cytosol was prepared as described above. 1 mg of protein dissolved in 500 µl of separation buffer was loaded onto a Superdex 75 column (Amersham Biosciences) previously equilibrated with separation buffer (10 mM imidazole, pH 6.8, 400 mM NaCl, 250 mM sucrose). The flow rate was 0.2 ml/min, and the fraction size was 500 µl. The fractions were precipitated with chloroform/methanol and resolved in 30 µl of Laemmli buffer. Fractions were analyzed by immunoblot for RhoA and GDI-1. Calibration of the column was performed with bovine serum albumin (66 kDa, fractions 4 and 5), chicken albumin (45 kDa, fractions 7 and 8), and chymotrypsin (25 kDa, fractions 11 and 12).
Cytosols (0.5 mg/ml) prepared in lysis buffer supplemented with 250 mM sucrose were incubated in the presence of 0.2 mg/ml PIP2 or buffer at 37 °C for 10 min and applied to a 30-kDa cut-off membrane (Microcon 30, Amicon, Beverly) at 7000 x g for 30 min at room temperature. Supernatant and filtrate were brought to the same protein concentration followed by immunoblot analysis for RhoA and GDI-1.
Pull-down Experiments with GST-C21—Pull-down experiments
were performed as described by Reid et al.
(29). Ice-cold lysis buffer
(500 µl; 50 mM NaCl, 20 mM Tris-HCl, pH 7.4, 3
mM MgCl2, 1% Nonidet P-40, 0.25% Triton X-100, 10
mM dithiothreitol, 100 µM phenylmethylsulfonyl
fluoride) was added to cell lysates containing 1 mg of protein in total. For
recombinant assay, 1 µg of GDP- or GTPS-loaded RhoA was added to 500
µl of lysis buffer. Samples were centrifuged for 10 min at 15,000 x
g, and the supernatant was used for pull-down assay. To this end, 20
µl of beads slurry of the Rho binding domain GST-C21 from rhotekin bearing
30 µg of fusion protein were added to each sample and rotated at 4
°C for 30 min. The beads were collected by centrifugation at 10,000 rpm
and washed twice with lysis buffer. To each sample 20 µl of Laemmli buffer
were added to the beads, and 12% SDS-PAGE with subsequent transfer of proteins
onto nitrocellulose was performed. RhoA was detected by Western blot using
specific antibody.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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S as well as ADP-ribosylated RhoA-GTPS stimulated ROK
activity, the latter to a lesser extent. Thus, ADP-ribosylation does not
interfere with Rho-dependent ROK activation per se.
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Subcellular Distribution of RhoA in C3-treated NIH3T3 Fibroblasts—In terms of the cytosol membrane model of the Rho-GTPase cycle, active Rho is translocated to the membranes where effector proteins are also present (30). To study whether ADP-ribosylated Rho does in fact bind to membranes, the subcellular distribution of Rho after treatment of NIH3T3 cells with the chimeric C3 toxin was determined (Fig. 2A). When cytosolic and membrane fractions of control NIH3T3 cells were adjusted to the same protein concentration, about one-half of the cellular Rho was localized in the cytosolic fraction and the other half in the membranes. ADP-ribosylation, however, resulted in a complete shift to the cytosol, and no Rho was detected anywhere in the membrane fractions (Fig. 2A). To exclude that the observed translocation of Rho was based on the reorganization of the actin cytoskeleton, that is the consequence of ADP-ribosylation of Rho, the actin filaments were depolymerized by C. botulinum C2 toxin, that directly ADP-ribosylates actin thereby rendering it incapable of polymerization (31, 32). Rho from these cells showed almost the same subcellular distribution as control cells (Fig. 2A). Thus, ADP-ribosylation rendered Rho incapable of binding to the membranes and led to its accumulation in the cytosol. The total localization of the Rho regulatory protein GDI-1 in the cytosol was altered neither by C3 nor by C2 toxin (Fig. 2A).
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Rho Activity State—To check the activity state of Rho within
the C3-treated cell, the amount of active ADP-ribosylated Rho was determined
by pull-down experiments using the Rho binding domain of rhotekin (C21)
(29). To exclude that
ADP-ribosylation hampers the Rho-C21 interaction, this experiment was first
carried out with recombinant proteins. As shown in
Fig. 2B, recombinant
GTPS-bound RhoA was pulled down with GST-C21 independently of the
ADP-ribosylation. Subsequently, Rho from lysates of C3-intoxicated cells was
subjected to the pull-down assay with C21. As expected, some active Rho was
present in fetal calf serum-cultured control cells, whereas Rho from
C3-treated cells was completely inactive
(Fig. 2B).
Binding of ADP-ribosylated RhoA to GDI-1—Because cellular
ADP-ribosylated Rho was completely cytosolic, we addressed the question
whether the modified Rho was monomeric or complexed with GDI-1
(33,
34). To separate monomeric
RhoA from the Rho-GDI complex, cytosolic fractions were passed through an
ultra-centrifugation membrane with a 30-kDa cut off. As shown in
Fig. 3A, both RhoA
(molecular mass 20 kDa) and GDI-1 (molecular mass 24 kDa) were
detected in the supernatant but not in the filtrate suggesting that RhoA was
completely complexed with GDI-1 (molecular mass of about 45 kDa). Addition of
PIP2 to control cytosol led to the detection of RhoA and GDI-1 in
the filtrate (Fig.
3A), indicating that PIP2 indeed released RhoA
from the complex and induced the formation of monomeric RhoA in the cytosol.
However, the ADP-ribosylated Rho-GDI complex in the cytosol persisted in the
presence of PIP2 (Fig.
3A). To directly prove the presence of the Rho-GDI
complex in the supernatant, gel permeation chromatography of cytosolic
fractions prepared from control and from C3-treated cells was performed. RhoA
from control cytosol eluted with an apparent molecular mass of 40–50 kDa
and a minority with 20–30 kDa (Fig.
3B). RhoA from C3-treated cells eluted completely at
40–50 kDa indicating that ADP-ribosylated RhoA was complexed with GDI-1
or other proteins but was not monomeric.
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Binding of ADP-ribosylated Rho to GDI-1 was also tested in a recombinant
system by nucleotide-dependent co-precipitation of Rho with GST-GDI-1
immobilized to Sepharose. As shown in Fig.
3C, unmodified as well as modified RhoA bound to GDI-1
when loaded with GDP but only faintly when bound to GTPS. The faint
binding of GTPS-loaded Rho reflects a low affinity binding of
RhoA-GTPS to GDI-1. ADP-ribosylated Rho bound to GDI-1 with higher
affinity in comparison to non-modified Rho
(Fig. 3C). A further
indication for higher affinity came from a precipitation experiment of RhoA
from cytosolic fractions using immobilized GST-GDI-1. Added GST-GDI-1 competed
with endogenous GDI-1 for RhoA binding by complete precipitation of cytosolic
Rho. However, GST-GDI-1 was incapable of precipitating modified RhoA from
cytosols prepared from C3-treated cells (data not shown). Thus, the
ADP-ribosylated RhoA-GDI-1 complex seemed to be more stable than the complex
with non-modified Rho.
Activitation of ADP-ribosylated Rho from Lysates—To confirm
that the increased stability of the ADP-ribosylated Rho-GDI complex in fact
hampers Rho activation, lysates were treated with and without C3 and NAD
followed by GTPS incubation. Active Rho was determined by rhotekin
pull-down. In contrast to the condition in
Fig. 2B, the pull-down
was carried out in the absence of any detergents. The reason for the latter
condition is not to disturb the stability of the Rho-GDI complex. As shown in
Fig. 4, unmodified
Rho-GTPS was active whereas Rho from C3-treated lysates turned out to
be inactive. This finding underlines that GTPS was capable of releasing
unmodified Rho but not ADP-ribosylated Rho from the GDI complex. As a control
the sequence of reaction was reversed: lysates were pretreated with
GTPS followed by ADP-ribosylation. This sequence of reaction guaranteed
the presence of monomeric Rho and GDI-1. Monomeric Rho-GTPS was then
ADP-ribosylated and pulled down by rhotekin
(Fig. 4). Thus, entrapment of
ADP-ribosylated Rho in the GDI complex effectively prevented Rho activation by
GTPS.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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C3-catalyzed ADP-ribosylation is thought to inactivate the cellular functions of Rho by blocking Rho-effector coupling, the crucial step in downstream signaling (40–42). ROK belongs to a class of Rho effector proteins that are characterized by an autoinhibitory intramolecular interaction released by binding to RhoA (43, 44). This regulatory concept is based on the findings that the Rho binding domain (RBD)-deficient ROK is constitutively active and that an antibody toward the RBD of ROK is sufficient for activating ROK, thus mimicking Rho (44). Based on the latter finding, mere binding of ADP-ribosylated RhoA should be sufficient for ROK activation as presented above in a recombinant system. However, direct inhibition of Rho-effector coupling is obviously not the mode of action for how Rho is inactivated by ADP-ribosylation in intact cells. The notion that ADP-ribosylated Rho is biologically active is further supported by the observation that sequential treatment of cells with C3 followed by CNF1 results in typical features of the CNF1 morphology (22, 24). ADP-ribosylated RhoAQ63E, likely loaded with cellular GTP, is obviously active, i.e. capable of downstream signaling. The ADP-ribose appears to be inert.
The prominent finding after treatment of cells with C3 was the altered
subcellular distribution of Rho, so that ADP-ribosylated cellular Rho was
completely localized to the cytosolic fraction. From this finding arises the
question whether cytosolic Rho is monomeric or bound to GDI-1. From the gel
filtration and the ultrafiltration experiments, it became clear that
ADP-ribosylated Rho is bound to GDI. Comparable data, the complexation of
ADP-ribosylated Rho with GDI, have been shown in smooth muscle strips using
immunoprecipitation techniques
(45). We confirmed this
finding directly, by the binding of recombinant ADP-ribosylated RhoA to
recombinant GDI-1 thereby excluding the involvement of any additional factors.
From the structure of the RhoA-GDI-1 complex can be deduced that the
ADP-ribose at Asn-41 is directed to the solvent and may not hamper the
formation of the complex (46).
ADP-ribosylation, however, changes the binding properties of Rho to GDI-1.
PIP2 and GTPS, which are reported to release Rho from the
complex (8,
9,
45), were unable to do so when
GDI-1 was complexed with ADP-ribosylated RhoA. Our findings on the stability
of the ADP-ribosylated Rho-GDI complex are an indication that the modified
complex escapes the release mechanisms. This notion is further supported by
the finding that recombinant GDI can compete with cellular GDI for Rho binding
but not when cellular GDI is bound to ADP-ribosylated Rho. Thus, modified Rho
seems to be trapped in the cytosolic GDI complex thereby escaping the
regulatory cycle.
It is generally accepted that the activation of Rho is accompanied by a translocation to the membranes and that membrane-bound Rho reflects the active one (47, 48). The ERM proteins are considered as displacement factors that release Rho from the GDI complex to allow subsequent interaction with the exchange factors (GEF) (11, 12). The GEFs are located at the plasma membrane as shown for the Rho-specific Lbc (49). Thus, ADP-ribosylation inhibits Rho activation by GEFs through prevention of its membrane binding. In a recombinant system, ADP-ribosylation reduced Lbc-catalyzed GTP loading of Rho (22). Both effects together may prevent GTP loading of ADP-ribosylated Rho. This notion is supported by our finding on the absence of any active ADP-ribosylated Rho from C3-treated cells as determined by rhotekin pull-down.
In terms of the cytosol-membrane model of Rho GTPase cycle a functional effector protein is characterized by membrane association. Because ADP-ribosylation entraps Rho in the cytosolic GDI complex, ADP-ribosylation indirectly blocks coupling of Rho to a membrane-bound effector protein. This notion meets the former hypothesis of blocked Rho-effector coupling by ADP-ribosylation; however, under special conditions such as the sequential intoxication with C3 and CNF1, ADP-ribosylated RhoAQ63E is likely to escape GDI-dependent inactivation due to the low affinity of modified Rho-GTP to GDI-1 (22, 24, 50).
Based on our current findings we propose a model of how ADP-ribosylation results in inactive Rho: ADP-ribosylated Rho-GDP binds strongly to GDI-1 and is trapped in this complex. The modified complex possesses altered properties so that ADP-ribosylated Rho is not released by activating signal inputs. ADP-ribosylated Rho, incapable of being translocated to the membranes, fails to meet membrane-associated players of the GTPase cycle such as exchange factors and effector proteins. ADP-ribosylation at Asn-41 captures Rho in the inactive form.
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FOOTNOTES |
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To whom correspondence should be addressed: Institut für Toxikologie,
Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, D-30625 Hannover,
Germany. Tel.: 49-511-532-2807; Fax: 49-511-532-2879; E-mail:
genth.harald{at}mh-hannover.de.
1 The abbreviations use are: GDI-1, guanine nucleotide dissociation
inhibitor; GTPS, guanosine 5'-O-(thiotriphosphate); C21,
Rho binding domain of rhotekin; C3, the chimeric C2IN-C3 transferase; RBD, Rho
binding domain; ROK, rhokinase; PIP2, phosphatidylinositol
4,5-bisphosphate; CNF, cytotoxic necrotizing factor; GST, glutathione
S-transferase; GEF, guanine nucleotide exchange factor.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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