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J Biol Chem, Vol. 274, Issue 41, 29050-29056, October 8, 1999
From the Institut für Pharmakologie und Toxikologie der
Universität Freiburg, Hermann-Herder-Strasse 5, D-79104
Freiburg, Germany
The small GTPases Rho, Rac, and Cdc42 are
monoglucosylated at effector domain amino acid threonine 37/35 by
Clostridium difficile toxins A and B. Glucosylation renders
the Rho proteins inactive by inhibiting effector coupling. To
understand the functional consequences, effects of glucosylation on
subcellular distribution and cycling of Rho GTPases between cytosol and
membranes were analyzed. In intact cells and in cell lysates,
glucosylation leads to a translocation of the majority of RhoA GTPase
to the membranes whereas a minor fraction is monomeric in the cytosol
without being complexed with the guanine nucleotide dissociation
inhibitor (GDI-1). Rho complexed with GDI-1 is not substrate for
glucosylation, and modified Rho does not bind to GDI-1. However, a
membranous factor inducing release of Rho from the GDI complex makes
cytosolic Rho available as a substrate for glucosylation. The binding
of glucosylated RhoA to the plasma membranes is saturable, competable
with unmodified Rho-GTP The Rho subfamily (Rho, Rac, and Cdc42) of low molecular mass
GTP-binding proteins are important regulators of the actin
cytoskeleton, phospholipid metabolism, membrane trafficking, smooth
muscle contraction, cell cycle progression, cell transformation,
apoptosis, and transcriptional activation (1-9). The Rho proteins are
molecular switches in intracellular signaling. In the GDP-bound form
they are switched off, and in the GTP-bound form they are switched on.
The on-off state is tightly governed by regulatory proteins. The
activation is catalyzed by guanine nucleotide exchange factors
(GEFs),1 which promote the
exchange with GTP. In the active GTP state, the Rho protein couples to
effector proteins, e.g. Ser/Thr kinases such as Rho kinase
or PAK for downstream signaling. The active state is terminated by the
GTPase-activating proteins (GAPs), which supply an arginine finger to
increase the GTPase activity, resulting in the formation of inactive
GDP-bound Rho (10). The cycling between the two nucleotide-bound states
is accompanied by cycling between the cytosolic fraction and the
membranes (11-17). The guanidine nucleotide dissociation inhibitors
(GDIs) are involved in this subcellular cycling. GDIs are unique for
the Rho and Rab subfamilies but not found in the other subfamilies of
the Ras superfamily. The GDIs bind to posttranslationally modified
(isoprenylated) Rho proteins, keep them in the GDP-bound form in the
cytosolic fraction, and inhibit nucleotide exchange (5, 18-22). The
GDP form is preferentially bound to GDI-1, but in the GTP-bound form GDI-1 inhibits interaction with GAP (23-26).
There are several reports that phosphatidylinositol bisphosphate
(PIP2) liberates Rho from the GDI complex but the
physiological significance of this finding is still unclear (27, 28).
Recently, the ezrin, moesin, and radixin (ERM) proteins have been
reported to release Rho from the GDI complex. The ERM proteins are
composed of a N-terminal membrane anchorage domain (binding to CD44 or ICAM) and a C-terminal F-actin binding domain allowing them to link the
cytoskeleton to the membrane (29, 30). The ERMs are inactive in the
cytosol through head to tail interaction (31). A so far unidentified
signal induces opening of the ERMs allowing the N-terminal part to bind
to GDI-1. ERM-GDI interaction results in the release of Rho, which
subsequently interacts with the cognate GEF to be activated for
downstream signaling (15, 32, 33). In this regard, the ERM family
functions as a displacement factor for Rho from the GDI complex
(8).
GDI-1 is exclusively localized in the cytosol and interacts with Rho,
Rac, and Cdc42 (12, 18). Ly-GDI (D4-GDI) exhibits the same properties
but is preferentially expressed in hematopoietic cell lines (34). In
addition to the cytosolic GDIs, GDI The Rho subfamily proteins are cellular targets of various bacterial
toxins to be covalently modified. CNF1 catalyzes deamidation of
glutamine 63 of Rho to generate constitutively active GTPase (35, 36).
Clostridium botulinum C3 exoenzyme inactivates RhoA, -B, and
-C but not Rac and Cdc42 by ADP-ribosylation at asparagine 41 (37, 38).
Clostridium difficile toxins A and B, which are cytotoxins
to cause disaggregation of the actin filament system, catalyze
monoglucosylation of Rho, Rac, and Cdc42, thereby inactivating the
GTPases (39, 40). The modification occurs at threonine 37 (Rho) and
threonine 35 (Rac, Cdc42), which is located in the effector loop.
Glucosylation does not significantly alter binding of GDP and GTP but
decreases the intrinsic GTPase activity by a factor of 5 and completely
blocks the GAP-stimulated GTPase activity. Binding to the effector
protein (PKN) for downstream signaling is completely inhibited (41).
Blockade of effector coupling fully explains how glucosylation renders
Rho inactive. However, it does not explain the dominant negative
activity of glucosylated Rho when microinjected into cell monolayers
(39). Therefore, we studied the subcellular localization of
glucosylated Rho and its interaction with the regulatory protein
GDI-1.
Materials and Chemicals--
RhoA and Rac1 were expressed in
Spodoptera frugiperda cells (Sf9 cells) and
purified from a Triton X-100-soluble fraction of cells as glutathione
S-transferase fusion protein. GDI-1 was purified as
glutathione S-transferase fusion protein from
Escherichia coli. The glutathione fusion proteins were
isolated by affinity purification with glutathione-Sepharose beads
(Amersham Pharmacia Biotech). The glutathione S-transferase
carrier was cleaved from RhoA, Rac1, and GDI-1 by thrombin (100 µg/ml
for 30 min at 22 °C). Thrombin was removed by precipitation with
benzamidine-Sepharose beads (Amersham Pharmacia Biotech).
C. difficile toxin B (42), C. botulinum C2 toxin
(43), and the chimeric C3 toxin (ADP-ribosyltransferase C3 is fused to the catalytically deficient C. botulinum C2 toxin) (44) were purified as described. UDP-[14C]glucose was purchased
from Bio Trend (Cologne, Germany).
Cell Culture--
NIH3T3 fibroblasts were grown in Dulbecco's
medium supplemented with 10% fetal calf serum, 4 mM
glutamine/penicillin/streptomycin.
Toxin Treatment and Lysis of Cells--
Cell cultures of 10-cm
dishes were treated with C. difficile toxin B (0.1 µg/ml),
C. botulinum C2 toxin (400 ng/ml C2II plus 200 ng/ml C2I) or
the chimeric C3 toxin (400 ng/ml C2II plus 200 ng/ml C2I-C3) for
different times as noted in the legends. The cells were rinsed with 5 ml of ice-cold phosphate-buffered saline and scraped off in 300 µl of
lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl,
2.5 mM MgCl2, 40 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml leupeptin, 80 µg/ml benzamidine) per dish. The cells were disrupted mechanically by
sonification (five times on ice), followed by centrifugation for 10 min
at 1,000 × g to remove the nuclear fraction and intact
cells. The supernatant (1 mg/ml protein) was used as cell lysate.
Fractionation of Cell Lysates--
Lysates were centrifuged at
100,000 × 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 with lysis
buffer and resuspended in the original volume of lysis buffer.
ADP-ribosylation Reaction--
Lysates from NIH3T3 fibroblasts
(1 mg/ml) were subjected to ADP-ribosylation by C3 toxin (0.1 µg/ml)
in the presence of 10 µM NAD and 10 mM
thymidine for 30 min at 37 °C. The reaction was determined by
addition of Laemmli sample buffer.
Glucosylation Reaction--
Lysates from NIH3T3 fibroblasts (1 mg/ml) were incubated with toxin B (1 µg/ml) in the presence of 30 µM UDP-[14C]glucose and 1 mM
MnCl2 at 37 °C for 1 h. Recombinant RhoA (2 µg)
dissolved in glucosylation buffer (50 mM HEPES, pH 7.4, 2 mM MgCl2, 1 mM MnCl2,
0.2 mM GDP, 1 mM dithiothreitol) was incubated with toxin B (1 µg/ml) and UDP-[14C]glucose (30 µM) at 37 °C for 30 min. Cytosols from NIH3T3
fibroblasts (0.8 mg/ml) were incubated with 0.2 mg/ml PIP2
or 50 µg/ml GDI-1 at 37 °C for 30 min followed by the
[14C]glucosylation reaction. The reaction was determined
by addition of Laemmli sample buffer.
SDS-Polyacrylamide Gel Electrophoresis--
SDS-polyacrylamide
gel electrophoresis was performed with 12.5% polyacrylamide gels. The
gels were analyzed by PhosphorImager SI from Molecular Dynamics.
Immunoblot Analysis--
Proteins were separated on 12.5%
polyacrylamide gels and transferred onto nitrocellulose for 2 h at
250 mA, followed by blocking with 5% (w/v) nonfat dried milk for
1 h. Blots were incubated for 2 h with the appropriate
primary antibody (diluted 1:3.000) in buffer B (50 mM
Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM KCl, 0.05% (w/v) Tween 20) and then for 45 min with a horseradish
peroxidase-conjugated secondary antibody.
Nucleotide Exchange Reaction--
Isoprenylated RhoA and Rac1
were incubated in buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 2.5 mM MgCl2) in the
presence of 100 µM GTP Sucrose Density Centrifugation--
100 µl of reaction mixture
were 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 at 4 °C (1 h at
30,000 × g). The supernatant was precipitated and
resolved in 30 µl of Laemmli sample buffer. The pellet was
dissolved in the same volume of sample buffer.
Binding of Isoprenylated RhoA [14C]Glucosylated to
NIH3T3 Membranes--
RhoA was [14C]glucosylated and
loaded with GTP RhoA Overlay--
RhoA (200 µg/ml) was glucosylated in the
presence of 100 µM non-radioactive UDP-glucose as
described above, followed by nucleotide exchange with 100 µM GDP or GTP Gel Permeation Chromatography of the Rho-GDI-1
Complex--
Cytosol was prepared as described above. 0.5 mg of
protein dissolved in 500 µl of separation buffer was loaded onto a
Superdex 75 column (Amersham Pharmacia Biotech) 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.
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 10 and 11).
Separation of Monomeric Rho from Rho-GDI Complex by 30-kDa
Cut-off Membrane Filters--
Cytosols (250 µg of protein dissolved
in 500 µl of separation buffer) were applied onto a 30-kDa cut-off
membrane (Microcon 30, Amicon, Beverly, MA) at 7,000 × g for 30 min at room temperature. Supernatant and filtrate
were brought to the same protein concentration, followed by immunoblot analysis.
Influence of the Glucosylation on the Subcellular Distribution of
RhoA--
The majority of RhoA (>90%) was localized in the cytosolic
fraction when lysates from NIH3T3 cells were fractionated by
ultracentrifugation (Fig. 1A).
After treatment of NIH3T3 cells with toxin B, the subcellular distribution changed and about 50% of RhoA was localized to the membranous fraction (Fig. 1A). To exclude that this
redistribution was merely based on disaggregation of the actin
cytoskeleton, C. botulinum C2 toxin was applied which
directly ADP-ribosylates monomeric actin thereby turning it incapable
of polymerization. As shown in Fig. 1A, disruption of the
actin filaments did not induce translocation of Rho to the membranes.
Furthermore, neither toxin B nor C2 toxin treatment of the cells led to
a change in GDI-1 localization (Fig. 1A). To prove whether
translocation of Rho to the membranes was due to the bound glucose
moiety, membrane binding of Rho ADP-ribosylated by C3 toxin was
studied. In contrast to toxin B, C3 selectively modifies Rho but not
Rac and Cdc42. ADP-ribosylated Rho was found in the cytosol (Fig.
1A) but not at the membranes, indicating that redistribution
of Rho was based on the glucose moiety and not on mere changes in
structure by posttranslational modification.
The translocation to the membranes was a time-dependent
process proceeding with the increase in Rho glucosylation (Fig.
1B). Glucosylation induced a shift of the majority of Rho to
the membranes, but a fraction (about 40%) remained in the cytosol.
Under these conditions, all cellular Rho was glucosylated in
vivo as was shown by a second [14C]glucosylation of
the lysates (Fig. 1C). That only a fraction of the
completely glucosylated cellular Rho bound to the membranes may be an
indication for limited binding capacity of the membranes.
Membrane Binding of Glucosylated Rho--
The effect of
glucosylation on the translocation of Rho was not only observed in
intact cells; but also when Rho was glucosylated in cell lysates (Fig.
2A). The failure of
ADP-ribosylated Rho to bind to membranes was also found in the in
vitro system. This finding indicated that the machinery of the
intact cell was not needed for translocation but merely the
modification with glucose.
To prove this hypothesis binding of [14C]glucosylated
RhoA in the GTP
Saturable binding of glucosylated and unmodified Rho indicated that the
binding was not merely mediated through lipid-lipid interaction by the
geranyl-geranyl moiety but rather through a protein-protein
interaction. To obtain more information on the binding site, we
performed an overlay assay. Membranous fractions from NIH3T3 cells were
electroblotted and then overlaid with unmodified and glucosylated RhoA
either bound to GDP or GTP Glucosylation of the Rho-GDI Complex--
The majority of Rho is
localized in the cytosol, and this cytosolic Rho is not monomeric but
bound to GDI-1 in a high affinity complex. The cytosolic Rho-GDI
complex is reported to be cleaved by PIP2 (27, 28).
Treatment of intact cells as well as cell lysates with toxin B resulted
in complete glucosylation of cellular Rho (Fig. 1C). This
observation implicated that also Rho from the GDI complex was modified.
Therefore, we tested the influence of GDI-1 on the glucosylation of
Rho. Toxin B-catalyzed glucosylation of Rho/Rac/Cdc42 from cytosolic
fractions was increased after addition of PIP2; conversely,
addition of recombinant GDI-1 to the cytosol completely abolished
glucosylation, an effect that was completely reversed by
PIP2 (Fig. 3A).
These data indicated that the Rho GTPases complexed with GDI-1 were not
substrates for toxin B. To test this assumption directly, we studied
glucosylation with recombinant proteins, using isoprenylated RhoA and
Rac1 from Sf9 cells because only isoprenylated Rho is
capable of binding to GDI-1. RhoA as well as Rac1 bound to GDI-1 were
not glucosylated by toxin B. However, addition of PIP2
turned RhoA and Rac1, respectively, glucosylatable (Fig.
3B). To exclude that PIP2 had no direct
stimulating effect on the enzyme reaction, increasing concentrations of
PIP2 were added to the glucosylation reaction of RhoA. As
shown in Fig. 3C, the phosphoinositide did not change the
glucosylation of recombinant RhoA, indicating that the effect of
PIP2 on the glucosylation of cellular Rho/Rac/Cdc42 was
indeed due to the cleavage of the Rho-GDI complex.
The finding that cellular Rho was completely glucosylated in intact
cells and cell lysates was apparently inconsistent with the observation
that the Rho-GDI complex was resistant to glucosylation. To solve this
contradiction, we tested whether membranes contained factors that
released Rho from the GDI complex. To this end cytosolic fractions were
incubated with increasing concentrations of membranes, followed by
toxin B-catalyzed glucosylation (Fig. 3D). The very low
glucosylation rate in the cytosol was enhanced by membranes that
stimulated glucosylation comparable to the addition of
PIP2. Thus, membranes contained a factor or factors that
make Rho available for glucosylation. PIP2 or the ERM
proteins both have been reported to be releasing factors for Rho (8,
32). In intact cells and also in cell lysates, Rho cycles between the
GDI-bound and the membrane-bound form; the intermediary free,
i.e. monomeric Rho, became substrate for glucosylation.
To test whether glucosylated RhoA was capable of binding to GDI-1,
coprecipitation experiments with GST-GDI-1 immobilized to Sepharose
were performed. As shown in Fig. 3E, RhoA-GDP binding to
GDI-1 was superior to binding of RhoA-GTP Extraction of Rho from Membranes--
One property of GDI-1 is to
extract Rho bound to membranes. Because glucosylated Rho lost its
property to bind to GDI-1, GDI-1 should not be able to extract modified
RhoA from membranes. Membranes from control and toxin B-intoxicated
cells were loaded with GDP, followed by extraction with GDI-1 or buffer
as control. Thereafter, the membranes were separated from the soluble
fraction by sucrose density centrifugation, and Rho was detected by
immunoblot. GDI-1 completely extracted unmodified Rho from the
membranes but was incapable of releasing glucosylated Rho (Fig.
5). Thus, glucosylation of Rho led to
trapping of modified Rho at the Rho binding site of the
membranes.
The Rho subfamily proteins Rho, Rac, and Cdc42 are
monoglucosylated at effector domain amino acid threonine 37 (Rho) and
threonine 35 (Rac/Cdc42) by C. difficile toxins A and B. The
glucose moiety located at switch I loop of Rho alters the GTPase cycle:
Activation of Rho by GEFs is reduced but not completely inhibited when
the properties of glucosylated Ras are transferred to that of
glucosylated Rho (45). The intrinsic GTPase activity of Rho is reduced
but the GAP-stimulated activity is completely inhibited (41). The pivotal step for signaling, the effector coupling, is completely blocked (41). From these biochemical data, it can be concluded that
glucosylated Rho should be trapped in the GTP-bound state but is
incapable of downstream signaling. In contrast to the Ras subfamily,
the Rho subfamily proteins are additionally regulated by GDIs, which
keep the Rho proteins in the GDP-bound form. The high affinity Rho-GDI
complex reflects the cytosolic pool of inactive Rho. Furthermore, GDI-1
extracts membrane-bound Rho and delivers it to the cytosolic pool.
Thus, GDI-1 is the key player of the cytosol-membrane cycling, which in
conjunction with GTP binding is important for downstream signaling (8,
46).
GDI-1 consists of a well structured C-terminal part with a pocket to
bind the isoprenyl moiety of the Rho proteins (47). The N-terminal part
is very flexible and is proposed to interact with the effector loop of
the Rho proteins (47). Rho in the GDI-1 complex is not substrate for
toxin B. This may be due to the inaccessibility of the toxin
recognition site on the Rho surface and/or the inaccessibility of the
acceptor amino acid threonine 37. The fact that glucosylated Rho does
not form a complex with GDI-1 strongly argues for the proposed
interaction of GDI-1 with the effector loop of Rho GTPases.
In intact cells and cell lysates, cellular Rho is completely
glucosylated and no unmodified is left. This finding is surprising because the majority of Rho is bound in the GDI-1 complex in which Rho
is not substrate for the toxins. The Rho-GDI complex, the recombinant
or cytosolic one, is cleaved by PIP2. Whether
PIP2 is the physiological release factor is unclear but
PIP2 is involved in the displacement of Rho from the GDI
complex by the ERM proteins (48). These proteins mediate the membrane
association of the actin filaments (30, 31, 49), and they displace Rho
from the Rho-GDI complex initiating the activation of Rho (32, 33). We
showed that membranes contain displacement activity to release Rho from
the GDI complex; released, monomeric Rho is then glucosylated by toxin
B. This finding explains why the complete quantity of cellular Rho is
glucosylated in intact cells as well as in lysates.
The incapability of glucosylated Rho to bind to GDI-1, shown by gel
filtration of intoxicated cytosol as well as by coprecipitation assay
using recombinant Rho and GDI-1, has a functional implication. It is
generally accepted that Rho is translocated to the membranes during the
activation process and that inactive Rho is extracted by GDI-1 to allow
new Rho to translocate. Glucosylated Rho accumulates at the membranes
for two reasons. (i) Rho is kept in the GTP-bound form. (ii) Rho cannot
be extracted by GDI-1 thereby inhibiting the translocation of
unmodified Rho to the membrane. The inhibition of the cytosol-membrane
cycling of Rho contributes to its functional inactivation.
It is very surprising that Rho with a glucose moiety at threonine 37 shows increased binding to the membranes. The binding of glucosylated
Rho to membranes is saturable and competable by unmodified Rho, as
shown in the membrane binding assay. The subfractionation experiments
showed that the majority of cellular Rho but not the complete amount of
glucosylated Rho translocated to the membranes. This finding also
argues for the existence of limited binding sites at the cytosolic face
of the membranes. These binding sites may be also shared with Rac, an
additional reason for the partial membrane binding of glucosylated Rho.
Furthermore, the binding takes place at a membrane protein with a
molecular mass of 70 kDa as shown in the overlay experiments. The
binding to p70 is also competed by unmodified Rho. Finally, the native
structure of glucosylated Rho is essential because denaturation by
removal of the Mg2+ ion completely abolishes membrane
interaction. These data argue for a specific binding to a membrane
protein, which is mediated rather by a protein-protein interaction than
by a nonspecific lipid-lipid interaction through the isoprenyl moiety.
Glucosylated Rho is unresponsive to GAP and should therefore be trapped
in the GTP-bound form, and GTP-bound glucosylated Rho should bind to
membranes as unmodified does. However, the experimental data, i.e. the membrane binding and the overlay assay, clearly
show that the binding of modified Rho is almost independent of the nucleotide-bound state. Thus, it is conceivable that glucosylated Rho does bind through a mechanism involving the glucose moiety to p70,
a mechanism that is different from the binding of unmodified Rho.
Because recombinant glucosylated RhoA in its native form binds to p70,
the glucose seems to change properties of RhoA. A direct involvement of
the glucose moiety at threonine 37 in binding is very unlikely. The
effector loop seems not to be involved in membrane binding because
membrane-bound Rho interacts with its effector proteins without being
released from the membranes.
The membranous p70 is not a selective binding site for toxin-modified
Rho. Normal cellular Rho in its GTP-bound form also binds to p70.
However, p70 seems to be not an effector protein for Rho because
glucosylated Rho does bind. Recently, saturable high affinity binding
of GTP-bound Rho to membrane preparations from erythrocytes has been
reported (50). It is tempting to speculate that the reported
saturable binding is mediated through p70. p70 may be a
membranous platform that binds Rho GTPases and displays them to
effector or GAP proteins for interaction. Alternatively, p70 may be a
multifunctional adapter protein.
The acceptor amino acid of glucosylation, threonine 37, resides in the
effector domain. However, it becomes more and more clear that there are
several domains that participate in the communication with the effector
proteins. Recently, it has been reported that Rho possesses accessory
domains that determine the effector specificity of Rho (51, 52). One
class (citron) solely interacts with the classical effector domain
covering amino acids 23-40, whereas the class of ROCK (Rho-associated
coiled-coiled-containing protein kinase) and the class of rhophilin
bind to two distinct regions, amino acids 23-40 (switch I) and amino
acids 75-92. Switch I seems to be indispensable for Rho effector
coupling, and therefore glucosylation at Thr-37 blocks communication
with every Rho effector.
Based on the data of the present study, we present a refined model on
the functional consequences of glucosylation of the Rho GTPases by
C. difficile toxins A and B (Fig.
6). Glucosylation at threonine 37/35
slows down activation by GEFs but completely inhibits downstream
signaling of GTP-bound Rho. Glucosylated Rho binds to a membranous
binding site but cannot be released from the membranes by GDI-1 as
unmodified Rho is, because of failure to interact with GAP and to bind
to GDI-1. Glucosylated Rho then accumulates at the membranes and
prevents translocation of unmodified Rho to the membranes for
signaling. Thus, glucosylation results in two functional consequences,
inhibition of effector coupling and of the cycling of the Rho GTPases
from cytosol to membranes and vice versa. The inhibition of
the subcellular cycling may explain why glucosylated Rho is dominant
negative in intact cells (39). Glucosylated Rho occupies the membrane
binding sites and inhibits membrane translocation of unmodified
cellular Rho.
We thank Jürgen Dumbach and Brigitte
Neufang for excellent technical assistance.
*
This work was supported by Deutsche Forschungsgemeinschaft
Project Ju231/3 and Grant SFB 388.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.
The abbreviations used are:
GEF, guanine
nucleotide exchange factor;
C3, C. botulinum C3 exoenzyme;
ERM, ezrin, moesin, radixin proteins;
GDI, guanine nucleotide
dissociation inhibitor;
PIP2, phosphatidylinositide
bisphosphate;
toxin B, C. difficile toxin B;
GAP, GTPase-activating protein;
PAGE, polyacrylamide gel
electrophoresis;
GST, glutathione S-transferase;
GTP
Monoglucosylation of RhoA at Threonine 37 Blocks
Cytosol-Membrane Cycling*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S guanosine
5'-O-(3-thiotriphosphate), and takes place at a membrane
protein with a molecular mass of about 70 kDa. Membrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is,
leading to accumulation of modified Rho at membranous binding sites.
Thus, in addition to effector coupling inhibition, glucosylation also
inhibits Rho cycling between cytosol and membranes, a prerequisite for
Rho activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and GDI-3 are membrane-bound
through a N-terminal extension. The latter GDIs are expressed in a more
tissue-specific manner, and they show more selective interaction with
Rho subfamily proteins (21, 22).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S or 100 µM GDP on
ice for 1 h.
S as described above. Complete glucosylation was
checked by [32P]ADP-ribosylation (data not shown). All
steps were carried out at 4 °C. Washed NIH3T3 membranes (0.35 mg/ml)
were incubated in binding buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 2.5 mM MgCl2, 0.1 mg/ml bovine serum albumin, 100 µM GTPS, 100 µM phenylmethylsulfonyl fluoride) with
[14C]glucosylated RhoA as indicated for 30 min on ice.
The mixture was fractionated by sucrose density centrifugation (as
described above), and the pellet was counted for radioactivity. The
amount found in the tips in the absence of membranes was set blank
(about 0.1%).
S on ice for 1 h. Complete
glucosylation was checked by [32P]ADP-ribosylation (data
not shown). After transfer of NIH3T3 membranes, the nitrocellulose was
blocked with 5% nonfat dried milk, followed by renaturation of
proteins in buffer B overnight at 4 °C. Binding was performed at
24 °C for 2 h with 2 µg/ml glucosylated or unmodified RhoA
(loaded either with GTPS or GDP) dissolved in buffer B supplemented
with 2.5 mM MgCl2. After washing four times
with buffer B for 10 min, the membranes were probed for RhoA as
described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
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Fig. 1.
Influence of glucosylation on the subcellular
distribution of RhoA. A, NIH3T3 fibroblasts were
treated with the indicated toxins for 2 h, and the lysates (1 mg/ml) were fractionated into cytosol and membranes as described under
"Experimental Procedures." Cytosol (cyt) and membranes
(mem) were analyzed for RhoA and GDI-1 by immunoblot
analysis. B, time-dependent translocation of
RhoA. NIH3T3 fibroblasts were treated with toxin B for the indicated
time periods. Cytosol and membranes were probed for RhoA and GDI-1 by
immunoblot analysis. C, complete glucosylation of Rho
proteins by toxin B. NIH3T3 fibroblasts were treated with toxin B or
phosphate-buffered saline for 2 h. Lysates (50 µg of protein)
were checked for complete modification by a second glucosylation
reaction by toxin B in the presence of 30 µM
UDP-[14C]glucose as described under "Experimental
Procedures." PhosphorImager data of 12.5% SDS-PAGE are shown.
View larger version (32K):
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Fig. 2.
Binding of glucosylated RhoA to plasma
membranes. A, glucosylation of Rho in lysates. NIH3T3
lysates (1 mg/ml) were treated with toxin B as described under
"Experimental Procedures," followed by fractionation into cytosol
(cyt) and membranes (mem). Fractions were
analyzed by immunoblot for RhoA and GDI-1. B, saturable
binding. NIH3T3 membranes (0.35 mg/ml) were incubated in binding buffer
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2.5 mM MgCl2, 100 µM GTP S, 0.1 mg/ml bovine serum albumin, 0.1 mM phenylmethylsulfonyl
fluoride) with the indicated concentrations of
[14C]glucosylated RhoA-GTPS (gluc
RhoA-GTP[S]) on ice for 30 min. C, competition of
glucosylated and nonmodified Rho for membrane binding. Binding assay
was carried out in the presence of 18 µg/ml
[14C]glucosylated RhoA-GTPS with increasing
concentrations of control RhoA-GTPS. Membranes were separated by
modified sucrose density centrifugation as described under
"Experimental Procedures" and counted for radioactivity. The
standard deviations from three experiments are given. D,
overlay of NIH3T3 membranes with glucosylated RhoA
(gluc-RhoA). Membranes from NIH3T3 cells (about 50 µg of
protein) were separated by SDS-PAGE and electroblotted onto
nitrocellulose filters. The filters were incubated with isoprenylated
RhoA as indicated, followed by immunoblot analysis for RhoA.
BSA, bovine serum albumin; mem, membranes.
S form to membranes from NIH3T3 cells freed from
endogenous Rho was performed. Bound Rho was separated from non-bound by
sucrose density centrifugation. Fig. 2B shows that
glucosylated RhoA-GTPS bound in a saturable manner to the membranes.
Maximal binding was about 2.5 µg of Rho/mg of membranes. Binding of
glucosylated RhoA was competed by unmodified RhoA-GTPS (Fig.
2C) but not by RhoA-GDP (data not shown), suggesting
comparable affinities to the membrane binding site. Glucosylated Rho
bound to GDP also showed binding but less compared with the GTPS
bound Fig. 2D.
S. Bound RhoA was detected with anti-RhoA.
Fig. 2D shows that unmodified RhoA bound when loaded with
GTPS, whereas in the GDP-bound form there was only a very faint
binding. The membrane protein to which RhoA bound exhibited an apparent
molecular mass of about 70 kDa (p70). Glucosylated RhoA bound to the
same protein; however, it bound in an almost nucleotide-independent
manner. Addition of RhoA-GTPS to the overlay with glucosylated
RhoA-GTPS clearly decreased the binding of glucosylated RhoA (data
not shown), corroborating the results of binding to native membranes
and indicating competition at the same binding site.
View larger version (33K):
[in a new window]
Fig. 3.
Glucosylation of the Rho-GDI complex.
A, effect of PIP2 and GDI-1 on the glucosylation
of cytosolic Rho. Cytosols of NIH3T3 cells (about 50 µg of protein)
were incubated with either 0.2 mg/ml PIP2 or 50 µg/ml
GDI-1 or buffer as indicated at 37 °C for 10 min, followed by
glucosylation reaction with toxin B in the presence of 30 µM UDP-[14C]glucose. B,
glucosylation of the recombinant Rho-GDI complex. RhoA and Rac1 (each 1 µg) were incubated with GST-GDI-1 (3 µg) immobilized to
glutathione-Sepharose at 4 °C for 1 h. The Sepharose beads were
washed and eluted with glutathione buffer (50 mM HEPES, pH
8.0, 10 mM glutathione, 2.5 mM
MgCl2), and glucosylation reaction was performed in the
absence and presence of 0.2 mg/ml PIP2. PhosphorImager data
of the 12.5% SDS-PAGE are shown. C, influence of
PIP2 on the glucosylation reaction. Recombinant RhoA (50 µg/ml) was [14C]glucosylated by toxin B in the presence
of the indicated concentrations of PIP2 or buffer.
PhosphorImager data of 12.5% SDS-PAGE are shown. D,
glucosylation of cytosol in the presence of membranes. Cytosols of
NIH3T3 cells (about 50 µg of protein) were incubated with increasing
amount of NIH3T3 membranes, followed by
[14C]glucosylation reaction with toxin B. The ratio
reflects milligrams of membranous proteins/mg of cytosolic proteins.
PhosphorImager data of the autoradiography are shown. mem,
membrane. E, nucleotide-dependent binding of
glucosylated RhoA to GDI-1. GST-GDI-1 (3 µg) immobilized to
glutathione-Sepharose was incubated with RhoA (1 µg) and glucosylated
Rho (glucRhoA, 1 µg) in binding buffer (50 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol), respectively,
loaded either with GDP or GTP S at 4 °C for 1 h. GST alone
was used as control. Beads were washed with binding buffer and eluted
with glutathione buffer. Eluted proteins were probed for RhoA by
immunoblot analysis.
S corroborating reported data (25). Glucosylated RhoA, however, showed no binding to GDI-1
independently of the nucleotide bound. The same results were found for
Rac1 with the exception that also Rac1-GTPS bound to GDI-1. Thus,
glucosylation inhibited binding to GDI-1 or significantly decreases the
affinity to GDI-1. From the finding that glucosylation prevented
binding to GDI-1, it can be deduced that glucosylated Rho, in contrast
to nonmodified Rho, should be monomeric in the cytosol from toxin
B-treated cells. Indeed, gel permeation chromatography of cytosol
prepared from control and toxin B-treated cells revealed that Rho from
control cells was eluted with an apparent molecular mass of about 50 kDa (22 kDa of Rho plus 23 kDa of GDI-1) and that glucosylated Rho was
eluted with a mass of 22 kDa consistent with the monomeric property of
modified Rho (Fig. 4A). The
same results were obtained when monomeric Rho was separated from the Rho-GDI complex by filtration through a membrane filter with a 30 kDa
cut-off (Fig. 4B).
View larger version (26K):
[in a new window]
Fig. 4.
Glucosylated RhoA is not complexed to GDI-1
in the cytosol. A, gel permeation chromatography.
Cytosols (about 0.5 mg of protein) from toxin B-treated and control
cells were chromatographed on Superdex 75 column. Fractions were
analyzed for RhoA by immunoblot analysis. RhoA from control cytosol
eluted in fractions 5-8, corresponding to an
apparent molecular mass of 40-50 kDa. Cytosolic Rho from toxin
B-treated cells eluted in fractions 11 and
12, corresponding to an apparent molecular mass of 20-30
kDa. B, fractionation of cytosol using a 30-kDa cut-off
membrane filter. Cytosols (about 250 µg of protein) from toxin
B-treated and control cells were separated by centrifugation at
7,000 × g for 30 min through a 30-kDa cut-off membrane
filter. Filtrate (F, <30 kDa) and supernatant
(S, >30 kDa) were analyzed for RhoA by immunoblot
analysis.
View larger version (27K):
[in a new window]
Fig. 5.
Incapability of GDI-1 to extract glucosylated
RhoA from plasma membranes. Membranes (50 µg of protein) from
toxin B-treated and control rat liver cells were loaded with GDP.
GST-GDI (2 µg) in reaction mixture (50 mM Tris-HCl, pH
7.4, 150 mM NaCl, 5 mM MgCl2, 200 µM GDP, 1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride) or reaction mixture alone were added to
the membranes and incubated at 37 °C for 30 min. Membranes
(mem) and soluble fraction (S) were fractionated
by sucrose density centrifugation and analyzed for RhoA by
immunoblot.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (32K):
[in a new window]
Fig. 6.
Glucosylation inhibits Rho cycling between
cytosol and membranes. A signal input leads to dissociation of Rho
from the GDI complex, translocation to the membrane, and interaction
with a GEF to be loaded with GTP. Communication of Rho-GTP with
effector proteins are terminated by GAP, and Rho-GDP is extracted from
the membrane by GDI to translocate Rho to the cytosol. Glucosylation
blocks binding to GDI and therefore extraction from the membrane
binding sites. Membrane-bound glucosylated Rho, however, does not
communicate with effector proteins but blocks the membrane binding
site, thereby inhibiting the cycling of nonmodified Rho from cytosol to
the membrane.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 49-761-2035301;
Fax: 49-761-2035311; E-mail: justingo@uni-freiburg.de.
ABBREVIATIONS
S, guanosine 5'-O-(3- thiotriphosphate).
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DISCUSSION
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