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INTRODUCTION |
Small molecular weight GTP-binding proteins of the Ras superfamily
play an integral role in eukaryotic signal transduction, controlling
processes such as cell differentiation and proliferation (1). Several
bacterial toxins covalently modify members of the Ras superfamily and
alter transduction of intracellular signals. Clostridium
botulinum C3 ADP-ribosylates Rho at Asn-41, inhibiting Rho-dependent differentiation (2). Clostridium
sordelii lethal toxin glucosylates Ras at Thr-37, inhibiting the
epidermal growth factor-stimulated p42/44 mitogen-activated protein
kinase signaling pathway (3). Escherichia coli cytolethal
necrotizing factor deaminates Gln-63 of Rho, inhibiting intrinsic
GTPase activity (4, 5).
Conditions such as cystic fibrosis, leukemia, neutropenia, and burn
wounds predispose individuals to infections by P. aeruginosa (6). Numerous factors, including the production of exoenzyme S
(ExoS1; 453 amino acids), may
contribute to the virulence of P. aeruginosa (7). ExoS, a
member of the family of bacterial ADP-ribosyltransferases (8), requires
the presence of a eukaryotic protein termed factor-activating exoenzyme
S (FAS), a 14-3-3 protein, for expression of ADP-ribosyltransferase activity (9). Unique to most bacterial toxins, ExoS does not have rigid
target protein specificity. In vitro, ExoS has been shown to
ADP-ribosylate a number of targets, including IgG3 (10), apolipoprotein A-I (10), vimentin (11), and several members of the Ras
superfamily (12). Analysis of chemical sensitivity indicated that ExoS
ADP-ribosylates these proteins at arginine residues (10, 13).
Initial studies implicated a role for ExoS in the dissemination of
P. aeruginosa from burn wounds (14) and in tissue
destruction in chronic lung infections (15). However, recent studies
have shown that the transposon mutant used in the earlier studies had a
disruption in the gene encoding a component of its type III secretion
system; thus, this mutant would have pleiotropic effects on the
expression of other type III secreted factors in addition to ExoS (16).
Two recent studies have implicated a role for the type III secretion
apparatus in the delivery of ExoS into eukaryotic cells. Olson and
co-workers (17) have shown that incubation of cultured cells with
strains of P. aeruginosa expressing ExoS resulted in the
ADP-ribosylation of Ras, whereas Forsberg and co-workers (18) have
demonstrated that ExoS is cytotoxic to cultured cells when delivered by
the type III secretion system of the heterologous host
Yersinia.
In this study, we examined the biochemical aspects of the
ADP-ribosylation of Ras by ExoS. We demonstrate that ExoS
ADP-ribosylates c-Ha-Ras at more than one site, with Arg-41 being the
preferred site of ADP-ribosylation. In addition, we show that infection of PC-12 cells by a strain of P. aeruginosa that produces
ExoS inhibits nerve growth factor (NGF)-stimulated neurite outgrowth, which is the first demonstration that ADP-ribosylation by ExoS disrupts
a Ras-mediated signal transduction pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
The following reagents were purchased from the
indicated manufacturers: [adenylate
phosphate-32P]NAD and [35S]methionine,
NEN Life Science Products; TnT7 quick coupled transcription/translation kit, Promega; Sculptor in vitro mutagenesis kit, Amersham
Pharmacia Biotech; bovine serum albumin, Pierce; and DNA oligomers,
Operon Technologies, Inc. Recombinant FAS and the pET16b c-Ha-Ras
vector were gifts from H. Fu (Emory University). P. aeruginosa 388 exoenzyme S was purified as described previously
(19). P. aeruginosa 388 and P. aeruginosa
388
ExoS were cultured as described previously (16).
Purification of His-tagged Ras Proteins--
Ras proteins were
expressed in E. coli and purified as described previously
(20). Briefly, protease inhibitors (phenylmethylsulfonyl fluoride and
aprotinin) were added to the concentrated cell suspensions, which were
disrupted with a French press. The lysate was centrifuged (30,000 × g for 20 min) and filtered (0.45-µm filter), and
His-tagged Ras proteins were purified by Ni2+ affinity
chromatography (20). His-tagged Ras proteins were eluted with elution
buffer containing 3 µM GTP and 10 mM
MgCl2, diluted to 40% glycerol, and stored at
20 °C.
These proteins were termed Ras
CAAX.
ADP-ribosylation of Ras
CAAX by ExoS--
Reaction mixtures
(25 µl) contained 0.2 M sodium acetate (pH 6.0), with the
indicated amount of [adenylate
phosphate-32P]NAD, and Ras
CAAX in the
presence and absence of FAS and/or ExoS or
N222 (a catalytic
deletion peptide of ExoS) (20) at the indicated concentrations.
Reactions were performed for 1 h at room temperature and stopped
with 0.5 volume of gel loading buffer containing
-mercaptoethanol
and boiling. Samples were analyzed by SDS-PAGE followed by
autoradiography.
Construction of M13mp18Ras
CAAX and Site-directed Mutagenesis
of Ras
CAAX--
Ras
CAAX was engineered by deletion
polymerase chain reaction mutagenesis of pet16b c-Ha-Ras using primers
containing EcoRI and BamHI restriction sites at
the 5'- and 3'-ends of the gene, respectively. The resulting polymerase
chain reaction product was digested with XbaI and
BamHI and ligated into the pET15b vector. The
EcoRI-BamHI fragment, encoding the
Ras
CAAX gene and the T7 promoter, was then cloned into
M13mp18. M13mp18Ras
CAAX was used as the template for
site-directed mutagenesis. Mutagenesis was performed essentially
following the instructions of Amersham Pharmacia Biotech. DNA primers
were constructed to be 10 base pairs complementary to the DNA template
flanking the mutation of interest and encoded a Arg-to-Ala substitution
(GC to CG). The presence of the mutation was confirmed by sequence
analysis. The mutants were designated RnA, where arginine at
residue n of Ras was changed to alanine.
ADP-ribosylation of in Vitro Transcribed/Translated M13ras by
ExoS--
M13mp18Ras
CAAX (M13ras) or mutated
M13mp18Ras
CAAX replicative form DNA was subjected to
coupled in vitro transcription/translation (Promega).
Transcription/translation reactions (12 µl) contained 0.5 µg of
replicative form DNA and [35S]Met (specific activity of
98 Ci/mmol). An aliquot of the transcription/translation reaction (2 µl) was added to 4 volumes of gel loading buffer containing
-mercaptoethanol and boiled for 5 min. This sample was subjected to
SDS-PAGE followed by autoradiography to monitor the electrophoretic mobility of intrinsically labeled
[35S]Met-Ras
CAAX. The
transcription/translation mixture was also subjected to
ADP-ribosylation by ExoS in reaction mixtures (25 µl) containing 4 µl of the transcription/translation mixture and 0.6 M
sodium acetate (pH 6.0) in the presence and absence of 0.1 µM FAS, 960 µM NAD, and either 12 or 1.2 nM ExoS as indicated. Reactions were stopped at the
indicated times by adding 2 volumes of gel loading buffer containing
-mercaptoethanol and boiling for 5 min. Samples were subjected to
SDS-PAGE followed by autoradiography. The electrophoretic mobility of
this sample was compared with the electrophoretic mobility of
intrinsically labeled [35S]Met-Ras
CAAX.
GTP Dissociation Kinetics of Wild-type Ras and
Ras
CAAXR41K--
The rate of GDP/GTP dissociation of wild-type
Ras
CAAX and Ras
CAAXR41K was measured
essentially as described previously (21). Briefly, 2 µM
Ras
CAAX was incubated in 4 mM EDTA, 50 mM Tris (pH 7.6), and 5 mM dithiothreitol for
10 min at 37 °C, and then 50 µM
[
-32P]GTP (specific activity of 1.4 Ci/mmol) and 10 mM MgCl2 were added. After 30 min at 37 °C,
a 40-fold excess of unlabeled GTP was added. At defined times, 25-µl
aliquots were removed, filtered through nitrocellulose filters, and
washed. Filter-bound radioactivity was determined by scintillation
counting.
PC-12 Cell Culture--
PC-12 cells were passaged in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated horse
serum and 5% fetal bovine serum. Twenty-four hours prior to an
experiment, ~4 × 103 cells were seeded per well in
a 12-well culture dish coated with poly-L-lysine. For
differentiation, the culture medium was changed to Dulbecco's modified
Eagle's medium containing 1% horse serum and 100 ng/ml NGF
(Boehringer Mannheim).
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RESULTS |
ADP-ribosylation of Ras
CAAX by ExoS--
ExoS ADP-ribosylates
several eukaryotic proteins in vitro, including vimentin,
IgG, and several members of the Ras superfamily (10, 13). In this
study, the ADP-ribosylation of c-Ha-Ras by ExoS was examined. To
facilitate expression and purification, a deletion peptide of Ras
(termed Ras
CAAX) was expressed as a His-tagged protein in
E. coli. Ras
CAAX possesses the complete GTP-binding domain and effector functions of Ras, but lacks the four
carboxyl-terminal residues, which comprises the farnesylation sequence
necessary for membrane targeting. ADP-ribosylation of Ras
CAAX by ExoS showed an absolute requirement for FAS
and NAD (Fig. 1). ADP-ribosylated
Ras
CAAX possessed a slower electrophoretic mobility as
determined by SDS-PAGE relative to non-ADP-ribosylated Ras
CAAX. Other experiments showed that at saturation,
ExoS had ADP-ribosylated Ras
CAAX at a stoichiometry of
~2 mol of ADP-ribose bound per mol of Ras
CAAX (Table
I). This suggested that ExoS ADP-ribosylated Ras
CAAX at potentially two sites. Similar
results were obtained when ExoS ADP-ribosylated full-length c-Ha-Ras
(data not shown).

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Fig. 1.
ADP-ribosylation by ExoS alters the
electrophoretic mobility of Ras CAAX.
Ras CAAX was ADP-ribosylated by ExoS in the presence (+)
or absence ( ) of the indicated reagents. The band with the fast
electrophoretic mobility corresponds to Ras CAAX, whereas
the band with slow electrophoretic mobility corresponds to
ADP-ribosylated Ras CAAX. Reaction mixtures (25 µl)
contained 0.2 M sodium acetate (pH 6.0), 300 µM NAD (specific activity of 1.6 Ci/mmol), and 30 µM histidine-Ras fusion protein in the presence and
absence of 1 µM FAS and/or 0.1 µM ExoS.
Reactions were performed for 1 h at room temperature and analyzed
by SDS-PAGE (upper panel) followed by autoradiography
(lower panel).
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ADP-ribosylation of in Vitro Transcribed/Translated Ras
CAAX by
ExoS--
Biochemical characterization of eukaryotic proteins that had
been ADP-ribosylated by ExoS indicated that arginine was the site of
ADP-ribosylation (10). Since the presence of two potential sites for
ADP-ribosylation within Ras could complicate a biochemical determination of the preferred site for ADP-ribosylation, a molecular approach was used to determine if ExoS ADP-ribosylated Ras at a
preferred arginine residue. Briefly, the gene encoding
Ras
CAAX was subcloned downstream of a T7 promoter, which
was then subcloned into M13mp18 (M13ras). The replicative
form of M13ras was isolated and subjected to in
vitro transcription/translation using [35S]Met as an
intrinsic label. The in vitro transcription/translation mixture, containing [35S]Met-Ras
CAAX, was
subjected to ADP-ribosylation by ExoS. ADP-ribosylated [35S]Met-Ras
CAAX was measured as a shift in
the electrophoretic mobility of
[35S]Met-Ras
CAAX. The requirements for the
ADP-ribosylation of in vitro translated
[35S]Met-Ras
CAAX by ExoS are shown in Fig.
2. In vitro translated [35S]Met-Ras
CAAX migrated as a distinct
28-kDa band on SDS-PAGE. Incubation of in vitro translated
[35S]Met-Ras
CAAX with exogenous NAD, FAS,
and ExoS resulted in the detection of two electrophoretic mobility
shifted forms of Ras
CAAX. This was consistent with ExoS
ADP-ribosylating Ras at two distinct sites. Ras
CAAX
showed little electrophoretic mobility shifting in reactions lacking
NAD, whereas two electrophoretic mobility shifted forms of
Ras
CAAX were observed in reactions lacking FAS. This
indicated that the reticulocyte lysates used for in vitro transcription/translation of Ras
CAAX contained a small
amount of NAD and saturating amounts of FAS relative to the
concentrations used in the ADP-ribosyltransferase assay.

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Fig. 2.
ADP-ribosylation of in vitro
transcribed/translated Ras CAAX requires the
addition of exogenous NAD and ExoS. In vitro transcribed/translated [35S]Met-Ras CAAX was
subjected to ADP-ribosylation by ExoS in the presence (+) or
absence ( ) of the indicated reagents for 10 or 120 min at room
temperature. ExoS (12 nM) was added to reaction mixtures as
indicated. Reaction mixtures were analyzed by SDS-PAGE followed by
autoradiography. Electrophoretic migrations are indicated as
Ras CAAX (arrow) and ADP-ribosylated
Ras CAAX (*, single-shifted Ras CAAX; **,
double-shifted Ras CAAX).
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ADP-ribosylation of Ras
CAAX Containing Single Arginine Mutations
by ExoS--
M13ras single-stranded DNA was subjected to
site-directed mutagenesis in which 12 individual Arg-to-Ala mutations
(each mutant was designated RnA (Arg at residue n
of Ras mutated to an Ala) were introduced into the ras open
reading frame. M13ras replicative form DNA, encoding the
wild type or an individual Arg-to-Ala mutation, was subjected to
in vitro transcription/translation. In vitro translated [35S]Met-Ras
CAAX was
ADP-ribosylated by ExoS at a concentration of ExoS such that a single
preferred site for ADP-ribosylation would be detected. Wild-type
[35S]Met-Ras
CAAX and 11 of the individual
Arg-to-Ala mutants of Ras
CAAX, with the exception of
Ras
CAAXR41A, showed an electrophoretic mobility shift
upon ADP-ribosylation by ExoS. In contrast, Ras
CAAXR41A did not show an electrophoretic mobility shift upon incubation with
ExoS. These data suggested that Arg-41 was the preferred site of
ADP-ribosylation by ExoS (Fig. 3).

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Fig. 3.
Arg-41 is the preferred site of
ADP-ribosylation of Ras by ExoS. Twelve site-directed Arg-to-Ala
mutants of Ras CAAX were produced (R41A, R169A, R68A,
R73A, R97A, R102A, R123A, R128A, R164A, R135A, R149A, and R161A).
In vitro transcribed/translated [35S]Met-Ras CAAX was ADP-ribosylated by
ExoS. Reaction mixtures were subjected to SDS-PAGE followed by
autoradiography. The - lanes represent the electrophoretic
mobility of [35S]Met-Ras CAAX proteins,
whereas the + lanes represent the electrophoretic mobility
of the [35S]Met-Ras CAAX proteins following
incubation with 1.2 nM ExoS and 960 µM NAD
for 1 h at room temperature. WT, wild type.
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ADP-ribosylation of in vitro translated
[35S]Met-Ras
CAAX (the wild type and
RnA mutants) was also assayed at higher concentrations of
ExoS such that multiple sites within Ras would be ADP-ribosylated (Fig.
4). With the exception of
Ras
CAAXR41A, ExoS ADP-ribosylated wild-type
Ras
CAAX and the RnA mutants, with the
appearance of two sequential electrophoretic mobility shifts,
suggestive of the presence of double ADP-ribosylated Ras. In contrast,
ExoS ADP-ribosylated Ras
CAAXR41A to only a single
electrophoretic mobility shift, which was consistent with the presence
of only a single site for ADP-ribosylation. These data also indicated that the second site of ADP-ribosylation was not at a specific arginine
residue and that more than one arginine could be ADP-ribosylated at
the second site. Since a third ADP-ribosylation event was not observed,
it appeared that ADP-ribosylation at the second site excluded a third
ADP-ribosylation event.

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Fig. 4.
Exoenzyme S does not ADP-ribosylate Ras at a
distinct secondary site. Twelve site-directed Arg-to-Ala mutants
of Ras CAAX were produced (R41A, R169A, R68A, R73A, R97A,
R102A, R123A, R128A, R164A, R135A, R149A, and R161A). In
vitro transcribed/translated [35S]Met-Ras CAAX was ADP-ribosylated by
ExoS. Reaction mixtures were subjected to SDS-PAGE followed by
autoradiography. The lanes represent the
electrophoretic mobility of
[35S]Met-Ras CAAX proteins, whereas the + lanes represent the electrophoretic mobility of
[35S]Met-Ras CAAX proteins following
incubation with 12 nM ExoS and 960 µM NAD for
1 h at room temperature. WT, wild type.
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ADP-ribosylation of Several Arg-41 Mutants of Ras by ExoS--
To
support the identification of Arg-41 as the preferred site for
ADP-ribosylation by ExoS, several more conservative site-directed mutations were engineered at residue 41, lysine
(Ras
CAAXR41K) and glutamine (Ras
CAAXR41Q).
As observed for Ras
CAAXR41A, low concentrations of ExoS
did not ADP-ribosylate either Ras
CAAXR41K or
Ras
CAAXR41Q (Fig. 5). A
second positive control, Ras
CAAXG12V, was ADP-ribosylated
by ExoS as measured by the generation of electrophoretic mobility
shifted Ras.

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Fig. 5.
ADP-ribosylation of wild-type
Ras CAAX and several Ras mutants by ExoS. In
vitro transcribed/translated
[35S]Met-Ras CAAX and
[35S]Met-Ras CAAX mutants (R41A, R41K, R41Q,
and G12V) were incubated alone ( ) or with (+) 1.2 nM ExoS
and 960 µM NAD for 1 h at room temperature. Reaction
mixtures were subjected to SDS-PAGE followed by autoradiography. The
asterisk denotes single-shifted
[35S]Met-Ras CAAX proteins.
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ADP-ribosylation of Ras
CAAX and Ras
CAAXR41K by
ExoS--
To address ExoS-mediated ADP-ribosylation of Ras at
Arg-41 in more detail, Ras
CAAX and
Ras
CAAXR41K were analyzed as targets for
ADP-ribosylation. Bacterially produced Ras
CAAX and
Ras
CAAXR41K were expressed and purified to similar levels
and reacted with polyclonal antisera to Ras by Western blotting (Fig.
6). To determine whether
Ras
CAAX and Ras
CAAXR41K were functional,
the GTP dissociation kinetics were measured. Both Ras
CAAX
and Ras
CAAXR41K exhibited similar GTP dissociation
kinetics (Table I), which indicated that the R41K mutation did not
alter the global functional properties of Ras. Others have reported
that mutations at Arg-41 did not alter the GTP dissociation kinetics
(21).

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Fig. 6.
Western blotting of wild-type
Ras CAAX and His-tagged
Ras CAAXR41K. Wild-type Ras CAAX
(WT), Ras CAAXR41K (K41), and soybean trypsin inhibitor (SBTI; 2 µg) were subjected to
SDS-PAGE. One gel was stained for protein (A), and proteins
in the second gel were transferred to nitrocellulose filters and
incubated with a Ras polyclonal antibody (Upstate Biotechnology, Inc.)
followed by incubation with 125I-protein A. The filter was
subjected to autoradiography (B).
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Next, the ADP-ribosylation of Ras
CAAX and
Ras
CAAXR41K by ExoS was studied. At saturation, ExoS
ADP-ribosylated wild-type Ras
CAAX to a stoichiometry of
~2 mol of ADP-ribose incorporated per mol of Ras (Table I), whereas
ExoS ADP-ribosylated Ras
CAAXR41K to a stoichiometry of
0.7 mol of ADP-ribose incorporated per mol of Ras. Examination of the
Coomassie Blue-stained gel and autoradiogram showed that, relative to
non-ADP-ribosylated Ras, ADP-ribosylated Ras
CAAX migrated
with an altered electrophoretic mobility on SDS-PAGE, whereas
ADP-ribosylated Ras
CAAXR41K did not have an altered
electrophoretic mobility on SDS-PAGE (Fig.
7). The fact that ADP-ribosylated
Ras
CAAXR41K did not have an altered electrophoretic mobility in this experiment is due to fact that this experiment was
performed using a 12% polyacrylamide gel, whereas the second ADP-ribosylation event was observed upon separation on a 15%
polyacrylamide gel, as seen in Fig. 4. Under linear velocity
conditions, ExoS ADP-ribosylated wild-type Ras
CAAX at a
faster velocity than Ras
CAAXR41K. A representative graph
depicting the velocity of the ADP-ribosylation of wild-type
Ras
CAAX and Ras
CAAXR41K by ExoS is shown in
Fig. 8. The average of three independent
experiments indicated that ExoS ADP-ribosylated wild-type
Ras
CAAX at 5-fold greater velocity than
Ras
CAAXR41K (Table I).

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Fig. 7.
ADP-ribosylation of wild-type Ras and
Ras CAAXR41K by ExoS. Wild-type Ras and
Ras CAAXR41K were ADP-ribosylated by N222. Reaction
mixtures (25 µl) contained 0.2 M sodium acetate (pH 6.0),
100 µM NAD (specific activity of 1.6 Ci/mmol), and 5 µM histidine-Ras fusion protein in the presence of 100 nM FAS and 50 nM N222. Reactions were
performed at room temperature and stopped at the indicated times, and
samples were analyzed by SDS-PAGE. The gel was stained for protein
(A) and subjected to autoradiography (B).
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Fig. 8.
Velocity of the ADP-ribosylation of
Ras CAAX and Ras CAAXR41K by ExoS.
Wild-type Ras and Ras CAAXR41K were ADP-ribosylated by
N222. Reaction mixtures (25 µl) contained 0.2 M sodium
acetate (pH 6.0), 30 µM NAD (specific activity of 12.8 Ci/mmol), and 2.5 µM histidine-Ras fusion protein in the
presence of 2.5 nM FAS and 2.5 nM N222.
Reactions were performed at room temperature and stopped after 7.5, 15, 30, and 60 min. Samples were analyzed by SDS-PAGE, and radioactivity
was quantitated by scintillation counting.
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Infection of PC-12 Cells with P. aeruginosa Expressing ExoS
Inhibits NGF-stimulated Neurite Formation--
Arg-41 is located next
to the switch 1 region of Ras (residues 30-39), a domain that
interacts with downstream effectors (Fig. 9) (22). Thus, ADP-ribosylation of Ras at
Arg-41 could potentially disrupt eukaryotic signal transduction by
inhibiting the interaction of Ras with downstream effectors. The
ability of ExoS to disrupt eukaryotic signal transduction was
determined by analysis of NGF-stimulated neurite outgrowth in PC-12
cells. NGF-stimulated neurite outgrowth in PC-12 cells follows a
Ras-mediated signal transduction pathway, although there are also
Ras-independent components to the pathway (23). Since ExoS is secreted
from P. aeruginosa via a type III secretion pathway (16),
ExoS was delivered into PC-12 cells by infection with P. aeruginosa. PC-12 cells were incubated alone, with a strain of
P. aeruginosa that produced ExoS (388), or with a strain of
P. aeruginosa that was genetically deleted for
exoS (388
ExoS). After 2 h, PC-12 cells were washed
to remove unbound bacteria and incubated alone or with NGF. At 24 h post-infection, PC-12 cells were examined by phase microscopy. PC-12
cells that were incubated in the absence of bacteria and NGF showed a
rounded morphology, typical of unstimulated cells (Fig.
10). In contrast, PC-12 cells that had
been incubated alone or with a strain of P. aeruginosa that
lacks exoS (388
ExoS) and then incubated with NGF had
undergone a morphological change, where the cells had spread and
exhibited neurite outgrowth. PC-12 cells that had been incubated with
the ExoS-expressing strain of P. aeruginosa followed by
incubation with NGF showed a rounded morphology, similar to the
morphology of non-infected, non-NGF-stimulated cells. These data
indicated that infection of PC-12 cells with an ExoS-producing strain
of P. aeruginosa inhibited NGF-stimulated signal
transduction leading to neurite outgrowth. In other experiments, we
observed that transient expression of ExoS from a cytomegalovirus
promoter also inhibited NGF-mediated neurite outgrowth in PC-12
cells.2 Together, these data
indicated that inhibition of NGF-mediated neurite outgrowth was due to
the ADP-ribosyltransferase activity of ExoS and not a general
cytotoxicity of ExoS or P. aeruginosa for PC-12 cells.

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Fig. 9.
Alignment of members of the family of small
molecular weight GTP-binding proteins with respect to Arg-41 of
Ras. Switch 1 and switch 2 are two domains of Ras that change
conformation with respect to the presence of GDP/GTP. Switch 1 also
represents a site of effector interaction within Ras.
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Fig. 10.
Uncoupling of NGF stimulation of neurite
formation by P. aeruginosa expression of ExoS. PC-12
cells were incubated alone or were infected with P. aeruginosa 388 or 388 ExoS at a multiplicity of infection of
10:1 (bacteria to PC-12 cells) for 3 h at 37 °C. Cells were
washed to remove unbound bacteria and incubated in differentiation
medium containing penicillin/streptomycin alone or containing 0.1 µg/ml NGF. After 24 h, the morphology of the PC-12 cells was
examined by phase microscopy.
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DISCUSSION |
Earlier studies predicted that ExoS ADP-ribosylates eukaryotic
proteins at arginine residues (10, 12). We initially pursued a
biochemical analysis of the site of ADP-ribosylated Ras. However, we
found that, during proteolysis, a considerable amount of radiolabel was
released as ADP-ribose and that the radiolabel that remained peptide-associated was present in several peptides.2 This,
in addition to the observation that ExoS ADP-ribosylated Ras to a
stoichiometry of ~2 (Table I), prompted a molecular approach for the
characterization of the site of ADP-ribosylation. The subsequent
determination that ExoS ADP-ribosylates Ras at multiple sites is
consistent with our inability to identify a specific arginine as the
preferred site of ADP-ribosylation by biochemical approaches.
Characterization of in vitro transcribed/translated forms of
Ras that each possessed one of 12 individual Arg-to Ala substitutions
identified Arg-41 as the preferred site for ADP-ribosylation since the
Arg-41 mutants of Ras were not ADP-ribosylated by low concentrations of
ExoS. Also, examination of the velocities of ADP-ribosylation of Ras by
ExoS showed that Arg-41 was essentially completely ADP-ribosylated
prior to the appearance of the second ADP-ribosylation event, which was
consistent with Arg-41 being the high affinity site of ADP-ribosylation
within Ras.2
Analysis of the single Arg mutants of Ras did not identify a specific
Arg as the second site for ADP-ribosylation since all of the single Arg
mutants of Ras, with the exception of Ras R41A, could be double
ADP-ribosylated. These data indicated that one of several arginine
residues could constitute the second site of ADP-ribosylation within
Ras. Since a third ADP-ribosylation event was not observed, it appeared
that the ADP-ribosylation at one arginine residue excluded a third
ADP-ribosylation event. This suggests that the arginines that are
targeted for the second ADP-ribosylation event are located in close
proximity in the three-dimensional structure of Ras. These data also
showed that the ADP-ribosylation at Arg-41 and that at the second
arginine were independent events and not the result of sequential,
ordered ADP-ribosylation reactions since the Arg-41 mutants could be
ADP-ribosylated at the second arginine residue. There is precedence for
the ADP-ribosylation of proteins at two sites. Aktories and co-workers
(24) recently showed that an endogenous eukaryotic
ADP-ribosyltransferase modifies actin at Arg-95 and Arg-372.
The ability of ExoS to ADP-ribosylate Ras at multiple arginine residues
assists in addressing an earlier report by Coburn et al.
(12) that described the ADP-ribosylation of several members of the Ras
superfamily by ExoS and the subsequent prediction that ExoS
ADP-ribosylated Ras at Arg-123 (13). Our data could be consistent with
Arg-123 being one of the secondary sites of ADP-ribosylation within
Ras.
Examination of the x-ray crystallographic structure of Ras (25)
predicts that Arg-41 lies adjacent to the switch 1 domain, which has
been shown to function in the interactions with downstream effectors,
particularly Raf (22). Alignment of the primary amino acid sequences of
members of the Ras superfamily of small molecular weight GTP-binding
proteins showed that Arg-41 is not a highly conserved residue. Thus, it
appears that ExoS can potentially ADP-ribosylate the Arg-41 homologue
of Ras, Ral, and Rap, but that members of the Rab or Rho family will
not be targeted for ADP-ribosylation by ExoS at the Arg-41 homologue.
However, the observation that ExoS may ADP-ribosylate Ras at a second
arginine residue poses the possibility that other members of the Ras
superfamily that lack the Arg-41 homologue, including Rho and Rab, may
be targeted for ADP-ribosylation by ExoS at an arginine residue that corresponds to the second site of ADP-ribosylation within Ras. The
ADP-ribosylation of other Ras superfamily members by ExoS is currently
under investigation.
Neurite outgrowth in PC-12 cells is a multistep process that is
initiated by NGF binding to the Trk membrane tyrosine kinase. NGF
binding stimulates Trk kinase activity, which results in the phosphorylation of several effector molecules that activate Ras. This
pathway has both Ras-dependent and -independent components. Several studies have suggested that Ras is a dominant element in the
transmission of NGF signals from Trk, including microinjection of
antibodies to Ras, which interferes with the signaling pathway (26), or
introduction of dominant inhibitory Ras alleles into PC-12 cells, which
inhibits NGF-induced neurite outgrowth. In addition, transfection of
constitutively active Ras, in the absence of NGF stimulation, is
sufficient to induce neurite outgrowth (26). The Ras switch 1 domain is
required for this signal transduction process since point mutations
within the switch 1 regions of constitutively active RasV12 interfere
with neurite outgrowth (21). The fact that ExoS preferentially modifies
Ras at a region adjacent to the switch 1 domain and that infection of
PC-12 cells with strains of P. aeruginosa expressing ExoS
results in an inhibition of neurite outgrowth in PC-12 cells is
consistent with the model that ExoS inhibits Ras-mediated signal
transduction in vivo. However, since ExoS has the potential
to modify a wide variety of targets in vitro, it is
conceivable that ExoS can disrupt PC-12 cell signal transduction by the
ADP-ribosylation of components of this signal transduction pathway.
Future studies will focus on whether ExoS disrupts PC-12 cell signal
transduction via modification of other components involved in PC-12
cell signal transduction.