From the Pharmakologisches Institut,
Universität Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany and the § Institut für
Pharmakologie, Freie Universität Berlin, Thielallee 67-73, 14195 Berlin, Germany
Received for publication, August 28, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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Pasteurella multocida toxin (PMT) is
a highly potent mitogen for a variety of cell types. PMT has been shown
to induce various cellular signaling processes, and it has been
suggested to function through the heterotrimeric G-proteins
Gq/G11. To analyze the role of
Gq/G11 in the action of PMT, we have studied
the effect of the toxin in G Pasteurella multocida is a small Gram-negative
coccobacillus present in the nasooropharynx and gastrointestinal
tract of many avian and mammalian species. Infections of P. multocida are associated with atrophic rhinitis in pigs (1) by
local dermatonecrosis, respiratory disease in cattle and rabbits (2),
and dermatonecrosis and bacteremia in humans (3, 4). P. multocida produces a 146-kDa protein that is its major
pathogenic factor (5-7). P. multocida toxin
(PMT)1 has little homology to
other known toxins. It binds to a ganglioside-type cell surface
receptor (8, 9) and is internalized through an endocytotic pathway to
act intracellularly after being processed via an acidic
compartment (10).
PMT has been shown to be a highly potent mitogen for various cell types
including fibroblasts and osteoblastic cells (10, 11). Exposure of
cells to PMT results in tyrosine phosphorylation of various proteins
including focal adhesion kinase and paxillin as well as in actin stress
fiber formation and focal contact assembly (9, 12, 13). Several lines
of evidence suggest that some of these effects are mediated by the
small GTP-binding protein Rho, which plays a major role in actin
cytoskeleton dynamics (14). Disruption of Rho function by the C3
exoenzyme of Clostridium botulinum, which ADP-ribosylates
and thereby inhibits Rho abolishes focal contact formation in response
to PMT, and incubation of endothelial cells with C3 exoenzyme blocks
PMT-induced actin stress fiber formation (12, 13).
PMT has also been shown to induce a robust increase in inositol
phosphate levels, mobilization of intracellularly stored calcium, production of diacyl glycerol, and activation of protein kinase C,
suggesting that it leads to an activation of phospholipase C (10,
15-18). PMT potentiates the production of inositol phosphates induced
by various agonists that function through receptors coupling to
G-proteins of the Gq/11 family, and PMT-induced formation
of inositol phosphates can be inhibited by guanosine
5'-O-( To determine the exact role of Gq/G11 in
various cellular responses of PMT, we have studied the effect of PMT in
G Materials--
Y-27632 was provided by Yoshitomi Pharmaceutical
Industries. The mutated Rho-binding domain of Rho kinase, RB/PH(TT),
was a gift from K. Kaibuchi (Ikoma, Japan). C. botulinum
C3-exoenzyme was a donation from I. Just and K. Aktories (Freiburg,
Germany) or was purchased from Upstate Biotechnology. PMT was purchased from Sigma.
Cell Culture--
Wild-type fibroblasts and fibroblasts lacking
both G-protein Microinjection--
For microinjection studies, cells were
seeded at a density of ~103 cells/mm2 on
glass coverslips imprinted with squares to facilitate the localization
of injected cells and grown overnight. To obtain quiescent and
serum-starved fibroblasts, cultures were rinsed in serum-free DMEM and
incubated in DMEM supplemented with 25% Ham's F-12 medium, 0.2%
NaHCO3, 10 mM Hepes, and 0.1% fetal bovine serum (modified DMEM) for 24 h, followed by a 48-h incubation in
modified DMEM devoid of fetal bovine serum. Plasmids were injected into
the nucleus together with Texas Red dextran (5 mg/ml; Molecular Probes)
to visualize injected cells. C. botulinum C3 exoenzyme was
comicroinjected with the cDNAs at a concentration of 100 µg/ml. About 150 cells/field were injected in each case, using a manual injection system (Eppendorf, Hamburg, Germany).
Visualization of Actin Cytoskeleton--
Microinjected cells
were stimulated with 100 ng/ml PMT overnight, fixed in 4%
paraformaldehyde for 20 min, and permeabilized in 0.2% Triton X-100
for 5 min. To visualize the cytoskeleton, cells were stained for
polymerized actin by incubation with 0.5 µg/ml fluorescein
isothiocyanate-phalloidin (Sigma) for 40 min. The coverslips were
mounted on glass slides and examined using an inverted microscope
(Zeiss Axiovert 100). Quantification of actin stress fibers was
performed as described (24).
Determination of Inositol Phosphate Levels--
Cells were
labeled for 20-24 h with 120 pmol of
myo-[2-3H]inositol (758.5 Gbq/mmole;
PerkinElmer Life Sciences)/well in the absence or presence of PMT. For
determination of receptor-mediated inositol phosphate
production, cells were washed with inositol-free medium and then
incubated for 10 min at 37 °C with 0.25 ml of inositol-free medium
containing 10 mM of LiCl. Thereafter, medium was aspirated,
the indicated agents were added in medium containing 10 mM
LiCl, and cells were incubated for 20 min. Inositol phosphate production was stopped by addition of 0.2 ml of 10 mM
ice-cold formic acid. After keeping the samples on ice for 20 min, 0.45 ml of 10 mM NH4OH was added, and the whole
sample was loaded onto a column containing 0.75 ml of anion exchange
resin (AG 1-X8; Bio-Rad) equilibrated with 5 mM borax and
60 mM sodium formate. Total inositol phosphates were then
separated and measured as described (25). If not stated otherwise,
measurements were done in triplicates.
Determination of Activated Cellular RhoA--
The amount of
activated cellular Rho was determined by precipitation with a fusion
protein consisting of GST and the Rho-binding domain of Rhotekin (amino
acids 7-89; GST-Rho-binding domain) as described (26). Cells were
washed with ice-cold Hank's buffer and lysed in RIPA buffer (50 mM Tris, pH 7.2, 1%, Triton X-100, 0.5% sodium
deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM
MgCl2, 10 µg/ml each of leupeptin and aprotinin, and 1 mM PMSF). Clarified cell lysates were incubated with
GST-Rho-binding domain (20 µg of beads at 4 °C for 45 min. The
beads were washed four times as described (26), and the precipitated
Rho was detected by Western blotting using a monoclonal antibody
against RhoA (Santa Cruz Biotechnology).
Determination of ERK Phosphorylation and c-Jun Kinase
Activity--
For determination of ERK phosphorylation, serum-starved
(48 h 0.5% fetal calf serum, 24 h 0.1% fetal calf serum) cells
grown in 12-well dishes were washed once with phosphate-buffered saline and lysed in Laemmli sample buffer. Cell lysates were separated by
SDS-polyacrylamide gelelectrophoresis, and phosphorylation of ERK was
determined by immunoblotting with an anti-phospho-ERK antiserum (New
England Biolabs). Blots were reprobed with an anti-ERK antiserum (New
England Biolabs).
c-Jun kinase activity was determined in a solid phase assay using
GST-c-Jun as a substrate (27, 28). GST-c-Jun phosphorylated in the
presence of [ For studies on the possible role of Gq/G11
in the cellular effects of PMT, we employed fibroblast cell lines
derived from mouse embryos deficient in either Gq/G
11
double-deficient fibroblasts as well as in fibroblasts lacking only
G
q or G
11. Interestingly, formation of
inositol phosphates in response to PMT was exclusively dependent on
G
q but not on the closely related G
11.
Although G
q/G
11 double-deficient and
G
q-deficient cells did not respond with any production
of inositol phosphates to PMT, PMT was still able to induce various
other cellular effects in these cells, including the activation of Rho,
the Rho-dependent formation of actin stress fibers and
focal adhesions, as well as the stimulation of c-Jun N-terminal kinase
and extracellular signal-regulated kinase. These data show that PMT
leads to a variety of cellular effects that are mediated only in part
by the heterotrimeric G-protein Gq.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thiodiphosphate) (18). It has therefore been
proposed that heterotrimeric G-proteins of the Gq/11 family
may be involved in the action of PMT. The Gq/11 family
contains four members, of which two, Gq and
G11, are expressed in almost all tissues of the mammalian
organism and couple heptahelical receptors in a stimulatory fashion to
-isoforms of phospholipase C (18). Further evidence for a possible role of Gq/G11 in cellular effects of PMT came
from studies in Xenopus oocytes. A PMT-induced
Ca2+-dependent chloride current could be
suppressed by injection of a G
q antisense RNA and an
antiserum recognizing both G
q and G
11
(20). In addition, PMT-induced phosphorylation of ERK-1 was reduced by
expression of a C-terminal peptide of G
q that is
believed to interfere with receptor-Gq interaction
(21).
q/G
11 double-deficient fibroblasts as
well as in fibroblasts lacking only G
q or
G
11. Surprisingly, we found that the formation of inositol phosphates in response to PMT is dependent on
G
q but not on the closely related G
11. In
addition, exposure of cells to PMT induced Rho activation,
Rho-dependent stress fiber formation, and activation of MAP
kinases in a manner independent of
G
q/G
11. These data show that PMT leads to
pleiotropic effects in a G
q-dependent and
-independent manner.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits were derived from embryonic day 10.5 mouse
embryos originating from intercrosses of G
q(
/+) and
G
11(
/+) mice. The generation of G
q and
G
11 mutant mice has been described previously (22, 23).
Fibroblasts lacking G-protein
-subunits were prepared and cultured
as described previously (24).
-32P]ATP was subjected to
SDS-polyacrylamide gelelectrophoresis, and phosphorylation of c-Jun was
determined by autoradiography of dried gels (29). JNK1 and JNK2 were
detected with an anti-JNK-antiserum (Santa Cruz Biotechnologies).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
q or
G
11 or lacking both G-protein
-subunits. The absence
or presence of G
q and G
11 was verified by
immunoblotting (Fig. 1). Treatment of
wild-type mouse fibroblasts with 100 ng/ml PMT for increasing time
periods resulted in a marked and time-dependent
accumulation of inositol phosphates that could be observed 8 h
after addition of the toxin and reached a maximum after about 20 h
(Fig. 2A). In contrast, incubation of G
q/G
11 double-deficient
fibroblasts for various time periods did not result in any increase in
the formation of inositol phosphates (Fig. 2A). Inositol
phosphate production in wild-type cells could be induced with 10 ng/ml
PMT and increased dose-dependently up to a concentration of
1000 ng/ml of the toxin (Fig. 2B). However, even at PMT
concentrations that were maximally effective in wild-type fibroblasts,
no effect on inositol phosphate levels could be observed in
G
q/G
11 double-deficient fibroblasts (Fig.
2B). This indicates that G-proteins of the
Gq/G11-family are indeed required for
PMT-induced inositol phosphate formation.
View larger version (14K):
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Fig. 1.
Expression of
G q and
G
11 in fibroblasts derived from
G
-deficient mouse embryos. Shown is an
immunoblot of cholate extracts from plasma membranes of fibroblasts
derived from wild-type, G
q-, G
11-, and
G
q/G
11-deficient embryos. Shown is an
auto-luminogram of a blot stained with an antiserum recognizing both
G
q and G
11. The position of the
respective G-protein
-subunit is indicated on the
left.
View larger version (17K):
[in a new window]
Fig. 2.
Effect of PMT on inositol phosphate
production in wild-type and
G q/G
11-deficient
embryonic fibroblasts. A, wild-type cells (closed
circles) and G
q/G
11-deficient cells
(open circles) were incubated for the indicated time periods
(abscissa) in the presence of 100 ng/ml PMT. B,
wild-type cells (closed circles) and
G
q/G
11-deficient cells (open
circles) were incubated for 16 h with the indicated
concentrations of PMT. PMT-dependent inositol phosphate
production was determined as described under "Experimental
Procedures." Shown are the mean values of triplicates ± S.D.
The -subunits of Gq and G11 are highly
homologous, and so far no functional differences between
G
q and G
11 either with regard to their
activation through receptors or their regulation of effectors have been
reported. To test whether both Gq and G11 are
involved in PMT-induced formation of inositol phosphates, we tested the
effect of PMT on inositol phosphate production in cells that lack only
G
q or G
11 (Fig.
3). The expression of either Gq or G11 was sufficient to mediate
receptor-dependent phospholipase C activation because
inositol phosphate production could be induced by thrombin and
bradykinin in G
q(
/
) as well as in
G
11(
/
) cells, but not in
G
q/G
11 double-deficient fibroblasts (Fig. 3). However, although G
11-deficient fibroblasts still
responded with an increased inositol phosphate production to PMT,
G
q-deficient cells behaved like
G
q/G
11 double-deficient fibroblasts and
were completely unresponsive, indicating that the effect of PMT was mediated solely by G
q and not by G
11.
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PMT has also been shown to induce a Rho-dependent actin
stress fiber formation and focal adhesion formation in fibroblasts and
endothelial cells (9, 12, 13). There are conflicting data as to the
ability of Gq/G11 to induce the formation of
actin stress fibers (30, 31). However, in some systems
Gq/G11 have been shown to be able to regulate
Rho-dependent processes (32-34). To study the involvement
of Gq/G11 in PMT-induced actin stress fiber
formation and focal adhesion assembly, we tested the effect of the
toxin on the actin cytoskeleton in wild-type and
Gq/G
11 double-deficient fibroblasts (Fig.
4). Actin filaments were visualized by
fluorescein isothiocyanate-labeled phalloidin, and focal adhesions were
stained with an anti-vinculin antibody. In serum-starved fibroblasts
lacking G
q and G
11, PMT induced a
pronounced formation of actin stress fibers and a formation of focal
adhesions indistinguishable from its effect in wild-type cells (Fig.
4). This indicates that the PMT-induced reorganization of the actin
cytoskeleton and focal adhesion assembly occurred independently of the
Gq/G11-mediated signaling pathway.
Preincubation of cells with pertussis toxin did not affect PMT-induced
actin stress fiber formation, indicating that G-proteins of the
Gi family were not involved. Induction of actin stress
fiber formation through various receptors in fibroblasts has been shown
to involve a Rho/Rho kinase-mediated signaling pathway (35-37). The
actin stress fiber formation by PMT in
G
q/G
11 double-deficient fibroblasts could
be blocked by cytosolic injection of C3 exoenzyme of C. botulinum, which ADP-ribosylates Rho at residue Asn41
in its effector domain resulting in the inactivation of Rho (38) (Figs.
4 and 5). Intranuclear injection of an
expression plasmid carrying a dominant negative mutant of Rho kinase
comprising a mutated Rho-binding and adjacent pleckstrin homology
domain of Rho kinase, RB/PH(TT) (39), and preincubation of cells with the Rho kinase inhibitor Y-27632 (40) strongly inhibited PMT-induced actin stress fiber formation (Figs. 4 and 5). These data suggest that
PMT engages a Rho/Rho kinase-mediated signaling pathway to induce actin
stress fiber formation.
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To test whether PMT indeed induces the activation of Rho, we directly
determined Rho activation by precipitation of endogenous GTP-bound Rho
using a fusion protein consisting of glutathione S-transferase and the Rho-binding domain of rhotekin (26).
As shown in Fig. 6 PMT induced a
pronounced activation of Rho that was indistinguishable in wild-type
and Gq/G
11 double-deficient cells,
indicating that PMT-induced Rho activation is independent of the
Gq/G11-dependent signaling pathway.
Similar to the Gq-mediated PMT-induced inositol phosphate
production, the Gq/G11-independent activation
of Rho could only be observed after a lag period of several hours
following exposure of cells to PMT (data not shown).
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Various Gq/G11-coupled receptors have been
demonstrated to mediate the activation of JNK and ERK (41-46), and
constitutively active mutants of Gq-family members have
been shown to stimulate JNK activity in some cellular systems (47, 48).
In addition, PMT has been shown to induce stimulation of ERK, an effect
that could be inhibited by a dominant negative Gq mutant
(21). To delineate the role of G
q/G
11 in
PMT-induced MAP kinase activation, we compared its effect on JNK and
ERK activity in wild-type and G
q/G
11
double-deficient fibroblasts (Fig. 7).
PMT induced activation of JNK as well as of ERK in both wild-type and
G
q/G
11 double-deficient fibroblasts,
demonstrating that PMT leads to JNK and ERK activation in a manner
independent of Gq.
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DISCUSSION |
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PMT has been shown to induce a variety of cellular effects. The precise molecular mechanism by which PMT acts is, however, still poorly defined. The toxin has only moderate homology to other proteins, and so far no enzymatic activity has been detected. Similar to many other toxins, PMT requires internalization and intracellular processing to exhibit cellular effects. This results in a lag period of a few hours between exposure of PMT to intact cells and the occurrence of cellular changes (16).
It has been suggested that G-proteins of the Gq/11 family
are involved in the cellular action of PMT. The two main members of
this family, Gq and G11, are structurally and
functionally highly homologous and couple receptors in a stimulatory
fashion to -isoforms of phospholipase C (19, 49). An antiserum
recognizing the
-subunits of both G
q and
G
11 blocks a PMT-induced
Ca2+-dependent Cl
current in
Xenopus oocytes, which involves PLC-
(20). Stimulation of
this current by PMT could also be inhibited by the injection of
G
q antisense RNA, whereas sense RNA potentiated the
effect of PMT (20). These data were obtained after injection of PMT into oocytes, which results in a rapid response within seconds after
injection. This, however, is a situation completely different from the
action of the toxin on intact cells that requires internalization and
processing of PMT. In a recent study using HEK-293 cells, it was shown
that PMT-induced activation of Erk-1 can be reduced by about 70-80%
upon expression of a C-terminal fragment of G
q that is
supposed to act in a dominant negative fashion (21). Although these
studies support an involvement of Gq/G11 in
some of the cellular effects of PMT, the evidence provided remains indirect.
To study the role of G-proteins of the Gq/G11
family in the cellular action of PMT, we used fibroblast cell lines
derived from mouse embryos that are deficient in
Gq/G
11. In wild-type embryonic
fibroblasts PMT induced a robust time- and dose-dependent increase in the production of inositol phosphates that could not be
observed in fibroblasts lacking both G
q and
G
11 (Figs. 2 and 3). Embryonic fibroblasts lacking only
G
q did not respond to PMT with inositol phosphate
production, whereas PMT lead to a strong response in
G
11-deficient cells, indicating that PMT-induced inositol phosphate production is mediated by Gq and not by
G11. This is surprising because evidence collected from
biochemical, pharmacological, and somatic cell genetic studies
suggested that G
q and G
11 have very
similar, if not identical, characteristics. G
q and
G
11 couple to the same set of seven transmembrane
receptors with the same effector specificity for phospholipase C-
isoforms (50-54).
Although our results in Gq/G
11-deficient
cells clearly show that the Gq/PLC-
pathway plays an
important role in the action of PMT, it has been suggested that PMT can
also act independently of PLC-mediated Ca2+ mobilization
and protein kinase C activation (12). We therefore tested whether PMT
can still induce other cellular effects in the absence of
Gq-dependent signaling. Exposure of
G
q/G
11-deficient embryonic fibroblasts to
PMT resulted in actin stress fiber formation and focal adhesion
assembly (Figs. 4 and 5). Actin stress fiber formation was inhibited by
C3 exoenzyme of C. botulinum as well as by a dominant
negative form of Rho kinase and the Rho kinase inhibitor Y-27632. This
indicates that a Rho/Rho kinase mediated but
Gq/PLC-
-independent pathway is involved in this cellular response to PMT. A Rho/Rho kinase-mediated pathway resulting in the
inhibition of myosin phosphatase and subsequent increase in myosin
light chain phosphorylation has recently been proposed to underlie
PMT-induced reorganization of the actin cytoskeleton in endothelial
cells (13). Actin rearrangement induced by PMT in endothelial cells
could be completely blocked by an inhibitor of the
Ca2+/calmodulin-regulated myosin light chain kinase,
suggesting that dual regulation of myosin light chain phosphorylation
through Ca2+-dependent myosin light chain
kinase activation and Rho/Rho kinase-mediated myosin phosphatase
inhibition is involved in the effect of PMT on the actin cytoskeleton.
Our data, however, suggest that the inhibition of myosin phosphatase
through Rho/Rho kinase is sufficient to induce a rearrangement of the
actin cytoskeleton in embryonic fibroblasts because it can be observed
in the absence of a Gq-mediated inositol phosphate
production and subsequent Ca2+ mobilization.
The involvement of Rho in PMT-induced cellular effects could be
directly demonstrated by precipitation of active Rho from cell lysates
of PMT-exposed wild-type and
Gq/G
11-deficient embryonic fibroblasts.
Thus, activation of Rho by PMT occurs independently of
G
q/G
11. The N-terminal half of cytotoxic
necrotizing factor (CNF) 1 and 2 from Escherichia coli show
moderate homology with N-terminal regions of PMT, and CNF1 has been
shown to inhibit the GTPase activity of RhoA by deamidation of
glutamine residue 63 resulting in constitutive activation of Rho (55,
56). However, the catalytic activity of CNF1 appears to reside in the
C-terminal part of the toxin (57, 58), and it is unlikely that PMT
functions analogous to CNF1 (12, 13, 20). PMT may act upstream of Rho
by regulating the activity of a guanine nucleotide exchange factor or a
GTPase-activating protein specific for Rho. G-proteins of the
G12 family that have been shown to be able to mediate Rho activation (30, 59, 60) are not involved because PMT-induced Rho
activation could also observed in fibroblasts lacking
G
12/G
13 (data not shown).
Various MAP kinases including JNK and ERK have been shown to be
regulated through Gq/G11-coupled receptors, and
PMT has been reported to activate ERK via a pathway involving the
epidermal growth factor receptor (21). Incubation of wild-type
fibroblasts with PMT resulted in a clear JNK and ERK activation. This
effect of PMT obviously did not involve Gq/G11
because Gq/G
11-deficient cells also
responded with activation of JNK and ERK after exposure to PMT to a
comparable extent as did wild-type cells (Fig. 7). It has previously
been suggested that PMT-induced ERK activation is mediated by
Gq/G11 (21). Our data demonstrate that
Gq/G11 are not required for JNK and ERK
activation by PMT in embryonic fibroblasts. However, we cannot exclude
the possibility that Gq contributes to PMT-induced MAP
kinase activation in wild-type cells.
In summary, we show that PMT induces a remarkable array of cellular
effects including the activation of phospholipase C, which is entirely
dependent on Gq but not on the closely related G-protein G11. However, the pleiotropic actions of PMT are only in
part mediated by Gq. Activation of Rho and MAP kinases can
be induced by PMT in a Gq/G11-independent
manner, suggesting that other, possibly G-protein independent processes
are involved in the cellular action of PMT.
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ACKNOWLEDGEMENTS |
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We thank Ursula Brandt and Birgit Klages for expert technical assistance and Dr. Andree Blaukat for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.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.
¶ Present address: Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
To whom correspondence should be addressed: Pharmakologisches
Institut, Universität Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany. Tel.: 49-6221-548246; Fax: 49-6221-548549; E-mail: Stefan.Offermanns@urz.uni-heidelberg.de.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M007819200
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ABBREVIATIONS |
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The abbreviations used are: PMT, P. multocida toxin; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; CNF, cytotoxic necrotizing factor.
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