Pleiotropic Effects of Pasteurella multocida Toxin Are Mediated by Gq-dependent and -independent Mechanisms

INVOLVEMENT OF Gq BUT NOT G11*

Alexandra ZywietzDagger §, Antje Gohla§, Milena Schmelz§, Günter Schultz§, and Stefan OffermannsDagger §||

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Galpha q/Galpha 11 double-deficient fibroblasts as well as in fibroblasts lacking only Galpha q or Galpha 11. Interestingly, formation of inositol phosphates in response to PMT was exclusively dependent on Galpha q but not on the closely related Galpha 11. Although Galpha q/Galpha 11 double-deficient and Galpha 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

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-(beta -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 beta -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 Galpha q antisense RNA and an antiserum recognizing both Galpha q and Galpha 11 (20). In addition, PMT-induced phosphorylation of ERK-1 was reduced by expression of a C-terminal peptide of Galpha q that is believed to interfere with receptor-Gq interaction (21).

To determine the exact role of Gq/G11 in various cellular responses of PMT, we have studied the effect of PMT in Galpha q/Galpha 11 double-deficient fibroblasts as well as in fibroblasts lacking only Galpha q or Galpha 11. Surprisingly, we found that the formation of inositol phosphates in response to PMT is dependent on Galpha q but not on the closely related Galpha 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 Galpha q/Galpha 11. These data show that PMT leads to pleiotropic effects in a Galpha q-dependent and -independent manner.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -subunits were derived from embryonic day 10.5 mouse embryos originating from intercrosses of Galpha q(-/+) and Galpha 11(-/+) mice. The generation of Galpha q and Galpha 11 mutant mice has been described previously (22, 23). Fibroblasts lacking G-protein alpha -subunits were prepared and cultured as described previously (24).

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 [gamma -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

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 Galpha q or Galpha 11 or lacking both G-protein alpha -subunits. The absence or presence of Galpha q and Galpha 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 Galpha q/Galpha 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 Galpha q/Galpha 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.



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Fig. 1.   Expression of Galpha q and Galpha 11 in fibroblasts derived from Galpha -deficient mouse embryos. Shown is an immunoblot of cholate extracts from plasma membranes of fibroblasts derived from wild-type, Galpha q-, Galpha 11-, and Galpha q/Galpha 11-deficient embryos. Shown is an auto-luminogram of a blot stained with an antiserum recognizing both Galpha q and Galpha 11. The position of the respective G-protein alpha -subunit is indicated on the left.



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Fig. 2.   Effect of PMT on inositol phosphate production in wild-type and Galpha q/Galpha 11-deficient embryonic fibroblasts. A, wild-type cells (closed circles) and Galpha q/Galpha 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 Galpha q/Galpha 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 alpha -subunits of Gq and G11 are highly homologous, and so far no functional differences between Galpha q and Galpha 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 Galpha q or Galpha 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 Galpha q(-/-) as well as in Galpha 11(-/-) cells, but not in Galpha q/Galpha 11 double-deficient fibroblasts (Fig. 3). However, although Galpha 11-deficient fibroblasts still responded with an increased inositol phosphate production to PMT, Galpha q-deficient cells behaved like Galpha q/Galpha 11 double-deficient fibroblasts and were completely unresponsive, indicating that the effect of PMT was mediated solely by Galpha q and not by Galpha 11.



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Fig. 3.   Effect of PMT and thrombin/bradykinin on inositol phosphate production in embryonic fibroblasts lacking Galpha q and/or Galpha 11. Wild-type cells, Galpha q, Galpha 11, or Galpha q/Galpha 11 double-deficient cells were incubated for 20 h in the absence or presence of 100 ng/ml PMT (upper panel) or for 20 min with a mixture of 5 µM bradykinin and 5 units/ml thrombin (lower panel). PMT-dependent inositol phosphate production was determined as described under "Experimental Procedures." Shown are the mean values of triplicates ± S.D.

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 Galpha q/Galpha 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 Galpha q and Galpha 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 Galpha q/Galpha 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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Fig. 4.   PMT-induced actin stress fiber formation and focal adhesion assembly in wild-type and Galpha q/Galpha 11-deficient embryonic fibroblasts. Wild-type cells (A, B, E, and F) or Galpha q/Galpha 11-double-deficient cells (C, D, and G-L) were incubated in the absence (A, C, E, and G; c, control) or presence of 100 ng/ml PMT (B, D, F, and H-L) for 16 h. Thereafter, cells were fixed and stained with fluorescein isothiocyanate-labeled phalloidin (A-D and I-L) or with an anti-vinculin antibody (E-H). Cells were treated with pertussis toxin (I, 100 ng/ml for 16 h) or with Y-27632 (L, 10 µM for 30-60 min before fixation). J and K, cells were injected cytoplasmatically with 100 µg/ml C3-exoenzyme (J) or intranuclearly with an expression plasmid carrying a dominant negative Rho kinase (K) shortly before exposure to PMT was started. To detect injected cells, cells were coinjected with Texas Red-labeled dextran. Injected cells are marked with arrows.



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Fig. 5.   Involvement of Rho and Rho kinase in PMT-induced actin stress fiber formation. Galpha q/Galpha 11-deficient embryonic fibroblasts were injected cytoplasmatically with 100 µg/ml C3-exoenzyme (C3) or intranuclearly with an expression plasmid carrying a dominant negative Rho kinase (dnROCK) or were treated with pertussis toxin (100 ng/ml for 16 h; PTX) or with Y-27632 (10 µM for 12 h before fixation). Injected cells were detected by coinjection of a fluorescent dye. After exposure to 100 ng/ml of PMT for 16 h, cells were fixed, and actin stress fibers were visualized with fluorescein isothiocyanate-phalloidin. Shown is the percentage of stress fiber positive cells. In each case, at least 120 cells were analyzed.

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 Galpha q/Galpha 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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Fig. 6.   Activation of Rho by PMT in wild-type and Galpha q/Galpha 11-deficient embryonic fibroblasts. Wild-type and Galpha q/Galpha 11-deficient cells were incubated for 20 h without and with 100 ng/ml PMT (-/+). Thereafter, cells were lysed and the amount of activated Rho was determined as described under "Experimental Procedures."

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 Galpha q mutant (21). To delineate the role of Galpha q/Galpha 11 in PMT-induced MAP kinase activation, we compared its effect on JNK and ERK activity in wild-type and Galpha q/Galpha 11 double-deficient fibroblasts (Fig. 7). PMT induced activation of JNK as well as of ERK in both wild-type and Galpha q/Galpha 11 double-deficient fibroblasts, demonstrating that PMT leads to JNK and ERK activation in a manner independent of Gq.



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Fig. 7.   Effect of PMT on JNK and ERK activity in wild-type and Galpha q/Galpha 11-deficient embryonic fibroblasts. Wild-type and Galpha q/Galpha 11-deficient cells were incubated for 20 h without and with 100 ng/ml PMT (-/+). Thereafter, cells were lysed, and the activity state of ERK (A) and JNK (B) was determined as described under "Experimental Procedures." A, shown is an immunoblot developed with an anti-phospho-ERK antibody (p-ERK; lower panel) as well as the same blot reprobed with an anti-ERK antibody (ERK; upper panel). Both antisera recognize the ERK1 (44 kDa) and ERK2 (42 kDa) isoform. B shows the result of an in vitro kinase assay using GST-c-Jun as a substrate (lower panel) as well as the amount of JNK in the employed lysates as detected with an anti-JNK antibody (upper panel). The anti-JNK antibody recognizes two JNK isoforms, JNK1 (46 kDa) and JNK2 (54 kDa).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -isoforms of phospholipase C (19, 49). An antiserum recognizing the alpha -subunits of both Galpha q and Galpha 11 blocks a PMT-induced Ca2+-dependent Cl- current in Xenopus oocytes, which involves PLC-beta (20). Stimulation of this current by PMT could also be inhibited by the injection of Galpha 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 Galpha 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 Galpha q/Galpha 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 Galpha q and Galpha 11 (Figs. 2 and 3). Embryonic fibroblasts lacking only Galpha q did not respond to PMT with inositol phosphate production, whereas PMT lead to a strong response in Galpha 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 Galpha q and Galpha 11 have very similar, if not identical, characteristics. Galpha q and Galpha 11 couple to the same set of seven transmembrane receptors with the same effector specificity for phospholipase C-beta isoforms (50-54).

Although our results in Galpha q/Galpha 11-deficient cells clearly show that the Gq/PLC-beta 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 Galpha q/Galpha 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-beta -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 Galpha q/Galpha 11-deficient embryonic fibroblasts. Thus, activation of Rho by PMT occurs independently of Galpha q/Galpha 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 Galpha 12/Galpha 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 Galpha q/Galpha 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.


    ACKNOWLEDGEMENTS

We thank Ursula Brandt and Birgit Klages for expert technical assistance and Dr. Andree Blaukat for critical reading of the manuscript.


    FOOTNOTES

* 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Ackermann, M. R., DeBey, M. C., Register, K. B., Larson, D. J., and Kinyon, J. M. (1994) J. Vet. Diagn. Invest. 6, 375-377[Medline] [Order article via Infotrieve]
2. Bisgaard, M. (1993) Zentralbl. Bakteriol. 279, 7-26[Medline] [Order article via Infotrieve]
3. Garcia, V. F. (1997) Pediatr. Rev. 18, 127-130[Free Full Text]
4. Morris, J. T., and McAllister, C. K. (1992) South Med. J. 85, 442-443[Medline] [Order article via Infotrieve]
5. Buys, W. E., Smith, H. E., Kamps, A. M., Kamp, E. M., and Smits, M. A. (1990) Nucleic Acids Res. 11, 2815-28166[Abstract]
6. Lax, A. J., and Chanter, N. J. (1990) Gen. Microbiol. 136, 81-87
7. Petersen, S. K. (1990) Mol. Microbiol. 4, 821-830[Medline] [Order article via Infotrieve]
8. Pettit, R. K., Ackermann, M. R., and Rimler, R. B. (1993) Lab. Invest. 69, 94-100[Medline] [Order article via Infotrieve]
9. Dudet, L. I., Chailler, P., Dubreuil, J. D., and Martineau-Doize, B. (1996) J. Cell. Physiol. 168, 173-182[CrossRef][Medline] [Order article via Infotrieve]
10. Rozengurt, E., Higgins, T., Chanter, N., Lax, A. J., and Staddon, J. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 123-127[Abstract]
11. Mullan, P. B., and Lax, A. J. (1998) Calcif. Tissue Int. 63, 340-345[CrossRef][Medline] [Order article via Infotrieve]
12. Lacerda, H. M., Lax, A. J., and Rozengurt, E. (1996) J. Biol. Chem. 271, 439-445[Abstract/Free Full Text]
13. Essler, M., Hermann, K., Amano, M., Kaibuchi, K., Heesemann, J., Weber, P. C., and Aepfelbacher, M. (1998) J. Immunol. 161, 5640-5646[Abstract/Free Full Text]
14. Hall, A. (1998) Science 279, 509-514[Abstract/Free Full Text]
15. Staddon, J. M., Chanter, N., Lax, A. J., Higgins, T. E., and Rozengurt, E. (1990) J. Biol. Chem. 265, 11841-11848[Abstract/Free Full Text]
16. Staddon, J. M., Barker, C. J., Murphy, A. C., Chanter, N., Lax, A. J., Michell, R. H., and Rozengurt, E. (1991) J. Biol. Chem. 266, 4840-4847[Abstract/Free Full Text]
17. Higgins, T. E., Murphy, A. C., Staddon, J. M., Lax, A. J., and Rozengurt, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4240-4244[Abstract]
18. Murphy, A. C., and Rozengurt, E. (1992) J. Biol. Chem. 267, 25296-25303[Abstract/Free Full Text]
19. Exton, J. H. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 481-509[CrossRef][Medline] [Order article via Infotrieve]
20. Wilson, B. A., Zhu, X., Ho, M., and Lu, L. (1997) J. Biol. Chem. 272, 1268-1275[Abstract/Free Full Text]
21. Seo, B., Choy, E. W., Maudsley, S., Miller, W. E., Wilson, B. A., and Luttrell, L. M. (2000) J. Biol. Chem. 275, 2239-2245[Abstract/Free Full Text]
22. Offermanns, S., Hashimoto, K., Watanabe, M., Sun, W., Kurihara, H., Thompson, R. F., Inoue, Y., Kano, M., and Simon, M. I. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14089-14094[Abstract/Free Full Text]
23. Offermanns, S., Zhao, L. P., Gohla, A., Sarosi, I., Simon, M. I., and Wilkie, T. M. (1998) EMBO J. 17, 4304-4312[Abstract/Free Full Text]
24. Gohla, A., Offermanns, S., Wilkie, T. M., and Schultz, G. (1999) J. Biol. Chem. 274, 17901-17907[Abstract/Free Full Text]
25. Offermanns, S., and Simon, M. I. (1995) J. Biol. Chem. 270, 15175-15180[Abstract/Free Full Text]
26. Ren, X. D., Kiosses, W. B., and Schwartz, M. A. (1999) EMBO J. 18, 578-585[Abstract/Free Full Text]
27. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, 2135-2148[Abstract]
28. Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., Johnson, G. L., and Karin, M. (1994) Science 266, 1719-1723[Medline] [Order article via Infotrieve]
29. Blaukat, A., Ivankovic-Dikic, I., Gronroos, E., Dolfi, F., Tokiwa, G., Vuori, K., and Dikic, I. (1999) J. Biol. Chem. 274, 14893-14901[Abstract/Free Full Text]
30. Buhl, A. M., Johnson, N. L., Dhanasekaran, N., and Johnson, G. L. (1995) J. Biol. Chem. 270, 24631-24634[Abstract/Free Full Text]
31. Kjoller, L., and Hall, A. (1999) Exp. Cell Res. 253, 166-179[CrossRef][Medline] [Order article via Infotrieve]
32. Sah, V. P., Hoshijima, M., Chien, K. R., and Brown, J. H. (1996) J. Biol. Chem. 271, 31185-31190[Abstract/Free Full Text]
33. Katoh, H., Aoki, J., Yamaguchi, Y., Kitano, Y., Ichikawa, A., and Negishi, M. (1998) J. Biol. Chem. 273, 28700-28707[Abstract/Free Full Text]
34. Hirshman, C. A., and Emala, C. W. (1999) Am. J. Physiol. 277, L653-L661[Abstract/Free Full Text]
35. Leung, T., Chen, X. Q., Manser, E., and Lim, L. (1996) Mol. Cell. Biol. 16, 5313-5327[Abstract]
36. Chihara, K., Amano, M., Nakamura, N., Yano, T., Shibata, M., Tokui, T., Ichikawa, H., Ikebe, R., Ikebe, M., and Kaibuchi, K. (1997) J. Biol. Chem. 272, 25121-25127[Abstract/Free Full Text]
37. Narumiya, S., Ishizaki, T., and Watanabe, N. (1997) FEBS Lett. 410, 68-72[CrossRef][Medline] [Order article via Infotrieve]
38. Aktories, K., Mohr, C., and Koch, G. (1992) Curr. Top. Microbiol. Immunol. 175, 115-131[Medline] [Order article via Infotrieve]
39. Amano, M., Chihara, K., Nakamura, N., Fukata, Y., Yano, T., Shibata, M., Ikebe, M., and Kaibuchi, K. (1998) Genes Cells. 3, 177-188[Abstract/Free Full Text]
40. Ishizaki, T., Uehata, M., Tamechika, I., Keel, J., Nonomura, K., Maekawa, M., and Narumiya, S. (2000) Mol. Pharmacol. 57, 976-983[Abstract/Free Full Text]
41. Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S. (1994) Nature 369, 418-420[CrossRef][Medline] [Order article via Infotrieve]
42. Coso, O. A., Chiariello, M., Kalinec, G., Kyriakis, J. M., Woodgett, J., and Gutkind, J. S. (1995) J. Biol. Chem. 270, 5620-5624[Abstract/Free Full Text]
43. Hawes, B. E., van Biesen, T., Koch, W. J., Luttrell, L. M., and Lefkowitz, R. J. (1995) J. Biol. Chem. 270, 17148-17153[Abstract/Free Full Text]
44. Zohn, I. E., Yu, H., Li, X., Cox, A. D., and Earp, H. S. (1995) Mol. Cell. Biol. 15, 6160-6168[Abstract]
45. Ramirez, M. T., Sah, V. P., Zhao, X. L., Hunter, J. J., Chien, K. R., and Brown, J. H. (1997) J. Biol. Chem. 272, 14057-14061[Abstract/Free Full Text]
46. Bogoyevitch, M. A., Ketterman, A. J., and Sugden, P. H. (1995) J. Biol. Chem. 270, 29710-29717[Abstract/Free Full Text]
47. Prasad, M. V., Dermott, J. M., Heasley, L. E., Johnson, G. L., and Dhanasekaran, N. (1995) J. Biol. Chem. 270, 18655-18659[Abstract/Free Full Text]
48. Heasley, L. E., Storey, B., Fanger, G. R., Butterfield, L., Zamarripa, J., Blumberg, D., and Maue, R. A. (1996) Mol. Cell. Biol. 16, 648-656[Abstract]
49. Strathmann, M., and Simon, M. I. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9113-9117[Abstract]
50. Wange, R. L., Smrcka, A. V., Sternweis, P. C., and Exton, J. H. (1991) J. Biol. Chem. 266, 11409-11412[Abstract/Free Full Text]
51. Blank, J. L., Ross, A. H., and Exton, J. H. (1991) J. Biol. Chem. 266, 18206-18216[Abstract/Free Full Text]
52. Wu, D., Katz, A., Lee, C.-H., and Simon, M. I. (1992) J. Biol. Chem. 267, 25798-25802[Abstract/Free Full Text]
53. Hepler, J. R., Kozasa, T., Smrcka, A. V., Simon, M. I., Rhee, S. G., Sternweis, P. C., and Gilman, A. G. (1993) J. Biol. Chem. 268, 14367-14375[Abstract/Free Full Text]
54. Offermanns, S., Heiler, E., Spicher, K., and Schultz, G. (1994) FEBS Lett. 349, 201-204[CrossRef][Medline] [Order article via Infotrieve]
55. Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., and Aktories, K. (1997) Nature 387, 725-729[CrossRef][Medline] [Order article via Infotrieve]
56. Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C., and Boquet, P. (1997) Nature 387, 729-733[CrossRef][Medline] [Order article via Infotrieve]
57. Schmidt, G., Selzer, J., Lerm, M., and Aktories, K. (1998) J. Biol. Chem. 273, 13669-13674[Abstract/Free Full Text]
58. Boquet, P. (1999) Ann. N. Y. Acad. Sci. 886, 83-90[Abstract/Free Full Text]
59. Gohla, A., Harhammer, R., and Schultz, G. (1998) J. Biol. Chem. 273, 4653-4659[Abstract/Free Full Text]
60. Seasholtz, T. M., Majumdar, M., and Brown, J. H. (1999) Mol. Pharmacol. 55, 949-956[Free Full Text]


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