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J Biol Chem, Vol. 275, Issue 3, 2239-2245, January 21, 2000


Pasteurella multocida Toxin Stimulates Mitogen-activated Protein Kinase via Gq/11-dependent Transactivation of the Epidermal Growth Factor Receptor*

Benjamin SeoDagger , Eric W. ChoyDagger , Stuart MaudsleyDagger , William E. MillerDagger , Brenda A. Wilson§, and Louis M. LuttrellDagger

From the Dagger  Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 and the § Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving Gq/11 family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha -thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of Gq/11 signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha q carboxyl-terminal peptide, Galpha q-(305-359). Like alpha -thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs Gq/11 family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pasteurella multocida is a wide ranging bacterium found in the respiratory tracts of over 60 avian and 40 mammalian species (1). It is a significant veterinary, and occasional human, pathogen and the cause of swine atrophic rhinitis, a condition characterized by osteoclastic bone resorption and severe progressive turbinate damage. The pathogenicity of P. multocida is related to the production of a 146-kDa toxin (2, 3) that exhibits little functional homology to other known toxins or proteins. Both native and recombinant PMT1 are potently mitogenic for several cell types in vitro (4). In Rat1 fibroblasts, rPMT produces anchorage-independent DNA synthesis and growth in soft agar (5). In primary osteoblastic cells in culture, rPMT induces cell proliferation and down-regulation of markers of osteoblast differentiation (6).

The mechanisms whereby rPMT exerts its mitogenic effects are poorly understood. The toxin interacts with a ganglioside-type cell surface receptor and is internalized via both coated and noncoated endocytic structures (7, 8). The mitogenic effects of rPMT require its internalization, since exposure of cells to rPMT at 4 °C or incubation with the weak base methylamine prevents the response (4). Exposure to rPMT stimulates the hydrolysis of inositol phospholipids, calcium mobilization, and phosphorylation of protein kinase C (PKC) substrates, including the myristoylated alanine-rich C kinase substrate protein (4, 5, 9). In addition, rPMT has been shown to stimulate tyrosine phosphorylation of several proteins, including the focal adhesion kinase p125FAK and paxillin, and to promote both focal adhesion assembly and the formation of actin stress fibers (8, 10).

Several lines of evidence suggest that rPMT exerts at least some its effects by modulating the activity of Gq/11 family heterotrimeric G proteins. Treatment of Swiss 3T3 cells with low doses of rPMT strongly potentiates inositol phosphate production following stimulation of Gq/11-coupled receptors, and PMT-induced phosphatidylinositol hydrolysis in permeabilized cells is blocked by guanosine 5'-O-(beta -thiodiphosphate) (11). In Xenopus oocytes, rPMT-induced calcium-dependent chloride currents are blocked by the injection of specific antisera against PLCbeta 1 or the alpha  subunit of Gq/11 and by Galpha q antisense RNA, and are dramatically enhanced by overexpression of Galpha q (12).

Recently, many heterotrimeric G protein-coupled receptors (GPCRs) have been shown to activate the Erk mitogen-activated protein kinase pathway. The mechanisms whereby these signals are transduced are characterized by significant heterogeneity. Depending upon cell type, GPCRs have been shown to mediate both Ras-independent Erk activation via stimulation of PKC isoforms, and Ras-dependent Erk activation via activation of receptor and nonreceptor tyrosine protein kinases (13, 14). Since rPMT is thought to mediate many of its effect via the regulation of heterotrimeric G proteins, understanding the mechanisms of PMT-induced mitogenesis might enhance our understanding of the roles of heterotrimeric G proteins in the regulation of cell growth. In this study, we have compared the mechanism of rPMT-mediated Erk activation to that employed by endogenous GPCRs in HEK-293 cells. We find that rPMT activates the Erk cascade via the stimulation of Gq/11 family G proteins. Subsequently, both rPMT and endogenous Gq/11-coupled alpha -thrombin receptors induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the EGF receptor. Thus, rPMT represents a novel bacterial strategy for inducing cell proliferation, by usurping heterotrimeric G protein-regulated mitogenic signals.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Recombinant P. multocida toxin (rPMT) expressed in Escherichia coli was prepared as described previously (15). Bis-indolylmaleimide I (GF109203X), EGF, and tyrphostin AG1478 were from Calbiochem. Bordetella pertussis toxin was from List Biologicals. Lysophosphatidic acid (LPA) and phorbol myristate acetate (PMA) were from Sigma. myo-[2-3H]Inositol was from NEN Life Science Products. The thrombin agonist hexapeptide H3N+-serine-phenylalanine-leucine-leucine-arginine-asparagine-COOH (SFLLRN) was synthesized at the Howard Hughes Medical Institute peptide facility (Duke University Medical Center, Durham, NC).

Eukaryotic plasmids for the expression of the cDNAs encoding Galpha q(Q209L), Galpha q-(305-359), GRK2(K220R), and mSos1(1071-1336) were prepared in our laboratory. The expression plasmid encoding HA epitope-tagged Erk 1 was provided by J. Pouyssegur (16). The expression plasmid encoding Ha-Ras(N17) was provided by D. Altschuler.

Cell Culture and Transient Transfection-- HEK-293 cells were from the American Type Culture Collection. HEK-293 cells were maintained in minimum essential medium with Earle's salts (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 50 µg/ml gentamicin. Transient transfections were performed using a modified calcium phosphate precipitation method as described previously (17). Monolayers of transfected cells were incubated in serum-free minimum essential medium supplemented with 10 mM HEPES, pH 7.4, 0.1% bovine serum albumin, and gentamicin for 16-20 h prior to stimulation.

Expression of transiently expressed mutants of GRK2, Galpha q, mSos1, and Ha-Ras was confirmed by protein immunoblotting of whole cell lysates. GRK2(K220R) was detected using a 1:3000 dilution of rabbit polyclonal anti-GRK2 (18), mSos1(1071-1336) was detected using a 1:1000 dilution of rabbit polyclonal anti-mSos1,2 (Transduction Laboratories), and Galpha q-(305-359) was detected using a 1:1000 dilution of rabbit polyclonal anti-Galpha q carboxyl terminus (Santa Cruz Biotechnology), each with horseradish peroxidase-conjugated anti-rabbit IgG (Jackson Laboratories) as secondary antibody. N17Ras was detected using a 1:500 dilution of monoclonal anti-Ras (Transduction Laboratories), with horseradish peroxidase-conjugated anti-mouse IgG (Jackson Laboratories) as secondary antibody. Proteins on nitrocellulose were visualized by enzyme-linked chemiluminescence (Amersham Pharmacia Biotech).

Inositol Phosphate Production-- HEK-293 cells on six-well plates were labeled overnight with 2 µCi/ml myo-[2-3H]inositol, washed once with phosphate-buffered saline (PBS), and placed in PBS supplemented with 10 mM LiCl and 1 mM CaCl2. Cells pretreated with rPMT were incubated for 45 min in Li+/Ca2+ PBS prior to lysis, while cells treated with agonist were preincubated for 15 min with Li+/Ca2+ followed by the addition of agonist for 30 min prior to lysis. Cells were lysed by the addition of 1.0 ml of 0.4 M perchloric acid, and a 0.8-ml aliquot of the resulting cell lysate was neutralized with 0.4 ml of 0.72 M KOH, 0.6 M KHCO3. Total inositol phosphates were determined by Dowex anion exchange chromatography as described previously (19).

Erk 1/2 Phosphorylation-- Treatment with rPMT or receptor agonist drugs was performed at 37 °C in serum-free medium following preincubation with inhibitors, as described in the figure legends. Erk 1/2 activation was assessed by measuring the phosphorylation state of endogenously expressed Erk 1/2 or of HA epitope-tagged Erk 1 immunoprecipitated from transiently transfected cells.

For determination of endogenous Erk 1/2 phosphorylation, cell monolayers on 12-well plates were washed once with ice-cold PBS and lysed in 200 µl/well Laemmli sample buffer. Approximately 15 µg of whole cell lysate protein/lane was resolved by SDS-polyacrylamide gel electrophoresis. Erk 1/2 phosphorylation was detected by protein immunoblotting using a 1:1000 dilution of rabbit polyclonal phosphospecific MAP kinase IgG (New England Biolabs) with horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham Pharmacia Biotech) as secondary antibody. Quantitation of Erk 1/2 phosphorylation was performed using scanning laser densitometry. After quantitation of Erk 1/2 phosphorylation, nitrocellulose membranes were stripped of immunoglobulin and reprobed using rabbit polyclonal anti-Erk1/2 IgG (Santa Cruz Biotechnology) to confirm equal loading of Erk2 protein.

In other experiments, transiently expressed HA epitope-tagged Erk 1 was immunoprecipitated from cells coexpressing putative dominant inhibitory mutant proteins. Following stimulation, monolayers on six-well plates were washed once with PBS and lysed in 1 ml of ice-cold immunoprecipitation precipitation buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 0.25% w/v sodium deoxycholate, 0.1% v/v Nonidet P-40, 1 mM NaF, 1 mM sodium pyrophosphate, 100 µM NaVO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Cell lysates were clarified by centrifugation and immunoprecipitation of HA-Erk1 was performed using 20 µl of HA.11 affinity matrix (Berkeley Antibody Co.) with 2 h agitation at 4 °C. Immune complexes were washed twice with ice-cold immunoprecipitation precipitation buffer and once with PBS, denatured in 2× Laemmli sample buffer, and resolved by SDS-polyacrylamide gel electrophoresis. Quantitation of HA-Erk 1 phosphorylation was determined by protein immunoblotting using rabbit polyclonal phosphospecific MAP kinase IgG (New England Biolabs) as described.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant PMT Mediates Erk 1/2 Activation via Pertussis Toxin-insensitive Gq/11 Proteins-- As shown in Fig. 1, treatment of HEK-293 cells with rPMT resulted in a dose- and time-dependent increase in Erk 1/2 phosphorylation. Erk 1/2 phosphorylation in rPMT-treated cells reached a maximum 6-10-fold over basal increase after 24-48 h of continuous exposure (Fig. 1A). Increasing concentrations of rPMT up to 100 ng/ml produced dose-dependent increases in Erk 1/2 phosphorylation (Fig. 1B), which approximately paralleled rPMT-induced increases in inositol phosphate hydrolysis (Fig. 1C).


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  		Fig. 1.   Effect of rPMT on Erk phosphorylation and inositol phosphate production in HEK-293 cells. A, time course of rPMT-induced Erk phosphorylation. HEK-293 cells in serum-free medium were incubated with rPMT (100 ng/ml) for the indicated time prior to the determination of Erk 1/2 phosphorylation as described. Upper panel depicts a representative immunoblot, while the lower panel presents the mean ± S.E. values for three independent experiments. B, dose dependence of rPMT-induced phosphorylation of Erk 1/2. Serum-starved HEK-293 cells were incubated for 24 h with the indicated concentration of rPMT prior to the determination of Erk 1/2 phosphorylation. Upper panel depicts a representative immunoblot, while the lower panel presents the mean ± S.E. values for four independent experiments. C, dose dependence of rPMT-stimulated inositol phosphate production. HEK-293 cells in serum-free medium were prelabeled with [3H]inositol and incubated for 24 h with the indicated concentration of rPMT prior to the determination of inositol phosphate production as described. The data shown represent mean ± S.D. values for triplicate determinations in one of three independent experiments.

Several lines of evidence suggest that the stimulation of inositol phosphate hydrolysis in response to rPMT results from toxin-induced activation of heterotrimeric G proteins (11, 12). To examine whether heterotrimeric G proteins contributed to rPMT-induced activation of the Erk cascade, we initially determined whether rPMT-induced Erk 1/2 activation was additive with that induced by endogenous Gi-coupled and Gq/11-coupled receptors. As shown in Fig. 2, stimulation of predominantly Gq/11-coupled alpha -thrombin receptors in HEK-293 cells increased Erk 1/2 phosphorylation 8-10-fold. Pretreatment with rPMT for 24 h prior to stimulation failed to produce an additive response. In contrast, rPMT-mediated Erk 1/2 phosphorylation was additive with that induced by acute stimulation of the predominantly Gi-coupled LPA receptor. Interestingly, the rPMT effect was also not additive with Erk 1/2 phosphorylation induced via the EGF receptor tyrosine kinase.


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  		Fig. 2.   Effect of rPMT pretreatment on acute thrombin, LPA, and EGF receptor-mediated Erk 1/2 phosphorylation. HEK-293 cells in serum-free medium were incubated for 24 h in the presence or absence of rPMT (100 ng/ml). Cells were stimulated for 5 min with the thrombin agonist peptide SFLLRN (10 µM), LPA (10 µM), or EGF (10 ng/ml) prior to the determination of Erk 1/2 phosphorylation.

The lack of additivity between rPMT and alpha -thrombin receptor-mediated Erk 1/2 activation suggests that a common G protein pool might mediate the response to both stimuli. To determine the role of heterotrimeric G proteins in rPMT-mediated Erk 1/2 activation, we employed three selective inhibitors of G protein function: B. pertussis toxin, which ADP-ribosylates and inactivates Gi/o family G proteins, and two inhibitory polypeptides GRK2(K220R) and Galpha q-(305-359). The catalytically inactive mutant of the G protein-coupled receptor kinase (GRK) 2, GRK2(K220R), which functions as an dominant negative antagonist of GPCR desensitization (20), has also been shown to directly block Gq/11-mediated inositol phosphate production in COS-1 cells (21). This effect results from the direct binding and sequestration of Galpha q/11 subunits by an RGS homology domain in the amino terminus of GRK2 (22). GRK2 also contains a carboxyl-terminal Gbeta gamma subunit binding motif, which inhibits G protein signaling by sequestering free Gbeta gamma subunits (23). The Galpha q-(305-359) peptide, derived from the carboxyl-terminal 55 amino acids of Galpha q, inhibits Gq/11-coupled, but not Gi-coupled, receptor-mediated PI hydrolysis in COS-7 cells (24).

As shown in Fig. 3, pertussis toxin treatment had no effect on rPMT-stimulated Erk 1/2 phosphorylation in HEK-293 cells. Stimulation of Erk 1/2 by endogenous alpha -thrombin receptors was minimally affected by pertussis toxin, consistent with Erk activation mediated predominantly via Gq/11 family G proteins. In contrast, LPA-stimulated Erk 1/2 phosphorylation was significantly reduced by pertussis toxin, consistent with a predominantly Gi-mediated response. EGF-stimulated Erk 1/2 activation was pertussis toxin-insensitive.


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  		Fig. 3.   Pertussis toxin-sensitivity of Erk 1/2 phosphorylation mediated by rPMT, and thrombin, LPA, and EGF receptors. HEK-293 cells in serum-free medium were incubated with or without pertussis toxin (100 ng/ml) for 24 h. Cells were either treated for 24 h with rPMT (100 ng/ml) or stimulated for 5 min with SFLLRN (10 µM), LPA (10 µM), or EGF (10 ng/ml) prior to the determination of Erk 1/2 phosphorylation. Upper panel depicts a representative immunoblot, while the lower panel represents the mean ± S.E. values for four independent experiments.

In contrast to the lack of pertussis toxin effect, cellular expression of GRK2(K220R) and Galpha q-(305-359) each significantly attenuated rPMT-mediated Erk 1/2 activation. To optimize the detection of signals originating from the transfected cell pool, HEK-293 cells in these experiments were transiently cotransfected with HA epitope-tagged Erk 1 (HA-Erk 1) along with the putative inhibitory peptide, and the phosphorylation state of immunoprecipitated HA-Erk 1 determined. As shown in Fig. 4A, expression of GRK2(K220R) significantly attenuated HA-Erk 1 phosphorylation in response to rPMT treatment, and to stimulation of alpha -thrombin and LPA receptors, with no effect on EGF-stimulated HA-Erk 1 activation. Phosphorylation of HA-Erk 1 resulting from cellular expression of a constitutively activated mutant of Galpha q (Galpha q(Q209L)) was also attenuated by GRK2(K220R) expression, consistent with direct inhibition of Galpha q-dependent signals by GRK2(K220R). Thus, inhibition by GRK2(K220R) is indicative either of a role for Gq/11 proteins or for Gbeta gamma subunits in rPMT-mediated Erk activation.


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  		Fig. 4.   Effect of GRK2(K220R) and Galpha q-(305-359) expression on Erk 1/2 phosphorylation mediated by rPMT, Galpha q(Q209L), and thrombin, LPA, and EGF receptors. A, effect of GRK2(K220R) expression on rPMT-stimulated Erk 1/2 phosphorylation. HEK-293 cells were transfected with plasmid cDNA encoding HA-Erk 1, plus GRK2(K220R) and Galpha q(Q209L) as indicated. Left panel shows a protein immunoblot demonstrating expression of GRK2(K220R). Serum-starved transfected cells were incubated for 24 h with rPMT (100 ng/ml) or stimulated for 5 min with SFLLRN (10 µM), LPA (10 µM), or EGF (10 ng/ml) as indicated. Monolayers were lysed in detergent buffer, and the phosphorylation state of immunoprecipitated HA-Erk 1 was determined as described. Upper right panel depicts a representative anti-phospho-HA-Erk 1 immunoblot, while the lower right panel presents the mean ± S.E. values for three independent experiments. B, effect of Galpha q-(305-359) expression on Gq/11-mediated inositol phosphate production. HEK-293 cells in six-well plates were transfected with plasmid cDNA encoding Galpha q(Q209L) and Galpha q(305-359) as indicated. Left panel shows a protein immunoblot demonstrating expression of Galpha q(305-359). Transfected cells in serum-free medium were prelabeled with [3H]inositol prior to the determination of basal and SFLLRN-stimulated inositol phosphate production as described. The right panel represents mean ± S.D. values for triplicate determinations in one of three independent experiments. C, effect of Galpha q-(305-359) expression on rPMT-stimulated Erk 1/2 phosphorylation. HEK-293 cells were transfected with plasmid cDNA encoding HA-Erk 1, plus Galpha q-(305-359) and Galpha q(Q209L) as indicated. Serum-starved transfected cells were incubated for 24 h with rPMT (100 ng/ml), or stimulated for 5 min with SFLLRN (10 µM), LPA (10 µM), or EGF (10 ng/ml) as indicated, and the phosphorylation state of immunoprecipitated HA-Erk 1 was determined as described. Upper panel depicts a representative anti-phospho-HA-Erk 1 immunoblot, while the lower panel presents the mean ± S.E. values for three independent experiments.

To discriminate between these alternatives, we employed the Galpha q-(305-359) peptide. Cellular expression of this peptide has been shown to inhibit Gq/11-dependent, but not Gi- or Gs-dependent signaling in COS-7 cells (24). As shown in Fig. 4B, expression of Galpha q-(305-359) significantly attenuated inositol phosphate production induced either by acute stimulation of alpha -thrombin receptor or by expression of Galpha q(Q209L), consistent with direct inhibition of Galpha q-dependent signals by Galpha q-(305-359). As shown in Fig. 4C, Galpha q-(305-359) expression markedly inhibited HA-Erk 1 phosphorylation in response to rPMT treatment, Galpha q(Q209L) expression, and stimulation of alpha -thrombin receptors. LPA-stimulated HA-Erk 1 phosphorylation was less sensitive to Galpha q-(305-359) expression, consistent with the greater contribution of pertussis toxin-sensitive G proteins to LPA-stimulated Erk activation in these cells (Fig. 3), and EGF-stimulated HA-Erk phosphorylation was insensitive to Galpha q-(305-359) expression.

Collectively, these data suggest that rPMT mediates Erk activation in HEK-293 cells via Gq/11 family G proteins. The sensitivity of sustained rPMT-mediated HA-Erk 1 phosphorylation to GRK2(K220R) and Galpha q-(305-359) expression parallels that of the constitutively active Galpha q(Q209L) mutant. In addition, the lack of additivity observed between rPMT-mediated Erk activation and that induced by the predominantly Gq/11-coupled alpha -thrombin receptor suggests that the two signals converge upon a common intermediate.

Recombinant PMT Induces Erk Activation via G Protein-dependent Transactivation of the EGF Receptor Tyrosine Kinase-- The mechanisms whereby GPCRs activate the Erk cascade are characterized by extensive heterogeneity (13, 14). Depending upon cell type, receptors coupled to Gq/11 proteins have been shown to mediate both Ras-independent Erk activation via stimulation of PKC isoforms, and Ras-dependent Erk activation via activation of receptor and nonreceptor tyrosine protein kinases. In the latter case, GPCR-induced activation of receptor tyrosine kinases, such as the EGF receptor (25, 26), or of focal adhesion kinases (FAK), such as the calcium-dependent FAK kinase Pyk2 (27, 28), serves to initiate a tyrosine phosphorylation cascade leading to Ras activation.

The absence of an additive effect of rPMT treatment on EGF-stimulated Erk 1/2 phosphorylation is consistent with a role for EGF receptors in mediating the rPMT response. In addition, rPMT is known to stimulate both inositol phosphate hydrolysis and to induce calcium- and PKC-independent tyrosine phosphorylation of several proteins, including p125FAK and paxillin (10). Thus, it is plausible that either mechanism could account for rPMT-stimulated Erk activation. To discriminate between these mechanisms, we assessed the sensitivity of rPMT-mediated Erk 1/2 activation to specific PKC and tyrosine kinase inhibitors. As shown in Fig. 5A, inhibition of classical PKC isoforms with GF109203X blocked acute phorbol ester-stimulated Erk phosphorylation, but had no effect on the rPMT, alpha -thrombin, and EGF receptor-mediated signals. Similar results were obtained using Ro31-8220, a chemically distinct PKC inhibitor with a similar spectrum (data not shown). As shown in Fig. 5B, inhibition of EGF receptor activity using the EGF receptor-specific tyrphostin AG1478 profoundly reduced rPMT, alpha -thrombin or EGF receptor-mediated Erk phosphorylation, with no effect on the response to phorbol ester. In contrast, treatment of cells with the platelet-derived growth factor receptor-specific tyrphostin AG1295 had no inhibitory effect (data not shown). Thus, rPMT-mediated Erk activation requires EGF receptor activity, but not the activity of classical PKC isoforms. Since rPMT-stimulated Erk activation, like rPMT-stimulated inositol phosphate hydrolysis (11) and calcium-dependent chloride currents (12), is sensitive to inhibitors of Gq/11 proteins, these data suggest that Gq/11-mediated EGF receptor transactivation accounts for the effects of rPMT on Erk 1/2 activity.


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  		Fig. 5.   Effect of PKC and EGF receptor inhibitors on Erk 1/2 phosphorylation mediated by rPMT, phorbol ester, and thrombin, LPA, or EGF receptors. A, effect of the protein kinase C inhibitor GF109203X on rPMT-stimulated Erk 1/2 phosphorylation. HEK-293 cells in serum-free medium were incubated with or without GF109203X (2 µM) for 24 h. Cells were either treated with rPMT (100 ng/ml) for 24 h, or stimulated for 5 min with SFLLRN (10 µM), EGF (10 ng/ml), or phorbol ester (PMA, 1 µM) prior to the determination of Erk 1/2 phosphorylation. Upper panel depicts a representative immunoblot, while the lower panel presents the mean ± S.E. values for three independent experiments. B, effect of the EGF receptor-specific tyrphostin AG1478 on rPMT-stimulated Erk 1/2 phosphorylation. HEK-293 cells in serum-free medium were incubated with or without tyrphostin AG1478 (100 nM) for 24 h. Cells were either treated with rPMT (100 ng/ml) for 24 h or stimulated for 5 min with SFLLRN (10 µM), EGF (10 ng/ml), or phorbol ester (PMA, 1 µM) prior to the determination of Erk 1/2 phosphorylation. Upper panel depicts a representative immunoblot, while the lower panel represents the mean ± S.E. values for three independent experiments.

Unlike direct PKC-mediated Erk activation (29), EGF receptor-dependent Erk activation is sensitive to dominant inhibitory mutants of mSos and Ha-Ras (30). As shown in Fig. 6, expression of the two dominant negatives, Sos-PRO and N17Ras, significantly inhibited rPMT, alpha -thrombin, or EGF receptor-mediated Erk phosphorylation, with no effect on the phorbol ester response. Thus rPMT-mediated Erk activation involves Gq/11-dependent EGF receptor transactivation and leads to the Ras-dependent activation of the Erk cascade. As depicted schematically in Fig. 7, these events are dissociated from rPMT-mediated effects on PKC, which contribute at most a minor component to rPMT-stimulated Erk activation.


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  		Fig. 6.   Effect of dominant negative mutants of mSos1 and Ha-Ras on Erk 1/2 phosphorylation mediated by rPMT, phorbol ester, and thrombin, LPA, or EGF receptors. A, expression of the Sos-PRO and N17 Ras constructs in HEK-293 cells. HEK-293 cells were transfected with plasmid cDNA encoding HA-Erk 1, plus Sos-PRO and N17 Ras as indicated. Protein immunoblots of whole cell lysates demonstrate expression of each construct. B, effect of Sos-PRO and N17 Ras expression on rPMT-stimulated Erk 1/2 phosphorylation. Serum-starved transfected cells were incubated for 24 h with rPMT (100 ng/ml), or stimulated for 5 min with SFLLRN (10 µM), LPA (10 µM), EGF (10 ng/ml), or PMA (1 µM) as indicated. Monolayers were lysed in detergent buffer, and the phosphorylation state of immunoprecipitated HA-Erk 1 was determined as described. Left panel depicts a representative anti-phospho-HA-Erk 1 immunoblot, while the right panel presents the mean ± S.E. values for three independent experiments.


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  		Fig. 7.   Model for the proposed mechanism of Erk 1/2 regulation by rPMT. Activation of Gq/11 proteins by either rPMT or Gq-coupled GPCRs such as the thrombin receptor results in activation of Gq/11 effectors such as phospholipase Cbeta (PLCbeta ) isoforms as well as transactivation of the EGF receptor tyrosine kinase. Subsequent activation of the Erk cascade is mediated predominantly via RTK transactivation, with no significant contribution derived from the PLCbeta -dependent activation of classical PKC activation protein kinase.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite growing evidence that many of the cellular effects of rPMT are mediated by the activation of Gq/11 family heterotrimeric G proteins, the detailed molecular mechanism of rPMT action remains poorly understood. Like other bacterial toxins with intracellular loci of action, binding to cell surface receptors and processing through endocytic vesicles appears to be the rate-limiting step for rPMT action on intact cells. A 3-4-h delay is observed between application of rPMT to intact cells and its maximal effects on inositol phospholipid hydrolysis (9), yet phospholipase Cbeta -mediated inositol 1,4,5-trisphosphate and calcium responses occur within seconds when the toxin is directly injected into Xenopus oocytes (12).

Unlike several bacterial toxins that modulate G protein activity, rPMT toxin lacks recognizable enzymatic activity. Microinjection of antisera directed against the amino terminus, but not the carboxyl terminus, of rPMT inhibits its biological activity in Xenopus oocytes (12). Although the full-length protein is required for biological activity on intact cells, the amino-terminal 500 residues of the toxin are active when microinjected (15). This region of PMT shares modest homology with the amino-terminal half of the cytotoxic necrotizing factors 1 and 2 from enteropathogenic E. coli (31, 32), which are thought to target Rho family small GTP-binding proteins (32). In the Xenopus system, the effect of rPMT on calcium-dependent chloride channel activation is markedly enhanced by Galpha q overexpression and specifically inhibited by microinjection of antisera against Galpha q (12). Oocytes injected with rPMT fail to respond to a second challenge, suggesting that rPMT may bind directly to Galpha q subunits and induce GTP exchange. Our results, demonstrating that cellular expression of peptide inhibitors of Gq/11 signaling block rPMT action in intact cells, are consistent with this model.

The activation of Gq/11 proteins by rPMT may be sufficient to account for its mitogenic effects, as well as its effects on inositol phospholipid turnover and PKC activation. Transient expression of a constitutively active mutant of Galpha q mimics the effects of rPMT, producing sustained increases in phosphatidylinositol hydrolysis and Erk activation that are blocked by peptide inhibitors of Gq/11 signaling. The mechanism of rPMT-stimulated Erk activation in HEK-293 cells parallels that employed by the predominantly Gq/11-coupled alpha -thrombin receptor. Both signals are independent of PKC, but dependent upon the catalytic activity of endogenous EGF receptors to activate Ras-dependent pathways.

Both rPMT exposure and acute stimulation of Gq/11-coupled receptors induce the tyrosine phosphorylation of multiple substrates. In Swiss 3T3 cells, rPMT stimulates tyrosine phosphorylation of p125FAK and paxillin in a cytochalasin D-sensitive manner (10). Similar results have been obtained following acute stimulation of Gq/11-coupled receptors in Swiss 3T3 (33), Rat 1 (34), and HEK-293 cells (35). Our data indicate that rPMT also mediates growth stimulatory effects via G protein-dependent activation of EGF receptors. Indeed, previous work has demonstrated that rPMT exposure promotes the loss of cell surface EGF receptors (36), an effect that may represent EGF receptor down-regulation following rPMT-induced transactivation.

Gq/11-coupled receptors have been shown to mediate two, apparently distinct, calcium-dependent tyrosine phosphorylation cascades that converge on Ras and its downstream effectors. In several cell types, GPCR-stimulated Erk activation involves the ligand-independent transactivation of receptor tyrosine kinases, such as the EGF receptor (25, 26). In PC12 cells, bradykinin (37) and m1 muscarinic acetylcholine (38) receptors activate EGF receptors via either calcium- or PKC-dependent mechanisms, respectively. In vascular smooth muscle, angiotensin II mediates calcium-dependent EGF receptor transactivation upstream of the Erk cascade (39). Alternatively, bradykinin-dependent Erk activation has been attributed to stimulation of the calcium-dependent FAK family kinase, Pyk2 (27, 28). Pyk2-dependent signals require either calcium or PKC (27) and are sensitive to cytochalasin D, suggesting a requirement for intact focal adhesions (40). Activated Pyk2, like p125FAK, associates with c-Src and provides docking sites for Grb2-Sos complexes (28).

Despite similarities in their mechanisms of activation (41), Pyk2-mediated signals appear to be independent of those mediated via EGF receptor transactivation. In PC12 cells, expression of a dominant negative mutant of Pyk2 does not inhibit bradykinin-induced transactivation of the EGF receptor, and expression of a dominant negative EGF receptor mutant has no effect on Pyk2 phosphorylation (42). In HEK-293 cells, treatment with tyrphostin AG1478 (35) and expression of a dominant negative mutant of Pyk2 (43) each cause a partial blockade of Gq/11-coupled receptor-mediated Erk activation, suggesting that both transactivated EGF receptors and Pyk2 contribute independently to Erk activation. While we find that rPMT-stimulated Erk activation is predominantly tyrphostin-sensitive, we cannot exclude a contribution from Pyk2 acting via a focal adhesion-based scaffold.

Study of the mechanisms of action of bacterial toxins has led to significant insights into the roles of heterotrimeric G proteins in cellular signaling. Our data suggest that rPMT employs Gq/11 proteins in a novel strategy for mitogenic regulation by a bacterial toxin, that of utilizing G proteins to initiate a tyrosine phosphorylation cascade leading to Ras activation. Given the potent mitogenic effects achieved by rPMT, these findings serve to underscore the importance of G protein-catalyzed mitogenic signals to growth regulation.

    ACKNOWLEDGEMENTS

We thank R. Lefkowitz for helpful discussion and critical reading of the manuscript. We thank D. Addison and M. Holben for excellent secretarial assistance.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK02352 and DK55524 (to L. M. L.) and AI38396 (to B. A. W.).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.

To whom correspondence should be addressed: Dept. of Medicine, Box 3821, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-2974; Fax: 919-684-8875; E-mail: luttrell@receptor-biol.duke.edu.

    ABBREVIATIONS

The abbreviations used are: PMT, P. multocida toxin; rPMT, recombinant P. multocida toxin; PKC, protein kinase C; MAP, mitogen-activated protein; GPCR, G protein-coupled receptor; EGF, epidermal growth factor; PMA, phorbol 12-myristate 13-acetate; LPA, lysophosphatidic acid; HA, hemagglutinin; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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