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• W2012106447 abstract "The intracellularly acting Pasteurella multocida toxin (PMT) is a potent mitogen that stimulates Gq-dependent formation of inositol trisphosphate. We show that PMT, a nontoxic mutant of PMT (PMTC1165S), and bombesin each stimulate time-dependent phosphorylation of Gαq at tyrosine 349. Although PMT and PMTC1165S each cause phosphorylation of Gαq, only the wild-type toxin activates Gq. Pretreatment of cells with wild-type or mutant PMT potentiated the formation of inositol phosphates stimulated by bombesin equally. These data show that PMT potentiates bombesin receptor signaling through tyrosine phosphorylation of Gq and distinguishes between the two proposed models of Gq activation, showing that tyrosine phosphorylation is not linked to receptor uncoupling. The intracellularly acting Pasteurella multocida toxin (PMT) is a potent mitogen that stimulates Gq-dependent formation of inositol trisphosphate. We show that PMT, a nontoxic mutant of PMT (PMTC1165S), and bombesin each stimulate time-dependent phosphorylation of Gαq at tyrosine 349. Although PMT and PMTC1165S each cause phosphorylation of Gαq, only the wild-type toxin activates Gq. Pretreatment of cells with wild-type or mutant PMT potentiated the formation of inositol phosphates stimulated by bombesin equally. These data show that PMT potentiates bombesin receptor signaling through tyrosine phosphorylation of Gq and distinguishes between the two proposed models of Gq activation, showing that tyrosine phosphorylation is not linked to receptor uncoupling. The Pasteurella multocida toxin (PMT) 1The abbreviations used are: PMT, P. multocida toxin; GPCR, G protein-coupled receptor; IP3, inositol trisphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; DMEM, Dulbecco's modified Eagle's medium; GRP, gastrin-releasing peptide.1The abbreviations used are: PMT, P. multocida toxin; GPCR, G protein-coupled receptor; IP3, inositol trisphosphate; GTPγS, guanosine 5′-3-O-(thio)triphosphate; DMEM, Dulbecco's modified Eagle's medium; GRP, gastrin-releasing peptide. is a highly potent mitogen for mesenchymal cells, including Swiss 3T3 fibroblasts (1Rozengurt E. Higgins T.E. Chanter N. Lax A.J. Staddon J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 123-127Crossref PubMed Scopus (108) Google Scholar). Although the primary molecular targets of this intracellularly acting toxin have not been identified, a prominent role for heterotrimeric G proteins has been elucidated (2Murphy A.C. Rozengurt E. J. Biol. Chem. 1992; 267: 25296-25303Abstract Full Text PDF PubMed Google Scholar, 3Wilson B.A. Xinjun Z. Ho M. Lu L. J. Biol. Chem. 1997; 272: 1268-1275Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 4Zywietz A. Gohla A. Schmelz M. Schultz G. Offermanns S. J. Biol. Chem. 2001; 276: 3840-3849Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The toxin affects several signal transduction pathways, resulting in increased inositol phosphate production, stimulation of protein kinase C activity, Ca2+ mobilization, actin rearrangements, and increased protein tyrosine phosphorylation (5Lax A.J. Grigoriadis A.E. Int. J. Med. Microbiol. 2001; 291: 261-268Crossref PubMed Scopus (36) Google Scholar). Heterotrimeric G proteins are guanine nucleotide-binding proteins that function as molecular switches that transduce signals from G protein-coupled receptors (GPCR) to effector proteins such as enzymes or ion channels (6Birnbaumer L. Abramowitz J. Brown A.M. Biochim. Biophys. Acta. 1990; 1031: 163-224Crossref PubMed Scopus (961) Google Scholar, 7Hepler J.R. Gilman A.G. Trends Biochem. Sci. 1992; 17: 383-387Abstract Full Text PDF PubMed Scopus (923) Google Scholar, 8Spiegel A.M. Schenker A. Weinstein L.S. Endocr. Rev. 1992; 13: 536-565Crossref PubMed Scopus (311) Google Scholar). The Gα proteins are divided into four families: Gαs, Gαi/o, Gαq, and Gα12 (8Spiegel A.M. Schenker A. Weinstein L.S. Endocr. Rev. 1992; 13: 536-565Crossref PubMed Scopus (311) Google Scholar, 9Simon M.I. Strathmann M.P. Gautam N. Science. 1991; 252: 802-808Crossref PubMed Scopus (1585) Google Scholar). The Gαq class are widely expressed and regulate various effector proteins including phospholipase Cβ and Bruton's tyrosine kinase (10Rhee S.G. Choi K.D. J. Biol. Chem. 1992; 267: 12393-12396Abstract Full Text PDF PubMed Google Scholar). Activation of GPCRs results in a conformational change in the Gα subunit, favoring the exchange of bound GDP for GTP. GTP binding results in the dissociation of Gα-GTP and βγ complexes, each of which can modulate effector proteins. The regulation of these processes in vivo has yet to be fully elucidated. Recently it has been demonstrated that the α subunit of Gq is a target for tyrosine phosphorylation. Interestingly, phosphorylation of Gαq increased its ability to activate phospholipase Cβ in an in vitro model, suggesting that phosphorylation may modulate the activity of the G protein in vivo (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar). Modulation of Gαq phosphorylation using chemical inhibitors of tyrosine kinases or tyrosine phosphatases had a profound effect on the production of inositol trisphosphates (IP3) in vivo (12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar, 13Umemori H. Hayashi T. Inoue T. Nakanishi S. Mikoshiba K. Yamamoto T. Oncogene. 1999; 18: 7399-7402Crossref PubMed Scopus (13) Google Scholar). Furthermore, transient expression of a dominant active mutant of the Fyn tyrosine kinase elevated the phosphorylation of Gαq but blocked receptor-stimulated IP3 production (12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar). Taken together, these results implied that cellular kinases and phosphatases coordinately regulate the activity of Gαq. The experiments presented here were designed to determine whether PMT stimulated activation and tyrosine phosphorylation of Gαq. We show that PMT is a potent stimulator of Gαq tyrosine phosphorylation but that this phosphorylation step is not a prerequisite for Gαq activation. Furthermore, using wild-type and mutant forms of PMT, we show that tyrosine phosphorylation of Gαq can potentiate signaling through the Gq-coupled bombesin receptor. Cell culture reagents were obtained from Invitrogen. All of the primary antisera were obtained from Santa Cruz Biotechnology, Inc. Horseradish peroxidase-conjugated donkey anti-rabbit and anti-mouse IgG were from Sigma-Aldrich. [γ-35S]GTPγS (1 mCi/ml) was obtained from Amersham Biosciences. myo-[2-3H]Inositol (1 mCi/ml) was obtained from New England Nuclear, Ltd. All other reagents were of the highest available grade from standard commercial sources. SYF–/– (CRL-2459) and YF–/– (CRL-2498) cells were purchased from the American Type Culture Collection. Gαq/11–/– double deficient fibroblasts were a generous gift from Professor Stefan Offermanns (Pharmakologisches Institut, Universität Heidelberg, Germany). Cell Culture—Cell culture procedures (14Rozengurt E. Mierzejewski K. Wigglesworth N. J. Cell. Physiol. 1978; 97: 241-252Crossref PubMed Scopus (44) Google Scholar, 15Rozengurt E. Sinnett-Smith J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2936-2940Crossref PubMed Scopus (371) Google Scholar), assays of mitogenicity by [3H]thymidine incorporation (16Dicker P. Rozengurt E. Nature. 1980; 287: 607-612Crossref PubMed Scopus (253) Google Scholar) or by cell number (15Rozengurt E. Sinnett-Smith J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2936-2940Crossref PubMed Scopus (371) Google Scholar), and measurements of total inositol triphosphates (17Nanberg E. Rozengurt E. EMBO J. 1988; 7: 2741-2748Crossref PubMed Scopus (86) Google Scholar) were performed as described previously. Immunoprecipitation of the Gα q Subunit—Quiescent cultures of confluent Swiss 3T3, Gαq/11–/–, or SYF–/–/YF–/– cells were incubated in DMEM containing PMT (70 pm), mutant PMTC1165S (70 pm), or bombesin (10 nm) as indicated and extracted at 4 °C with RIPA buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 1 mm Na3VO4, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, COMPLETE™ protease inhibitors; Roche Applied Science). The lysates were incubated for 18 h at 4 °C with rabbit anti-Gq/11 (2 μg) antibody coupled to 50 μl of protein G-Sepharose. The immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blotting. Preparation of Swiss 3T3 Cell Membranes—Quiescent cultures of Swiss 3T3 cells were rinsed twice with cold phosphate-buffered saline and scraped into phosphate-buffered saline containing 1 mm sodium orthovanadate and proteinase inhibitors. Following centrifugation (200 × g, 10 min, 4 °C), washed cell pastes were frozen at –70 °C until required. The frozen cell pastes were thawed on ice and suspended in 5 ml of buffer A (10 mm Tris-HCl, 10 mm MgCl2, 0.1 mm EDTA, pH 7.5) containing 1 mm sodium orthovanadate and proteinase inhibitors. The cells were ruptured by 25 passes through a 23-gauge needle, and the resulting homogenate was centrifuged at 800 × g for 10 min to remove unbroken cells and nuclei. The supernatants were transferred to fresh tubes and centrifuged at 50,000 × g for 10 min. The pellet was washed and suspended in 10 ml of buffer A containing inhibitors. After a second centrifugation step the membrane pellet was suspended in buffer A to a protein concentration of 3 mg/ml and stored at –70 °C. Determination of GDP-GTP Exchange on Gq—Determination of GTPγS binding was essentially as described previously (18Panchalingam S. Undie A.S. Neurochem. Res. 2000; 25: 759-767Crossref PubMed Scopus (26) Google Scholar). Briefly, membranes (30 μg) were suspended in 100 μl of assay buffer (50 mm Hepes, pH 7.4, 120 mm NaCl, 20 mm MgCl2, 2 mm KCl, 1 mm deoxycholate, 20 μm GDP, 0.2% bovine serum albumin) and incubated for 10 min at 37 °C. Following incubation, an equal volume of assay buffer containing either toxins (140 pm) or bombesin (40 nm) and 2 nm [35S]GTPγS (∼100,000 cpm/tube) was added, and incubation at 37 °C was continued. The reactions were quenched by the addition of 1 ml of ice-cold assay buffer followed by centrifugation. The membrane pellets were solubilized in 50 μl of solution containing 1.5% (v/v) Triton X-100, 0.2% (w/v) SDS and then diluted to 1 ml with immunoprecipitation buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 20 mm MgCl2,2mm KCl, 1 mm EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, protease inhibitors). The solubilized lysate was incubated for 18 h at 4 °C with rabbit anti-Gq/11 (2 μg) antibody coupled to 40 μl of protein G-Sepharose. Immunoprecipitates were recovered by centrifugation and washed six times with immunoprecipitation buffer. The complexes were solubilized by boiling in 5% SDS, and the bound GTPγS was quantitated using a Wallac BetaRack scintillation counter. Purification of Wild-type and Mutant PMT—Recombinant PMT and inactive, mutant PMTC1165S were expressed and purified as described previously (19Ward P.N. Miles A.J. Sumner I.G. Thomas L.H. Lax A.J. (1998) Infect. Immun. 1998; 66: 5636-5642Crossref PubMed Google Scholar). Expression of the Bombesin/GRP Receptor in SYF – / – and YF – / – Cells—Membranes were prepared from confluent cultures of cells as outlined above. The membrane proteins (50 μg) were fractionated by SDS-PAGE, transferred to nitrocellulose, and Western blotted with a rabbit polyclonal antiserum to GRP receptor as described previously (20Kroog G.S. Sainz E. Worland P.J. Akeson M.A. Benya R.V. Jensen R.T. Battey J.F. J. Biol. Chem. 1995; 270: 8217-8224Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). G Protein Mutation—Tyrosine residue 349 of Gq was converted to phenylalanine using a QuikChange kit (Stratagene) in accordance with the manufacturer's instructions. The constructs were confirmed by restriction digest and full nucleotide sequencing using a Beckman Coulter CEQ 2000XL automated sequencer. Transfection of Gαq/11–/– Cells—Gαq/11–/– cells (21Offermanns S. Zhao L.-P. Gohla A. Sarosi I. Simon M.I. Wilkie T.M. EMBO J. 1998; 17: 4304-4312Crossref PubMed Scopus (204) Google Scholar) were grown in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum, l-glutamine (1 mm), and 1× modified Eagle's medium nonessential amino acids in a 95% air and 5% CO2 atmosphere at 37 °C. The cells were plated at 5 × 104 cells/well in 33-mm dishes, and after a 24-h growth period they were transfected using LipofectAMINE Plus™ (Invitrogen BV) according to the manufacturer's instructions. After6hthe cells were washed twice with Opti-MEM I and then cultured in DMEM for a further 18 h. The cells were washed twice with DMEM/nutrient mixture F-12 3:1 (v/v) and then incubated in the same medium for a further 48 h to induce quiescence. A total of 2 μg of pcDNA3 containing the relevant Gαq construct was used to transfect each dish. Statistical Analysis—Data for production of inositol phosphates in Fig. 5 are presented as the means and standard errors from seven independent experiments with each point repeated in triplicate. Statistical analysis was carried out using the STATA 7 program (STATA Corp.). The significance of differences between various treatments was analyzed using the Mann-Whitney U test. PMT Stimulates Tyrosine Phosphorylation of Gα q—Treatment of Swiss 3T3 cells with mitogenic concentrations of PMT potently stimulated the tyrosine phosphorylation of the α subunit of Gq (Fig. 1a). Increases in tyrosine phosphorylation were concentration-dependent, reaching a maximum at a PMT concentration of ∼70 pm (Fig. 1a). The increase in phosphorylation occurred after a lag period of 1 h and peaked around 4–6 h after exposure to PMT (Fig. 1b, upper panel). The lag period is consistent with the time required for binding and internalization of the toxin to occur. Mutation of cysteine 1165 to serine in PMT (PMTC1165S) leads to a complete loss of mitogenic activity and toxicity without grossly affecting toxin structure (19Ward P.N. Miles A.J. Sumner I.G. Thomas L.H. Lax A.J. (1998) Infect. Immun. 1998; 66: 5636-5642Crossref PubMed Google Scholar). Surprisingly, PMTC1165S was found to stimulate an increase in Gαq phosphorylation with kinetics matching those of the wild-type toxin (Fig. 1b, lower panel). Treatment of Swiss 3T3 cells with the GPCR agonist bombesin also stimulated phosphorylation of Gαq (Fig. 1c). However, in contrast to PMT, phosphorylation of Gαq stimulated by bombesin was highly transient, peaking at 1 min post exposure and returning to basal levels within 5 min. The increase in Gαq phosphorylation stimulated by PMT or PMTC1165S, but not by bombesin, could be effectively blocked by preincubation of the cells with either methylamine or a PMT antiserum (Fig. 1, d and e). This strongly suggested that the induced phosphorylation was a specific effect that followed toxin internalization. PMT Stimulates Phosphorylation of Tyrosine 349 —Umemori et al. (12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar) previously demonstrated that GPCR agonists promoted tyrosine phosphorylation of Gα11 at tyrosine residue 352. It was therefore important to identify the site of Gαq/11 phosphorylation in response to stimulation with PMT. Murine Gαq (1–353) was cloned into the eukaryotic expression vector pcDNA3 and the C-terminal tyrosine residue (349), analogous to Gα11 tyrosine 352, was changed to phenylalanine by site-directed mutagenesis. The constructs were confirmed by restriction digestion and nucleotide sequencing. Embryonic fibroblasts deficient in both Gαq and Gα11 (Gαq/11–/– cells) were transfected with either wild-type or mutant (Y349F) Gαq, and the expression of the Gα subunit was determined. In agreement with previous studies (28Liu S. Carrillo J.J. Pediani J.D. Milligan G. J. Biol. Chem. 2002; 277: 25707-25714Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), an anti-Gαq/11 antiserum directed against the C terminus of the G protein failed to detect the mutant Y349F Gαq. However, probing of the membranes with an antiserum directed against an internal sequence of Gαq (115–133) revealed equivalent expression of both constructs (data not shown). Because the C-terminal antiserum fails to recognize mutant Y349F Gαq, it was not possible to use this antiserum for immunoprecipitation. It was therefore decided to carry out Gαq immunoprecipitation using an anti-phosphotyrosine antibody and probe blots with the antiserum directed against the internal sequence of Gαq. Wild-type or mutant (Y349F) Gαq was expressed in Gαq/11–/– cells, and cells were stimulated either with a mixture of bradykinin and thrombin or with wild-type or mutant PMT. In cells expressing wild-type Gαq treatment with GPCR agonists or toxins resulted in increased tyrosine phosphorylation of Gαq. In contrast, mutant Y349F Gαq was not immunoprecipitated using an antibody directed against phosphotyrosine in either untreated or treated cells (Fig. 2a). Parallel experiments revealed that the observed changes in phosphorylation were not due to major differences in the expression of either wild-type or mutant Gαq (Fig. 2b). This indicated that Gαq Tyr349 represents the major site of tyrosine phosphorylation in response to either GPCR or toxin stimulation. PMT, but Not PMTC1165S, Stimulates Activation of Gq—It had been assumed that mutant PMT (PMTC1165S) failed to activate pathways associated with wild-type toxin, although this had never been demonstrated. We decided to determine whether PMT and PMTC1165S each stimulated activation of Gq. The initial steps of G protein activation in response to either bombesin or toxins were determined by analyzing binding of the GTP analog GTPγS. Because PMT is predicted to act enzymatically, standard GTPγS binding assays were carried out at 37 °C for 1 h. The use of an in vitro assay system means prolonged incubation steps are not required because the toxin does not require cellular binding and internalization. PMT and bombesin each stimulated concentration-dependent binding of GTPγS to Swiss 3T3 membrane fractions (Fig. 3a). PMT potently stimulated GTPγS binding at concentrations (picomolar range) 100-1000-fold lower than the GPCR agonist bombesin. In contrast, PMTC1165S did not stimulate a significant increase in GTPγS binding in parallel experiments (Fig. 3a) under a variety of conditions (longer incubation periods or higher toxin concentrations; data not shown). To determine whether Gq was directly affected by both bombesin and PMT, we performed GTPγS binding assays followed by immunoprecipitation with antisera against Gαq. Membranes from Swiss 3T3 cells were stimulated with 70 pm PMT or PMTC1165S or 20 nm bombesin as indicated. Nonspecific binding was determined in the presence of excess GTPγS and by using normal rabbit serum. Bombesin and PMT each stimulated direct GTPγS binding to Gq (Fig. 3b). The kinetics of GTPγS binding differed between bombesin and PMT. As previously reported, bombesin stimulated a rapid increase in the levels of bound GTPγS, which peaked 5–10 min after addition (22Offermanns S. Heiler E. Spicher K. Schultz G. FEBS Letts. 1994; 349: 201-204Crossref PubMed Scopus (64) Google Scholar). By comparison, increases in GTPγS binding stimulated by PMT occurred gradually, peaking 40–50 min after addition. This result would support the concept that PMT has an enzymatic action, with GTP binding occurring downstream of a toxin-catalyzed event. PMTC1165S had no effect on GTPγS binding to Gαq (Fig. 3b). Thus despite stimulation of Gαq tyrosine phosphorylation by PMTC1165S, this mutant failed to stimulate this key indicator of Gq activation. To further investigate the functional role of tyrosine phosphorylation in G protein activation, the production of inositol phosphates was determined. Activation of G protein-coupled receptors linked to members of the Gαq subfamily stimulates phospholipase Cβ-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. As previously reported (1Rozengurt E. Higgins T.E. Chanter N. Lax A.J. Staddon J.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 123-127Crossref PubMed Scopus (108) Google Scholar), bombesin and PMT were potent activators of this pathway. In contrast, we found PMTC1165S did not stimulate inositol phosphate production (Fig. 3c). A wide variety of experimental conditions (longer incubation periods or higher toxin concentrations) failed to elicit increases in inositol phosphates in response to PMTC1165S (data not shown). These findings demonstrated that PMT and bombesin but not PMTC1165S could activate Gq and that tyrosine phosphorylation of Gαq did not necessarily lead to its activation. PMT Activation of Gq Is Not Dependent on Gq Tyrosine Phosphorylation—The ability of PMTC1165S to promote tyrosine phosphorylation of Gαq without leading to its activation conflicts with the model proposed by Umemori et al. (13Umemori H. Hayashi T. Inoue T. Nakanishi S. Mikoshiba K. Yamamoto T. Oncogene. 1999; 18: 7399-7402Crossref PubMed Scopus (13) Google Scholar) suggesting that tyrosine phosphorylation occurred downstream of GTP binding. We decided to investigate whether phosphorylation of Gαq was required for activation of phospholipase C. Cultures of Swiss 3T3 cells were treated with the broad spectrum tyrosine kinase inhibitor genistein to block phosphorylation of Gαq. Genistein blocked tyrosine phosphorylation stimulated by PMT in a concentration-dependent manner (Fig. 4a). The increased phosphorylation of Gαq in response to either PMT or bombesin was completely blocked by prior exposure to 50 μm genistein (Fig. 4b). Daidzein, an analog of genistein, which lacks tyrosine kinase inhibitory activity, had no effect on the phosphorylation of Gαq (data not shown). Application of 20 nm bombesin or 70 pm PMT to Swiss 3T3 cells pretreated with the solvent Me2SO or daidzein resulted in the production of inositol phosphates. However, at concentrations that blocked tyrosine phosphorylation, genistein only inhibited inositol phosphate production stimulated by bombesin and did not inhibit the stimulation of inositol phosphate production by PMT (Fig. 4c). This result demonstrated that phosphorylation of Gq is not an absolute requirement for phospholipase C activation in vivo. Furthermore, these data strongly argue against PMT acting directly as a tyrosine kinase. PMT Enhances Inositol Phosphate Production in Response to Bombesin—It was previously reported that pretreatment of Swiss 3T3 cells with subsaturating concentrations of PMT enhanced the production of IP3 in response to neuropeptides but not platelet-derived growth factor (2Murphy A.C. Rozengurt E. J. Biol. Chem. 1992; 267: 25296-25303Abstract Full Text PDF PubMed Google Scholar). To further investigate this effect, we asked whether PMTC1165S could also enhance the production of IP3. As previously reported, PMT was able to facilitate the production of IP3 in response to bombesin treatment (Fig. 5). Although treatment of Swiss 3T3 cells with PMTC1165S alone did not stimulate production of IP3, pretreatment of Swiss 3T3 cells with PMTC1165S significantly enhanced the production of IP3 in response to bombesin (p < 0.01) (Fig. 5). The degree of potentiation stimulated by either wild-type or mutant PMT (approximately 140 and 142%, respectively, of additive values) was comparable (Table I) and thus argues that PMT facilitates the production of IP3 in response to bombesin through phosphorylation of Gαq tyrosine 349.Table IPMT and PMTC1165S potentiate inositol phosphate production in response to bombesin.TreatmentInositol phosphatesPotentiationcpm%Control186 ± 14NABombesin519 ± 28NAPMTC1165S187 ± 15NAPMT1294 ± 20NABombesin with PMTC1165S pretreatment659 ± 35141.6Bombesin with PMT pretreatment2206 ± 150140.2 Open table in a new tab Role of Src Family Kinases in Gq/11 Phosphorylation—The differential effects of wild-type and mutant PMT on the stimulation of phosphorylation and inositol phosphate production suggested that a cellular kinase phosphorylated Gαq. Src family kinases (subsequently referred to as Src kinases) have been postulated to be possible regulators of this process (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar, 12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar, 13Umemori H. Hayashi T. Inoue T. Nakanishi S. Mikoshiba K. Yamamoto T. Oncogene. 1999; 18: 7399-7402Crossref PubMed Scopus (13) Google Scholar). To clarify the role of Src kinases, we utilized the recently described Src/Yes/Fyn-deficient (SYF–/–) cell line (23Klinghoffer R.A. Sachsenmaier C. Cooper J.A. Soriano P. EMBO J. 1999; 18: 2459-2471Crossref PubMed Scopus (646) Google Scholar). We compared activity in these cells with SYF–/– cells rescued with a retroviral vector expressing murine c-Src (YF–/– cells). PMT potently stimulated DNA synthesis in both cell lines, indicating that the cells were responsive to toxin (Fig. 6, a and b). PMTC1165S did not stimulate DNA synthesis in either cell line under the same experimental conditions. Interestingly, bombesin only stimulated DNA synthesis in the Src expressing control cells (YF–/–). This suggested that Src kinases might be required for GPCR stimulated DNA synthesis. To ensure differences were not due to a lack of receptor expression, we analyzed membrane fractions for the presence of the bombesin/GRP receptor. Both cell lines expressed the receptor (Fig. 6c). The ability of PMT to induce phosphorylation of focal adhesion kinase (p125FAK) was investigated to check the phenotype of the cell lines. PMT stimulated tyrosine phosphorylation of p125FAK in YF–/– cells but not in SYF–/– cells (Fig. 6d). Finally, we investigated the effect of toxins and bombesin on the phosphorylation of Gαq in YF–/– and SYF–/– cells (Fig. 6, e and f, respectively). Treatment of quiescent YF–/– cells with PMT, PMTC1165S, or bombesin stimulated tyrosine phosphorylation of Gαq. By comparison, no changes in tyrosine phosphorylation of Gαq could be observed in quiescent SYF–/– cells, although there was a high basal level of tyrosine phosphorylation of Gαq. Although it is not clear why Gαq was highly phosphorylated in these cells, the data demonstrate that Src kinases mediate the phosphorylation of Gαq. Tyrosine phosphorylation of proteins can modulate their activity and/or promote interaction with other molecules (24Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2225) Google Scholar). Several neuropeptides that regulate cell growth and differentiation induce tyrosine phosphorylation of Gαq. Recent studies have indicated that Gαq phosphorylation forms part of a novel cycle in which tyrosine kinases and phosphatases regulate Gq activation (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar, 12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar, 13Umemori H. Hayashi T. Inoue T. Nakanishi S. Mikoshiba K. Yamamoto T. Oncogene. 1999; 18: 7399-7402Crossref PubMed Scopus (13) Google Scholar). The aim of the present study was to further investigate the role of phosphorylation using PMT. The results presented here show for the first time that PMT induces both dose- and time-dependent increases in tyrosine phosphorylation of Gαq. A nontoxic mutant of PMT (PMTC1165S) also stimulated phosphorylation, with kinetics matching those of wild-type PMT. Phosphorylation of Gαq/11 in response to wild-type or mutant PMT occurs at Gαq tyrosine residue 349. Previous studies indicated that this tyrosine residue is also phosphorylated in response to GPCR agonists, an event confirmed in these studies. Our data demonstrate that the induction of tyrosine phosphorylation by either wild-type or mutant PMT is a specific event following toxin internalization. Methylamine, an agent that increases endosomal and lysosomal pH (25Middlebrook J.L. Dorland R.B. Microbiol. Rev. 1984; 48: 199-221Crossref PubMed Google Scholar) and therefore inhibits the entry and processing of many toxins, selectively blocked the induction of tyrosine phosphorylation by PMT. Similarly, the early addition of neutralizing antisera to PMT selectively blocked phosphorylation of Gαq. The addition of PMT to cell membranes induced an increase in binding of GTPγS to Gαq. This is the first description of an in vitro assay for PMT activity. PMTC1165S neither affected GDP/GTP exchange nor stimulated an increase in levels of inositol phosphates. Thus the stimulation of tyrosine phosphorylation by PMTC1165S does not lead to activation of Gq. These data demonstrate that stimulation of Gαq tyrosine phosphorylation can occur in the absence of G protein activation and confirm that phosphorylated Gαq is not constitutively active in the basal GDP bound state (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar). Lui et al. (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar) reported that in Rat-1 fibroblasts transformed with the v-src oncogene, inositol phosphate production stimulated by endothelin-1 was increased 6-fold, without changes in the number of receptors. This increased response was mediated through Gq, which was phosphorylated on tyrosine residue(s). Moreover, when extracted G protein was reconstituted with exogenous phospholipase C, ALF4--stimulated Gq activity was significantly increased in extracts from v-src transformed cells. These data implied that phosphorylation of Gq may have a regulatory role in vivo. Subsequent studies by Umemori et al. (12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar, 13Umemori H. Hayashi T. Inoue T. Nakanishi S. Mikoshiba K. Yamamoto T. Oncogene. 1999; 18: 7399-7402Crossref PubMed Scopus (13) Google Scholar) have further investigated the role of Gq tyrosine phosphorylation in regulating G protein activity. These authors demonstrated that Gq was phosphorylated upon ligand activation. This phosphorylation was suggested to prevent interaction of the GPCR with Gq and was essential for the activation of Gq by receptor stimulation (12Umemori H. Inoue T. Kume S. Sekiyama N. Nagao M. Itoh H. Nakanishi S. Mikoshiba K. Yamamoto T. Science. 1997; 276: 1878-1881Crossref PubMed Scopus (127) Google Scholar). However, the data conflict with the previous report of Lui et al. (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar), who clearly demonstrated that phosphorylated Gq in v-src transformed cells could still interact with and be activated by the endothelin-1 receptor. Indeed, in such cells the production of IP3 was potentiated by the phosphorylation of Gq. Similarly, overexpression of c-Src in mouse fibroblasts potentiates both agonist-induced signaling through β-adrenergic receptors and cAMP accumulation in response to cholera toxin (26Bushman W.A. Wilson L.K. Luttrell D.K. Moyers J.S. Parsons S.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7462-7466Crossref PubMed Scopus (36) Google Scholar, 27Moyers J.S. Linder M.E. Shannon J.D. Parsons S.J. Biochem. J. 1995; 305: 411-417Crossref PubMed Scopus (35) Google Scholar). Analysis of the in vitro sites of phosphorylation catalyzed by c-Src identified residues Tyr37 and Tyr377 (27Moyers J.S. Linder M.E. Shannon J.D. Parsons S.J. Biochem. J. 1995; 305: 411-417Crossref PubMed Scopus (35) Google Scholar). Tyr37 lies near the site of Gβγ binding in the N terminus, whereas Tyr377 is located in the extreme C terminus, within a region of Gαs important for receptor interaction. Moreover, phosphorylation of Gαs by immune-complexed c-Src resulted in enhanced rates of receptor-mediated GTPγS binding and GTP hydrolysis (29Hausdorff W.P. Pitcher J.A. Luttrell D.K. Linder M.E. Kurose H. Parsons S.J. Caron M.G. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5720-5724Crossref PubMed Scopus (87) Google Scholar). These data support the findings of Lui et al. (11Liu W.W. Mattingly R.R. Garrison J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8258-8263Crossref PubMed Scopus (34) Google Scholar) and suggest that tyrosine phosphorylation of Gα subunits does not, in itself, prevent interaction of the G protein with the GPCR. Most recently, Liu et al. (28Liu S. Carrillo J.J. Pediani J.D. Milligan G. J. Biol. Chem. 2002; 277: 25707-25714Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) reported that an aromatic group is required for efficient information transfer from an agonist occupied receptor to Gα11. However, their data established that tyrosine 352 could be substituted with phenylalanine or tryptophan without abolishing G protein activity. These findings, together with the data presented here, clearly demonstrate that tyrosine phosphorylation of Gαq/11 is not a requirement for G protein activation in vivo. To further address this problem we investigated the effects of wild-type and mutant PMT on the production of IP3 in response to bombesin. As reported previously (2Murphy A.C. Rozengurt E. J. Biol. Chem. 1992; 267: 25296-25303Abstract Full Text PDF PubMed Google Scholar), wild-type PMT facilitated the production of IP3 in response to bombesin. Interestingly, we have now shown that mutant PMT can also potentiate signaling via the bombesin receptor. Wild-type and mutant PMT each stimulated phosphorylation of Gq, but importantly, only the wild-type toxin activated the G protein. These findings imply that phosphorylation of Gq alone is not sufficient to uncouple the receptor from the G protein. The mechanism through which tyrosine phosphorylation of Gq is induced remains unclear. Our data support the suggestion that a cellular kinase is activated early in the G protein cycle to phosphorylate Gq. Umemori et al. (13Umemori H. Hayashi T. Inoue T. Nakanishi S. Mikoshiba K. Yamamoto T. Oncogene. 1999; 18: 7399-7402Crossref PubMed Scopus (13) Google Scholar) proposed that a kinase is activated in response to GTP-GDP exchange on Gq. However, the findings that mutant PMT can stimulate phosphorylation of Gq in the absence of GTP-GDP exchange suggest that this model may be an oversimplification. To further understand the mechanisms through which phosphorylation of Gαq is regulated, the putative kinase must be identified. Previous work has indicated Src family kinases are critical mediators of this pathway. Our data using Src-deficient cell lines suggest that Src mediates phosphorylation of Gq in response to PMT in vivo. However, because basal phosphorylation of Gq is high in these cells, we cannot exclude the possibility that other kinases may also be involved. In summary, these studies have conclusively demonstrated that PMT activates members of the Gq family of heterotrimeric G proteins. Utilizing GPCR agonists and a mutant form of PMT, we have confirmed that tyrosine phosphorylation of Gαq can be dissociated from G protein activation. Moreover, the finding that wild-type and mutant PMT can potentiate GPCR signaling highlights as yet undefined roles for tyrosine phosphorylation of Gαq. We thank Drs. H. Cox and A. Grigoriadis (King's College London) for helpful comments on the manuscript and Dr. J. F. Battey, Jr. (NIDCD, National Institutes of Health) for the generous gift of the rabbit anti-GRP serum. We also acknowledge Dr. Ron Wilson (King's College London) for help with the statistical analysis." @default.
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• W2012106447 title "Pasteurella multocida Toxin Facilitates Inositol Phosphate Formation by Bombesin through Tyrosine Phosphorylation of Gαq" @default.
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