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Mammalian Target of Rapamycin Complex 1 (mTORC1) Plays a Role in Pasteurella multocida Toxin (PMT)-induced Protein Synthesis and Proliferation in Swiss 3T3 Cells*

  • Hammou Oubrahim
    Correspondence
    To whom correspondence may be addressed: Bldg. 50, Rm. 2130, 50 South Dr., MSC-8012, Bethesda, MD 20892-8012. Tel.: 301-496-8862; Fax: 301-496-0599;
    Affiliations
    From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8012
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  • Allison Wong
    Affiliations
    From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8012
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  • Brenda A. Wilson
    Affiliations
    the Department of Microbiology, University of Illinois at Urbana Champaign, Urbana, Illinois 61801
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  • P. Boon Chock
    Correspondence
    To whom correspondence may be addressed: Bldg. 50, Rm. 2134, 50 South Dr., MSC-8012, Bethesda, MD 20892-8012. Tel.: 301-496-2073; Fax: 301-496-0599;
    Affiliations
    From the Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-8012
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  • Author Footnotes
    * This work is supported, in whole or in part, by the intramural Research Program of the National Heart, Lung, and Blood Institute and by National Institutes of Health and Grant AI038396 (to B. A. W.) from the NIAID.
Open AccessPublished:December 07, 2012DOI:https://doi.org/10.1074/jbc.M112.427351
      Pasteurella multocida toxin (PMT) is a potent mitogen known to activate several signaling pathways via deamidation of a conserved glutamine residue in the α subunit of heterotrimeric G-proteins. However, the detailed mechanism behind mitogenic properties of PMT is unknown. Herein, we show that PMT induces protein synthesis, cell migration, and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (rpS6), in quiescent 3T3 cells. The extent of the phosphorylation is time and PMT concentration dependent, and is inhibited by rapamycin and Torin1, the two specific inhibitors of the mammalian target of rapamycin complex 1 (mTORC1). Interestingly, PMT-mediated mTOR signaling activation was observed in MEF WT but not in Gαq/11 knock-out cells. These observations are consistent with the data indicating that PMT-induced mTORC1 activation proceeds via the deamidation of Gαq/11, which leads to the activation of PLCβ to generate diacylglycerol and inositol trisphosphate, two known activators of the PKC pathway. Exogenously added diacylglycerol or phorbol 12-myristate 13-acetate, known activators of PKC, leads to rpS6 phosphorylation in a rapamycin-dependent manner. Furthermore, PMT-induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö6976. Although PMT induces epidermal growth factor receptor activation, it exerts no effect on PMT-induced rpS6 phosphorylation. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gαq/11/PLCβ/PKC pathway. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that these processes are in part mediated by the mTORC1 pathway.
      Background: Pasteurella multocida toxin (PMT) is a highly mitogenic bacterial protein whose detailed mechanism is not well understood.
      Results: PMT deamidates and activates Gαq/11 leading to mTORC1 activation.
      Conclusion:q/11/PLCβ/PKC-mediated mTORC1 activation is at least partially responsible for PMT-induced cell growth, migration, and proliferation in Swiss 3T3 cells.
      Significance: Understanding PMT-induced mTORC1 activation could help elucidate mechanisms of oncogene regulation.

      Introduction

      Toxin-producing strains of Pasteurella multocida are known to cause pasteurellosis in humans and animals and atrophic rhinitis in swine (
      • Wilson B.A.
      • Ho M.
      Cellular and molecular action of the mitogenic protein-deamidating toxin from Pasteurella multocida.
      ), a pathology characterized by bone loss in the dorsal and ventral nasal turbinates. The gene (toxA) encoding P. multocida toxin (PMT),
      The abbreviations used are: PMT
      Pasteurella multocida toxin
      mTORC1
      mammalian target of rapamycin complex 1
      S6K1
      ribosomal S6 kinase
      DAG
      diacylglycerol
      IP3
      inositol trisphosphate
      RTK
      receptor tyrosine kinase
      RSK
      ribosomal S6 kinase
      EGFR
      epidermal growth factor receptor
      PIP2
      phosphatidylinositol 4,5-bisphosphate
      rpS6
      ribosomal protein S6
      PLCβ
      phospholipase C β
      4E-BP1
      eIF4E-binding protein 1
      PI3K
      phosphatidylinositol 3-kinase
      PMA
      phorbol 12-myristate 13-acetate
      rpS6
      ribosomal S6 protein.
      acquired by horizontal transmission (
      • Pullinger G.D.
      • Bevir T.
      • Lax A.J.
      The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage.
      ), has been cloned and sequenced (
      • Buys W.E.
      • Smith H.E.
      • Kamps A.M.
      • Kamp E.M.
      • Smits M.A.
      Sequence of the dermonecrotic toxin of Pasteurella multocida ssp. multocida.
      ). It is a single polypeptide of 146 kDa whose C-terminal activity domain structure has been solved (
      • Kitadokoro K.
      • Kamitani S.
      • Miyazawa M.
      • Hanajima-Ozawa M.
      • Fukui A.
      • Miyake M.
      • Horiguchi Y.
      Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin.
      ). In addition to its mitogenic properties for certain types of cells, including quiescent fibroblast and osteoclast cells, PMT is a strong inducer of anchorage-independent growth (
      • Rozengurt E.
      • Higgins T.
      • Chanter N.
      • Lax A.J.
      • Staddon J.M.
      Pasteurella multocida toxin. Potent mitogen for cultured fibroblasts.
      ,
      • Lax A.J.
      • Grigoriadis A.E.
      Pasteurella multocida toxin. The mitogenic toxin that stimulates signalling cascades to regulate growth and differentiation.
      ,
      • Higgins T.E.
      • Murphy A.C.
      • Staddon J.M.
      • Lax A.J.
      • Rozengurt E.
      Pasteurella multocida toxin is a potent inducer of anchorage-independent cell growth.
      ). Proliferative properties of PMT have been observed in vivo. Injection of purified recombinant toxin into animals induced proliferation in bladder epithelium without evidence of an inflammatory reaction (
      • Hoskins I.C.
      • Thomas L.H.
      • Lax A.J.
      Nasal infection with Pasteurella multocida causes proliferation of bladder epithelium in gnotobiotic pigs.
      ). PMT also inhibits in vitro differentiation, e.g. bone cells and preadipocytes, where it prevents the formation of mineralized bone nodules and important adipocyte markers, respectively (
      • Aminova L.R.
      • Wilson B.A.
      Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin. Suppression of Notch1, stabilization of β-catenin and pre-adipocyte factor 1.
      ). These properties, growth stimulation and inhibition of cell differentiation, suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections (
      • Lax A.J.
      Opinion. Bacterial toxins and cancer. A case to answer?.
      ).
      Recently, PMT has been shown to exert some of its biological effects through the activation of heterotrimeric G-proteins, which involves Gαq-, Gα11-, Gα12/13-, and Gαi-dependent pathways, via the deamidation of a conserved glutamine residue in the α subunit (
      • Orth J.H.
      • Preuss I.
      • Fester I.
      • Schlosser A.
      • Wilson B.A.
      • Aktories K.
      Pasteurella multocida toxin activation of heterotrimeric G proteins by deamidation.
      ,
      • Kamitani S.
      • Ao S.
      • Toshima H.
      • Tachibana T.
      • Hashimoto M.
      • Kitadokoro K.
      • Fukui-Miyazaki A.
      • Abe H.
      • Horiguchi Y.
      Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated α subunit of the heterotrimeric GTPase Gq.
      ). Abnormal G protein signaling induced by bacterial toxins may lead to diverse biological consequences. Through Gαq activation, PMT activates signaling pathways known to be affected by proto-oncogenes, including those associated with phospholipase C, protein kinase C, ERK1/2 MAPKs, calcium mobilization, and STATs (
      • Staddon J.M.
      • Chanter N.
      • Lax A.J.
      • Higgins T.E.
      • Rozengurt E.
      Pasteurella multocida toxin, a potent mitogen, stimulates protein kinase C-dependent and -independent protein phosphorylation in Swiss 3T3 cells.
      ,
      • Staddon J.M.
      • Barker C.J.
      • Murphy A.C.
      • Chanter N.
      • Lax A.J.
      • Michell R.H.
      • Rozengurt E.
      Pasteurella multocida toxin, a potent mitogen, increases inositol 1,4,5-trisphosphate and mobilizes Ca2+ in Swiss 3T3 cells.
      ,
      • Murphy A.C.
      • Rozengurt E.
      Pasteurella multocida toxin selectively facilitates phosphatidylinositol 4,5-bisphosphate hydrolysis by bombesin, vasopressin, and endothelin. Requirement for a functional G protein.
      ,
      • Seo B.
      • Choy E.W.
      • Maudsley S.
      • Miller W.E.
      • Wilson B.A.
      • Luttrell L.M.
      Pasteurella multocida toxin stimulates mitogen-activated protein kinase via Gq/11-dependent transactivation of the epidermal growth factor receptor.
      ,
      • Zywietz A.
      • Gohla A.
      • Schmelz M.
      • Schultz G.
      • Offermanns S.
      Pleiotropic effects of Pasteurella multocida toxin are mediated by Gq-dependent and -independent mechanisms. involvement of Gq but not G11.
      ,
      • Wilson B.A.
      • Aminova L.R.
      • Ponferrada V.G.
      • Ho M.
      Differential modulation and subsequent blockade of mitogenic signaling and cell cycle progression by Pasteurella multocida toxin.
      ). In addition, PMT has been shown to induce Rho activation, Rho-dependent stress fiber formation, and FAK phosphorylation in a Gα12/13-dependent manner (
      • Thomas W.
      • Pullinger G.D.
      • Lax A.J.
      • Rozengurt E.
      Escherichia coli cytotoxic necrotizing factor and Pasteurella multocida toxin induce focal adhesion kinase autophosphorylation and Src association.
      ,
      • Orth J.H.
      • Lang S.
      • Taniguchi M.
      • Aktories K.
      Pasteurella multocida toxin-induced activation of RhoA is mediated via two families of Gα proteins, Gαq, and Gα12/13.
      ). However, the effects of PMT on signaling pathways associated with stimulation of protein synthesis and cell proliferation have not been studied.
      The mammalian target of rapamycin (mTOR), a key Ser-Thr kinase highly conserved from yeast to mammals, exists intracellularly in two functionally distinct complexes, mTORC1 and mTORC2 (
      • Jacinto E.
      What controls TOR?.
      ,
      • Ma X.M.
      • Blenis J.
      Molecular mechanisms of mTOR-mediated translational control.
      ,
      • Zoncu R.
      • Efeyan A.
      • Sabatini D.M.
      mTOR. From growth signal integration to cancer, diabetes and ageing.
      ). mTORC1 consists of the mTOR catalytic subunit and associated proteins raptor, PRAS40, and mLST8/GβL. This complex is involved in the regulation of protein synthesis, cell growth, proliferation, and autophagy in a nutrient- and energy-responsive manner. It has been shown that activation of mTORC1 leads to the rapamycin-sensitive phosphorylation of S6K1, which in turn phosphorylates ribosomal S6 protein (rpS6) (
      • Jacinto E.
      What controls TOR?.
      ,
      • Ma X.M.
      • Blenis J.
      Molecular mechanisms of mTOR-mediated translational control.
      ,
      • Zoncu R.
      • Efeyan A.
      • Sabatini D.M.
      mTOR. From growth signal integration to cancer, diabetes and ageing.
      ,
      • Pullen N.
      • Thomas G.
      The modular phosphorylation and activation of p70S6k.
      ,
      • Choo A.Y.
      • Yoon S.O.
      • Kim S.G.
      • Roux P.P.
      • Blenis J.
      Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation.
      ). The mTORC2 is activated by growth factors via a mechanism involving mTOR, rictor, mLST8/GβL, mSin1, and protor. Active mTORC2 activates Akt/PKB, PKCα, and regulates actin cytoskeletal organization.
      Here we show that PMT stimulates protein synthesis, ATP production, and cell proliferation and migration in serum-starved Swiss 3T3 fibroblast cells. Concomitantly, PMT also activates mTORC1, monitored by the phosphorylation of rpS6. To elucidate the role of mTORC1 in PMT-induced protein synthesis, we investigated the effect of rapamycin and Torin1, the specific inhibitor of mTORC1, on PMT-induced activation of S6K1 and protein synthesis. Our results reveal that PMT activates mTORC1 via a PKC-mediated pathway, Furthermore, our data also indicate that PMT-induced protein synthesis is mediated in part by the mTORC1 pathway.

      Acknowledgments

      We thank Dr. Shigeki Kamitani and Prof. Stefan Offermanns for providing the 3G3 monoclonal antibody that recognizes the deamidated form of Gαq and MEF wild type and Gαq/11-deficient cells, respectively.

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