Mechanism of PMT-Induced Anchorage-Independent Growth and mTOR Signaling

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Pasteurella multocida toxin (PMT) is an intracellular acting bacterial protein known for its potent mitogenic properties in vitro and in vivo and its ability to induce strong anchorage-independent growth for certain type of cells. These properties suggest that PMT might have the potential to act as a tumor promoter especially in the case of chronic infections. The detailed mechanism behind mitogenic properties of PMT is unknown. Recent reports show that PMT exerts its biological effects, in part, via the deamidation of a conserved glutamine residue in the alpha subunit of heterotrimeic G proteins, including Gaq, Gai, Ga12, and Ga13, and leads to a constitutively active phenotype of the G proteins. We showed that rPMT induces protein and ATP synthesis, cell migration and proliferation in serum-starved Swiss 3T3 cells. Concomitantly PMT induces a sustained phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, ribosomal S6 protein (S6). This phosphorylation is inhibited by rapamycin and Torin1, two specific inhibitors of mammalian target of rapamycin (mTOR). The PMT-mediated mTOR activation was observed in MEF WT but not in MEF Gaq/11 knockout cells, consistent with our results indicating that PMT-induced mTOR activation proceeds via the deamidation of Gaq/11 and leads to the activation of PLCβ to generate diacylglycerol (DAG) and inositol trisphosphate (IP3), two known activators of PKC pathway. Exogenously added DAG or PMA, activators of PKC, leads to S6 phosphorylation in a manner dependent on rapamycin. Furtheremore, PMT-induced S6 phosphorylation is inhibited by PKC inhibitor, Go6976. Together, our findings reveal for the first time that PMT activates mTORC1 through the Gaq/11/PLCβ/PKC pathway. PMT also induces EGF receptor activation and glucose receptor I (Glut1) upregulation. However, they exert no effect on S6 phosphorylation. The fact that PMT-induced protein synthesis and cell migration is partially inhibited by rapamycin indicates that PMT could likely stimulate additional signaling cascades. In addition, an increasing body of evidence supports the idea that extracellular matrix (ECM) proteins are major players in the global control of intercellular communication and integration of environmental signals. It was not known whether rPMT-treated cells are able to express and secrete into the medium a substrate(s) capable of activating autocrine and/or paracrine signaling. We found that the conditioned medium from rPMT-treated cells activates mTOR and MAPK signaling, but not membrane-associated tyrosine kinase signaling. Surprisingly, this diffusible factor(s) is capable of activating mTOR and MAPK pathways even in MEF Gaq/11 double knockout cells. Microarray analysis identified connective tissue growth factor (CTGF) mRNA as the most upregulated gene in 3T3 cells, along with other genes involved in cell proliferation and cancer biology. The elevation of CTGF, an ECM protein upregulated in certain cancers and in fibrosis, was confirmed by RT-PCR and Western blot analysis. In accord with rPMT-induced mTOR activation, upregulation of CTGF was mediated by deamidation of Gaq/11, and was independent of TGFβ, a well known inducer of CTGF. Furthermore, MEK/ERK but not mTOR regulates rPMT-induced upregulation of CTGF at the translational level. Importantly, overexpression of CTGF in mammalian cells leads to S6 phosphorylation, a readout of mTOR activation. These findings reveal that CTGF plays an important role, but there are additional factors involved in the mitogenic action of PMT.