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

多杀性巴氏杆菌毒素（PMT）是一种细胞内作用的细菌蛋白，以其在体外和体内的强有丝分裂特性以及诱导某些类型细胞的强非锚定生长的能力而闻名。这些特性表明PMT可能具有作为肿瘤促进剂的潜力，特别是在慢性感染的情况下。PMT有丝分裂特性背后的详细机制尚不清楚。最近的报道表明，PMT发挥其生物学作用，部分是通过在异三聚体G蛋白（包括Gaq， Gai， Ga12和Ga13）的α亚基中保守的谷氨酰胺残基的脱酰胺作用，并导致G蛋白的组成活性表型。我们发现rPMT诱导血清饥饿的Swiss 3T3细胞的蛋白质和ATP合成、细胞迁移和增殖。同时，PMT诱导核糖体S6激酶（S6K1）及其底物核糖体S6蛋白（S6）的持续磷酸化。这种磷酸化被雷帕霉素和Torin1这两种哺乳动物雷帕霉素靶蛋白（mTOR）的特异性抑制剂所抑制。在MEF WT中观察到pmt介导的mTOR激活，而在MEF Gaq/11敲除细胞中没有观察到，这与我们的研究结果一致，表明pmt诱导的mTOR激活通过Gaq/11的脱酰胺进行，并导致plc的激活产生二酰基甘油（DAG）和肌醇三磷酸（IP3），这是PKC途径的两种已知激活剂。外源添加的DAG或PMA， PKC的激活剂，以依赖雷帕霉素的方式导致S6磷酸化。此外，pmt诱导的S6磷酸化被PKC抑制剂Go6976抑制。总之，我们的研究结果首次揭示了PMT通过Gaq/11/PLC&#946；/PKC途径激活mTORC1。PMT还诱导EGF受体激活和葡萄糖受体1 （Glut1）上调。然而，它们对S6磷酸化没有影响。事实上，PMT诱导的蛋白质合成和细胞迁移被雷帕霉素部分抑制，这表明PMT可能刺激额外的信号级联反应。

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.