Department of Pathology, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS One. 2012;7(12):e51735. doi: 10.1371/journal.pone.0051735. Epub 2012 Dec 14.
Tetrameric α(2)-macroglobulin (α(2)M), a plasma panproteinase inhibitor, is activated upon interaction with a proteinase, and undergoes a major conformational change exposing a receptor recognition site in each of its subunits. Activated α(2)M (α(2)M*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of α(2)M* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells.
Employing immunoprecipitation techniques and Western blotting as well as kinase assays, activation of the mTORC1 and mTORC2 complexes, as well as down stream targets were studied. RNAi was also employed to silence expression of Raptor, Rictor, or GRP78 in parallel studies.
Stimulation of cells with α(2)M* promotes phosphorylation of mTOR, TSC2, S6-Kinase, 4EBP, Akt(T308), and Akt(S473) in a concentration and time-dependent manner. Rheb, Raptor, and Rictor also increased. α(2)M* treatment of cells elevated mTORC1 kinase activity as determined by kinase assays of mTOR or Raptor immunoprecipitates. mTORC1 activity was sensitive to LY294002 and rapamycin or transfection of cells with GRP78 dsRNA. Down regulation of Raptor expression by RNAi significantly reduced α(2)M*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. α(2)M*-treated cells demonstrate about a twofold increase in mTORC2 kinase activity as determined by kinase assay of Akt(S473) phosphorylation and levels of p-Akt(S473) in mTOR and Rictor immunoprecipitates. mTORC2 activity was sensitive to LY294002 and transfection of cells with GRP78 dsRNA, but insensitive to rapamycin. Down regulation of Rictor expression by RNAi significantly reduces α(2)M*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates.
Binding of α(2)M* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.
四聚体 α(2)-巨球蛋白（α(2)M）是一种血浆泛蛋白酶抑制剂，在与蛋白酶相互作用时被激活，并经历主要构象变化，在每个亚基中暴露受体识别位点。激活的 α(2)M（α(2)M*）与癌细胞表面的 GRP78 结合，并触发增殖和抗凋亡信号。我们研究了 α(2)M* 在调节人前列腺癌细胞中 mTORC1 和 TORC2 信号的作用。
采用免疫沉淀技术和 Western blot 以及激酶测定法，研究 mTORC1 和 mTORC2 复合物以及下游靶标的激活情况。还采用 RNAi 沉默 Raptor、Rictor 或 GRP78 的表达，进行平行研究。
细胞刺激 α(2)M以浓度和时间依赖性方式促进 mTOR、TSC2、S6-激酶、4EBP、Akt(T308)和 Akt(S473)的磷酸化。Rheb、Raptor 和 Rictor 也增加。α(2)M处理细胞可提高 mTORC1 激酶活性，如通过 mTOR 或 Raptor 免疫沉淀物的激酶测定法测定。mTORC1 活性对 LY294002 和雷帕霉素敏感，或用 GRP78 dsRNA 转染细胞。RNAi 下调 Raptor 表达可显著降低 α(2)M*-诱导的 Raptor 免疫沉淀物中 S6-激酶在 T389 的磷酸化和激酶活性。用激酶测定法测定 Akt(S473)磷酸化和 mTOR 和 Rictor 免疫沉淀物中 p-Akt(S473)水平，α(2)M*-处理的细胞显示 mTORC2 激酶活性增加约两倍。mTORC2 活性对 LY294002 和用 GRP78 dsRNA 转染细胞敏感，但对雷帕霉素不敏感。RNAi 下调 Rictor 表达可显著降低 Rictor 免疫沉淀物中 α(2)M*-诱导的 Akt(S473)磷酸化。
α(2)M*与前列腺癌细胞表面的 GRP78 结合可上调 mTORC1 和 mTORC2 的激活，并促进前列腺癌细胞中的蛋白质合成。
