Receptor-Recognized α2-Macroglobulin Binds to Cell Surface-Associated GRP78 and Activates mTORC1 and mTORC2 Signaling in Prostate Cancer Cells

Objective: Tetrameric alpha(2)-macroglobulin (alpha M-2), 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 alpha M-2 (alpha M-2*) binds to cancer cell surface GRP78 and triggers proliferative and antiapoptotic signaling. We have studied the role of alpha M-2* in the regulation of mTORC1 and TORC2 signaling in the growth of human prostate cancer cells. Methods: 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. Results: Stimulation of cells with alpha M-2* 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. alpha M-2* 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 alpha M-2*-induced S6-Kinase phosphorylation at T389 and kinase activity in Raptor immunoprecipitates. alpha M-2*-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 alpha M-2*-induced phosphorylation of Akt(S473) phosphorylation in Rictor immunoprecipitates. Conclusion: Binding of alpha M-2* to prostate cancer cell surface GRP78 upregulates mTORC1 and mTORC2 activation and promotes protein synthesis in the prostate cancer cells.