Historically, mechanistic target of rapamycin (mTOR) was purified from mammalian cells using mild nonionic detergents such as NP-40 and or Triton-X100 that resulted in dissociation of core regulatory components essential for its native kinase activity. Consequently, these older kinase assays required MnCl2 to artificially enhance the weak phosphotransfer activity observed (Bai et al., 2007; Kim et al., 2002). With the use of the zwitterionic detergent 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), the mTOR complex 1 (mTORC1) containing Regulatory-associated protein of mTOR (Raptor) and Lst8 (also known as GbetaL) can be successfully purified as a complex. This in vitro kinase assay allows for purification of mTORC1 that resembles its physiological state and retains kinase activity under physiological MgCl2 concentrations (Sancak et al., 2007). The activity of mTORC1 can be further enhanced through the use of hyperactive mutations within the kinase domain of mTOR or inclusion of GTP-bound RAS enriched in brain (Rheb) that is supplemented into the in vitro kinase assays. Rheb is a small-G-protein that binds to and activates mTORC1 to phosphorylate downstream substrates, such as eukaryotic initiation factor 4E-BP1 (4E-BP1) (Burnett et al., 1998), ribosomal protein S6 kinase 1 (S6K1) (Kim et al., 2002), Signal transducer and activator of transcription 3 (STAT3) (Dodd et al., 2015), and proline-rich Akt substrate of 40 kDa (PRAS40) (Dunlop et al., 2009).
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