|
|导读： An In Vitro Assay for the Kinase Activity of mTOR Complex 2
The mTOR complex 2 (mTORC2) is a protein kinase complex involved in many important physiological processes through the regulation of its substrates, such as Akt, SGK1, and conventional PKC. Its activity is modulated negatively by interaction with DEPTOR and positively by the TSC1–TSC2 protein complex. To study the regulation of mTORC2 activity, it is a common practice to examine the phosphorylation of its substrates in vivo and verify the fi ndings with an in vitro assay of kinase activity. This kinase assay measures the phosphorylation of exogenously derived Akt by mTORC2 immunoprecipitates isolated from cells and can be modifi ed to study the effect of small molecules on mTORC2 activity.
|背景介绍： The serine/threonine kinase, mTOR, is a member of the PIKK (phosphatidylinositol kinase-related kinase) family and functions in at least two well-defined multiprotein complexes.In mTOR complex 1 (mTORC1), mTOR associates with RAPTOR (regulatory associated protein of mTOR) and mLST8 (mammalian lethal with sec18 protein 8). The mTOR complex 2 (mTORC2) is biochemically distinct from mTORC1.In this complex, mTOR associates with RICTOR (rapamycin-insensitive companion of mTOR), mSIN1 (stress-activated protein kinase interacting protein-1), and mLST8 and is implicated in the phosphorylation of two conserved motifs in some members of the AGC family of kinases. The most well-characterized substrate of mTORC2 is S-473 on the hydrophobic motif of Akt.Similar motifs on protein kinase C a (PKC a, Ser657) and serum- and glucocorticoid-induced protein kinase-1 (SGK1, Ser422) have also been shown to be phosphorylated by mTORC2.The phosphorylation of the “turn” motifs of Akt (T450) and some isoforms of PKC (e.g., T497 of PKC a) are also mediated by mTORC2. Through the regulation of these substrates, mTORC2 play a significant role in malignant transformation, fat metabolism and growth.Recent studies have demonstrated that the activity of mTORC2 is negatively regulated by DEPTOR, and positively regulated by the TSC1–TSC2 protein complex, and these effects are mediated through direct interaction with mTORC2.On the contrary, phosphorylation of RICTOR by ribosomal protein S6 kinase-1 (S6K1) and interaction of mTORC2 with PROTOR have no effect on mTORC2 kinase activity. Although currently known chemical inhibitors of mTOR kinase lack specificity and affect both mTORC1 and mTORC2, efforts to discover other regulatory mechanisms may reveal those that can be targeted by small molecules to modulate mTORC2 activity specifically. For these studies, measuring mTORC2 activity in vitro should be useful as a biochemical tool to complement the detection of substrate phosphorylation in the determination of the functional status of this protein kinase complex.
Fig. 1. Kinase activity of mTORC2 is stimulated by insulin and inhibited in vitro by LY294002. HEK293 cells were serum starved overnight and stimulated with insulin (Ins, 100 nM) for 15 min before lysis and immunoprecipitation (IP) with control IgG or antiRICTOR antibodies (Ab). Where indicated, LY294002 (LY; 15 m M) was added to the kinase reaction. Kinase activity of the RICTOR immunoprecipitates was assessed by determining the increase of Akt (S473) phosphorylation in the inactive Akt substrate used. Some basal Akt phosphorylation can be detected in the Akt substrate when the fi lm is exposed for a longer duration.
|
|
