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 findings 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 modified to study the effect of small molecules on mTORC2 activity.