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. Author manuscript; available in PMC: 2013 Apr 19.
Published in final edited form as: Oncogene. 2011 Sep 12;31(16):2115–2120. doi: 10.1038/onc.2011.404

Figure 3.

Figure 3

The charged amino acids replacing Gly-934 residue of rictor prevent the rictor’s binding to Sin1. (a) The myc-tagged wild-type rictor and its G934E, G934D, G934K, G934L, G934Q mutants and Sin1-V5 cDNAs were transiently expressed in 293T cells. To equalize the level of expression for non-interacting mutants, the indicated increased amounts of DNA (5 ×) for both rictor and Sin1 were applied for transfection. After 48 h, the transfected cells were harvested and rictor/Sin1 complexes were immunoprecipitated as described in Figure 1b. Whenever non-polar Gly-934 is mutated to any charged amino acid, rictor/Sin1 interaction is impaired. Non-charged amino acids at this site have the wild-type phenotype. (b) The summary table indicating the property of the rictor mutants to interact with Sin1. In our study, we developed and analyzed seven mutants of rictor: G934E, G934D, G934K, G934L, G934Q, L923E and L943E. (c) To assemble mTORC2, 293T cells were transfected with 1 μg myc-rictor, 500 ng HA-mTOR, 400 ng Sin1-V5 and 100 ng HA-mLST8 cDNAs. For G934E mutant 5 μg rictor was used. Following cell lysis, mTOR complexes were immunoprecipitated and used for in vitro kinase assay with full-length GST-Akt as a substrate, as has been previously described (Sarbassov et al., 2005b). 15 μl of kinase buffer containing 500 ng inactive Akt1-GST and 500 μm ATP were added to the immunoprecipitates and incubated at 37 °C for 20 min. The reaction was stopped by the addition of 200 μl ice-cold enzyme dilution buffer (20 mm 3-(N-morpholino)propanesulfonic acid, pH 7.0, 1 mm EDTA, 0.3% CHAPS, 5% glycerol, 0.1% 2-mercaptoethanol and 1 mg/ml bovine serum albumin). After a quick spin the supernatant was removed from the protein G-agarose and a 15 μl portion of the former was analyzed by immunoblotting for phospho-S473 Akt and total Akt levels. The pelleted protein G-agarose beads were also analyzed to determine the levels of rictor, Sin1 and mTOR in the immunoprecipitates. These data indicate that rictor G934L mutant is assembled into the functional mTOR complex 2 as determined by in vitro kinase assay.