FIGURE 3.

Disassociation of mTOR and raptor by Triton X-100 and disruption of the TOS motif in PRAS40 are unable to eliminate phosphorylation of PRAS40. 3T3-L1 adipocytes were incubated with insulin (60 nm) for 30 min. A, mTORC1 and Akt1 were isolated from adipocyte extracts in the presence of 0.2% Tween 20 by using raptor and Akt1 antibodies. The indicated detergents (0.2% final concentration) were added to the washed complexes, which were then incubated with [γ-³²P]ATP and PRAS40 or 4E-BP1. ³²P-Labeled PRAS40 and 4E-BP1 were detected by phosphorimaging. The amounts of incorporated ³²P in PRAS40 and 4E-BP1 relative to the level of phosphorylation in the presence of Tween 20 with insulin treatment (%TW) were determined (mean ± S.E. from three experiments). TW-20, Tween 20; TX-100, Triton X-100. B, mTORC1 and Akt1 kinase assays were conducted using PRAS40 wild type (wt) and the indicated mutative proteins as substrates. The effect on ³²P incorporation into PRAS40 (corrected for substrate level) is expressed as a percentage relative to wild type (mean ± S.E. from three experiments), and a t test was conducted between wild type and mutants. IP, immunoprecipitation.