FIGURE 1.

Identification of Ser-212 and Ser-221 as in vitro mTORC1-catalyzed phosphorylation sites in PRAS40. 3T3-L1 adipocytes were incubated with insulin (60 nm) for 30 min (A and D). A, immune complex kinase assays were performed with [γ-³²P]ATP and PRAS40 wild type (wt) and the indicated mutative proteins as substrates after conducting immunoprecipitations (IP) of raptor, Akt1, and S6K1. After SDS-PAGE, a phosphor image of ³²P-labeled PRAS40 (left) and immunoblots of mTOR, raptor, PRAS40, Akt1, and S6K1 (right) were prepared, and substrates were stained with 0.5% Ponceau S, as shown with substrate level. 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). B, mTOR wild type, kinase-dead (S2338A, KD), and constitutive active (deleting 2433–2451, ΔRD) mutants were co-expressed with raptor in HEK293 cells. mTOR complexes were isolated with antibodies toward FLAG-tagged mTOR, and then mTOR kinase assays were performed with PRAS40 as substrate. C, PRAS40 wild type and mutants as indicated were phosphorylated in vitro by mTORC1 immune complexes (raptor IP) from insulin-stimulated HEK293 cells. After SDS-PAGE and transferring, ³²P-incorporated PRAS40 was excised from the polyvinylidene difluoride membrane, digested with trypsin, and then subjected to two-dimensional phosphopeptide mapping. The results were visualized by a PhosphorImager. The pattern of migration for the three identified sites is shown in the last panel. D, phosphorylation of the indicated PRAS40 mutants in kinase assays with mTORC1 and Akt1 immune complexes were assessed by ³²P incorporation. 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). E, washed raptor antibody immune complexes isolated from HEK293 cells were incubated in kinase reaction mixtures containing no additions (None) for 20 min or the following: 10 μm LY294002 (LY), 5 μm rapamycin (rapa), 5 μm GST-FKBP12 (BP12), or 5 μm GST-FKBP12 plus 5 μm rapamycin (rapa + BP12). Phosphor images of ³²P-labeled PRAS40 wild type and mutants of S212A and S183A/S221A are presented.