FIGURE 6.

Rheb promotes multisite raptor phosphorylation in Ser(P)⁸⁶³-dependent and -independent manners. A, raptor Ser(P)⁸⁶³ is required for Rheb-induced supershift of Myc-raptor on SDS-PAGE. HEK293 cells were co-transfected with WT or S863A Myc-raptor (0.5 μg) and AU1-mTOR (2 μg) with or without FLAG-Rheb (2 μg). Cells were serum deprived, WCLs were immunoprecipitated (IP) with Myc antibodies, and immunoprecipitates were resolved and immunoblotted (IB) as indicated. WCL was also immunoblotted directly to confirm expression of the transfected proteins as well as the expected activation of mTORC1 signaling by FLAG-Rheb. Note, immunoprecipitates were resolved on 8% SDS-PAGE and run at 35 mA for 8 (long run) or 4 h (short run). B, Rheb promotes raptor phosphorylation on multiple sites, with Ser⁸⁵⁵ and Ser⁸⁵⁹ phosphorylation occurring in a Ser(P)⁸⁶³-dependent manner. HEK293 cells were transfected and analyzed as in A, employing WT, S863A, and S863D Myc-raptor alleles. Myc-raptor immunoprecipitates from serum-deprived cells were immunoblotted with our panel of raptor phosphospecific antibodies (e.g. Ser(P)⁸⁶³, Ser(P)⁸⁵⁹, Ser(P)⁸⁵⁵, Ser(P)⁶⁹⁶, Thr(P)⁷⁰⁶, and Ser(P)⁸⁷⁷) as well as with a commercially available phosphospecific antibody (e.g. Ser(P)⁷⁹²). Note: immunoprecipitates were resolved on 6% SDS-PAGE and run at 35 mA for ∼2.5 h. C, phospho-specificity of P-raptor antibodies. HEK293 cells were co-transfected, treated, and immunoprecipitated as in A. In lanes 4, 7, and 10, Myc-raptor immunoprecipitates were incubated with λ-phosphatase in vitro and immunoblotted as indicated. WCL was also immunoblotted directly to confirm expression of the transfected proteins as well as the expected activation of mTORC1 signaling by FLAG-Rheb.