FIGURE 2.

Identification of Ser⁸⁶³ as a site of raptor phosphorylation in intact cells and generation of phosphospecific Ser⁸⁶³ antibodies. A, localization of cluster 1 (Ser⁶⁹⁶/Thr⁷⁰⁶) and cluster 2 (Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) P-sites within the domain structure of raptor. The six P-sites identified map to a central, poorly conserved region of raptor that localizes between the N-terminal RNC (raptor N-terminal conserved) domains and HEAT repeats and the C-terminal WD40 repeats. B, low energy, collision-induced dissociation spectrum of the doubly charged raptor Ser(P)⁸⁶³ phosphopeptide. HEK293 cells were transfected with Myc-raptor, cultured in DMEM/FBS, and WCL was immunoprecipitated (IP) with Myc antibodies. A Coomassie-stained band of Myc-raptor (upper panel) was digested with trypsin after SDS-PAGE. Liquid chromatography-MS/MS and data analysis were conducted as described under “Experimental Procedures.” Note that the y₄–y₇ ion series clearly places the site of phosphorylation on Ser⁸⁶³. C, Ser(P)⁸⁶³ antibodies are site-specific. HEK293 cells were transfected with vector control (−) or with WT or S863A Myc-raptor alleles, as indicated. WCL was immunoprecipitated (IP) with Myc antibodies and immunoblotted (IB) with the indicated antibodies. D, Ser(P)⁸⁶³ antibodies are phosphospecific. HEK293 lysate was immunoprecipitated with preimmune (PI) antibodies (lane 1) or with anti-raptor antibodies (lanes 2–4). The immunoprecipitates were resuspended immediately in sample buffer (lanes 1 and 2) or washed in phosphatase (PPase) buffer and incubated in the absence (lane 3) or presence (lane 4) of λ-phosphatase. The immunoprecipitates were immunoblotted as indicated.