(A and B) Schematic of mTOR (A) and RAPTOR (B) illustrate the domains and locations of candidate PKA sites. (C) myc-mTOR (left panel) and myc-RAPTOR (right panel) were transfected into HEK293 cells for 24 hours; myc-tagged proteins were IP from cell lysates with anti–c-myc–conjugated beads, washed twice with the lysis buffer, and incubated with PKA catalytic (cPKA) subunit, followed by Western blotting for PKA substrate and myc detection. (D) HEK293 cells transfected with myc-mTOR plasmid and treated 24 hrs later with the PKA inhibitor H89 (15 μM) for 30 minutes, followed by Fsk (10 μM) or Iso (10 μM) for 30 minutes treatment. Myc-mTOR was IP from cell lysates with anti–c-myc–conjugated beads, and Western blots were probed with PKA substrate antisera or anti-myc antisera as indicated. (E) HEK293 cells expressing myc-RAPTOR were treated and analyzed as in panel D. (F and G) HEK293 cells were transfected with either myc-mTOR plasmid (F) or myc-RAPTOR plasmid (G) and treated 24 hours later with Iso (1 μM) or Ins (100 nM) for 30 min, followed by process as in D. (H) HEK293 cells were transfected with myc-mTOR or triple Ser mutant (S3A: S1276A/S1288A/S2112A), followed by the procedure as in C. (I) WT myc-mTOR or the S3A vector was expressed in HEK293 cells, treated as in D, and processed to measure PKA motif phosphorylation or myc as indicated. (J) HEK293 cells were transfected with myc-RAPTOR or S791A plasmid for 24 hrs and proceeded as in C. (K) WT myc-RAPTOR and the Ser mutants S791A and S791A/S792A were expressed in HEK293 cells, treated as in D, and processed to measure PKA motif phosphorylation or myc as indicated. The sample indicated as Iso* are nontransfected cells treated with Iso, revealing a nonspecific band (*). GAPDH served as loading input control.