FIGURE 5.

ERK1/2 phosphorylate Raptor on Ser⁶⁹⁶ and Ser⁸⁶³in vivo. A, cells were transfected with myc-tagged Raptor and stimulated with 50 ng/ml PMA following pretreatment with 10 μm U0126 or vehicle for 30 min. Immunoprecipitated Raptor was then assayed for phosphorylation using phosphospecific antibodies. B, cells were transfected with myc-tagged Raptor and aui-tagged mTOR, serum-starved, and stimulated with 50 ng/ml PMA or 100 nm insulin following pretreatment with 10 μm PD184352 or 25 nm rapamycin and treated as in A. C, endogenous Raptor phosphorylation at Ser⁶⁹⁶ and Ser⁸⁶³ was determined in U2OS and HeLa cells. Both cell types were serum-starved overnight, and total cell lysates were assayed for Raptor phosphorylation using phosphospecific antibodies against Ser⁶⁹⁶ and Ser⁸⁶³. D, phosphorylation of endogenous Raptor in HEK293 cells stimulated with 50 ng/ml PMA or 25 μg/ml EGF for 20 and 10 min, respectively, is shown. Cells were either transfected with an siRNA with a scrambled sequence (Scr), or targeting both ERK1 and ERK2. Phosphorylation of endogenous Raptor in total cell lysates was performed as in A.