FIGURE 2.

ERK1/2 phosphorylate Raptor in vitro. A, immunoprecipitated HA-tagged WT ERK1 or HA₆-tagged ERK2 from serum-starved or PMA-stimulated cells lysed in a Nonidet P-40 containing buffer was incubated with immunopurified Raptor in a kinase reaction with [γ-³²P]ATP. The resulting samples were subjected to SDS-PAGE, and the dried Coomassie Blue-stained gel was autoradiographed. B, samples were treated as in A, but kinase reactions were performed without radioactivity. The resulting samples were immunoblotted for Raptor phosphorylation on pS/T-P consensus sites. C, as in A, but ERK2 was immunoprecipitated from cells treated with 50 ng/ml PMA in the presence or absence of 10 μm U0126. The levels of ³²P incorporation into Raptor were quantified and are shown below the autoradiogram. D, recombinant activated ERK1 was incubated with immunopurified WT or S3A Raptor in a kinase reaction, and the resulting samples were immunoblotted for Raptor phosphorylation on pS/T-P consensus sites.