Fig. 2.

RSK1 directly phosphorylates tuberin both in vitro and in vivo. (A) HEK293 cells were transfected with either HA-RSK1, kinase inactive RSK1 (K112/464R), or control vector (pRK7), serum starved for 16–18 h, and stimulated with epidermal growth factor (25 ng/ml) for 10 min. Immunoprecipitated RSK1 was incubated with HEK293-derived Flag-tuberin/hamartin, and incubated for 15 min at 30°C in a kinase reaction containing [³²P]ATP. The resulting samples were subjected to SDS/PAGE, and the dried gel was autoradiographed. (B) In a similar experiment, a kinase assay reaction without [³²P]ATP was performed for 60 min, and samples were immunoblotted for tuberin phosphorylation on RXRXXpS/T sites. (C) HEK293 cells were transfected with tuberin/hamartin, and either RSK1, myrRSK1, or RSK1 K112/464R, and treated as indicated. Tuberin/hamartin immunoprecipitates and total cell extracts were immunoblotted for tuberin levels and phosphorylation, RSK1 (anti-avian), and ERK1/2. The data presented are representative of at least three independent experiments.