PMC

Model depicting that both ERK/RSK1- and PI3K/Akt-dependent phosphorylation of tuberin regulates mTOR signaling. Activation of both pathways results in the phosphorylation of tuberin on RXRXXpS/T consensus sites, which include Ser-939, Thr-1462, and Ser-1798.

RSK1 and Akt interact with tuberin and hamartin in cells. (A) HEK293 cells were cotransfected with Flag-tuberin/hamartin, and either HA-tagged RSK1, Akt, or S6K1, and serum starved for 16–18 h before harvesting. Immunoprecipitates of tuberin/hamartin were immunoblotted for the presence of RSK1, Akt, and S6K1. All protein levels were determined by immunoblotting whole cell lysates. (B). HEK293 cells were transfected as above with wild-type RSK1 or kinase-inactive RSK1, and assayed for their association to tuberin/hamartin after PMA stimulation. Immunoprecipitates and total cell extracts were immunoblotted for tuberin/hamartin, RSK1, and ERK1/2.

Ser-1798 is the major regulatory phosphorylation site stimulated by PMA. HEK293 cells were transfected with either tuberin wild-type or various point mutants, and treated with either PMA (A) or insulin (100 nM) (B). Immunoprecipitated tuberin/hamartin were immunoblotted for protein levels and tuberin phosphorylation on RXRXXpS/T sites. Total cell lysates were immunoblotted by using antibodies against ERK1/2 and phosphorylated Akt on Ser-473. (C) HEK293 cells were transfected with HA-S6K1 and either tuberin (wt), tuberin S1798A, tuberin S939A/T1462A (SATA), or tuberin SATA S1798A mutants, and treated as in A and B. Immunoprecipitated S6K1 was assayed for activity as in , and results from three independent experiments are represented in a histogram as the means ± SE. Total cell lysates were immunoblotted for tuberin/hamartin levels, S6K1, and ERK1/2.

Analysis of tuberin phosphorylation by using MS. (A) HEK293 cells were transfected with Flag-tagged tuberin/hamartin and treated as indicated. Immunoprecipitated tuberin/hamartin were immunoblotted for tuberin phosphorylation by using 5% of the total sample. The remainder of the sample was subjected to SDS/PAGE and prepared to be analyzed by liquid chromatography tandem MS (LC-MS/MS). (B) The positions of the identified RXRXXpS/T phosphorylation sites on tuberin are illustrated in a schematic (Ser-939, Ser-981, Ser-1420, and Ser-1798). (C) MS/MS spectrum of the C-terminal peptide containing phosphorylated Ser-1798 characterized in this study. The doubly charged precursor ion had an m/z of 733.6. (D) Shown is a primary alignment of the C termini of tuberin from human, rat, Takifugu rubripes (fish), and Drosophila (fly).

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.

Activation of the Ras/ERK signaling cascade induces tuberin phosphorylation. (A) S6K1 and tuberin/hamartin-transfected HEK293 cells were serum-starved for 16–18 h, pretreated with either wortmannin (100 nM; Wort), U0126 (10 μM), or rapamycin (25 nM; RAP) for 30 min, and then stimulated with either insulin (100 nM) or PMA (100 ng/ml) for an additional 15 min. Immunoprecipitated tuberin/hamartin were immunoblotted for protein levels and tuberin phosphorylation on RXRXXpS/T motif [α-(P)tuberin]. Total cell lysates were immunoblotted for S6K1, Akt phospho-T308, and ERK1/2. Part of the initial lysates was used to immunoprecipitate S6K1 by using anti-HA antibodies and assayed in vitro for S6K1 kinase activity by using GST-S6 as substrate. A typical autoradiogram is shown, and results from three independent experiments are represented in a histogram as the means ± SE. (B) HEK293 cells were cotransfected with tuberin/hamartin, and constructs expressing activated MEK1 (MEK-DD) and Ras (Ras61L), or dominant negative Ras (Ras17N). Cell extracts were immunoprecipitated and immunoblotted as in A.(C) Endogenous tuberin was immunoprecipitated from cells treated as in A and with bisindolylmaleimide I (5 μM; BIM), and immunoblotted for phosphorylation on RXRXXpS/T motifs and total levels by using an anti-tuberin antibody. Total cell extracts were immunoblotted for phosphorylated ERK1/2, RSK, and S6K1, and for corresponding total proteins.