Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase

Abstract

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.

Fig. 1.

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.

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.

Fig. 3.

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.

Fig. 4.

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).

Fig. 5.

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 Fig. 1, 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.

Fig. 6.

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.