Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products hamartin and tuberin, respectively. Here, we show that hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.

Figure 1

Hamartin and tuberin inhibits PI3K-dependent 4E-BP1 phosphorylation. HEK293E cells coexpressing Flag-tagged hamartin (Ham) and tuberin (Tub), where indicated, with HA-tagged 4E-BP1 were serum-starved and pretreated with 20 nM rapamycin (rap) for 30 min before being stimulated with insulin (100 nM) or PMA (100 ng/ml) for 30 min, where indicated. (A) The levels and Thr-308 phosphorylation of Akt and the levels and phosphorylation of the MAPK isoforms (p44 and p42) were determined. (B) Hamartin and tuberin protein levels were accessed from the cell lysates (Sol) and the insoluble fraction (Non-Sol) as described in Materials and Methods by using the anti-Flag antibody. Thr-1462 tuberin phosphorylation was analyzed by using a tuberin Thr-1462 phospho-specific antibody. (C) The phosphorylation of exogenous 4E-BP1 was determined with an anti-HA antibody and phospho-specific antibodies for 4E-BP1 at Thr-37 and/or 46, Ser-65, and Thr-70, as indicated. The α-, β-, and γ-species of 4E-BP1 are labeled accordingly. (D) Cell extracts were subjected to affinity chromatography on m⁷GTP-Sepharose, as described in Materials and Methods. The levels of eIF4E and exogenous 4E-BP1 that was copurified were determined.

Figure 2

Hamartin and tuberin inhibit 4E-BP1 phosphorylation and S6K1 activity within proliferating cells. U20S cells overexpressing 4E-BP1 (A) or S6K1 (B) with or without hamartin (Ham) and tuberin (Tub), where indicated, were grown in serum and treated with 20 nM of rapamycin (Rap) for 30 min, as indicated. Hamartin, tuberin, and MAPK isoform (as a loading control) protein levels are shown. The α-, β-, and γ-species of 4E-BP1 are labeled accordingly. S6K1 kinase assays were carried out as described in Materials and Methods. The total levels of S6K1 are shown. Incorporation of ³²P label into GST-S6 was assessed, and an autoradiograph of the gel is presented. The ratios of ³²P label incorporated into GST-S6 were normalized against the empty vector (pRK7). The data presented are representative of at least three experiments. (C) HEK293E cells overexpressing 4E-BP1 with or without hamartin (Ham), tuberin (Tub), and the tuberin K599M mutant [Tub(K599M)], where indicated, were serum-starved and then stimulated with 100 nM insulin for 30 min, where indicated. Hamartin and tuberin expression and the extent of phosphorylation of 4E-BP1 was analyzed as for Fig. 1C.

Figure 3

Tuberin-hamartin inhibition of S6K1 depends on mTOR. (A) Diagrammatic representation of the S6K1 constructs. (B) HEK293E cells overexpressing these S6K1 proteins with or without hamartin (Ham) and tuberin (Tub) were serum-starved, pretreated with 20 nM rapamycin for 30 min before being stimulated with 100 nM insulin for 30 min, where indicated. Expression of hamartin, tuberin, and the protein levels and extent of Akt phosphorylation at Thr-308 were determined. S6K1 kinase assays were carried out as for Fig. 2. The graphs show the activity of S6K1 that is standardized to 1 for the insulin-treated sample for each of the S6K1 constructs. The data are representative of three individual experiments.

Figure 4

Hamartin and tuberin inhibit amino acid-mediated signaling through mTOR to S6K1. U20S (A) and HEK293E (B) cells overexpressing hamartin (Ham) and tuberin (Tub) with S6K1 were nutrient-deprived (D-PBS), as described in Materials and Methods. Cells were pretreated with either 20 nM rapamycin or 100 nM wortmannin for 30 min before the re-addition of amino acids in the continued presence of inhibitors, where indicated. HEK293E cells were treated for 30 min with 100 nM insulin as indicated. Levels of hamartin, tuberin, and Akt, as well as the phosphorylation of Akt at Thr-308, where determined. S6K1 kinase assays were carried out as for Fig. 2. The activity of S6K1 is standardized to 1 for the amino acid withdrawal-only sample. The data presented here are representative of three individual experiments.

Figure 5

Hamartin and tuberin inhibits S6K1 activity that is independent of PI3K signaling. HEK293E cells overexpressing wild-type or F5A-ΔCT S6K1 with or without hamartin (Ham) and tuberin (Tub) were serum-starved, pretreated with 20 nM rapamycin or 100 nM wortmannin for 30 min, and then stimulated with 100 nM insulin for 30 min, where indicated. Expression of hamartin and tuberin and the protein levels and extent of phosphorylation of Akt at Thr-308 were determined. S6K1 kinase assays were carried out as for Fig. 2. The graphs show the activity of S6K1 that is standardized to 1 for the insulin-stimulated sample for each of the S6K1 constructs.

Figure 6

Model showing that tuberin-hamartin complexes modulate PI3K-dependent signaling through mTOR to both 4E-BP1 and S6K1. Activation of PI3K leads to inactivation of the tuberin-hamartin complex by Akt-mediated phosphorylation of tuberin at Ser-939 and Thr-1462 (8). Inactivation of the tuberin-hamartin complex releases the inhibition of mTOR and allows nutrient-dependent signaling from mTOR to S6K1 and the 4E-BP1-eIF4E complex. As a result, cap-dependent and 5′terminal oligopyrimidine tract (5′-TOP) mRNA-mediated translation are increased. The dotted arrow depicts the finding that Akt phosphorylates mTOR (32).