Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase

Abstract

During the evolution of metazoans and the rise of systemic hormonal regulation, the insulin-controlled class 1 phosphatidylinositol 3OH-kinase (PI3K) pathway was merged with the primordial amino acid-driven mammalian target of rapamycin (mTOR) pathway to control the growth and development of the organism. Insulin regulates mTOR function through a recently described canonical signaling pathway, which is initiated by the activation of class 1 PI3K. However, how the amino acid input is integrated with that of the insulin signaling pathway is unclear. Here we used a number of molecular, biochemical, and pharmacological approaches to address this issue. Unexpectedly, we found that a major pathway by which amino acids control mTOR signaling is distinct from that of insulin and that, instead of signaling through components of the insulin/class 1 PI3K pathway, amino acids mediate mTOR activation by signaling through class 3 PI3K, hVps34.

Fig. 1.

Amino acids and TSC1/TSC2 signaling. (A) HEK293 cells were transfected with either TSC1/TSC2 or Rheb 48 h before measuring the amino acid concentrations (as described in Materials and Methods). (B) HEK293 cells were deprived of serum overnight followed by a 2-h amino acid deprivation. Cells were stimulated with 200 nM insulin in the absence or presence of amino acids for 30 min. (C) HeLa cells were transfected with 16 nM TSC2 siRNA. Two days posttransfaction, cells were deprived of serum overnight followed by stimulation with 200 nM insulin for 30 min or, after serum withdrawal, cells were deprived of amino acids for 2 h and then stimulated with 2× amino acids for 30 min. (D) TSC2^+/+ and TSC2^-/- MEFs were treated as in C.In B-D, cell lysates were analyzed by Western blot analysis with the indicated antibodies.

Fig. 2.

Rheb GTP and a wortmannin-sensitive pathway. (A and B) HeLa cells were transfected with 8 nM Rheb siRNA, and, 2 days posttransfection, cells were deprived of serum overnight followed by stimulation with 200 nM insulin for 30 min or, after serum withdrawal, cells were deprived of amino acids for 2 h and then stimulated with 2× amino acids for 30 min. (C Upper) Serum-deprived TSC2^-/- MEFs were depleted of amino acids for 2 h and subjected to Western blot analysis. (C Lower) Parallel dishes were [³²P]orthophosphate-labeled, and the ratio of endogenous GTP to GDP-bound Rheb was determined as described in ref. . (D) TSC2^-/- MEFs were serum-starved overnight and deprived of amino acids for 2 h before being stimulated with 2× amino acids for 30 min in the presence of 100 nM wortmannin or 20 nM rapamycin for 15 min before amino acid stimulation. (E and F) HeLa cells were transfected with 16 nM PI3Kα siRNA, and 2 days posttransfection, cells were deprived of serum overnight followed by stimulation with 200 nM insulin for 30 min or, after serum withdrawal, were deprived of amino acids for 2 h and then stimulated with 2× amino acids for 30 min. (A-E) Cell lysates were analyzed by Western blot analysis with the indicated antibodies.

Fig. 3.

Amino acid-induced PI3P production and hVps34 activation. (A-D) TSC2^+/+ and TSC2^-/- MEFs were deprived of serum overnight and then deprived of amino acids for 2 h before being stimulated with 2× amino acids for 30 min. Staining with PI3P antibody was carried out as described in Materials and Methods. (E) In vitro PI3P production in TSC2^+/+ and TSC2^-/- MEFs was assayed under conditions of serum deprivation, amino acid deprivation, and amino acid stimulation.

Fig. 4.

hVps34 is required for amino acid-induced S6K1 activation. (A) HEK293 cells were transfected with myc-S6K1 and increasing amounts of gst-hVps34 expression vectors. Two days posttransfection, cells were serum deprived overnight and further deprived of amino acids for 2 h and then stimulated with 2× amino acids for 30 min. (B and C) HeLa cells were transfected with 16 nM of either hVps34 siRNA 920 or 923, and 2 days posttransfection, cells were deprived of serum overnight followed by stimulation with 200 nM insulin for 30 min or, after serum withdrawal, cells were deprived of amino acids for 2 h and then stimulated with 2× amino acids for 30 min. (D) As in C, except cells were only transfected with 16 nM hVps34 920 siRNA. (A-D) Cell lysates were analyzed by Western blot analysis with the indicated antibodies.

Fig. 5.

PI3P is required for mTOR signaling, and hVps34 associates with mTOR. (A) HeLa cells were transfected with 16 nM hVps15 siRNA and analyzed as in Fig. 4C.(B) HEK293 cells were transfected with myc-S6K1 and a GFP-FYVE expression vector and analyzed as in Fig. 4A.(C) Model depicting two signaling pathways converging on the mTOR/raptor/GβL signaling complex.