Figure 4. Ragulator-null and -depleted cells are highly deficient in the activation of mTORC1 signaling by amino acids.
(A) p14 is necessary for the activation of the mTORC1 pathway by amino acids and serum. p14-null or control cells were starved of amino acids or serum for 50 minutes, or starved and re-stimulated with amino acids or serum for 10 minutes. Immunoblot analyses were used to measure the levels of the indicated proteins and phosphorylation states.
(B) p18 is necessary for the activation of the mTORC1 pathway by amino acids and serum. p18-null or control cells were treated and analyzed as in (A).
(C) Partial knockdown of MP1 blunts mTORC1 pathway activation by amino acids. HEK-293T cells expressing a control shRNA or two distinct shRNAs targeting MP1 were starved for amino acids for 50 minutes, or starved and stimulated with amino acids for 10 minutes and analyzed as in (A).
(D) p14 and p18 are not necessary for mTORC2 pathway activity. p14-null or control cells were starved for serum, or starved and then re-stimulated with serum as in (A). p18-null or control cells were grown in complete media. Cell lysates were prepared and analyzed by immunobloting for the levels of Akt1 and Akt phosphorylation at the S473 site phosphorylated by mTORC2.
(E) Decreased p14 expression impairs amino acid-induced mTORC1 activation in human cells. Cells derived from patients with lower p14 expression or healthy individuals were treated and analyzed as in (A).
(F) Cells lacking Ragulator are smaller than control cells. Cell size distributions of p14-null or p18-null cells are overlaid with those from corresponding control cells.
(G) Ragulator function is conserved in Drosophila cells. Drosophila S2 cells were transfected with a control dsRNA, or dsRNAs targeting dRagC, dMP1, dp14, or dp18, starved of amino acids for 90 minutes, or starved and restimulated with amino acids for 30 minutes. Levels of indicated proteins and phosphorylation states were analyzed by immunobloting.
See also Fig S4.