Supplementary Figure 7: Recurrent follicular lymphoma RagC mutants activate the mTORC1 pathway.
From: Recurrent mTORC1-activating RRAGC mutations in follicular lymphoma
(a) Follicular lymphoma RagC mutants (RagCS75F, RagCS75N, RagCT90N and RagCW115R) dramatically increase mTORC1 binding (mTOR and raptor), whereas RRAGC mutations identified in solid cancers (p.M121V, p.Y165C, p.D202G, p.L217R and p.R396Q) did not coimmunoprecipitate mTORC1 as strongly. Anti-FLAG immunoprecipitates were collected and analyzed as in Figure 3a. (b) All four RagC mutants coimmunoprecipitate more raptor than wild-type RagC in Raji cells. Anti-FLAG immunoprecipitates from Raji cells stably expressing the indicated proteins were collected and analyzed as in Figure 3a. (c) Three RagC mutants (RagCS75N, RagCS75F and RagCW115R) coimmunoprecipitate more raptor than wild-type RagC when overexpressed in OCI-Ly7 cells. Anti-FLAG immunoprecipitates from OCI-Ly7 cells stably expressing the indicated proteins were collected and analyzed as in Figure 3a. (d) Four RagC mutants increase raptor binding over wild-type RagC in OCI-Ly8 cells. Anti-FLAG immunoprecipitates from Ly8 cells stably expressing the indicated proteins were collected and analyzed as in Figure 3a. (e) Stable overexpression of RagCS75N, RagCS75F, RagCT90N and RagCW115R renders the cells partially insensitive to full amino acid deprivation. HEK293T cells that stably expressed the indicated proteins were starved of amino acids for 50 min and restimulated with amino acids for 10 min. The cell lysates were analyzed as in Figure 3b. (f) Quantification of the amount of phosphorylated S6K1 in Figure 3c, under leucine starvation or starvation followed by restimulation in HEK293T cells stably expressing the indicated proteins. (g) Quantification of the amount of phosphorylated S6K1 in Figure 3d, under arginine starvation or starvation followed by restimulation in HEK293T cells stably expressing the indicated proteins. (h) Quantification of the amount of phosphorylated S6K1 in Figure 3e, under leucine starvation or starvation followed by restimulation in Karpas-422 cells stably expressing the indicated proteins.
