Fig. 4

Src kinase disrupts the interaction of GATOR1 with the Rags. a Src kinase interacts with the GATOR1 complex. HEK293 cells, transiently transfected with GFP-Depdc5, were lysed and subjected to immunoprecipation with antibodies against Src or IgG (negative control) followed by immunoblotting for the indicated proteins. Lysates were probed with antibodies as indicated. b HEK cells, transiently transfected with scramble shRNA or shRNAs targeting DEPDC5, NPRL2, and NPRL3 genes for 48 h prior to starvation and treatment with vehicle (DMSO) or PP2 for the last 2 h of starvation and then stimulated with amino acids (30 min). Immunoblot analyses were performed to measure the levels of the indicated proteins and phosphorylation states. c HEK293 cells, transiently transfected with the components of GATOR1 and constitutively active Src (CA-Src), were lysed and coIP analyses were performed to test interaction of GATOR1 with RagA, RagB, or RagC. d HEK293 cells, transiently transfected with GATOR1 components, CA-Src or kinase-dead Src (K295R), were lysed and coIP analyses were performed to test interaction of GATOR1 with RagA or RagC. e HEK293 cells, transiently transfected with the components of GATOR1 and wild-type Src (WT-Src), were lysed and subjected to RagC immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. Lysates were probed with antibodies as indicated. f Inhibition of Src kinase promotes GATOR1–Rag interaction. HEK293 cells transfected as in e were treated with PP2 (5 μM, 24 h) prior to lysis, and then subjected to RagC immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. GAPDH was used as a loading control in all immunoblot assays