(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (T398). Drosophila S2 cells treated with or without each indicated RNAi were amino acids (AA) starved for 1 h followed by AA stimulation for 30 min. Phosphorylation and protein levels of dS6K were determined by immunoblotting with indicated antibodies.
(b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells treated with or without each indicated RNAi were AA starved for 1 h and stimulated with AA for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC: negative control RNAi.
(c) dRagA and dRagC function parallel to TSC2 and PTEN. dRagA or/and dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (T398) and this increase was compromised by dRagA or/and dRagC RNAi.

(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC, or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant negative mutants (RagA T21N, RagB T54N, RagC S75N, RagD S76N), and constitutively active mutants (RagA Q66L, RagB Q99L, RagC Q120L, RagD Q121L), were also tested. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated. Molecular weights of markers are indicated on the right.
(b) RagA has a dominant role over RagC in regulating S6K phosphorylation. 200 ng of each indicated Rag constructs were co-transfected with 20 ng of HA-S6K. The different Rag mutants used in the transfection are indicated on the top of each lane.
(c) Rag regulates TORC1 but not TORC2 activity. 200 ng of each indicated Rag constructs were co-transfected with 20 ng of HA-S6K, 20 ng of Myc-4EBP1, or 100 ng of GST-AKT. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated.

(a) RagA Q66L and RagC S75N activate TORC1 in the absence of AA. 200 ng of each indicated Rag constructs was co-transfected with HA-S6K (20 ng) into HEK293 cells. Cells were AA starved for 1 h before harvest. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated. Molecular weights of markers are indicated on the right.
(b) RagA T21N and RagC Q120L block S6K phosphorylation in response to AA stimulation. 200 ng of pcDNA3 or each indicated Rag construct was co-transfected with HA-S6K into HEK293 cells. Cells were AA starved for 1 h (−AA) and either remained in AA starvation media or stimulated with AA containing media for 30 min (+AA) before harvest. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated.
(c) RagA T21N and RagC Q120L suppress the insulin stimulation on S6K phosphorylation. 200 ng of pcDNA3 or each indicated Rag construct was co-transfected with HA-S6K (20 ng) or GST-AKT (100 ng) into HeLa cells. Cells were serum starved overnight and stimulated with insulin (400 nM) for 30 min. Phosphorylation and protein levels were determined by immunoblotting with appropriate antibodies, as indicated.

(a) dRagA positively regulates wing compartment size. Wild type or mutant dRagA transgenes were expressed in posterior compartments with the en-GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area is significantly increased in response to dRagA Q61L expression and decreased in response to dRagA T16N expression. P values: *7.32e-04 (n=7), **6.18e-04 (n=12); Student’s 2-tailed t-test, where n represents number of adult wings analyzed.
(b) dRagA positively regulates wing cell size. Average area of posterior compartment cells from en-GAL4 UAS-dRagA adult wings is shown. dRagA Q61L expressing cells are significantly larger and dRagA T16N expressing cells are smaller than controls. P values: *1.15e-03 (n=7), **0.025 (n=12); Student’s 2-tailed t-test, where n represents number of adult wings analyzed.
(c, d) dRag GTPases positively regulate larval fat body cell size, (c) Cell area of clonally-induced dRagA expressing cells or dRagC homozygous mutant cells relative to neighboring wild type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 hr starved larvae. Expression of dRagA WT or dRagA Q61L significantly increases relative cell area under starvation but not fed conditions. dRagA T16N expressing cells and dRagC loss-of-function cells are significantly smaller than control cells only under nutrient replete conditions. Results are graphed as mean ± standard deviation in n samples. P values: *2.04e0-3 (n=5), **2.94e-06 (n=14), ***3.79e-07 (n=14), ****1.36e-0 (n=30)5; Student’s 2-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. dRagA transgene expressing cells are marked by expression of GFP in the two left panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.

(a) Rag acts through TORC1 to regulate S6K phosphorylation. transfected with constructs as indicated. Co-expression of 600 ng of mTOR kinase dead (mTOR KD) construct or rapamycin treatment (20 nM, 30 min) abolished the effect of RagA Q66L and RagC S75N on S6K phosphorylation. Protein level of mTOR KD was determined by immunoblotting with anti-mTOR antibody. Molecular weights of markers are indicated on the right.
(b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or TSC constructs as indicated. Amino acid starvation for 1 hour (−AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.
(c) TSC2 and RagA/B independently affect S6K phosphorylation. 20 ng of HA-S6K was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated.
(d) RagA T21N and RagC Q120L do not block the Rheb-induced S6K phosphorylation. RagA T21N and RagC Q120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.

(a, b) dRagC is not required for Rheb-induced cell growth. (a) Area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighboring control cells which were assigned a value of 1. Overexpression of Rheb leads to a significant increase in cell area in both control and dRagC mutant backgrounds. *P=3.4e-02 (n=5), **P=6.1e-03 (n=5); Student’s 2-tailed t-test, where n represents number of experimental samples.. (b) A representative example of Rheb overexpressing cells in dRagC mutant background. Rheb transgene expressing cells are marked by co-expression of GFP. Cell boundaries are labeled by phalloidin staining in red; nuclei are labeled by DAPI in blue. Scale bar represents 50 μm.
(c, d) Expression of dRagA Q61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb^26.2 homozygous mutant cells in a control background and in animals expressing dRagA Q61L throughout the fat body. Clonally induced Rheb^26.2 homozygous mutant cells are significantly smaller than neighboring control cells both in wild type and in dRagA Q61L expressing backgrounds. *P=2.91e-04 (n=7), **P=2.59e-08 (n=5); Student’s 2-tailed t-test; fed conditions where n represents number of experimental samples. (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS-dRagA Q61L. GFP-positive control cells in this experiment are a mixture of Rheb^+/− and Rheb^+/+. Scale bar represents 50 μm.

(a–d) dRagA Q61L suppresses autophagy. (a) Drosophila fat body cells clonally expressing dRagA Q61L (marked in green by GFP expression) fail to accumulate autolysosomes (evident in surrounding control cells by punctate Lysotracker Red staining) in response to 4-hr starvation. Nuclei are marked in blue by DAPI. (b–d) Induction of autophagosomes in response to 4-hr starvation is evident by the punctate pattern of GFP-Atg8a expression in control fat body cells (b), but not in cells expressing dRagA Q61L (c). Average number of GFP-Atg8a-marked autophagosomes per cell in control and dRagA Q61L-expressing clones is shown in (d) (*P=2.91E-06, Students two-tailed t-test for 33 fat body samples imaged per genotype). Scale bars represent 25 μm in each panel.
(e) RagA regulates LC3 conversion in mammalian cells. Myc-LC3 was co-transfected with RagA QL and RagC SN or RagA SN and RagC QL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino acid sufficiency medium (+AA) or amino acid depleted medium (−AA) for 4 hours before harvesting. Western blotting for Myc-LC3 and HA-Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form is blocked by active RagA and stimulated by dominant negative RagA.

(a, b) dRagA activation increases sensitivity to starvation. Expression of dRagA Q61L using the fat body-specific Cg-GAL4 driver significantly decreases survival of adult female flies under starvation (a) conditions but not fed (b) conditions, relative to controls (Cg-GAL4 alone). Asterisks indicate time points with significant difference compared to controls (*P<0.05; Students two-tailed t-test; n = 150 flies/genotype/treatment).
(c) A proposed model of Rag GTPase in regulation of TORC1 activity. Rag GTPases act independently of and in parallel to TSC-Rheb to activate TOR signaling, possibly by transducing a nutrient-dependent signal. The mechanism of TOR regulation by Rag GTPases is indirect, and likely involves additional unidentified factors.