Volume 58, Issue 5, Pages 804-818 (June 2015) The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation  Lu Deng, Cong Jiang, Lei Chen, Jiali Jin, Jie Wei, Linlin Zhao, Minghui Chen, Weijuan Pan, Yan Xu, Hongshang Chu, Xinbo Wang, Xin Ge, Dali Li, Lujian Liao, Mingyao Liu, Li Li, Ping Wang  Molecular Cell  Volume 58, Issue 5, Pages 804-818 (June 2015) DOI: 10.1016/j.molcel.2015.03.033 Copyright © 2015 Elsevier Inc. Terms and Conditions

Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 1 The K63-Linked Ubiquitination of RagA by RNF152 (A and B) The ubiquitination of RagA is increased upon amino acid starvation. HEK293T cells were transfected and starved of amino acids for 50 min. The ubiquitinated proteins were pulled down under denaturing conditions using Ni-NTA agarose beads and were analyzed via western blotting. The asterisk indicates a band that may correspond to RagA nonspecifically bound to the Ni-NTA beads. (C and D) The ubiquitination of RagA is reduced upon amino acid stimulation. HEK293T cells were transfected, starved of amino acids for 50 min, and then supplemented with amino acids for 15 min. RagA ubiquitination was analyzed as in (A). (E–H) RNF152 promotes RagA ubiquitination in a manner that depends on its RING domain in HEK293T; RagA ubiquitination was analyzed as in (A). (I) RNF152 ubiquitinates RagA in vitro. (J and K) RNF152 promotes the K63-linked polyubiquitination of RagA. (L) Knockdown UBC13 blocks RagA ubiquitination in HEK293T cells. (M) RNF152 promotes the K63-linked ubiquitination of RagA in vitro. See also Figure S1. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 2 RNF152 Interacts with RagA in an Amino-Acid-Dependent Manner (A) A coIP assay revealed that endogenous RagA formed a complex with RNF152 in HEK293T cells. (B) GST pull-down assays indicated that RNF152 interacts with RagA directly. (C–F) RagA interacts with RNF152 in an amino-acid-dependent manner in HEK293T cells. (G and H) RNF152 preferentially binds to the inactive RagAGDP in HEK293T. (I) RNF152 preferentially promotes the ubiquitination of inactive RagAGDP (T21N) in HEK293T. (J) The interaction between RNF152 and RagA is independent of its E3 ligase activity in HEK293T. (K) The schematic diagram of RNF152. (L) The domain of RNF152 involved in its interaction with RagA. See also Figure S2. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 3 RNF152 Acts as a Negative Regulator of mTORC1 Activation (A and B) RNF152 knockdown enhances amino-acid-dependent mTORC1 signaling in HEK293T. (C) RNF152 negatively regulates the localization of mTOR to the lysosome in H1299 cells. The quantification was carried out on at least ten cells per condition from three independent experiments. Results are shown as means ± SEM (∗∗∗p < 0.001). (D) H1299 cells were transfected with the indicate siRNA. FACS analysis were performed to determine the cell size. The relative size was siNC: 483, siRNF152: 535, siTSC1: 527. (E and F) RNF152 promotes autophagy in H1299 cells. Shown are average values of triplicated results with means ± SEM (∗p < 0.05; ∗∗p < 0.01). (G and H) RNF152 induces the formation of LC3 puncta in HeLa cells. The data are presented as the means ± SEM (∗p < 0.05, n = 50). See also Figure S3. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 4 RNF152 Ubiquitinates RagA at Multiple Sites to Inhibit mTORC1 (A–C) RNF152 regulates mTORC1 activation upstream of RagA in HEK293T. (D) RNF152 affects mTORC1 activation dependent on RagA in H1299. (E and F) In HEK293T cells RNF152 affects the activation of RagA by using γ-amino-hexyl-GTP beads. (G) DUB3 reverses the effect of RNF152 on RagA in HEK293T. (H) Summary of the ubiquitination sites on RagA as identified via MS. (I) The 4KR mutant of RagA displays reduced RNF152-mediated ubiquitination in HEK293T. (J) 4KR RagA mutant more strongly induces mTORC1 activation; shown are average values of triplicated results with means ± SEM (∗p < 0.05; ∗∗p < 0.01). (K and L) 4KR RagA-activated mTORC1 is resistant to RNF152 in HEK293T. (M) The 4KR RagA mutant displays reduced formation of LC3 puncta. The data are presented as the means ± SEM (∗p < 0.05; ∗∗p < 0.01). See also Figure S4. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 5 The Ubiquitination of RagA by RNF152 Promotes Its Interaction with GATOR1 (A and B) RNF152 promotes the interaction between RagA and the GATOR1 complex in HEK293T. (C) DUB3 reduces the interaction between RagA and the GATOR1 complex in HEK293T. (D) The interaction between RagA and the GATOR1 complex was reduced in the absence of RNF152 in HEK293T. (E) The 4KR RagA mutant shows a reduced binding affinity to the GATOR1 complex in HEK293T. (F) DUB3 reverses the binding affinity of WT but not 4KR mutant RagA to the GATOR1 complex in HEK293T. (G) GATOR1 inhibits the GTP hydrolysis of WT, but not 4KR mutant RagA in HEK293T. (H) In HEK293T cells DEPDC5 binds to the polyubiquitinlated of RagA. (I) Schematic depicting the relationship between RNF152 and GATOR1 in their regulation of RagA. See also Figure S5. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 6 The Ubiquitination of RagA by RNF152 Promotes Its Interaction with the TSC (A) RNF152 knockdown enhances amino-acid-dependent mTORC1 signaling in H1299. The quantification was carried out as Figure 4J. (B) RNF152 affects the translocation of TSC2 to lysosome in H1299 cells. The quantification was carried out as Figure 3C. (C) RNF152 affects the interaction between TSC2 and RagA in HEK293T. (D) RNF152 enhance the interaction between TSC2 and RagA in HEK293T. (E) The RagA 4KR mutant displays reduced binding affinity to TSC2 in HEK293T. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions

Figure 7 RNF152 Deficiency Promotes mTORC1 Activation in MEF Cells (A) RNF152 deficiency promoted amino-acid-induced mTORC1 activation in MEF cells. The quantification was carried out as Figure 4J. (B) RNF152 deficiency affected the lysosomal localization of mTOR in MEF cells. The quantification was carried out as Figure 3C. (C) RNF152 deficiency enhanced amino-acid-induced RagA activation in MEF cells. (D) RNF152 deficiency delayed amino-acid-deprivation-induced mTORC1 inactivation in MEF cells. (E) RNF152 deficiency affected the lysosomal localization of TSC2 in MEF cells. The quantification was carried out as Figure 3C. (F and G) RNF152 affected autophagy in MEF cells. The quantification was carried out as Figure 3F. See also Figure S6. Molecular Cell 2015 58, 804-818DOI: (10.1016/j.molcel.2015.03.033) Copyright © 2015 Elsevier Inc. Terms and Conditions