The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation

Article

The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation Graphical Abstract

Authors Lu Deng, Cong Jiang, ..., Li Li, Ping Wang

Correspondence [email protected]

In Brief Deng et al. demonstrate that the lysosome-localized E3 ubiquitin ligase RNF152 negatively regulates mTORC1 function by targeting RagA GTPase for K63 polyubiquitination, which promotes the recruitment of its inhibitor GATOR1, a GAP complex for Rag GTPases. The regulation of RNF152-mediated RagA ubiquitination is controlled by amino acids.

Highlights d

Amino acid signaling regulates RagA ubiquitination

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RNF152 targets RagA for K63-linked ubiquitination

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RagA ubiquitination by RNF152 inhibits RagA and mTORC1 activation

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RagA ubiquitination promotes its binding to GATOR1

Deng et al., 2015, Molecular Cell 58, 1–15 June 4, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.03.033

Please cite this article in press as: Deng et al., The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.03.033

Molecular Cell

Article The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation Lu Deng,1,5 Cong Jiang,1,5 Lei Chen,1,5 Jiali Jin,1 Jie Wei,1 Linlin Zhao,1 Minghui Chen,1 Weijuan Pan,1 Yan Xu,1 Hongshang Chu,1 Xinbo Wang,1 Xin Ge,4 Dali Li,1 Lujian Liao,1 Mingyao Liu,1 Li Li,3 and Ping Wang1,2,* 1Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China 2Department of Central Laboratory, Shanghai Tenth People’s Hospital of Tongji University, School of Life Science and Technology, Tongji University, Shanghai 200072, China 3Institute of Aging Research, Hangzhou Normal University, Hangzhou 311121, China 4Department of Clinical Medicine, Shanghai Tenth People’s Hospital of Tongji University, Tongji University, Shanghai 200072, China 5Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.03.033

SUMMARY

mTORC1 is essential for regulating cell growth and metabolism in response to various environmental stimuli. Heterodimeric Rag GTPases are required for amino-acid-mediated mTORC1 activation at the lysosome. However, the mechanism by which amino acids regulate Rag activation remains not fully understood. Here, we identified the lysosome-anchored E3 ubiquitin ligase RNF152 as an essential negative regulator of the mTORC1 pathway by targeting RagA for K63-linked ubiquitination. RNF152 interacts with and ubiquitinates RagA in an amino-acid-sensitive manner. The mutation of RagA ubiquitination sites abolishes this effect of RNF152 and enhances the RagA-mediated activation of mTORC1. Ubiquitination by RNF152 generates an anchor on RagA to recruit its inhibitor GATOR1, a GAP complex for Rag GTPases. RNF152 knockout results in the hyperactivation of mTORC1 and protects cells from aminoacid-starvation-induced autophagy. Thus, this study reveals a mechanism for regulation of mTROC1 signaling by RNF152-mediated K63-linked polyubiquitination of RagA. INTRODUCTION Mechanistic target of rapamycin complex 1 (mTORC1) is an essential signaling pathway that integrates several environmental stimuli to regulate cell growth, protein synthesis, nutrient transport, autophagy, and lipid metabolism (Howell and Manning, 2011; Laplante and Sabatini, 2012). In response to amino acids, mTORC1 translocates from the cytosol to the lysosomal surface and is activated (Kim et al., 2008; Sancak et al., 2008). The activated mTORC1 phosphorylates substrates such as S6K, 4EBP1, ULK1, and TFEB (Martina et al., 2012; Martina and Puertollano, 2013; Settembre et al., 2012). Deregulated

mTORC1 signaling is closely associated with various diseases, including cancer, metabolic diseases, and developmental disorders (Laplante and Sabatini, 2012). mTORC1 senses diverse signals primarily via two kinds of Ras-related small G proteins, the Rag and Rheb GTPases, both of which act directly upstream of mTORC1 (Garami et al., 2003; Kim et al., 2008). Amino acids are regarded as one of the major signals that activate mTORC1 (Efeyan and Sabatini, 2013; Jewell et al., 2013). The amino-acid-dependent activation of mTORC1 requires the activation of the evolutionarily conserved Rag GTPases. To date, four mammalian Rag GTPases have been identified: RagA, RagB, RagC, and RagD. These GTPases are anchored to the lysosomal membrane via their interaction with the Ragulator complex and form heterodimeric complexes consisting of RagA or RagB and RagC or RagD (Efeyan et al., 2012; Jewell et al., 2013). In response to amino acids, RagA or RagB in the Rag complex binds to GTP, and RagC or RagD in the Rag complex binds to GDP. The Rag/Regulator complex recruits mTORC1 to the lysosomal membrane, where it is activated by Rheb via the direct interaction of Rag heterodimers with Raptor (Sancak et al., 2008). The activation of Rag GTPases is tightly regulated by the GEF, GAP, and GDI proteins. The lysosomal Ragulator complex functions as a GEF for RagA/B in response to amino acid stimulation (Bar-Peled et al., 2012). Sestrins, including Sestrin1, Sestrin2, and Sestrin3, negatively regulate the amino-acid-sensing pathway either by interacting with GATOR2 or by functioning as a GDI protein for Rag GTPases to regulate mTORC1 signaling (Chantranupong et al., 2014; Parmigiani et al., 2014; Peng et al., 2014). Multiple GAPs have also been identified for the Rag proteins, including the GATOR1 complex for RagA/B (Bar-Peled et al., 2013), the FLCN-FNIP complex for RagC/D (Tsun et al., 2013), and leucyl-tRNA synthase for RagD (Han et al., 2012). GATOR1, consisting of DEPDC5, Nprl2, and Nprl3, negatively regulates amino-acid-induced mTORC1 activation (Bar-Peled et al., 2013). However, the mechanism by which GATOR1 interacts with and regulates the Rag GTPase complex via amino-acid-sensing pathways remains unknown. Ubiquitination is an essential posttranslational modification that is catalyzed by an enzymatic cascade including Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc. 1

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a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3) (Deshaies and Joazeiro, 2009). E3 ligases are critical for determining the specificity of the ubiquitinated substrates. According to their structural characteristics, two major E3 ubiquitin ligase subtypes have been identified: RING and HECT E3s (Ardley and Robinson, 2005). Based on their conjugation style, eight types of ubiquitin linkages have been identified: K6, K11, K27, K29, K33, K48, K63, and linear ubiquitination (Kim et al., 2007; Komander and Rape, 2012). These distinct polyubiquitin chains are involved in a wide variety of cellular functions. For instance, K48-linked polyubiquitin chains function as a proteolytic signal by targeting substrates for proteasome-mediated degradation. By contrast, K63-linked polyubiquitin chains typically serve as a signal to regulate the activity of the target protein (Kulathu and Komander, 2012). In the current study, we found that RagA is modified by polyubiquitination in an amino-acid-sensitive manner. RNF152 was identified as a lysosomal E3 ubiquitin ligase targeting RagA for K63-linked polyubiquitination. RNF152 interacts with RagA, and their interaction is regulated by stimulation with amino acids. The ubiquitination of RagA recruits GATOR1 to the Rag complex, thereby inactivating mTORC1 signaling. RESULTS RagA Is Ubiquitinated in an Amino-Acid-Sensitive Manner Accumulating evidence shows that ubiquitination plays essential roles in various signaling pathways, such as the NF-kB and Wnt pathways (Husnjak and Dikic, 2012). Although mTOR undergoes K63-linked poly-ubiquitination by TRAF6 in response to amino acids, which regulates mTORC1 activation (Linares et al., 2013), the role of ubiquitination in the mTORC1 pathway remains largely unknown. In this study, we examined whether RagA GTPase is regulated by ubiquitination in response to various amino acid conditions. We found that RagA was significantly ubiquitinated when expressed in HEK293T cells and that its ubiquitination was markedly increased in response to amino acid starvation (Figure 1A). Similar results were obtained for endogenously expressed RagA (Figure 1B). Because the amino-acid-induced activation of mTORC1 requires the formation of heterodimers of RagA with RagC or RagD (Sekiguchi et al., 2001), we determined whether RagA ubiquitination is affected by the presence of RagC. Our data showed that RagA ubiquitination was not affected by the presence of RagC (Figure S1A). We also found that the ubiquitination of both ectopically and endogenously expressed RagA was significantly reduced in response to amino acid treatment (Figures 1C, 1D, and S1B). Together, these data suggest that RagA is ubiquitinated in an amino-acid-sensitive manner. RNF152 Is an E3 Ubiquitin Ligase for RagA We next aimed to identify the E3 ubiquitin ligase that targets RagA. Because RagA is localized to the lysosomal surface, we screened a panel of E3 ligases, including b-Trcp1, FBW7, TRIM34, FBXL6, KHLH17, BACURD, and KHLH22, as well as E3 ligases localized to the lysosomal membrane, including 2 Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc.

RNF152 and RNF167. Interestingly, we found that RNF152 displayed the strongest E3 ubiquitin ligase activity on RagA among the E3 ligases that we screened (Figure S1C). The expression of RNF152 dramatically promoted the ubiquitination of both endogenous and transfected RagA (Figures 1E and S1D). RNF152 is a RING family E3 ligase whose activity is dependent on the RING domain (Zhang et al., 2010). To examine whether RNF152-dependent RagA ubiquitination is dependent on its E3 ligase activity, we generated RNF152 mutants by either replacing four cysteines with serines (CS) or deleting the RING domains (DR), both of these alterations disrupt the E3 ubiquitin ligase activity of RNF152. Our data indicated that either mutating or deleting the RING domains significantly reduced the E3 ligase activity of RNF152 to ubiquitinate RagA (Figure 1F). These observations revealed that RNF152 targets RagA for ubiquitination in a manner that depends on its E3 ligase activity. We also determined whether RNF152 is involved in amino acid withdrawal-induced RagA ubiquitination and found that the expression of RNF152 significantly enhanced amino-acidinduced RagA ubiquitination (Figures S1E and S1F). Depletion RNF152 using a specific shRNA markedly blocked the ubiquitination of both endogenous (Figure 1G) and transfected RagA (Figure 1H), suggesting that RNF152 physiologically acts on RagA as an E3 ubiquitin ligase. Previous studies have revealed that RNF152 is anchored to the lysosomal surface by a transmembrane domain (Zhang et al., 2010). We confirmed that wild-type (WT) RNF152 is indeed localized at the lysosomal surface (Figure S1G). The transmembrane-domain-deleted RNF152 mutant (DTM) failed to localize to the lysosomal surface (Figure S1H) and also nearly completely lost its ability to ubiquitinate RagA (Figure S1I), suggesting that the transmembrane-domain-mediated lysosomal localization of RNF152 is required for RNF152 to promote RagA ubiquitination. Next, we examined whether RNF152 acts on RagA as a direct E3 ubiquitin ligase by employing an in vitro ubiquitination assay. Our data clearly indicated that RNF152 promotes RagA ubiquitination in a RING domain-dependent manner in vitro (Figure 1I). Together, our data indicated that RNF152 acts on RagA as a direct E3 ubiquitin ligase. RNF152 Targets RagA for K63-Linked Polyubiquitination Ubiquitination can regulate the function, localization, or stability of its target proteins (Weissman, 2001). Overexpression of RNF152 exerted little effect on the protein abundance of RagA (Figure S1J), suggesting that RNF152 does not target RagA for degradation. We then examined the linkage specificity of RNF152-induced RagA ubiquitination and found that RagA ubiquitination was dramatically reduced when co-transfected with a lysine-free ubiquitin mutant (Ub-7KR), in which all of the lysines were replaced with arginines (Figure 1K). These data suggested that RNF152 primarily promotes the polyubiquitination of RagA. Moreover, RNF152 strongly promoted the K63- and K33-linked, but not the K6-, K11-, K27-, K29-, or K48-linked, polyubiquitination of RagA using lysine ubiquitin mutants that contained only one lysine (Figure S1K). By contrast, the mutation of K63, but not the other lysines, including K33R, in ubiquitin markedly reduced RNF152-induced RagA ubiquitination (Figure 1J). These data indicated that RNF152 promotes the

Please cite this article in press as: Deng et al., The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.03.033

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.

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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.

K63-linked polyubiquitination of RagA in vivo. Previous studies have shown that UBC13 is the predominant E2 for K63-linked ubiquitination in vivo (Komander and Rape, 2012). Our data indicated that RagA polyubiquitination was significantly blocked by UBC13 depletion (Figure 1L). These data support our conclusion that RNF152 promoted the K63-linked polyubiquitination of RagA in vivo. We also confirmed that RNF152 targets RagA for K63-linked polyubiquitination in vitro (Figure 1M). RNF152 Directly Interacts with RagA We next evaluated the interaction between RNF152 and RagA via a co-immunoprecipitation (coIP) assay and found that both the tagged and the endogenous RagA interacted with RNF152 4 Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc.

in HEK293T cells (Figures 2A and S2A). RNF152 also directly interacted with RagA examined using a GST pull-down assay (Figure 2B). Because RNF152 ubiquitinates RagA in an aminoacid-sensitive manner (Figure 1), we examined whether their interaction is also regulated by amino acid signaling. Interestingly, the binding between the endogenous RNF152 and RagA was significantly increased in response to amino acid removal (Figure 2C), whereas stimulation with amino acids reduced their interaction (Figure 2D). Similar result was obtained for the interaction between the transfected RNF152 and RagA (Figures 2E and 2F). These data indicated that RNF152 and RagA bind to each other and that their interaction is regulated by amino acid signaling.

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As a GTPase, RagA binds to either GTP or GDP, and its nucleotide-bound status is tightly regulated by amino acid signaling (Bar-Peled et al., 2012, 2013). After amino acid stimulation, RagA is converted from the GDP-bound form to the GTP-bound form, which is a key process for mTORC1 activation (Sancak et al., 2010). Our data indicated that RNF152 strongly bound to the GDP-bound (RagAT21N), but not to the GTP-bound (RagAQ66L), form of RagA (Figures S2E and S2F). The similar result was obtained in presence of RagC (Figure 2G). Our data from in vitro GST pull-down assays indicated that GST-RNF152 preferentially interacted with the GDP-loaded His-HA-RagA (Figure 2H). Consistently, RNF152 significantly promoted the ubiquitination of the inactive form (RagA-GDP/RagC-GTP) but not the active form (RagA-GTP/RagC-GDP) of RagA (Figure 2I). Moreover, our data also show that RNF152 colocalized with mTOR and Rags on lysosome via confocal microscopy (Figures S2G and S2H). Together, these findings indicated that the interaction between RNF152 and RagA is regulated by the nucleotide-bound status of RagA. We next sought to determine the domains of RNF152 that are required for its interaction with RagA. Our data indicated that the CS mutant or RING domain is not required for the interaction of RNF152 with RagA (Figures 2J–2L). The deletion of the transmembrane domain fully disrupted the ability of RNF152 to interact with RagA (Figure 2L), suggesting that the transmembrane domain of RNF152 is required for the RNF152-RagA interaction. The fragment from 105 to 165 of RNF152 is essential for the interaction of RNF152 with RagA because mutants in which this fragment was deleted (D2) lost the ability to interact with RagA (Figure 2L). Consistently, the D2 mutant failed to target RagA for ubiquitination (Figure S2I). Our data further indicated that this effect is not due to the impairment of its lysosomal localization (Figure S2J). These data suggest that the binding of RNF152 to RagA is essential for RNF152-mediated RagA ubiquitination. RNF152 Regulates mTORC1 Activation, Lysosome Translocation, and Autophagy Given that amino acid significantly blocked RNF152-mediated RagA ubiquitination (Figure 1), we were particularly interested in examining whether RNF152 exerts any effect on amino-acidinduced mTORC1 activation. To this end, endogenous RNF152 was depleted in non-small cell lung cancer cells (H1299) using four different specific siRNAs, and the activation of mTORC1 was monitored by measuring S6K1 phosphorylation at Thr389, an mTORC1-dependent phosphorylation site. We found that the depletion of RNF152 significantly enhanced S6K1 phosphorylation at Thr389 (Figure S3A). Importantly, the upregulation of S6K1 phosphorylation strongly correlated with the efficiency of RNF152 knockdown (Figure S3A), indicating that RNF152 knockdown-induced mTORC1 activation was not due to an off-target effect. Amino-acid-induced S6K phosphorylation was also significantly increased at various time points when RNF152 was knocked down in HEK293T cells (Figure 3A), HeLa cells (Figure S3B), or H1299 cells (Figure S3C). Moreover, RNF152 knockdown induced a significant increase in S6K1 phosphorylation at Thr389 in HEK293T cells after stimulation with amino acids at a broad range of concentrations (Figure 3B).

In addition, the phosphorylation of S6, which is a target of S6K, strongly correlated with the phosphorylation status of S6K1 (Figure 3A), indicating that RNF152 depletion also promotes S6K1 kinase activity in cells. To confirm that the effect of RNF152 on S6K phosphorylation is due to its effect on mTORC1 activation, we treated H1299 cells with the mTORC1-specific inhibitor rapamycin, which blocks the mTORC1-dependent phosphorylation of S6K and ULK1 (Kim et al., 2011). Our data indicated that the RNF152-depletioninduced elevation of the phosphorylation of S6K, S6, and ULK1 was completely ameliorated by rapamycin (Figure S3D), suggesting that the RNF152-depletion-induced elevation of S6K activation is dependent on the activation of mTORC1. Together, these data indicated that RNF152 acts as an endogenous negative regulator of mTORC1 activation in response to amino acids. Consistent with the negative effect of RNF152 on mTORC1 signaling, we found that the ectopic expression of WT RNF152 significantly suppressed amino-acid-induced mTORC1 activation (Figures S3E–S3G). This inhibition was dependent on the E3 ligase activity of RNF152, as the deletion of the RING domain and the CS mutation failed to inhibit mTORC1 activation (Figure S3H). The deletion of the transmembrane domain or amino acids 105–165 of RNF152 blocked the ability to inhibit mTORC1 activation (Figures S3I and S3J). Taken together, these data suggested that RNF152 acts as a negative regulator of aminoacid-induced mTORC1 activation in a manner that depends on the E3 ligase activity of RNF152 and its interaction with RagA. Previous studies have shown that amino-acid-induced mTORC1 activation requires the translocation of mTOR from the cytosol to the lysosome (Kim et al., 2002; Kim et al., 2008). Therefore, we measured whether RNF152 affects the lysosomal translocation of mTOR. As shown in Figure 3C, the depletion of RNF152 significantly increased the amino-acid-induced colocalization of mTOR with the lysosomal marker LAMP2. Because the translocation of mTORC1 to the lysosome is dependent on the interaction between Raptor and RagA (Sancak et al., 2008), we evaluated whether RNF152 affects the interaction between Raptor and RagA. We found that the co-expression of RNF152 significantly blocked the interaction between Raptor and RNF152 (Figure S3K). Next, to determine whether the effects of RNF152 depletion on mTORC1 signaling are physiologically significant, we measured the effect of RNF152 on cell size, which is a critical phenotypic characteristic of mTORC1 activation (Fingar et al., 2002). Our data showed that the RNF152-depleted cells were larger than the control cells (Figure 3D). Taken together, these data indicated that RNF152 acts as a negative regulator of mTOR. It has been demonstrated that amino acid starvation induces autophagy due to mTORC1 inhibition (Kim et al., 2011). We found that the overexpression of RNF152 almost completely suppressed the phosphorylation of both ULK1 and TFEB (Figures S3L and S3M) and promoted the nuclear transport of TFEB (Figure S3N), which is a marker of autophagy (Kim et al., 2011; Martina et al., 2012). Moreover, amino-acid starvation-induced autophagy was significantly reduced by RNF152 depletion and increased by RNF152 overexpression through measuring the Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc. 5

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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.

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production of LC3II (Figures 3E and 3F). This result was confirmed by detecting the appearance of GFP-LC3II puncta (Figures 3G and 3H). Together, these data indicated that RNF152 acts as a negative regulator of amino-acid-starvationinduced autophagy. RNF152 Negatively Regulates RagA Activation by Targeting RagA for Ubiquitination at Multiple Sites Next, we examined the underlying mechanisms by which RNF152 inhibits mTORC1 activation. Because RNF152 promotes RagA ubiquitination, we determined whether the inhibitory effect of RNF152 on mTORC1 signaling occurs via RagA ubiquitination. To this end, we examined the effect of RNF152 on mTOCR1 activation induced by a Raptor-Rheb15 mutant in which a lysosome-targeting signal from Rheb1 (C-terminal 15 amino acids) was fused to the C terminus of Raptor. Consistent with previous studies (Sancak et al., 2010), the expression of Raptor-Rheb15 strongly activated mTORC1 activation. However, the overexpression of RNF152 failed to inhibit RaptorRheb15-mediated mTORC1 activation (Figure 4A), suggesting that RNF152 regulates mTORC1 activation upstream of mTORC1. Moreover, our data also indicated that the dominant negative form of RagA significantly blocked RNF152 knockdown-induced mTORC1 activation (Figure 4B). However, RNF152 failed to inhibit mTORC1 activation in response to the constitutively active form of RagA (Figure 4C). These data suggest that RNF152 regulates mTORC1 activation upstream of RagA. To confirm this finding, we knocked down RagA using siRNA and found that RagA knockdown significantly blocked RNF152-depletion-induced mTORC1 activation (Figure 4D). Together, these data suggest that RNF152 inhibits amino-acidinduced mTORC1 activation in a manner that is dependent on RagA. As a GTPase, RagA converted from the GDP- to the GTPbound form to activate mTORC1 in response to amino acid stimulation (Kim et al., 2008; Schu¨rmann et al., 1995). We thus used a GTP activation assay to examine whether RNF152 exerts any effect on RagA activation by pulling down the GTP-bound form of RagA. We first confirmed that this assay was suitable for measuring RagA activation (Figures S4A and S4B). Then, we determined whether RNF152 affects mTORC1 activation based on the pull-down assay. We transfected HEK293T cells with RNF152 and analyzed the GTPbound form of RagA via western blotting. Our data revealed that the co-expression of RNF152 significantly blocked RagA activation in the presence of amino acids (Figure 4E), whereas knocking down RNF152 increased RagA activation (Figure 4F). These data strongly indicated that RNF152 inhibits the activation of the GTPase RagA. Ubiquitination is a reversible process, and the removal of ubiquitin-chain from a target protein is catalyzed by a family of deubiquitinating enzymes (Heride et al., 2014). To confirm that RNF152 affects RagA activation in a ubiquitination-dependent manner, we screened the deubiquitinating enzymes that may deubiquitinate RagA. We identified DUB3 as a strong deubiquitinating enzyme that removes the ubiquitin chains attached to RagA (Figure S4C). We further examined whether DUB3 exerts any effect on RagA activation. Our data clearly indicated that

the co-expression of DUB3 significantly reversed the inhibitory effect of RNF152 on RagA activation (Figure 4G), confirming that RNF152 inhibits RagA activation by targeting RagA for ubiquitination. Next, we examined whether RNF152 negatively regulates RagA/mTOR activation by targeting RagA for ubiquitination. To this end, we first aimed to identify the site(s) in RagA that undergo RNF152-mediated ubiquitination. RagA contains 18 lysines. The mutation of all of these lysine residues completely abolished RNF152-mediated RagA ubiquitination (Figure S4D), suggesting that RNF152 attaches ubiquitin chains to the lysine residues of RagA. We used mass spectrometry (MS) analysis to identify the RNF152-mediated ubiquitination sites of RagA. A total of five lysine resides were identified as ubiquitination sites based on the tandem MS (MS/MS) spectra (Figures 4H and S4E). To examine whether these residues are indeed RNF152-mediated ubiquitin-targeting sites, we replaced each lysine with arginine individually and examined RNF152-mediated RagA ubiquitination. We found that the mutation of K142, K220, K230, or K244, but not K171, reduced RNF152-mediated ubiquitination (Figure S4F). Interestingly, the mutation of K142, K220, K230, and K244, but not K171, increased the RagA-mediated activation of mTORC1 (Figure S4G), suggesting that the ubiquitination of K142, K220, K230, and K244, but not K171, negatively regulates mTORC1 activation. Moreover, RNF152-mediated RagA ubiquitination was markedly reduced when we simultaneously mutated these four residues to arginines (4KR) (Figure 4I). The 4KR RagA mutant displayed a similar affinity to RNF152 as WT RagA (Figure S4H), indicating that the binding of RNF152 to RagA is independent of RagA ubiquitination. Collectively, we concluded that RNF152 targets RagA for ubiquitination on at least at four sites, including K142, K220, K230, and K244. Then, we examined whether RagA ubiquitination affects its ability to activate mTORC1. We found that the 4KR RagA mutant, which is resistant to RNF152-mediated ubiquitination, more strongly induces S6K1 phosphorylation at Thr389 than WT RagA in response to amino acids at various time points (Figure 4J). Moreover, 4KR RagA-activated mTORC1 is resistant to RNF152 (Figure 4K). These data suggest that RNF152 regulates mTORC1 activation via RagA ubiquitination. To determine whether RNF152-mediated ubiquitination affects RagA activation, we performed a GTP activation assay. We found that 4KR RagA bound more GTP than WT RagA in cells (Figures S4I and 4L). Moreover, RNF152 did not reduce the level of the GTP-bound form of 4KR RagA (Figure 4L). Consistent with the concept that 4KR mutant displays increased GTP-binding activity, we found that the 4KR mutant displayed an enhanced binding affinity to Raptor (Figure S4J). Together, these data indicated that the RNF152-induced reduction of the GTP-bound form of RagA in cells occurs via RagA ubiquitination. We also examined the effect of WT and 4KR RagA on aminoacid-starvation-induced autophagy. To this end, we depleted endogenous RagA using siRNA against the 30 UTR of RagA, and WT or 4KR RagA was reintroduced into the RagA-depleted cells. We found that the 4KR RagA mutant more strongly blocked amino-acid-starvation-induced autophagy than WT RagA (Figures 4M and S4K). These data strongly suggest that the Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc. 7

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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 g-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.

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ubiquitination of RagA at these four residues is essential for RNF152-mediated RagA inhibition. Taken together, these data suggest that RNF152 inhibits mTORC1 activation primarily by targeting RagA for ubiquitination. The Ubiquitination of RagA by RNF152 Promotes Its Interaction with GATOR1 Next, we examined the mechanism by which RNF152-mediated ubiquitination regulates RagA activation. We first examined whether RNF152 affects the ability of RagA to bind to GTPgS. To this end, RagA was expressed with or without RNF152 in HEK293T cells. Our data indicated that RNF152 exerted little effect on the GTP-binding activity of RagA using an in vitro GTPgS binding assay (Figure S5A). Previous studies revealed that RagA activation is regulated by GEF, GAP, and GDI proteins (Bar-Peled et al., 2013; Bar-Peled et al., 2012; Peng et al., 2014). All of these regulators need to interact with Rag GTPases to regulate mTORC1 activation. To understand the mechanism by which RNF152 regulates RagA activation, we examined whether RNF152 affects the interaction of RagA with its regulators. Our data indicated that the expression of RNF152 exerted a minor effect on the interaction of RagA with the Ragulator complex (Figure S5B) and with the GDI protein Sestrin2 (Figure S5C). In strong contrast, the overexpression of RNF152 significantly enhanced the binding between RagA and GATOR1 (Figures 5A and 5B), which is a complex that displays GAP activity, by promoting the GTPase activity of RagA. As a control, the CS mutant of RNF152 exerted a minor effect on the binding of RagA to GATOR1 (Figure S5D), suggesting that the effect of RNF152 on the binding between GATOR1 and RagA depends on the E3 ligase activity of RNF152. Next, we determined whether RNF152-mediated RagA ubiquitination is involved in the interaction between RagA and GATOR1. To this end, we co-expressed RagA with DUB3, which significantly blocks RNF152-mediated RagA ubiquitination (Figure S4C), and detected the interaction between RagA and GATOR1. We found that the co-expression of DUB3 markedly blocked the binding of RagA to GATOR1 (Figure 5C). Moreover, the knockdown of RNF152 significantly reduced the interaction between endogenous RagA and GATOR1 (Figure 5D). Similar results were obtained for the interaction between ectopically expressed RagA and GATOR1 (Figure S5E). These data suggest that RNF152 regulates mTORC1 activation by affecting the interaction between RagA and GATOR1. We next examined whether RagA ubiquitination affects its binding to GATOR1. To this end, we compared the binding affinity between GATOR1 and 4KR and WT RagA. Our data indicated that the 4KR mutant displayed a weaker binding affinity to GATOR1 than WT RagA (Figure 5E). The co-expression of DUB3 significantly reduced the binding between GATOR1 and WT RagA, but not the 4KR RagA mutant (Figure 5F). These data indicated that RNF152 negatively regulates the interaction between RagA and GATOR1 by targeting RagA for ubiquitination. To confirm that the RNF152-mediated inhibition of mTORC1 is dependent on the GATOR1 complex, we depleted the GATOR1 complex and examined the effect of RNF152 on mTORC1 activation. We found that RNF152 exerted little effect on

amino-acid-induced mTORC1 activation once DEPDC5 or Nprl2, two key components of GATOR1, was depleted (Figure S5F), confirming that RNF152 regulates mTORC1 via GATOR1. Moreover, we found that GATOR1 failed to inhibit the activation of 4KR RagA (Figure 5G). Results from an in vitro pull down assay showed that ubiquitinated RagA possesses a severely increased ability to bind to DEPDC5 (Figures S5G and 5H), indicating that the ubiquitination of RagA by RNF152 promotes its binding to GATOR1, thereby inactivating RagA (Figure 5I). RNF152 Negatively Regulates TSC Translocation by Targeting RagA mTORC1 is rapidly inactivated in response to amino acid withdrawal. Because we demonstrated that amino acid withdrawal promotes RNF152-mediated RagA ubiquitination, we examined whether RNF152 regulates amino-acid-deprivation-induced mTORC1 inactivation. As shown in Figure 6A, the depletion of RNF152 significantly delayed amino acid withdrawal-induced mTORC1 inactivation. Recent studies have indicated that the recruitment of TSC2 to the lysosome, which is induced by amino acid withdrawal, is a major mechanism that inactivates mTORC1. The translocation of TSC2 to the lysosome is dependent on its binding to inactive RagA (Demetriades et al., 2014). Therefore, we examined whether RNF152 affects the translocation of TSC2 to the lysosome in response to amino acid starvation. As shown in Figure 6B, the knockdown of RNF152 significantly blocked the translocation of TSC2 to the lysosome in response to amino acid withdrawal. Consistently, the overexpression of RNF152 promoted, whereas the knockdown of RNF152 inhibited the interaction between TSC2 and RagA (Figures 6C and 6D). Moreover, we found that the 4KR RagA mutant displayed a weaker binding affinity to TSC2 than WT RagA (Figure 6E). Therefore, our data indicated that RNF152 is also involved in the inactivation of mTORC1 by targeting RagA for ubiquitination. RNF152 Deficiency Enhances the Activation of mTOR and RagA To confirm that endogenous RNF152 is essential for mTORC1 activation, we generated an RNF152 knockout (KO) mouse using the CRISPR-Cas9 technique (Figures S6A–S6D). The mice deficient in RNF152 were born at the expected Mendelian ratios (data not shown), suggesting that RNF152 is not required for embryonic development. To investigate whether RNF152 is indeed involved in mTORC1 regulation, we derived MEFs from RNF152-deficient embryos and examined amino-acid-induced mTORC1 activation (Figure 7A). The lack of RNF152 protein expression was confirmed via immunoprecipitation and western blotting (Figure S6E). To further confirm this result, we examined whether RNF152 deficiency affects the lysosomal translocation of mTOR. We found that amino-acid-induced mTORC1 translocation was significantly increased in the RNF152-deficient MEFs compared to the WT MEFs when amino acids were added to the medium for various periods (Figure 7B). Moreover, RNF152 deficiency increased amino-acid-induced RagA activation in MEFs (Figures 7C and S6F). Consistent with this result, the binding of Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc. 9

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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.

RagA to GATOR1 was reduced in the RNF152-deficient MEFs compared with the WT MEFs (Figure S6G). We also examined whether RNF152 deficiency affects amino-acid-deprivation-induced mTORC1 inactivation. Our data indicated that RNF152 deficiency significantly delayed amino10 Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc.

acid-deprivation-induced mTORC1 inactivation (Figure 7D). Consistently, RNF152 deficiency markedly inhibited the aminoacid-withdrawal-induced translocation of TSC2 to the lysosome (Figure 7E). Moreover, the binding of RagA to TSC2 was reduced in the RNF152-deficient cells (Figure S7H).

Please cite this article in press as: Deng et al., The Ubiquitination of RagA GTPase by RNF152 Negatively Regulates mTORC1 Activation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.03.033

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.

Next, we examined whether endogenous RNF152 regulates amino-acid-induced autophagy and found that nutrient-deprivation-induced autophagy was also reduced in RNF152-deficient MEFs, as detected by either the production of LC3II or the presence of GFP-LC3II puncta (Figures 7F, 7G, and S7I). Taken together, our data indicated that RNF152 physiologically acts as a negative regulator of the mTORC1 pathway.

DISCUSSION Rag proteins are central regulators that connect amino acid sensing with mTORC1 signaling (Kim et al., 2008). In the current study, we unexpectedly found that polyubiquitination is an essential mechanism by which RagA activity is regulated. We identified the lysosomal surface-anchored E3 ubiquitin Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc. 11

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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.

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ligase RNF152 as an essential E3 ligase that targets RagA for ubiquitination. RNF152 attaches a K63-linked polyubiquitin chain to RagA. The K63-linked polyubiquitination of RagA promotes its binding to GATOR1, which acts as a GAP on Rag proteins to negatively regulate mTORC1 activation and signaling in response to amino acids (Bar-Peled et al., 2013). Therefore, we identified a mechanism by which RagA polyubiquitination mediates amino-acid-induced mTORC1 activation. RagA Is Posttranslationally Modified by K63-Linked Polyubiquitination As a key component of amino-acid-induced mTORC1 activation, RagA is tightly regulated by its subcellular localization and its binding targets. For example, RagA must be localized to the lysosome via its binding to Ragulators to regulate mTORC1 activation (Bar-Peled et al., 2012; Sancak et al., 2010). However, it remained unknown whether RagA is regulated by posttranslational modification. In this study, we have provided the evidence that RagA is modified by K63-linked polyubiquitin chains. Importantly, we found that the polyubiquitination of RagA is tightly regulated by the amino acid status. The starvation of amino acids strongly induces the polyubiquitination of RagA, whereas amino acid stimulation inhibits RagA ubiquitination. Our data further indicated that RagA polyubiquitination negatively regulates RagA-mediated mTORC1 activation. These data have also provided evidence that K63-linked polyubiquitination is involved in the regulation of amino-acid-induced mTORC1 activation. RNF152 Acts on RagA as an E3 Ubiquitin Ligase We identified RNF152 as an important E3 ligase for RagA ubiquitination. Based on a screen of various E3 ligases, we identified the lysosomal E3 ligase RNF152 as an E3 ubiquitin ligase that targets RagA. Although we cannot exclude the possibility that other E3 ligases ubiquitinate RagA, our data indicated that RNF152 acts as an important negative regulator of RagA activation in vivo. The overexpression of RNF152 strongly inhibited RagA-mediated mTORC1 activation, whereas RNF152 deficiency increased amino-acid-induced mTORC1 activation. As a lysosome-localized E3 ubiquitin ligase, RNF152 requires its transmembrane domain to mediate RagA ubiquitination. This finding is consistent with the evidence that RagA is localized to the lysosomal surface. We found that RNF152 interacts with RagA in a nucleotide-bound state. RNF152 appears to prefer binding to the GDP-bound form of RagA. Once activated by amino acids, RagA binds to GTP and disassociates from RNF152. This result is consistent with our findings that amino acid treatment inhibited RagA ubiquitination. By contrast, amino acid deprivation results in the binding of RagA to GDP, which promotes the interaction between RagA and RNF152. Consequently, the binding of RNF152 to RagA promotes RagA ubiquitination. The exact mechanism by which the nucleotide status of RagA affects the binding of RagA to RNF152 remains to be identified. RNF152 is a lysosome-anchored E3 ubiquitin ligase whose biological function is unknown. Our study indicated that RNF152 KO mice give birth at the expected Mendelian ratios.

Several previous reports have indicated that the deficiency of the negative regulator of the Rag complex may result in embryonic or neonatal lethality (Nada et al., 2009). Because we observed that mTORC1 is hyperactivated in MEFs from RNF152-deficient mice, RNF152 may be specifically expressed at different stages. On the other hand, other potential E3 ligases may compensate for the function of RNF152. mTORC1 is involved in various physiological or pathological conditions, such as cancer, aging, and metabolic diseases (Laplante and Sabatini, 2012). It will be interesting to investigate whether RNF152 acts as a regulator of mTORC1 in various diseases, such as obesity, diabetes, and other liver-related diseases, using RNF152-KO mice. RNF152-Mediated RagA Polyubiquitination Enhances the Binding of RagA to GATOR1 GATOR1 is a complex that consists of DEPC5, Nprl3, and Nprl2 that acts as a GAP protein for Rag proteins (Bar-Peled et al., 2013). The mechanism by which amino acids regulate of RagA via GATOR1 remained largely unknown. In this study, we proposed a model in which K63-linked polyubiquitination is essential for the binding of GATOR1 to RagA. However, the exact mechanism by which RagA polyubiquitination affects the binding of RagA to GATOR1 remains unknown. Accumulating evidence indicates that K63 polyubiquitination chains can function as a platform for protein-protein interactions (Komander and Rape, 2012). Therefore, the polyubiquitination chain on RagA may serve as a platform for the recruitment of GATOR1. Proteins that interact with ubiquitin typically contain ubiquitin-binding domains. However, we did not identify any typical ubiquitin interaction motifs in the components of the GATOR1 complex. Alternatively, the polyubiquitination of RagA may recruit certain ubiquitin-binding proteins to RagA, thereby promoting the binding of RagA to the GATOR1 complex. RagA Is Regulated by Deubiquitination Ubiquitination is a reversible process, and the removal of ubiquitin from a ubiquitin chain or a target protein is catalyzed by a family of deubiquitinating enzymes (Heride et al., 2014). We found that polyubiquitin chains are removed from RagA in response to amino acid stimulation, suggesting that RagA ubiquitination is regulated by deubiquitination. Based on a screen for the DUBs involved in RagA deubiquitination, we identified DUB3 as an efficient candidate enzyme that removes ubiquitin chains from RagA. The overexpression of DUB3 significantly deubiquitinated RagA and rescued RagA activation (Figures S4C and 4G). However, we could not detect any interaction between DUB3 and RagA in cells (data not shown), suggesting that DUB3 indirectly or nonspecifically affects RagA deubiquitination. Therefore, it will be important to examine the mechanism by which RagA deubiquitination is regulated by amino acids and to confirm whether DUB3 physiologically acts on RagA as a deubiquitinating enzyme in future studies. Taken together, we propose a model (Figure S6J) in which in response to amino acid withdrawal, GAPs of the Rag complex, such as GATOR1, stimulate the intrinsic GTPase activity of RagA on the lysosomal surface, thereby converting RagA into Molecular Cell 58, 1–15, June 4, 2015 ª2015 Elsevier Inc. 13

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the GDP bound form. This produces a strong binding affinity of RagA for the E3 ubiquitin ligase RNF152. Consequently, RNF152 ubiquitinates RagA at the lysosomal surface, and ubiquitinated RagA displays increased binding affinity to GATOR1, thereby connecting GATOR1 with the Rag complex to prevent amino-acid-induced mTORC1 activation. Upon acute amino acid stimulation, RNF152 disassociates from the Rag complex, and the ubiquitin chain on RagA is removed by a deubiquitinating enzyme, thereby releasing GATOR1 from the RagA complex. Thus, the RagA complex rapidly becomes activated. In this state, the deubiquitinating enzyme activity that mediates RagA deubiquitination will be interesting to explore in future studies. Such a model helps to explain the rapid change in RagA ubiquitination in response to amino acid signaling. EXPERIMENTAL PROCEDURES All animal experiments were approved by the East China Normal University Center for Animal Research. Plasmids, Antibodies, Cell Lines, Transfection, and Immunofluorescence This information is provided in the Supplemental Experimental Procedures. Amino Acid Starvation and Stimulation HEK293T or H1299 cells in culture dishes were rinsed with and incubated in amino-acid-free DMEM medium for the indicated duration. Then, the cells were stimulated with amino acids for 5 to 30 min. The final concentration of amino acids in the medium was the same as that in DMEM medium. GTP Pull-Down Assay Cells were lysed in lysis buffer G (1 3 PBS, pH 7.4, 1% Triton X-100, 1 3 phosphatase inhibitor cocktail) by rotating at 4 C for 0.5 hr, and the lysates were purified via centrifugation for 15 min at 4 C. The supernatants were incubated with rotation in 30 ml of g-amino-hexyl-GTP-Sepharose suspension (Jena Bioscience) at 4 C for 2 hr. Then, the beads were washed three times with 1 ml of lysis buffer G and once with PBS. Statistical Analysis Statistical analyses were performed with a two-tailed unpaired Student’s t test. The data are presented as the means ± SEM. The values of p < 0.05 were considered statistically significant.

Ministry of Education of China (20130076110022), and the Science and Technology Commission of Shanghai Municipality (13QH1401300). The authors declare no conflicts of interest. Received: January 22, 2015 Revised: March 17, 2015 Accepted: March 27, 2015 Published: April 30, 2015 REFERENCES Ardley, H.C., and Robinson, P.A. (2005). E3 ubiquitin ligases. Essays Biochem. 41, 15–30. Bar-Peled, L., Schweitzer, L.D., Zoncu, R., and Sabatini, D.M. (2012). Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1. Cell 150, 1196–1208. Bar-Peled, L., Chantranupong, L., Cherniack, A.D., Chen, W.W., Ottina, K.A., Grabiner, B.C., Spear, E.D., Carter, S.L., Meyerson, M., and Sabatini, D.M. (2013). A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106. Chantranupong, L., Wolfson, R.L., Orozco, J.M., Saxton, R.A., Scaria, S.M., Bar-Peled, L., Spooner, E., Isasa, M., Gygi, S.P., and Sabatini, D.M. (2014). The Sestrins interact with GATOR2 to negatively regulate the amino-acidsensing pathway upstream of mTORC1. Cell Rep. 9, 1–8. Demetriades, C., Doumpas, N., and Teleman, A.A. (2014). Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 156, 786–799. Deshaies, R.J., and Joazeiro, C.A. (2009). RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434. Efeyan, A., and Sabatini, D.M. (2013). Nutrients and growth factors in mTORC1 activation. Biochem. Soc. Trans. 41, 902–905. Efeyan, A., Zoncu, R., and Sabatini, D.M. (2012). Amino acids and mTORC1: from lysosomes to disease. Trends Mol. Med. 18, 524–533. Fingar, D.C., Salama, S., Tsou, C., Harlow, E., and Blenis, J. (2002). Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev. 16, 1472–1487. Garami, A., Zwartkruis, F.J., Nobukuni, T., Joaquin, M., Roccio, M., Stocker, H., Kozma, S.C., Hafen, E., Bos, J.L., and Thomas, G. (2003). Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11, 1457–1466. Han, J.M., Jeong, S.J., Park, M.C., Kim, G., Kwon, N.H., Kim, H.K., Ha, S.H., Ryu, S.H., and Kim, S. (2012). Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 149, 410–424.

SUPPLEMENTAL INFORMATION

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Supplemental Information includes six figures and Supplemental Experimental Procedures and can be found with this article online at http://dx.doi.org/10. 1016/j.molcel.2015.03.033.

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AUTHOR CONTRIBUTIONS L.D., C.J., L.C., and P.W. conceived the project and designed experiments. L.D., C.J., L.C., J.J., J.W., L.Z., M.C., W.P., Y.X., and H.C. performed experiments. L.D., C.J., L.C., X.W., X.G., D.L., L.L., M.L., L.L., and P.W. analysis the data. L.D., X.G., and P.W. wrote the manuscript. ACKNOWLEDGMENTS We thank Dr. Kun-Liang Guan for kindly providing reagents. We thank Dr. Dianqing Wu,Kun-Liang Guan and Dangsheng Li for critical reading of the manuscript. We also thank members of the Wang lab for their assistance. This work was supported by grants from the National Basic Research Program of China (973 program 2012CB910404), the National Natural Science Foundation of China (30971521, 91440104 and 31171338), the Doctoral Fund of the

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