A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1 - PubMed (original) (raw)

A Tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1

Liron Bar-Peled et al. Science. 2013.

Abstract

The mTOR complex 1 (mTORC1) pathway promotes cell growth in response to many cues, including amino acids, which act through the Rag guanosine triphosphatases (GTPases) to promote mTORC1 translocation to the lysosomal surface, its site of activation. Although progress has been made in identifying positive regulators of the Rags, it is unknown if negative factors also exist. Here, we identify GATOR as a complex that interacts with the Rags and is composed of two subcomplexes we call GATOR1 and -2. Inhibition of GATOR1 subunits (DEPDC5, Nprl2, and Nprl3) makes mTORC1 signaling resistant to amino acid deprivation. In contrast, inhibition of GATOR2 subunits (Mios, WDR24, WDR59, Seh1L, and Sec13) suppresses mTORC1 signaling, and epistasis analysis shows that GATOR2 negatively regulates DEPDC5. GATOR1 has GTPase-activating protein (GAP) activity for RagA and RagB, and its components are mutated in human cancer. In cancer cells with inactivating mutations in GATOR1, mTORC1 is hyperactive and insensitive to amino acid starvation, and such cells are hypersensitive to rapamycin, an mTORC1 inhibitor. Thus, we identify a key negative regulator of the Rag GTPases and reveal that, like other mTORC1 regulators, Rag function can be deregulated in cancer.

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Figures

Figure 1

Figure 1

GATOR is a Rag-interacting complex, whose Mios component is necessary for the activation of mTORC1 by amino acids. (A) Mios interacts with endogenous RagA and RagC. HEK-293T cells were transfected with the indicated cDNAs in expression vectors. Cells were treated with a cell permeable chemical cross-linker, lysates were prepared and subjected to Flag immunoprecipitation (IP) followed by immunoblotting for the indicated proteins. (B) Mios is necessary for the activation of the mTORC1 pathway by amino acids. HEK-293T cells expressing shRNAs targeting GFP or Mios were starved of amino acids for 50 min or starved and then re-stimulated with amino acids for 10 min. Cell lysates were analyzed for the phosphorylation state of S6K1. (C) S2 cells treated with dsRNAs targeting Mio or GFP were starved of amino acids for 90 min or starved and re-stimulated with amino acids for 30 min. The indicated proteins were detected by immunoblotting. (D) Cell size histogram of S2 cells after dsRNA-mediated depletion of Mio. (E)–(F) GATOR is an octomeric complex defined by two distinct subcomplexes and interacts with the Rag GTPases. HEK-293T cells were transfected and processed as in (A) with the exclusion of the cross-linking reagent, and cell lysates and FLAG-immunoprecipitates were subjected to immunoblotting. (G) HEK-293T cells stably expressing FLAG-tagged DEPDC5 or WDR24 were lysed and cell lysates and FLAG immunoprecipitates were analyzed by immunoblotting for endogenous RagA, RagC, Mios and Nprl3. (H) Schematic summarizing GATOR-Rag interactions. GATOR2 (Mios, Seh1L, WDR24, WDR59 and Sec13) interacts with GATOR1 (DEPDC5, Nprl2 and Nprl3), which likely then binds the Rags.

Figure 2

Figure 2

The GATOR complex is required for the regulation of the TORC1 pathway by amino acids. (A) shRNA-mediated depletion of the GATOR2 components Seh1L, WDR24, or WDR59 in HEK-293T cells inhibits amino acid-induced S6K1 phosphorylation. (B) In HEK-293Ts expressing shRNAs targeting the GATOR1 components DEPDC5, Nprl2, and Nprl3, S6K1 phosphorylation is insensitive to amino acid withdrawal. In (A) and (B) cells were starved of amino acids for 50 min or starved and restimulated with amino acids for 10 min. Cell lysates were immunoblotted for the phosphorylation state of S6K1. dsRNA-mediated depletion in S2 cells of (C) dSeh1L; (D) dWDR59 and dWDR24; and (E) dDEPDC5, dNprl2, and dNprl3. S2 cells were treated with the indicated dsRNAs and were starved of amino acids for 90 min or starved and restimulated with amino acids for 30 min. Immunoblotting was used to detect the phosphorylation state of dS6K. (F) S2 cell sizes after dsRNA-mediated depletion of dSeh1L and dDEPDC5. (G) GATOR2 functions upstream of GATOR1. S2 cells were treated with the indicated combinations of dsRNAs and starved and restimulated with amino acids and analyzed as in (C)–(E). (H) Schematic depicting the relationship between GATOR1 and GATOR2 in their regulation of mTORC1.

Figure 3

Figure 3

GATOR regulates mTORC1 localization to the lysosomal surface and functions upstream of the Rag GTPases. (A) RNAi-mediated depletion of the GATOR2 components Mios and Seh1L prevents amino acid-induced mTOR lysosomal translocation. HEK-293T cells expressing the indicated shRNAs were starved or starved and restimulated with amino acids for the specified times prior to co-immunostaining for mTOR (red) and Lamp2 (green). (B) Reduced expression of DEPDC5 in HEK-293T cells results in constitutive mTOR localization to the lysosomal surface. HEK-293T cells treated with the indicated lentiviral shRNAs were processed as described in (A). (C) Images of HEK-293T cells stably expressing FLAG-DEPDC5 starved for or starved and restimulated with amino acids. Cells were processed as described in (A). In all images, insets show selected fields that were magnified five times and their overlays. Scale bar equals 10 μM. (D) GATOR1 functions upstream of the nucleotide binding state of the Rags. HEK-293T cells transfected with the indicated cDNAs in expression vectors were starved of amino acids for 50 min or starved and restimulated with amino acids for 10 min. The indicated proteins were detected by immunoblotting.

Figure 4

Figure 4

GATOR1 is a GTPase activating protein complex for RagA and RagB. (A)–(D) GATOR1 stimulates GTP hydrolysis by RagA and RagB. RagA-RagCX, RagB-RagCX, RagC-RagBX or the control GTPase Rap2a were loaded with [α-32P] GTP and incubated with GATOR1 (20 pmols) or the control Leucyl tRNA synthetase (LRS) (20 pmols). GTP hydrolysis was determined by thin layer chromatography (see methods). Each value represents the normalized mean ± SD (n = 3). (E) GATOR1 increases GTP hydrolysis by RagB in a time dependent manner. RagB-RagCX was loaded with [γ-32P]GTP, incubated with GATOR1 or a control and hydrolysis was determined by phosphate capture (see methods) Each value represents the normalized mean ± SD (n = 3). (F) GATOR1 preferentially interacts with the dominant active Rag heterodimer. In vitro binding assay in which FLAG-GATOR1 was incubated with immobilized HA-GST-tagged RagBT54N-RagCQ120L (dominant negative), RagBQ99L-RagCS75N (dominant active) or Rap2a. HA-GST precipitates were analyzed by immunoblotting for the levels of FLAG-GATOR1.

Figure 5

Figure 5

GATOR1 components are mutated in cancer and GATOR1-null cancer cells are hypersensitive to the mTORC1 inhibitor rapamycin. (A) Table summarizing genomic alterations in DEPDC5 and NPRL2 and their frequencies in glioblastoma and ovarian cancer. The ratios of nonsense and frameshift mutations to missense mutations in DEPDC5 (p-value = 0.00015) and NPRL2 (p-value = 0.00342) in glioblastoma differ significantly from the ratio of all nonsense and frameshift mutations to missense mutations in glioblastoma genomes as determined by a fisher exact test. (B)–(C) Mutations and deletions identified in DEPDC5 in glioblastomas and ovarian cancers. (D) In GATOR1-null cancer cells the mTORC1 pathway is resistant to amino acid starvation. Cells were starved of amino acids for 50 min and starved and restimulated with amino acids for 10 min. Cell lysates were analyzed by immunoblotting for levels of the indicated proteins. (E) Cancer cells were starved or starved and restimulated with amino acids for the specified times prior to co-immunostaining for mTOR (red) and Lamp2 (green). In all images, insets show selected fields that were magnified five times and their overlays. Scale bar equals 10 μM. (F) Re-introduction of Nprl2 into the SW780 cell line (NPRL2−/−) restores amino acid-dependent regulation of mTORC1. Cells stably expressing a control protein or Nprl2 were treated and analyzed as in (D). (G) GATOR1-null cancer cells are values for indicated cancer cell lines. hypersensitive to Rapamycin. Rapamycin IC50 Values are presented as mean ± SD (n = 3). (H) Model for the role of the GATOR complex in the amino acid sensing branch of the mTORC1 pathway. GATOR2 is a negative regulator of GATOR1, which inhibits the mTORC1 pathway by functioning as a GAP for RagA.

Comment in

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