GCN2
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The document summarizes research finding that the kinase GCN2 sustains mTORC1 suppression during amino acid deprivation by inducing the stress response protein Sestrin2. GCN2 activates the transcription factor ATF4 during amino acid starvation. ATF4 then transcriptionally upregulates Sestrin2, which is required for mTORC1 suppression. Chromatin immunoprecipitation confirms that ATF4 directly binds to the Sesn2 promoter. Sestrin2 depletion prevents mTORC1 suppression and reduces cell survival during glutamine withdrawal. Therefore, the GCN2-ATF4 pathway induces Sestrin2 to repress mTORC1 activity and promote cell survival during amino acid deprivation.
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GCN2
- 1. GCN2 Sustains mTORC1 Suppression Upon Amino Acid Deprivation By Inducing Sestrin2 Ye, et al., 2015. Genes & Development Supervisor: Dr. Moorhead Brooke Rackel & Ahmad R. Vahab
- 2. Amino Acid Sensory Mechanism Eukaryotes have 2 major regulatory mechanisms for amino acid sensing: 1. mTORC1 signaling pathway, which is activated in the presence of amino acids 2. GCN2 signaling pathway, which is activated by the absence of amino acids.
- 3. mTORC1 Substrates Amino Acid mTORC1 Complex S6K Protein Production 4EBP Cap-dependent mRNA translation ULK1 Autophagy Rag GTPase Rheb P P P P Activation Inhibition P
- 4. Magnuson, et al., Biochem. Jour., 2012
- 5. Magnuson, et al., Biochem. Jour., 2012
- 6. Magnuson, et al., Biochem. Jour., 2012
- 7. Stress Response Proteins - Sestrins Low Amino Acid Levels High Amino Acid Levels Chantranupong et al.
- 8. General Control Nonderepressible 2 (GCN2) 3 Major Domains: 1. Pseudo-Kinase Domain 2. Kinase Domain 3. Histidyl tRNA Synthetase-Like Domain
- 9. General Control Nonderepressible 2 (GCN2) Cellular Amino Acid Levels
- 10. Sustained mTORC1 Repression During Leucine Deprivation Requires The GCN2–ATF4 Pathway A B
- 11. GCN2–ATF4 Pathway Represses mTORC1 Activity By Inhibiting Its Lysosomal Localization
- 12. ATF4 is Necessary For the Transcriptional Upregulation of Sestrin2 under AAD
- 13. mRNA Expression of Sestrins By qPCR
- 14. ChIP Confirms ATF4 Binding To the Sesn2 or Sesn1 Promoter.
- 15. Effect of Individual Amino Acid Deprivation on mTORC1 Suppression
- 16. The Kinetics Of Sestrin2 Induction Resembled Those of an Established ATF4 Target PSAT1
- 17. Inhibition By Stress-Inducing Agent, Thapsigargin Thapsigargin specifically inhibits the fusion of autophagosomes with lysosomes; the last step in the autophagic process.
- 18. Amino Acid Starvation In Human Cell Lines Depends On ATF4
- 19. Sestrin 2 is Required for mTORC1 Suppression Upon Amino Acid Deficiency
- 20. Sestrin 2 is Required for mTORC1 Suppression Upon Amino Acid Deficiency
- 21. Sestrin2-Dependent mTORC1 Suppression is Necessary for Cell Survival during Glutamine Withdrawal
- 22. Sestrin2-Dependent mTORC1 Suppression is Necessary for Cell Survival during Glutamine Withdrawal
- 23. Sestrin2-Dependent mTORC1 Suppression is Necessary for Cell Survival during Glutamine Withdrawal
- 24. Sestrin2-Dependent mTORC1 Suppression is Necessary for Cell Survival during Glutamine Withdrawal
- 26. Questions?
- 27. Chromatin Immunoprecipitation (ChIP) DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo. Isolate the chromatin. Shear DNA along with bound proteins into small fragments. Bind antibodies specific to the DNA- binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins. Use PCR to amplify specific DNA sequences to see if they were precipitated with the antibody.
Editor's Notes
- mTORC1 regulates cell growth, protein translation and metabolism by sensing oxygen and growth factors. The growth factors stimulate mTORC1 but AA are needed for full activity
- When AA present, Rags GTPases along with Rheb promote the localization of mTORC1 to lysosome. When activated, mTORC1 phosphorylates ribosomal S6 kinase (S6K), unc-51-like autophagy-activating kinase 1 (ULK1), and eIF4E-binding protein 4EBPs (eIF4E is Eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs) – 4EBPs - Family of translation repressor proteins.
- mTORC1 is a major environmental sensor. It responds to a plathora of signals from various sources like amino acids, mitogens, energy stresses and hypoxia. Even if a cell has the proper energy for protein synthesis, if it does not have the amino acid building blocks for proteins, no protein synthesis will occur.
- mTORC1 is a major environmental sensor. It responds to a plathora of signals from various sources like amino acids, mitogens, energy stresses and hypoxia. Even if a cell has the proper energy for protein synthesis, if it does not have the amino acid building blocks for proteins, no protein synthesis will occur.
- Model for initiation of cap-dependent translation by the mTORC1–4EBP1 and mTORC1–S6K1 axes (A) In the absence of mTORC1-activating stimuli (i.e. growth factors/mitogens, amino acids and energy), hypophosphorylated 4EBP1 binds to eIF4E on the mRNA 5′-cap to suppress assembly of the pre-initiation complex. (B) In response to mTORC1-activating stimuli, mTORC1 docks to eIF3, localized at the 5′-cap, whereby it phosphorylates 4EBP1 and S6K1, inducing 4EBP1 release from eIF4E and S6K1 release from eIF3. (C) Dissociation of 4EBP1 enables eIF4G to dock to eIF4E, thus initiating assembly of the eIF4F complex (eIF4E, eIF4G and eIF4A). Upon release, S6K1 phosphorylates eIF4B, which induces eIF4B binding to eIF4A, an event that enhances eIF4A helicase activity. S6K also phosphorylates and inactivates PDCD4, which functions as an eIF4A inhibitor. (D) Assembly of these factors enables binding of the 40S ribosome and the ternary complex (eIF2, Met-tRNA and GTP) at the 5′-cap and thus formation of the pre-initiation complex (PIC) to initiate cap-dependent translation.
- Sestrins are a family of proteins that are known to accumulate in cells under stressful conditions. They are highly conserved across species, but lack domain structures and their physilogical functions are not well defined. There are 3 Sestrins expressed in mammals, all of which exhibit oxidoreductase activity, as well as inhibition of TOR. It is known that they inhibit the Rag complex by inhibiting Rag’s upstream GAP (GTPase activating proteins) proteins, Gator1/2, which in turn disrupts mTORC1 localization to the lysosome, preventing mTORC1 from initiating transcription and translation. The mechanism of inhibition as well as the regulation of the sestrin proteins by amino acid availability is not fully understood and is one of the topics explored in this paper.
- GCN2 is a Ser/Thr protein kinase that is directly responsible for sensing amino acid levels within the cell and eliciting the appropriate response. The protein consists of 3 major domains. The first is the pseudo-kinase domain which although similar to the kinase domain lacks the catalytic residues required to phosphorylate a protein. These domains are often involved in regulating protein activity. The second domain is the kinase domain. This is the typical protein kinase domain, ~300 aa in length and with the catalytic residues necessary to phosphorylate is substrate, in the case of GCN2, eIF2 It is an unusual protein kinase as its third domain is a histidyl-tRNA synthetase like-domain. This domain is able to bind uncharged tRNAs in the cell. Allowing it sense the overall amino acid level.
- When amino acid levels are low, uncharged tRNA’s bind to GCN2 and promote dimerization and autophosphorylation which leads to an activation of the protein. Once activated, GCN2 is then able to phosphorylate eIF2alpha which leads to a dead end complex. eIF2 is a GTP-binding protein with an intrinsic GTPase activity. It functions to directly initiate protein synthesis by binding Methionine charged tRNAs and carrying them to the 40s subunit of the ribosome, allowing translation to begin. The phosphorylation of this initiation factor prevents the eIF2 protein from having GDP exchanged for GTP, which is required for its activity. This phosphorylation event ultimately leads to the prevention of global protein synthesis. Certain genes are still able to be translated under eIF2 inhibition, including transcription factor GCN4 in yeast, or ATF4 in humans. ATF4 allows the expression of many genes that increase amino acid metabolism. As you can see, this pathway acts in direct opposition to the mTORC1 complex. So in this paper they explored the connection between GCN2 and mTORC1
- Genetic experiments have suggested that GCN2 activation can contribute to mTORC1 inhibition following leucine depletion. repression of mTORC1 activity, as assessed by decreased S6K phosphorylation (T389), was detected within 30 min, followed by a brief recovery phase. ATF4 induction was monitored as a measure of GCN2 activity, which inversely correlated with mTORC1 activity during prolonged starvation. B) suggesting that GCN2 is required for long-term mTORC1 suppression but not short-term suppression Sustained mTORC1 repression during leucine deprivation requires the GCN2–ATF4 pathway. (A) Immunoblots of lysates from wild-type MEFs that were cultured in leucine-free medium for up to 24 h. (B) Immunoblots of lysates from wild-type, Gcn2−/−, and Atf4−/− MEFs that were cultured in leucine-free medium for up to 24 h.
- Since amino acids regulate mTORC1 activity by inducing its recruitment to lysosomal membranes, they examined whether the GCN2–ATF4 pathway was required to block lysosomal localization of mTORC1 during AAD. In wild-type MEFs cultured in full medium, mTOR was present in punctate structures that colocalized with the lysosomal membrane protein LAMP2. After 24 h of leucine starvation, mTOR was distributed throughout the cytoplasm. In contrast, in both Gcn2−/− and Atf4−/− MEFs, mTOR remained in punctate structures upon leucine starvation. Together, these data show that the GCN2–ATF4 pathway represses mTORC1 activity by inhibiting its lysosomal localization.
- The lysosomal localization of mTORC1 requires the Rag GTPases, which localize to the lysosomal surface and are responsible for mTORC1 recruitment in the presence of amino acids . There is emerging evidence that members of the Sestrin family of stress response proteins are negative regulators of the Rag GTPases. B) Since Sestrins function upstream of Rag GTPases, they generated Rag A/B knockout HEK293T cells to determine whether Rag signaling is necessary for the sustained mTORC1 suppression upon leucine deprivation. In contrast to control cells, the Rag A/B knockout cells displayed sustained S6K phosphorylation even after 24 h of leucine starvation, indicating that long-term mTORC1 suppression is dependent on the Rag GTPases. ATF4 is necessary for the transcriptional up-regulation of Sestrin2 under AAD. (A) Immunoblots of lysates from wild-type and Atf4−/− MEFs that were cultured in leucine-free medium for 0, 8, and 16 h. (B) Immunoblots of lysates from control and Rag A/B knockout HEK293T cells that were cultured in leucine-free medium for up to 24 h.
- Since ATF4 is a transcription factor, they next examined whether ATF4 up-regulates Sestrin2 transcriptionally. In- deed, Sestrin2 mRNA was significantly induced during leucine deprivation in a time-dependent manner in wild- type MEFs but not in Gcn2−/− or Atf4−/− MEFs . The kinetics of Sestrin2 induction resembled those of an established ATF4 target, phosphoserine amino transferase 1 (PSAT1) C) Quantitative PCR (qPCR) measuring mRNA expression in wild-type, Gcn2−/−, and Atf4−/− MEFs cultured in leucine-free medi- um for 0, 8, and 16 h.
- To confirm that ATF4 played a direct role in Sestrin2 induction, they tested the promoter regions in response to leucine deprivation by performing ATF4- specific ChIP. They found that ATF4 was potently enriched at the Sesn2 promoter at regions that contained multiple ATF4 consensus binding sequences upon leucine deprivation. In contrast, ATF4 was not enriched at the Sesn1 promoter.
- To determine whether mTORC1 suppression and Sestrin2 induction by the GCN2–ATF4 pathway were specific to leucine deprivation or general consequences of AAD, we next examined mTORC1 activity during isoleucine, lysine, or arginine withdrawal. Immunoblots of lysates from wild-type, Gcn2−/−, and Atf4−/− MEFs that were cultured in media lacking individual amino acids as indicated for 24 h. All deprivation conditions reduced mTORC1 activity significantly in wild-type MEFs. In contrast, Gcn2−/−, and Atf4−/− MEFs maintained mTORC1 activity under these starvation conditions. Withdrawal of each of these three amino acids (I, K, or R) increased Sestrin2 protein and mRNA levels in wild-type MEFs but not in Gcn2−/−, and Atf4−/− MEFs (Fig. 2D,E). These data suggest that GCN2– ATF4-dependent Sestrin2 induction and mTORC1 suppression are general consequences of AAD and are not caused by the absence of a specific amino acid
- The kinetics of Sestrin2 induction resembled those of an established ATF4 target, phosphoserine amino transferase 1 (PSAT1). qPCR for measuring mRNA expression in wild-type, Gcn2−/−, and Atf4−/− MEFs that were cultured in media lacking individual amino acids as indicated for 24 h.
- Thapsigargin specifically inhibits the fusion of autophagosomes with lysosomes; the last step in the autophagic process. The inhibition of the autophagic process in turn induces stress on the endoplasmic reticulum which ultimately leads to cellular death. The results indicating that GCN2-dependent Sestrin2 induction specifically responds to AAD but not other stress conditions that induce ATF4 . IB of lysates from wild type, Gcn2−/−, and Atf4−/− MEFs that were treated with 3μM thapsigargin (Tg) for up to 24 h.
- Using two human cancer cell lines (HT1080 - fibrosarcoma cell line and DLD1 - colorectal adenocarcinoma) with stable expression of an ATF4 shRNA, they demonstrated that the induction of Sestrin2 upon amino acid starvation in these human cell lines also depended on ATF4. A) q-PCR for Sestrin2 expression measurement in HT1080 or DLD1 cells expressing either shRNA against ATF4 (shATF4) or a control shRNA (shNT) cultured in +/- leucine media for 24 h. PHGDH, the rate-limiting enzyme of serine synthesis, which is upregulated by the GCN2-ATF4 pathway during AAD, is used as a positive control.
- In order to see if Sestrin2 was required for mTORC1 suppression during leucine withdrawl, WT or Sesn2 knockout cells were grown in the absence of leucine for up to 24h. As you can see in the blot, the KO cells were unable to suppress mTORC1 function as indicated by the presence of p-S6K. This result indicates the need for Sestrin2 for long-term mTORC1 suppression in low amino acid conditions. In order to ensure, it was not just leucine deficiency causing these results, they repeated the experiment with Isoleucine, Lysine or Arginine lacking from the media. As you can see here in the Sesn2 KO’s, p-S6K as well as p-ULK1, anoth one of mTORC1’s substrates were present which shows that mTORC1 activity is not suppressed with out Sesn2 present. The ATF4 levels remained constant in both the WT and the KO cells which shows that ATF4 levels are not affected by Sesn2 deletion.
- When looking at the localization of mTOR, you can see it remained as near the lysosome in Sesn2 KO cells, similar to those seen in the GCN2 KO cells. This is directly in contrast to the WT cells where the mTOR is not seen around the lysosomes when depleted from Leu for 24h. Based on these results we can conclude that Sesn2 is needed for GCN2-ATF4-mediated mTORC1 suppression during amino acid deficiency.
- Glutamine is a non-essential amino-acid that can activate mTORC1 through mechanisms that are different that the activation by essential amino-acids. Unlike during leucine deprivation, glutamine deprivation only repressed mTORC1 after 24h and no acute repression was observed. As we can see in the blot here, with WT cells, it took nearly 24h for mTORC1 suppression to be seen through the reduced levels of p-S6K. Explain blot. To see whether the mTORC1 repression was through Sesn2 induction, WT, GCN2 KO and ATF4 KO cells were deprived of Glutamine for 24h. Similar as to what was seen with Leu deprivation, during Glu deprivation, Sesn2 was induced through GCN2-ATF4 and repressed mTORC1. GCN2 and ATF4 are both required as Sesn2 was not induced in the KO cells.
- GCN2-ATF4 signaling has previously been shown to promote cell-survival during Glutamine deprivation, the investigators looked at whther Sesn2 was required for cell survival when Glutamine was lacking. Interestingly as we can see in the graph here, Sesn2 KO cells were extremely sensitive to glutamine deprivation with 80% of cells dying after 48h. This is a much greater number than the 30% seen in the WT deprived of glutamine. When looking for the apoptosis marker cleaved caspase3, it was confirmed that the cause of death in these cells was indeed apoptosis.
- To further confirm that mTORC1 repression is a crucial downstream function of Sesn2, the researchers examined whether mTORC1 repression could suppress death of Sesn2 KO cells during glutamine deprivation. As you can see from the graph on the right, when rapamycin concentrations were increased to 20mM, cell death was partially rescued. Inhibition of mTORC1 by rapamycin also reduced caspase3 activity and therefore rescued Sesn2 KO cells from cell death during glutamine deprivation. As you can see in the blot on the right here cleaved caspase is not present in the Sesn2 KO cells when deprived of glutamine and rescued by rapamycin. Walk through blot on right. Rapamycin inhibition of mTORC1 was also seen to rescue Gcn2 KO cells during glutamine deprivation.
- Bright field images of wild type and Sesn2-/- MEFs cultured in +/- glutamine media with/without 50nM rapamycin for 48 h. Further showing the rescue of the Sesn2 KO cells with the addition of Rapamycin.
- It has been reported that Leu, Arg, and Glu are all major amino acids required for the activation of mTORC1 through various mechanisms. The results of this study show that the deprivation of any of these amino acids results in the GCN2-dependent induction of Sesn2 in order to maintain long-term repression of mTORC1. The ability of GCN2 to become activated by uncharged tRNAs links the deficiency of amino acids to mTORC1-dpeendent translation. Explain the model. GCN2 senses low amino-acid levels within the cell by binding uncharged tRNAs. GCN2 then dimerizes and autophosphorylates activating the protein and allowing it to phosphorylate eIF2alpha. This phosphorylated protein activates ATF4 which leads to increased levels of transcription of Sestrin2. Sesn2 expression is then able to inhibit mTORC1 by preventing it from localizing to the lysosome through the Rag proteins. In conclusion, this paper has found the link between the amino acid sensing function of both GCN2 and mTORC1 during amino acid starvation through the stress protein Sestrin2 , connecting some of the dots in the protein translation signaling pathway.
- To confirm that ATF4 played a direct role in Sestrin2 induction, they examined a publicly available data set of genome-wide sequencing of ATF4 chromatin immunoprecipitation (ChIP). ATF4 was potently enriched at the Sesn2 promoter at regions that contained multiple ATF4 consensus binding sequences