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mTORC1 clearly is a key regulator of protein synthesis but that begs the question as to how it determines that protein synthesis is required. How does it sense that? Obviously regulators of mTORC1 might be amino acids in cells, but who would have thought that the master regulator would be leucine, a simple branched chain hydrophobic amino acid.
It would be nice if free leucine bound directly to mTORC1, but it's not that simple. Rather, it binds to a "leucine" receptor, sestrin 2 (SESN2). The figure below shows the binding interactions between Leu (spacefill) and key side chains in sestrin 2 (5dj4).
The Leu is rather buried, which suggests a conformational change ensues on binding to the protein. Saxton et al (2016) describe three types of sestrin2 side chains involved in the interaction:
- Lid: Thr374, Thr377, and Thr386 form H bonds with the Leu amine and carboxyl group. Leucine is represented as a stick model (orange).
- Latch: Tyr375 and His86 forms hydrogen bonds to the Leu. Note that these residues are distal in the chain and are probably pulled together during the conformational changes which occurs after binding to form a latch to sequester the bound Leu.
- Floor: F447 and W444 which interact with the nonpolar side chain of Leu.
Jmol: Sestrin 2 Jmol14 (Java) | JSMol (HTML5)
What happens after leucine binds? It's a complicated but understandable process described below in words and an images. But first a quick review. Kinases must be regulated to be turned on and off at the right time. They are often regulated by phosphorylation, as mTOR is. In addition, they can be regulated by binding proteins as mTOR is (by Raptor, FKBP, etc). They can also be regulated by small G proteins (like Ras) which are active when bound to GTP and inactive when bound to GDP. Of course whether small G proteins have GTP bound depends in part if they interact with GAPs (GTPase activating protein which inactivates small G proteins) or GEFs (which facilitate exchange of GDP for GTP and activate them). ISuch a master regulator of growth as mTORC1 is regulated by all of these, in addition to the presence of abundant leucine.
In the absence of leucine, sestrin 2 is bound to a protein called GATOR2 (GTPase-activating protein - GAP - activity toward Rags 2). The binding of leucine to sestrin 2 causes the dissociation of GATOR2. This is shown in the figure below;
Free GATOR2 is a GAP that regulated mTORC1. Specifically, it regulates the activity of a heterodimer of small GTP binding proteins, RagA/B:RagC/D (see pathway above) which are associated with the outer leaflet of the lysosome. There they interact with a membrane protein, SLC38A9 and a protein that regulates the Rag proteins, which of course is named Ragulator. Active RagA/B:RagC/D recruits mTORC1, presumably through the Raptor subunit) from the cytoplasm to the lysosome membrane. Small G proteins like Ras, when activated by exchanging bound GDP for GTP, can interact with and activate kinases (like the Raf kinase for Ras). When mTORC1 binds to active RagA/B:RagC/D, it becomes activated.
We often think of activating a protein by ligand binding, which promotes a conformational change, or by post-translational modification, which can provide a binding interaction or conformational change to activate the protein. Another way is to inhibit an inhibitor of a protein, as shown below. Y inhibits Z as denoted by the blunt ended arrow. If X inhibits Y, the Y can't inhibit Z, which is now active. This is analogous to the quote that "the enemy of my enemy is my friend", which has been attributed to Kautilya (from India) in the 4th century BCE.
Leucine binding to sestrin 2 leads to free GATOR, which activates mTORC1 by blocking downstream inhibitors as shown in two different figures below.
The figure immediately below (after Buel and Blenis, 2016) shows the interactions from an activation (arrow) or inhibition (blunt arrow) perspective.
The figure above shows the involvement of multiple proteins in the lysosome membrane that are involved in mTORC1 activation. There is yet another way that the RagA/B and RagC/D protein are regulated (other than by the GATOR GAP activity. The main one appears to be Ragulator, which is a GEF for the Rag proteins. Here is a summary of components of this lysosomal membrane recruitment center for mTORC1.
- Ragulator (what a great name) binds and recruits the small G proteins Rag to the lysosome membrane where Ragulator acts as a GEF for RagA/B
- SLC38A9 is a weak amino acid transporter in the lysosome membrane, with preferences towards polar amino acids. More likely it is yet another sensor of amino acids, particularly of arginine, which has a high concentration in the lysosome. The protein has a high Km for transport of Arg. It has a Ragulator binding domain and is hence part of the complex that recruits mTORC1 to the lysosome
- vacuolar adenosine triphosphatase (v-ATPase): function unclear
These interactions, which involve multiple activations and inhibitions, are difficult to follow even with a diagram. The actions of small G proteins can be especially difficult to understand since the G protein is biologically INACTIVE in its GDP-bound form towards its target binding protein. This occurs when the GTPase activity of the G protein is ACTIVE. The arrows and blunt end arrows in the figure above represent the activity of the protein towards its target protein.
Here are two alternative ways to make sense out of the interactions:
- Stepping backwards from Rag A/B, Gator 1 (a GAP) inhibits the ACTIVITY of the protein Rag A/B as it acts as a GAP to leave Rag A/B in the inactive GDP-bound state. Paradoxically this occurs as the inherent GTPase activity of the protein is activated as described above). Free Gator 2 (also a GAP) appears to inhibit the GAP activity of Gator 1 (through an unknown mechanism), thereby increasing the amount of GTP-bound Rag A/B, which then can activate mTORC1. Free Gator 2 does this only if Sestrin 2 is bound to Leu which allowed the Gator 2 to dissociate from the inactive sestrin 2:Gator 2 complex.
- The diagram above shows that in the absence of leucine, three blunt end (inhibition) arrows occur between Sestrin 2 and Rag A/B. One blunt arrow denotes inhibition, two activation (inhibition of inhibition), and hence three net inhibition Hence in the absence of Leu (when Sestrin is bound to Gator 2, Rag A/B is inhibited in its ability to activate mTORC1 as Rag A/B is in the GDP-bound state. However, free leucine unblocks the inhibitor action of sestrin 2 as Gator 2 is now free and active on its own.
Amino acids (especially arginine which is abundant) in the lumen of the lysosome activate, through the v-ATPase and SLC38A9, the GEF activity of Ragulator. When Rag A/B has sufficient GTP, some conformational changes must ensue to allow mTORC1 recruitment to the lysosomal membrane.
- Prof. Henry Jakubowski (College of St. Benedict/St. John's University)
In this study, we explored the coordinate regulation of mTORC1 by insulin and amino acids. Rat livers were perfused with medium containing various concentrations of insulin and/or amino acids. At fasting (1×) or 2× (2×AA) concentrations of amino acids, insulin maximally stimulated Akt phosphorylation but had no effect on global rates of protein synthesis. In the absence of insulin, 4×AA produced a moderate stimulation of protein synthesis and activation of mTORC1. The combination of 4×AA and insulin produced a maximal stimulation of protein synthesis and activation of mTORC1. These effects were accompanied by decreases in raptor and PRAS40 and an increase in RagC associated with mTOR (mammalian target of rapamycin). The studies were extended to a cell culture model in which mTORC1 activity was repressed by deprivation of leucine and serum, and resupplementation with the amino acid and insulin acted in an additive manner to restore mTORC1 activation. In deprived cells, mTORC1 was activated by expressing either constitutively active (ca) Rheb or a caRagB·caRagC complex, and coexpression of the constructs had an additive effect. Notably, resupplementation with leucine in cells expressing caRheb or with insulin in cells expressing the caRagB·caRagC complex was as effective as resupplementation with both leucine and insulin in non-transfected cells. Moreover, changes in mTORC1 activity correlated directly with altered association of mTOR with RagB/RagC, Rheb, raptor, and PRAS40. Overall, the results suggest that amino acids signal through the Rag complex and insulin through Rheb to achieve coordinate activation of mTORC1.
This work was supported, in whole or in part, by National Institutes of Health Grants DK13499 and DK15658 (to L. S. J.).
The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Tables S1–S4.
D4. Regulation of mTORC1 by Leucine - Biology
Cellular amino acid levels tightly control the activity of the master growth regulator mTORC1.
The emergence of molecular details on how the mTORC1 pathway senses amino acids is an important advance in the field.
The amino acid sensing pathway is composed of several multicomponent complexes that act in concert to convey changes in amino acid levels to mTORC1.
The mechanistic target of rapamycin complex I (mTORC1) is a central regulator of cellular and organismal growth, and hyperactivation of this pathway is implicated in the pathogenesis of many human diseases including cancer and diabetes. mTORC1 promotes growth in response to the availability of nutrients, such as amino acids, which drive mTORC1 to the lysosomal surface, its site of activation. How amino acid levels are communicated to mTORC1 is only recently coming to light by the discovery of a lysosome-based signaling system composed of Rags (Ras-related GTPases) and Ragulator v-ATPase, GATOR (GAP activity towards Rags), and folliculin (FLCN) complexes. Increased understanding of this pathway will not only provide insight into growth control but also into the human pathologies triggered by its deregulation.
In 2020, the population over 60 years old in China was in excess of 250 million and accounted for 18.1% of the total population, becoming the first country in the world with an elderly population in excess of 100 million. Among them, the number of disabled and partially disabled elderly (i.e., core activity restrictions in self-care and mobility, such as feeding, dressing, transfer from bed, getting around inside the home, and bathing) exceeds 40 million. Age-related sarcopenia is a common disease in the elderly, and it is also a major predictor of disability. Sarcopenia is operationally defined as low muscle strength and is considered severe when low muscle quantity, quality, and low muscular strength are all present (Cruz-Jentoft et al., 2019). Age-related sarcopenia is characterized by the progressive decline of skeletal muscle mass and resultant impairment of physical and physiological function (Csete, 2021). With the accelerated growth of the older population, age-related sarcopenia will place an increasing burden on economic and social resources. Presently, there are no recognized consensus standards or guidelines for the clinical treatment of senile muscular atrophy, and no pharmacological interventions fully mitigate its occurrence and development (Chen et al., 2020). Therefore, clarifying the pathophysiological mechanisms of this disease and identifying potential intervention targets is important to promote the development of clinical intervention programs and new therapeutic drugs.
The maintenance of skeletal muscle mass depends on the dynamic balance between the rate of muscle protein synthesis (MPS) and degradation (MPB). The algebraic difference (MPS minus MPB) between the two indicates the state of net protein balance (NPB) (Tipton and Phillips, 2013 Murach and Bagley, 2016). When NPB is positive, protein deposition increases, which eventually induces muscle hypertrophy on the contrary, when NPB is negative, protein deposition decreases and atrophy occurs (Holwerda et al., 2019). The decline in skeletal muscle mass as a result of age is primarily due to a decrease in MPS in response to anabolic stimuli such as amino acids and exercise, also referred to as anabolic resistance (Rong et al., 2020). Thus, how to effectively ameliorate the decline in NPB and protein deposition of aging skeletal muscle has become an important area of research in biomedical sciences. Exercise can enhance the sensitivity of aging skeletal muscle to dietary amino acid and protein and thus has important potential in promoting skeletal muscle protein deposition (Rebelo-Marques et al., 2018 Ebner et al., 2020). Previous studies have confirmed that resistance or aerobic exercise combined with dietary protein and/or amino acid ingestion induces greater MPS rates, hypertrophy and muscular performance, than nutritional support alone (Xia et al., 2016 Phillips and Martinson, 2019). Leucine, an essential amino acid (and one of the three branched-chain amino acids), is recognized as a key anabolic stimulus and can be directly involved in the regulation of MPS both on a substrate and signal transduction level. Additional studies have shown that leucine sensing has an important effect on MPS in aging skeletal muscle (Devries et al., 2018 Lehman and Abraham, 2020).
As previously discussed, anabolic resistance is a key factor in age-related sarcopenia, and leucine sensing and response play an important role in the regulation of MPS. Therefore, enhancing the sensing and the response of aging skeletal muscle to anabolic stimuli such as leucine to counteract anabolic resistance is an important and timely topic of research in the field of aging. This paper, therefore, will review the latest research to explore new mechanisms of age-related sarcopenia and new strategies for its prevention and treatment and provide theoretical rationale for the application of related targets in clinical translational medicine.
MTORC1 promotes lipid biosynthesis through SREBPs
In addition to protein synthesis, actively growing cells require a substantial amount of lipids to support membrane biogenesis (reviewed by Menendez and Lupu, 2007). Over the last few years, several reports showed that mTORC1 plays a fundamental role in promoting lipid biogenesis by regulating the expression of many lipogenic genes.
One important group of transcription factors that are involved in lipid synthesis are the sterol-regulatory-element-binding proteins (SREBPs). SREBPs are basic helix-loop-helix (bHLH) transcription factors that regulate lipid homeostasis by controlling the expression of lipogenic genes (reviewed by Horton et al., 2002). Three members of the SREBP family have been described in mammals, SREBP1a and SREBP1c (hereafter referred to as SREBP1) and SREBP2. SREBP1 is involved in insulin-mediated fatty acid synthesis, whereas SREBP2 mainly controls cholesterol biosynthesis (reviewed by Horton et al., 2002). Insulin increases SREBP1 expression and cleavage, which allows the release of a mature form of SREBP1 that translocates into the nuclei to regulate gene expression. On the other hand, cholesterol depletion induces the expression of SREBP2 and its subsequent cleavage, thus promoting its activity.
mTORC1 positively regulates the activation of SREBPs through several mechanisms (reviewed by Bakan and Laplante, 2012). Blocking of mTOR signaling reduces the mRNA and protein levels of SREBPs in several experimental models (Li et al., 2010 Li et al., 2011 Owen et al., 2012 Wang et al., 2011 Yecies et al., 2011). Although differences exist between cell types, some of these studies have reported that mTORC1 regulates transcription of SREBPs through a mechanism that is independent of the mTORC1 substrate S6K1. In addition to promoting expression of SREBPs, mTORC1 induces the processing and the nuclear accumulation of the mature and active form of these transcription factors (Düvel et al., 2010 Owen et al., 2012 Porstmann et al., 2008 Wang et al., 2011 Yecies et al., 2011). Studies have revealed that S6K1 plays a crucial in promoting processing of SREBPs downstream of mTORC1 but the exact mechanism involved is still unknown. Finally, it has been shown that mTORC1 promotes the activation of SREBPs by inducing their nuclear accumulation through a mechanism that requires Lipin 1, a phosphatidic acid phosphatase that also serves as a transcriptional coactivator (Peterson et al., 2011). When active, mTORC1 phosphorylates Lipin 1, which results in its exclusion from the nucleus. Upon mTORC1 inhibition, Lipin 1 accumulates in the nucleus, which promotes the association of SREBPs to the nuclear matrix and impairs their ability to bind target genes (Peterson et al., 2011). The fact that mTORC1 regulates activation of SREBPs at multiple levels suggests that the control of lipid synthesis must be intimately coupled to nutrient and growth factor signaling to maintain cellular homeostasis.
An increase in lipogenesis is a hallmark of proliferating cancer cells (reviewed by Menendez and Lupu, 2007). As mTORC1 is often hyperactivated in cancers, it is possible that it could have a role in driving tumorigenesis by promoting lipid synthesis through the activation of SREBPs. In support of this idea, Manning’s group recently reported that depletion of SREBPs blocks proliferation in cells with constitutively active mTORC1 (Düvel et al., 2010). The mTORC1–SREBPs axis might also play a role in the development of nonalcoholic fatty liver disease (NAFLD), a condition that is characterized by excessive accumulation of lipids in the liver that can lead to cirrhosis and liver cancer. Obesity and nutrient overload, which are linked to NAFLD, exacerbate mTORC1 activity in the liver (Khamzina et al., 2005 Tremblay et al., 2007), which, in turn, might promote NAFLD by activating SREBP1. Consistent with this idea, liver-specific deletion of mTORC1 impairs SREBP1 function and makes mice resistant to western-diet-induced NAFLD (Peterson et al., 2011).
Regulation by amino acids
It should come as no surprise that the availability of the basic building blocks of protein control its synthesis. When a cell is deprived of amino acids, mTOR can be found throughout the cytoplasm, whereas addition of amino acids rapidly localizes mTOR to the peri-nuclear region of the cell, to large vesicular structures, or to both . The amino acid-induced locatization is similar to that of Rab7, a late endosome-/lysosome-associated small GTPase. This suggests that amino acids might stimulate mTORC1 activity by localizing it to lysosomal surface where it can be activated by Rheb-GTP. The Ragulator-Rag complex was found responsible for targeting mTORC1 to the lysosomal surface . At the lysosomal surface, mTORC1 associates with Ras-related GTPases (Rags). There are four different Rags: RagA, RagB, RagC and RagD. RagA and RagB (RagA/B) bind to RagC and RagD (RagC/D) to form heterodimeric pairs. Rags, in turn, associate with the protein complex Ragulator which is anchored in the lysosomal membrane. The interaction of Rags with mTORC1 is dependent on their guaninenucleotide binding state. In an amino acid-deprived cell, the RagA/B are bound to GDP, and the RagC/D are bound to GTP. The addition of amino acids induce a nucleotide exchange favoring the GTP bound state of RagA/B and the GDP bound state of RagC/D. The Ragulator, anchored in the lyosomal membrane, associates with Rags, therefore localizing them to the lysosomal membrane. Importantly, the Ragulator functions as a guanine nucleotide exchange factor (GEF) for RagA/B , thereby facilitating the exchange of GDP bound RagA/B for GTP bound RagA/B (the active form). The GEF activity of Ragulator is regulated by v-ATPase . v-ATPase consumes ATP in order to pump hydrogens up their concentration gradient into the lysosome in order to maintain its acidic environment. Ragulator is associated with v-ATPase and amino acids induce a conformational change to the protein which then acts to activate Ragulator’s GEF activity. As of yet it is unclear how amino acids induce this conformational change, but the signal appears to originate from inside the lysosome due to accumulation of amino acids in its lumen (Fig. 3) .
Regulation of mTORC1 by amino acids. a The Rags are found in their inactive state under low amino acid conditions and therefore are unable to recruit mTORC1 to the lysosomal membrane for activation by Rheb-GTP. Ragulator and v-ATPase are in their inactive state, whereas GATOR1 exerts GAP-activity towards RagA/B, ensuring an inactive state of these Rags. b An increase in the amino acid concentration triggers a conformational change in v-ATPase and Ragulator, which initiates GEF activity towards RagA/B of the latter. FLCN and its binding partners exhibit GAP activity towards RagC/D and thereby activating them as well. Additionally, GATOR1 its GAP activity is inhibited due to inhibition of GATOR2. These actions lead to the active heterodimer of GTP-bound RagA/B and GDP-bound RagC/D, which then recruit mTORC1 to the lysosomal surface where it can be activated by Rheb-GTP. Figure based on 
Whereas Ragulator acts as a GEF for RagA/B, the GAP activity towards Rags (GATOR1) complex functions as a GAP towards RagA/B . The GATOR1 complex thus exchanges the GTP for GDP of RagA/B, leading to deactivation of the Rags and subsequently inhibition of mTORC1. Another protein complex dubbed GATOR2 is responsible for inhibiting GATOR1 activity  and therefore relieves mTORC1 from its inhibition. The inhibiting effect of GATOR2 on GATOR1 is mediated by Sestrin proteins in response to amino acids . However, it is unknown how GATOR2 mediates its inhibiting effect and how amino acids regulate the complex.
Lastly, there is evidence that the guanine nucleotide binding state of RagC/D is regulated by leucyl tRNA-synthetase (LRS), the enzyme responsible for loading tRNA with leucine. The enzyme acts as a GAP for RagD GTPase, in a leucine depedent manner . However, a later study found that purified LRS did not act as a GAP for any of the Rags . Instead the authors propose that folliculin tumor suppressor (FLCN) and its binding partners act as Rag-interacting proteins with GAP activity for RagC/D, leading to mTORC1 activation. Moreover, leucine specifically appears to regulate mTORC1 through Sestrin2 .
Role of the Induction of the Methylation of PP2A by Sam in mTORC1 Activation
The study performed by Sutter et al. also showed that methionine regulates the mTORC1 signaling pathway and autophagy through the regulation of the methylation status of phosphatase 2A (PP2A) in yeast (Sutter et al., 2013 Laxman et al., 2014). In the presence of high levels of intracellular SAM, Ppm1 induces the methylation of the catalytic subunit of PP2A in response to SAM concentration. PP2A is activated by its methylation thereafter, methylated PP2A can suppress Npr2 through its dephosphorylation, which results in mTORC1 activation and the suppression of autophagy (Figure 2A). The complex consisting of Npr2, Npr3, and Iml1 (NPRL2, NPRL3, and DEPDC5 in mammals, respectively) is termed SEACIT in yeast (GATOR1 in mammals, as described above) (Panchaud et al., 2013) and functions as a negative regulator of mTORC1 via a GAP activity toward the yeast Rag orthologs, that is, Gtr1/2 (Rags family in mammals) (Gao and Kaiser, 2006). Therefore, suppression of SEACIT by the dephosphorylation of Npr2 induced by the activation of PP2A results in the activation of mTORC1. In contrast, lower SAM levels in cells reduce the methylation levels of PP2A and promote the phosphorylation of Npr2, which results in the suppression of mTORC1 activity and the induction of autophagy (Figure 2B). In mammalian cells, the methylation of PP2A is catalyzed by a specific S-adenosyl methionine (SAM)pendent methyltransferase, the leucine carboxyl methyltransferase 1 (LCMT1) (Stanevich et al., 2011). Activated PP2A possibly dephosphorylates NPRL2 and results in mTORC1 activation in mammalian cells however, no report has shown whether PP2A is directly involved in the regulation of the phosphorylation state of NPRL2. Therefore, further studies are necessary to clarify this issue.
Figure 2. Regulation of mTORC1 via the methylation of PP2A in response to SAM. (A) In conditions of high levels of intracellular SAM in yeast, Ppm1 induces the methylation of the catalytic subunit of PP2A in response to SAM concentration. The activated (methylated) form of PP2A suppresses Npr2 through its dephosphorylation. The complex consisting of Npr2, Npr3, and Iml1 (SEACIT) is a negative regulator of mTORC1 therefore, the suppression of SEACIT via the dephosphorylation of Npr2 results in the activation of mTORC1. In mammalian cells, LCMT1 induces the methylation of the catalytic subunit of PP2A in response to SAM concentration, leading to the activation of mTORC1, possibly through the activation of GATOR1. Moreover, in mammalian cells, PP2A possibly regulates the phosphorylation levels of NPRL2 in response to SAM levels. (B) Lower SAM levels reduce the methylation levels of PP2A in yeast and mammalian cells and promote the activation of Npr2 via its phosphorylation, which results in the suppression of mTORC1 activity. In mammalian cells, PP2A possibly regulates the phosphorylation levels of NPRL2 in response to SAM levels. mTORC1, mechanistic target of rapamycin complex 1 PP2A, phosphatase 2A SAM, S-adenosyl methionine GAP, GTPase-activating protein LCMT1, leucine carboxyl methyltransferase 1.
We also reported that a low-protein diet ameliorates diabetes-induced kidney injury and that dietary methionine abrogates the beneficial effects of a low-protein diet in diabetic kidneys (Kitada et al., 2020). More specifically, diabetic rats that were fed a low-protein + methionine diet exhibited increased expression of LCMT1 and methyl-PP2A compared with control (standard-diet) and low-protein-diet diabetic rats, which was accompanied by an increase in renal SAM levels. Although the expression of glycine N-methyltransferase (Gnmt), which is a SAM-converted enzyme, was decreased in diabetic rat kidneys, changes in renal SAM levels were dependent on the dietary methionine content (Kitada et al., 2020). Consistent with the alteration of LCMT1 and methyl-PP2A, mTORC1 activation and autophagy suppression were observed in standard-diet and low-protein + methionine diabetic rats. Furthermore, we also used cultured human kidney-2 cells to confirm that the administration of SAM-induced methylated PP2A increased the expression of methyl-PP2A and activated mTORC1 (Kitada et al., 2020). However, the involvement of SAM-induced methylated PP2A in mTORC1 activation through NPRL2 and the activation of the negative regulator of mTORC1 by its increased phosphorylation, such as that observed for Npr2 in yeast, remain unknown.
The authors thank all members of the Sabatini laboratory for helpful discussions, with particular gratitude to K. J. Condon and J. M. Orozco for their insightful comments on this manuscript and K. Shen for his assistance with Fig. 1. This work was supported by grants from the National Institutes of Health (NIH) (R01 CA103866, R01 CA129105 and R37 AI047389) and the Lustgarten Foundation to D.M.S and by fellowship funding from the NIH (T32 GM007287 and F31 CA232340) to G.Y.L. D.M.S. is an Investigator at the Howard Hughes Medical Institute and an American Cancer Society Research Professor.
Results and Discussion
Screening for Rabs that are involved in autophagy
To comprehensively screen for Rabs that regulate mammalian autophagy, we generated effective short interfering RNAs (siRNAs) targeting each mouse Rab isoform (supplementary Fig S1A,B online supplementary Table S1 online) and proceeded to use these siRNAs to perform screening in two steps. In the first step of our screening procedure, we searched for Rabs, whose knockdown in mouse embryonic fibroblasts (MEFs) altered the amount of LC3-II, a lipidated form of LC3/Atg8 that specifically associates with autophagosomes, under starved conditions (Fig 1A supplementary Fig S1C online) [ 6 , 7 ]. Of the 58 Rabs we tested, 4 Rab siRNAs (Rab1A, 1B, 6A and 11A) significantly increased the amount of LC3-II in comparison with the control siRNA, and 7 Rab siRNAs (Rab9B, 12, 17, 18, 32, 40B and 40C) significantly decreased it (Fig 1A). If these candidate Rabs were actually involved in autophagy, the amount of an autophagic substrate should also be affected by the same siRNAs. In the second step of our screening procedure, we therefore investigated whether knockdown of the candidate Rabs identified in the first step influenced the amount of p62 protein, a specific substrate for autophagic degradation, under starved conditions [ 8 ]. As shown in Fig 1B and supplementary Fig S1D online, six Rab siRNAs (Rab1A, 1B, 11A, 12, 18 and 40B) significantly increased the amount of p62 protein, but the other five Rab siRNAs had no effect. It should be noted that some of the final candidate Rabs, for example, Rab1 and Rab11, have previously been reported to be involved in autophagy, thereby validating our two-step screening procedure. In the present study, we focused on Rab12, whose knockdown resulted in the greatest increase in the amount of p62 protein among the final candidates identified (Fig 1B).
Rab12 regulates the efficiency of autophagy
The impact of Rab12 knockdown on autophagy was further evaluated by several independent approaches [ 9 ] in which two different Rab12 siRNAs were used. Both siRNAs clearly reduced the number of dots that were positive for LC3 and dots that were positive for Atg16L1 (an isolation membrane marker) under both nutrient-rich conditions and starved conditions (supplementary Fig S2 online Fig 1C,D). Similarly, the amount of p62 protein was increased by these siRNAs under both conditions (Fig 1E,F). It should be noted, however, that starvation still increased the number of LC3-positive and Atg16L1-positive dots and reduced the amount of p62 protein even in the Rab12-depleted cells in comparison with nutrient-rich conditions, the same as in the control cells (Fig 1C–F). In addition, Rab12 knockdown did not affect autophagic flux as revealed by LC3 turnover assays (Fig 1G) [ 9 ], indicating that Rab12 knockdown did not affect lysosomal functions. Moreover, monomeric strawberry (mStr)-tagged Rab12 did not colocalize with Atg16L1 or LC3 at all (Fig 1H). Taken together, these results suggested that Rab12 modulates the signals involved in initiation of autophagy.
To determine the mechanism by which Rab12 regulates initiation of autophagy, we first focused on mTORC1, a well-known upstream negative regulator of autophagy [ 10 , 11 , 12 , 13 , 14 , 15 ], because several Rab isoforms, including Rab5, have been shown to regulate mTORC1 localization and/or activity [ 16 , 17 ]. Under nutrient-rich conditions, mTORC1 is targeted to the lysosomal surface by a Ragulator–Rag GTPases complex and activated by Rheb GTPase, and its activation regulates growth and metabolism as well as inhibits autophagy [ 18 , 19 , 20 , 21 , 22 , 23 , 24 ]. As we previously found that Rab12 regulates a membrane traffic pathway from recycling endosomes to lysosomes to degrade transferrin receptor (TfR, a recycling endosome marker) [ 25 ], we investigated whether the lysosomal targeting of mTORC1 is influenced by Rab12 knockdown (Fig 2A). However, the results showed that under nutrient-rich conditions, Rab12 knockdown did not affect the lysosomal targeting of mTOR, although the signals of mTOR targeting to lysosomes were clearly increased in Rab12-depleted cells in comparison with the control cells under nutrient-rich conditions. This result led us to consider two possibilities: the possibility that Rab12 knockdown leads to increased mTORC1 activity and the possibility that Rab12 knockdown increases the amount of mTOR protein itself, and we performed immunoblotting analyses to determine whether either one of them was correct (Fig 2B). The results showed that Rab12 knockdown increased phosphorylation of ribosomal protein S6 kinase, a readout of mTORC1 activity, without affecting the total amount of mTOR protein. Interestingly, dissociation of mTOR from lysosomes and decreased mTORC1 activity as a result of starvation seemed to occur normally in Rab12-depleted cells (Fig 2A,B), and treatment with rapamaycin, an mTORC1 inhibitor, reduced mTORC1 activity and induced autophagy (that is, p62 degradation and LC3-dot formation) even in the Rab12-depleted cells (supplementary Fig S3 online). By contrast, overexpression of a constitutive active mutant of Rab12 (Rab12-QL) reduced mTORC1 activity (supplementary Fig S4 online). These results taken together indicated that Rab12 functions upstream of mTORC1.
We also found that Rab12 knockdown increased mTORC1 activity independent of Akt activity (Fig 2C–E). As mTORC1 is activated by certain amino acids independent of the PI3K-Akt signalling pathway [ 19 , 26 , 27 , 28 ], we hypothesized that Rab12 knockdown increases the intracellular amino-acid concentration. To test our hypothesis, we measured the intracellular L -amino-acid concentration in Rab12-depleted cells with an L -Amino-Acid Quantitation Kit (supplementary Fig S5 online). As predicted in our hypothesis, the intracellular L -amino-acid concentration was much higher in the Rab12-depleted cells than in the control cells, suggesting that Rab12 regulates mTORC1 activity through modulation of the intracellular amino-acid concentration.
Rab12 controls the degradation of PAT4
As the intracellular amino-acid concentration is regulated by amino-acid transporters, we hypothesized that Rab12 regulates lysosomal localization or degradation of amino-acid transporters. To test this hypothesis, we focused on the PAT family, because two members of the PAT family that are ubiquitously expressed, PAT1/Slc36a1 and PAT4/Slc36a4, have been shown to affect mTORC1 activity [ 29 , 30 , 31 , 32 ]. PAT1, in particular, is specifically localized at lysosomes (supplementary Fig S6A online) and has been reported to regulate mTORC1 activity through export of amino acids from the lysosome lumen into the cytosol [ 29 , 30 ]. However, detailed colocalization analyses in MEFs indicated that PAT1 only partially colocalized with Rab12 (supplementary Fig S6B online), whereas PAT4 colocalized well with Rab12 and TfR but it did not colocalize with Lamp-1 (Fig 3A,B). We therefore selected PAT4 as the prime candidate for the cargo of Rab12. As both TfR and PAT4 are localized at recycling endosomes, it appeared highly possible that PAT4 cycles between the plasma membrane and recycling endosomes, the same as TfR does. As anticipated, surface biotinylation assays revealed that PAT4 is actually present in the plasma membrane (Fig 3C). However, as Rab11A knockdown, which inhibited the recycling pathway to the plasma membrane, clearly resulted in a reduction in the amount of plasma membrane-localized TfR protein but did not affect the amount of plasma membrane-localized PAT4 protein (supplementary Fig S7 online), unlike TfR protein, PAT4 is not actively recycled back to the plasma membrane by Rab11. Although PAT4 was originally described as a proton-coupled amino-acid transporter, recent evidence indicated that when expressed in Xenopus laevis oocytes [ 33 ], PAT4 is most functional at neutral pH, not at low pH, suggesting that PAT4 imports extracellular amino acids into cells through the plasma membrane. As Rab12 regulates lysosomal degradation of TfR through the pathway from recycling endosomes to lysosomes without affecting lysosome function [ 25 ], we wondered whether Rab12 also regulates PAT4 degradation. Rab12 knockdown was found to dramatically increase the amount of HA-PAT4 protein in MEFs stably expressing HA-PAT4 (Fig 3D,E) without affecting the PAT4 messenger RNA concentration (Fig 3F). Furthermore, the results of exposure to a lysosomal inhibitor showed that PAT4 is constitutively degraded in lysosomes (supplementary Fig S6C online) and that PAT4 trafficking to lysosomes is inhibited by Rab12 knockdown (supplementary Fig S6D online), indicating that Rab12 regulates constitutive degradation of PAT4 in lysosomes. It was particularly noteworthy that Rab12 knockdown increased the amount of plasma membrane-localized PAT4 (Fig 3G), which is likely to contribute to the rise in the intracellular amino-acid concentration, and thereby result in upregulation of mTORC1 activity and inhibition of autophagy.
Rab12 regulates mTORC1 activity and autophagy
If accumulation of PAT4 in Rab12-depleted cells is the primary cause of the increased mTORC1 activity and decreased autophagic activity, overexpression of PAT4 should mimic Rab12 deficiency. As anticipated, increased phosphorylation of S6K and fewer LC3-positive dots were also observed in HA-PAT4-overexpressing cells (supplementary Fig S8A–D online). Conversely, increased phosphorylation of S6K and a reduced number of autophagosomes, both of which were induced by Rab12 knockdown, were completely rescued by simultaneous knockdown of PAT4 (Fig 4A–C), although PAT4 knockdown alone had little effect on mTORC1 activity or autophagy under our experimental conditions (supplementary Fig S8E–J online). Furthermore, addition of certain L -amino acids, for example, Pro and Trp, both of which have been found to be high-affinity substrates of PAT4 when expressed in Xenopus oocytes [ 33 ], to Rab12(QL)-overexpressing MEFs restored phosphorylation of S6K (supplementary Fig S4C,D online). These results allowed us to conclude that Rab12 regulates mTORC1 activity and autophagy through trafficking of PAT4.
The results of this study revealed an unexpected role of Rab12 in the regulation of mTORC1 activity and autophagy: Rab12 regulates constitutive degradation of amino-acid transporter PAT4, which indirectly modulates mTORC1 activity and autophagy through uptake of amino acids (see the schematic model in Fig 4D). As mTORC1 activity induces translation of cell-division-related genes and inhibits programmed cell death, upregulation of mTORC1 (and/or autophagy dysfunction) is closely associated with cancer/tumour [ 34 , 35 , 36 ]. Intriguingly, PAT4 is broadly expressed in many cancer cell lines [ 30 ]. Hence, the Rab12-regulating mechanism that controls mTORC1 activity and autophagy through quality control of PAT4 that we discovered in this study might provide a new target for the treatment of cancer and tumorigenesis.
D5. Regulation of mTORC1 by Energy Availability - AMP Kinase
- Contributed by Henry Jakubowski
- Professor (Chemistry) at College of St. Benedict/St. John's University
Believe it or not, another small G protein with GTPase activity, Rheb (Ras homolog enriched in brain), is involved in both mTORC1 recruitment to the lysosomal membrane and activation of mTOR. This interaction is also shown in the figure above. Mostly, Rheb is involved in activation of the kinase activity of the mTORC1 complex and specifically the phosphoryation by mTOR of the substrates S6K1 and EIF4EBP1. In the presence of growth factors, Rheb is localized to the membrane by a lipid anchor (a farnesyl group). The mTORC1 kinase-activating activity of Rheb stands in contrast to the role of the Rag G proteins which appears to be chiefly recruitment.
How is the small G protein Rheb regulated? Of course by its interaction with yet another GAP, named the tuberous sclerosis complex (TSC). In the absence of growth factors, TSC binds to Rheb and, acting as a GAP, promotes GTP hydrolysis. This inactivates Rheb, inhibiting mTOR kinase activity.
How then is Rheb regulated? One way is through phosphorylation by AMP Kinase, an enzyme that is itself regulated by the energy level of the cells (see Chapter 9C-10: AMP Kinase). AMPK phosphorylates and activates the TSC, which, acting as a GAP, inactivates the small G protein Rheb complex (TSC complex). Sestrins 1 and 2 may also regulate AMPK.
The figure below shows a more complete pathway of activation, regulation, and activity of AMPK. The illustration is used with courtesy of Cell Signaling Technologies (www.cellsignal.com)
The figure also shows that AMPK phosphorylates and inhibits mTORC1.
AMPK activates processes when energy is needed (glycolysis, lipolysis) and inhibits those when energy is abundant or when cell growth and proliferation occurs (fatty acid and protein synthesis). Hence AMPK activates catabolism while mTORC1 activates anabolism. Both can be viewed as master regulators of metabolism.
Amino acid signaling is a mitogenic pathway that controls growth and metabolic processes (1, 2). Although leucine is known to be the most effective amino acid for mTORC1 activation, glutamine and arginine can also activate mTORC1 via independent routes (27, 30). Our results unveiled a unique position of LRS in the control of the RagD–mTORC1 axis. Although LRS and Sestrin2 share a common role in the mediation of leucine signal for mTORC1 activation, their working mechanisms in the Rag GTPase cycle are idiosyncratic. Perhaps multiple leucine sensors are required for fine control of Rag GTPases in response to different nutritional environments. Whereas LRS is a positive regulator of the Rag GTPase cycle by functioning as a GAP for RagD, Sestrin2 is a negative regulator of the Rag GTPase cycle by inhibiting GATOR2. Thus, LRS and Sestrin2 could work as “on” and “off” switches, respectively, throughout the entire Rag GTPase cycle (Fig. 7H). Namely, during leucine signaling, LRS initiates the Rag GTPase cycle via RagD whereas Sestrin2 terminates the Rag GTPase cycle by controlling RagA–RagB GAP activity of GATOR1 via GATOR2 inhibition. Since the Km value of LRS for leucine in the amino acid activation reaction and the Kd of leucine for Sestrin2 are similar (22, 31), whether LRS and Sestrin2 regulate Rag GTPases independently or cooperatively needs further investigation.
This work also unveiled the kinetic difference and functional hierarchy among Rag GTPases. Interestingly, RagD seems to be functionally dominant among the four Rag GTPases (Fig. 4 C and D). Since the kinetics of S6K phosphorylation is well-correlated with that of RagB GTP (Fig. 3 A–D), the GTP–GDP status of RagB seems to be directly involved in mTORC1 activation. The Rag heterodimer that contains RagB GTP directly interacts with Raptor of mTORC1 (6). The GTP–GDP status is rate-limiting for RagB-mediated mTORC1 activation (7). In addition, our results support the notion that GTP hydrolysis of RagD by LRS is critical for leucine-induced RagB GTP formation (Fig. 4 C and D), which may explain the differential role of RagD and RagB in the Rag heterodimer. LRS-mediated GTP hydrolysis of RagD may control GTP loading of RagB via the recruitment of Ragulator, which is a RagB-GEF, leading to a direct interaction of GTP-loaded RagB with Raptor, thereby activating mTORC1.
The entire Rag GTPase cycle is affected by knockdown of LRS and RagD (Fig. 4 C and F) or overexpression of RagD GDP (Fig. 4D), which is possible because the Ragulator complex binds to RagA as well as RagB (Fig. 2C), albeit with a different binding affinity (Fig. 4 A and B). Consistent with these data, Ragulator is known to possess GEF activity toward RagA and RagB (24). Perhaps a conformational change induced by GTP hydrolysis of RagD causes a structural change in Ragulator, leading to activation of its GEF activity. Recently, the structure of the Ragulator complex was revealed, showing that the nucleotide binding, or G domain, of the Rag GTPase is distal from the LAMTOR components of the Ragulator complex (32 ⇓ –34). Thus, the driving force of the nucleotide exchange of RagB by Ragulator may require the GTP hydrolysis of RagD.
It is known that LRS is a component of the multi-tRNA synthetase complex (MSC), which serves as a signaling hub for its component enzymes and factors (35). LRS was also shown to interact with Vps34 in a leucine-dependent manner to activate the mTORC1 pathway (36). It is unclear how cellular localization and target interactions of LRS are regulated at this time. By analogy to the behavior of other MSC components such as EPRS, KRS, and AIMPs (37) and considering that the cellular level of LRS is unchanged by leucine concentration, it is speculative that cellular localization and interaction could be specifically controlled by context-dependent posttranslational modifications of MSC-associated LRS. However, we do not exclude the possibility that freely existing LRS could be primarily recruited for leucine-induced mTORC1 activation.
Our results suggest that the RagD–RagB and RagC–RagA heterodimers play differential roles in the process of mTORC1 activation. However, the exact roles of the RagD–RagB and RagC–RagA heterodimers remain unclear. Since amino acid or leucine supplementation, LRS knockdown, BC-LI-0186 treatment, or RagD GDP overexpression affected the change of all the Rag GTPases (Fig. 4 D, F, and G and SI Appendix, Fig. S3 A and B) and knockdown of RagA or RagC also blocked leucine-induced S6K phosphorylation (Fig. 4C), the RagC–RagA and RagD–RagB heterodimers are somehow involved in the mTORC1 activation process. One possibility is that the RagD–RagB heterodimer directly controls lysosomal translocation of mTORC1 while the RagC–RagA heterodimer affects the TSC–Rheb pathway, since there is a high degree of reciprocal interaction between RagC and TSC1 (38). Amino acids induce lysosomal translocation of mTORC1 and allow it to encounter its activator Rheb on the lysosome (11). Therefore, the RagC–RagA heterodimer may control Rheb inhibition by the TSC complex, although its role in the regulation of mTORC1 requires further investigation. This proposed link between the RagC–RagA heterodimer and TSC–Rheb pathway could provide a possible explanation for why mTORC1 activation occurs only when both Rag GTPases and Rheb are active.