mTORC2: The other mTOR in autophagy regulation

1.1. Autophagy and endosomal traffiking in human disease and aging

Defective lysosomal function is the cause of a group of more than 50 rare inherited metabolic disorders designated as lysosomal storage diseases (LSDs). These disorders are characterized by mutations that usually result in deficiency of a single enzyme required for the metabolism of substrates, leading to enlarged lysosomal vacuoles filled with undegraded material often localized adjacent to the nucleus. They are further classified, depending on the substrate that accumulates into lipid storage disorders, mucopolysaccharidoses, or glycoprotein storage disorders. Pompe disease, for example, is a LSD characterized by an accumulation of glycogen in the lysosomes of muscle cells, nervous system, and liver. To date, there are no cures for LSDs and treatments are mostly symptomatic.

Recently, increasing attention has been given to a potential link between abnormal lysosomal function and neurodegeneration and the induction of autophagy as a potential therapeutic target for conditions such as Huntington's disease, Parkinson's disease, and Alzheimer's disease, where pathogenic protein aggregation is a shared characteristic hallmark. These conditions have been attributed to changes in, among others, mTORC1, dynein, and Ras‐related protein‐7 (RAB7) activity (Caviston et al., 2011; Erie et al., 2015; Wen et al., 2017), preventing degradation of protein deposits by the autolysosomal pathway through a mechanism not yet fully elucidated. Accumulation of lysosomes that disturb the normal morphology of neuronal axons due to blockade of lysosomal proteolysis and vesicle transport has been described in Alzheimer's disease (Lee et al., 2011).

Understanding the role of the autophagy response in cancer biology is crucial in the search for novel effective therapeutics for this group of disorders. Anticancer agents have been shown to activate autophagy which can serve as a means for promoting their survival through efficient use of available nutrients, handling of oxidative stress, and limiting DNA damage. Activated autophagy can be detected at the most hypoxic regions of the tumor where there is considerable metabolic stress, conferring a survival advantage. Autophagy inhibition has been shown to improve the performance of anticancer drugs, including the response to chemotherapy in several human tumors and in preclinical mouse models. Inhibition has been shown to enhance the cytotoxic activity of effector T lymphocytes and natural killer cells against tumoral cells (Amaravadi et al., 2011). Human pancreatic tumors and pancreatic cancer cell lines are dependent on elevated basal levels of autophagy compared to normal pancreatic cells (Yang et al., 2011). Consistent with this, autophagy inhibition has been reported to induce apoptosis and to halt pancreatic tumor growth (Marchand et al., 2015). During oncogenic transformation, lysosomes can undergo changes in pH, subcellular localization, or composition; reductions in RAB7 expression promote lysosomal relocation to the cell periphery which has been documented to occur in prostate cancer and associated with increased tumor invasiveness (Steffan et al., 2014). The authors proposed that the mechanism behind this involves the unloading of proteolytic enzymes into the extracellular space that then digest the extracellular matrix. In addition, the endosomal network can incorporate proteins such as the transmembrane type 1 matrix metalloproteinase into the plasma membrane invadopodia, facilitating tumor migration, invasion, and metastasis (Macpherson et al., 2014; Monteiro et al., 2013). Peripheral lysosomal relocation has also been associated with increased expression of integrins in the plasma membrane, enhancing migration, and cell adhesion necessary for the establishment of new tumor populations (Dozynkiewicz et al., 2012). Importantly, tumor cells that undergo the process of autophagy, cease cell division and movement, and enter a dormant state, while maintaining the capacity to regenerate and resume proliferation when proper growth conditions are restored. Tumor regeneration and re‐emergence from latency is a key issue in cancer management (White & DiPaola, 2009).

Paradoxically, a tumor‐suppressive role for autophagy in line with its known role in clearing damaged organelles and cellular components has also been documented. This role is particularly prominent in hepatocellular carcinoma. Mice carrying deletions of Atg5 or Atg7 display an inactivation of autophagy and a build‐up of cellular waste. Defective autophagy leads to increased DNA‐damaging reactive oxygen species production, genomic instability, and generalized inflammation which can promote tumor initiation (Komatsu et al., 2007; Takamura et al., 2011). Monoallelic loss of Beclin‐1, a gene required for autophagic induction, has been reported in both ovarian and breast cancer (Liang et al., 1999). Beclin‐1 deletions induce chromosome instability and tumor initiation (Mathew et al., 2007). A very common mutagenesis event in a wide variety of human cancers involve mutations that lead to abnormal activity of the PI3K and mTOR pathways. Hyperactive phosphoinositide 3‐kinase (PI3K) inhibits autophagy and simultaneously stimulates uncontrolled cell growth and proliferation, regardless of nutrient and growth signal availability, which eventually leads to extreme metabolic stress and cell death (Jin et al., 2007), perhaps in a delicate balance that is still advantageous to the tumor. Due to its various known effects, autophagy has been referred to as a double‐edged sword in regard to whether it contributes to the overall suppression or promotion of tumorigenesis (White & DiPaola, 2009). Current efforts are directed to understand how pharmacological modulation of autophagy in cancer could lead to the development of novel, effective therapies depending on the particular autophagy context and whether it is activated or suppressed.

Current research on therapies that aim to prolong lifespan shows that the autophagy pathway is one of those implicated during the aging process. Age‐related impairments in autophagy have been reported in the rat liver and studies in model organisms including Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster demonstrate that deficiency in key autophagy genes (Atgs) results in a reduced lifespan (Martinez‐Lopez et al., 2015). In mammals, protein levels of LAMP2A have been shown to decline in aging mice, correlating with decreased CMA activity. Interestingly, inducible liver overexpression of the LAMP2A receptor helped maintaining functional CMA in the murine aged liver (Zhang & Cuervo, 2008). The effects of pharmacological approaches directed to promote longevity including caloric restriction, sirtuin‐1 (SIRT1) activation, mTOR, and insulin growth factor (IGF) inhibition all require autophagy activation (Rubinsztein et al., 2011). However, there are other documented effects of these approaches that are autophagy‐independent. A better understanding of the role that autophagy plays in the complex biological process of aging will therefore be essential in order to reach the goal of boosting cellular rejuvenation and lifespan extension.

2.1. The role of the mTORC1 pathway in autophagy

The mTORC1 complex has been shown to be regulated by multiple input signals such as hormones, growth factors, amino acids, and fatty acids. One of the main effects of activation of mTORC1 is the phosphorylation of S6K1 and eukaryotic translation initiation factor 4E‐binding protein‐1 (4EBP1), which facilitates the translation of mRNA and positively controls downstream anabolic processes. The tuberous sclerosis complex (TSC) is the main upstream negative regulator of mTORC1, integrating these upstream signals (Inoki & Guan, 2009; Swiatkowski et al., 2017). It stimulates the GTPase activity of the ras homolog enriched in brain (RHEB) protein that hydrolyzes RHEB‐GTP to RHEB‐GDP, maintaining mTORC1 in a switched‐off state. TSC can be inhibited by the AKT, MAPK, and Wnt pathways, whereas it is activated via phosphorylation by AMP‐activated protein kinase (AMPK) (Dalle Pezze et al., 2016), a kinase that becomes active at low ATP:AMP ratio. An important TSC‐independent mechanism of mTORC1 regulation is its inhibition under conditions of amino acid deprivation, connecting cellular nutritional status to the modulation of cell metabolism. Of note, the particular circulating amino acids responsible for the activation of the complex remain elusive. Arginine and leucine have been shown to be required for mTORC1 activation, however, they are not sufficient (Hara et al., 1998). Importantly, in addition to its role as an activator of the mTORC1 inhibitor TSC, AMPK has been shown to become activated by amino acid sufficiency, and subsequently able to place an inhibitory phosphorylation on Raptor (Gwinn et al., 2008). The concurrent activation of mTORC1 and AMPK under nutrient‐rich conditions may suggest a role for AMPK in maintaining autophagy and metabolic homeostasis and an intricate cross‐regulation of the AMPK‐mTORC1 axis (Dalle Pezze et al., 2016). Recent studies support a two‐step mechanism for the activation of mTORC1 by two distinct groups of amino acids, namely priming and activating amino acids (Dyachok et al., 2016). When amino acids are abundant, a conformational change in Rag‐GTPase heterodimers is induced. These heterodimers interact with Raptor, a member of the mTORC1 complex, which in turn localizes the complex to the surface of late endosomes and lysosomes where it is activated by RHEB‐GTP (Martina & Puertollano, 2013; Sancak et al., 2008). This highlights the notion that the endosome‐lysosomal network is a central hub for intracellular signaling and not just merely as a degradation station (Perera & Zoncu, 2016).

Importantly, upon integration of these multiple environmental and chemical cues, mTORC1 is known to play a pivotal role as a negative regulator of catabolic processes such as autophagy in a transcription‐dependent and independent manner. Under a sufficient supply of nutrients, growth factors, and cellular signaling stimuli, mTORC1 is activated at the lysosomal surface and, through phosphorylation, inhibits proteins that are important for autophagy and lysosomogenesis. These include TFEB (Martina et al., 2012; Roczniak‐Ferguson et al., 2012) and transcription factor E3 (TFE3) (Martina et al., 2014), two important transcriptional regulators of these cellular processes, and Unc‐51 like autophagy activating kinase 1 (ULK1) and ATG13 (Hosokawa et al., 2009), proteins that are directly involved in the formation of autophagosomes (Figure 1).

TFEB contains a number of amino acids that undergo phosphorylation by several different kinases. Serine 142 of TFEB has been shown to be phosphorylated by ERK and mTORC1, the latter which also targets serine 211 of TFEB. This takes place at the outer lysosomal membrane and affects the subcellular localization of the protein. Phosphorylated TFEB at the lysosomal membrane is bound by 14‐3‐3 proteins that mask a nuclear localization signal in the vicinity, thereby retaining TFEB in the cytoplasm (Pena‐Llopis et al., 2011; Roczniak‐Ferguson et al., 2012; Settembre et al., 2012). Similarly, mTORC1 phosphorylates TFE3 and microphthalmia‐associated transcription factor (MITF) and regulates their activity through the same mechanism of cytoplasmic retention (Martina et al., 2014; Martina & Puertollano, 2013). Inactivation of the complex due to lack of nutrients or mitogens switches off the mTORC1 and MAPK/ERK pathways, leading to nuclear translocation of these factors and upregulation of target genes. Unphosphorylated TFEB and TFE3 shuttle to the nucleus, where they bind to Coordinated Lysosomal Expression and Regulation (CLEAR) consensus sequences, whereas nuclear MITF has been shown to activate genes involved in melanogenesis in MNT‐1 melanoma cells (Bentley et al., 1994; Hah et al., 2012). Inactive mTORC1‐dependent shuttling of microphthalmia/transcription factor E (MiT/TFE) proteins to the nucleus establishes a negative feedback loop, turning on expression of genes required for mTORC1 activity including folliculin interacting protein 2 (FNIP2), RagC/D, and vATPase and promoting lysosomogenesis and autophagy thereby increasing amino acid pools (Palmieri et al., 2011; Zhang et al., 2015). Nuclear shuttling of TFEB and subsequent enhancement of its transcriptional activity has been shown to lead to induction of autophagy and clearance of intra‐lysosomal aggregates in a model of neurological Batten disease (Palmieri et al., 2017). Interestingly, mTORC1 can be positively regulated through the MAPK/ERK axis by two different routes, pointing to a crosstalk between these two distinct pathways in the regulation of the CLEAR network. Firstly, ERK1/2 downstream of the BRAF/MAPK pathway can phosphorylate RSK which, in turn, activates mTORC1 in melanoma cells and its downstream targets, promoting increased protein translation, growth, and proliferation (Romeo et al., 2013; Xue et al., 2017). Secondly, MAPK signaling leads to phosphorylation of raptor on proline sites prior to raptor‐mediated scaffolding and recruitment to the lysosomes of active mTORC1 (Carriere et al., 2011).

3.1. The mTORC2‐AKT axis in autophagy

Novel findings link mTORC2 activity with the regulation of the autophagy process. In addition to the plasma membrane, nucleus, mitochondria, and other cytoplasmic structures, mTORC2 has been found to localize to the lysosomes (Berchtold et al., 2012; Ebner et al., 2017; Zinzalla et al., 2011). Approximately 29% of endogenous lysosomal‐associated membrane protein‐1 (LAMP1) co‐localizes with mTORC2, indicating that a subgroup of lysosomes are mTORC2‐positive (Jia & Bonifacino, 2019). At the lysosomal level, mTORC2 and AKT have been shown to regulate CMA machinery recruitment (Arias et al., 2015).

Similar to mTORC1, mTORC2 has been shown to be a negative regulator of autophagy. AKT itself has been reported as an mTORC1‐independent negative regulator of autophagy. AKT can directly phosphorylate Beclin‐1 at Ser234 and Ser295, promoting binding of Beclin‐1 to intermediate filaments and autophagy suppression (Mostowy, 2014; Wang et al., 2012). IGF‐1 can inhibit autophagy via activation of mTORC2 and AKT, linking glucose metabolism as a nutritional cue able to modulate autophagy processes through these proteins (Wang & Gu, 2018). In colorectal cancer cells with constitutively activated mTORC1, mTORC2 appears to regulate the levels of basal autophagy and participate in the maintenance of signaling vesicles and tumor cell survival (Lampada et al., 2017, 2017).

A regulatory feedback loop between starvation‐induced autophagy and mTORC2 has been suggested, as starvation initially triggers reduced levels of phosphorylated Ser473, followed by re‐phosphorylation upon prolonged starvation (Bernard et al., 2014). Along these same lines, chronic autophagy has been shown to increase the activity of mTORC2 via the induction of reactive oxygen species (Bernard et al., 2020).

A very compelling study by Arias and colleagues (Arias et al., 2015) investigated the effects of AKT inhibition on chaperone‐mediated autophagy (CMA) in fibroblasts. Treatment with cell‐permeable AKT inhibitors abrogated the phosphorylation of Ser473 of Akt induced by platelet‐derived growth factor (PDGF) which, in turn, activated basal CMA. In this context, the Pleckstrin homology (PH) domain and leucine‐rich repeat protein phosphatase 1 (PHLPP1) were the key phosphatase counteracting phosphorylation of AKT by mTORC2 (Arias et al., 2015). Whereas mTORC2 activity is reduced during starvation and hence inhibits CMA, it increases upon prolonged starvation and the action of PHLPP1 is required to maintain basal CMA (Arias, 2015). Sustaining a balanced CMA activity is especially relevant for metabolic homeostasis as this degradatory pathway is important for regulating levels of key metabolic enzymes involved in carbohydrate and lipid metabolism (Schneider et al., 2014). Remarkably, treatment of peripheral blood mononuclear cells with an mTORC1/2 inhibitor has been shown to inhibit phosphorylation of Ser473 of AKT and enhance association of AKT with PHLPP1 which may further contribute to the inactivation of AKT (Gupta et al., 2012). Another study suggests that inhibition of mTORC2 via the ATP synthase inhibitor mycotoxin citreoviridin inhibits mTORC2, resulting in apoptosis of cardiomyocytes through lysosomal membrane permeabilization and detrimental autophagy (Feng et al., 2019).

Glycogen synthase kinase 3 beta (GSK3β), a kinase known to participate in several pathways connected with autophagy regulation, can phosphorylate Ser1235 of RICTOR and abrogate binding and phosphorylation of AKT by mTORC2 (Chen et al., 2011). ShRNA‐mediated silencing of RICTOR or its downregulation through the action of microRNAs such as miR211 or miR15a and miR16 have been reported to activate autophagy, presumably through the downstream inactivation of mTORC1 (Huang et al., 2015; Ozturk et al., 2019). Active mTORC2 induces the phosphorylation of AKT at Ser473, a step needed for full activation of AKT (Sarbassov et al., 2005), enhancing its activity in a substrate‐dependent manner. Deletion of RICTOR or mTOR‐associated protein, LST8 homolog (mLST8) ablated insulin‐induced phosphorylation of Forkhead box class O 3 (FOXO3), whereas this did not affect phosphorylation of TSC2 or Ser9 of GSK3β (Guertin et al., 2006). These findings suggest that although Ser473 phosphorylation is required for AKT‐mediated phosphorylation of FOXO3 or PKCα, it is not required for that of TSC2, a well‐described mTORC1 inhibitor (Saxton & Sabatini, 2017; Webb & Brunet, 2014). This is an important observation that suggests that the proposed mTORC2‐AKT‐TSC2 pathway thought to regulate mTORC1 may not be valid (Gaubitz et al., 2016), and that a different pool of phosphorylated AKT is responsible for phosphorylation of TSC2 (Huang & Manning, 2009) (Figure 2).

3.2. SIN1‐RB

Sin1 is a protein component specifically found inTORC2 (absent in mTORC1) and is required for the kinase activity of the complex in multiple ways. Sin1 is required for docking the complex to the plasma membrane (Yuan & Guan, 2015). Sin1 knockdown triggers the dissociation of RICTOR from the complex (Yang et al., 2006); however, Sin1 is also responsible for recruiting substrates such as AKT and SGK to mTORC2 for subsequent phosphorylation (Jhanwar‐Uniyal et al., 2017; Liu, Gan et al., 2015; Yang et al., 2006; Yuan & Guan, 2015). Sin1 binds through its PH domain to mTORC2 blocks access to its substrate AKT, whereas PIP₃ binding to Sin1‐PH releases the tight interaction with mTORC2 and allows the kinase to access substrates such as AKT (Liu, Gan et al., 2015). Accordingly, ablating the particular residues of Sin1 mediating interaction with PIP₃ inactivates mTORC2 (Liu, Gan et al., 2015). Interestingly, the retinoblastoma (Rb) protein has been found to bind the Sin1‐PH domain, suppressing access of AKT to mTORC2 and its subsequent phosphorylation (Zhang et al., 2016). Conversely, in Rb‐deficient cells, the activity of mTORC2 is enhanced as shown by an increase in the phosphorylation of its downstream targets FOXO and PRAS40 (Zhang et al., 2016). Thus, Rb functions as a positive modulator of autophagy via this interaction and its silencing leads to blockage of autophagic flux in glioblastoma cells, sensitizing them to the action of chemotherapeutic agents (Biasoli et al., 2013).

3.3. PKC

One of the first identified substrates of mTORC2 was PKCα, a modulator of actin cytoskeleton reorganization and cell migration (Dos et al., 2004; Hernández‐Negrete et al., 2007; Jacinto et al., 2004,). Whereas deletion of mLST8 or RICTOR, which specifically inactivate mTORC2, compromised the phosphorylation and stability of PKCα (Guertin et al., 2006), mTORC2 has been indicated to be a positive regulator of PKCδ (Gan et al., 2012). The role of different PKC isozymes is under investigation, as contrasting studies indicate that they might be relevant modulators of autophagy (Kaleli et al., 2020). PKCα has been shown to suppress autophagy in breast cancer cells (Wong et al., 2017) and during gestational diabetes, where its action may trigger neural tube defects (Wang et al., 2017). Rapamycin treatment, and resulting inhibition of mTORC2, reduced the phosphorylation and activity of several PKC isozymes, in particular PKCβ, and ameliorated mitochondrial disease and neuropathology in a mouse model of Leigh syndrome (Martin‐Perez et al., 2020). PKCβ pharmacological inhibition or genetic depletion increased autophagy and mitochondrial health (Patergnani et al., 2013). In a contrasting study, PKCα/δ induce phosphorylation of Ser9 and Ser21 in GSK3β residues associated with an inactive form of this kinase (Li et al., 2016). Inactive GSK3β has been shown to contribute to nuclear translocation and activation of TFEB, resulting in autophagy induction (Li et al., 2018; Marchand et al., 2015). An additional mechanism by which PKCδ may be involved in the positive regulation of autophagy is via activation of c‐Jun N‐terminal kinases (JNK) and p38 MAPK which subsequently phosphorylate zinc finger with KRAB And SCAN domains 3 (ZKSCAN3), resulting in cytoplasmic translocation and inactivation of this transcriptional repressor of autophagy (Li et al., 2016). Taken in total, these data suggest that in the context of autophagy regulation, the interaction between the mTORC2 and different PKC isozymes is complex and likely dependent on crosstalk with other connected signaling pathways.

3.4. SGK‐1

Serum and glucocorticoid‐induce protein kinase is another member of the AGC subfamily of kinases which has significant sequence homology (~54%) with AKT (Kobayashi & Cohen, 1999; Liu et al., 2017). SGK‐1 is phosphorylated at Ser422 in its hydrophobic region by mTORC2 (Luo et al., 2018) and functions as an effector modulated by the insulin/IGF‐1 pathway, involved in the stress response through the negative regulation of the skinhead 1 (skn‐1)/nuclear factor erythroid 2‐related factor 2 (NRF2) pathway (Aspernig et al., 2019; Heimbucher et al., 2020; Mizunuma et al., 2014).

Due to its high structural homology, SGK‐1 has been shown to display significant overlap with AKT in the subset of target proteins that they both regulate via phosphorylation. Some of these shared downstream effectors include GSK3β (Kobayashi & Cohen, 1999) and FOXO3 (Brunet et al., 2001). As a putative negative regulator of FOXO3, the role of SGK‐1 in autophagy is the subject of current investigation. Pharmacological inhibition of SGK‐1 results in an increase in protein expression of ULK1, a major initiator of the autophagy process. Although the mechanism for the increased expression of ULK1 upon SGK‐1 inhibition remains elusive, it has been hypothesized that it takes place through the inhibitory phosphorylation of FOXO3 by SGK‐1 (Maestro et al., 2020; Zuleger et al., 2018). Interestingly, genetic disruption of the PDK1 interacting fragment (PIF)‐pocket catalytic domain of PDK1, which abrogates activation of SGK‐1, RSK, and S6K but not AKT, do not result in overall reduced phosphorylation of GSK3 or FOXO (Collins et al., 2003), suggesting that SGK‐1 might not be the key driver in their activation, possibly due to functional overlap with other AGC kinases. Defining the specific targets of SGK‐1 activity and their biological effects has been challenging due to the lack of effective and specific inhibitors (Di Cristofano, 2017). Downregulation of sgk‐1 activity through direct knockout/knockdown of sgk‐1 or through ablation of rict‐1 and associated mTORC2 inhibition in C. elegans induces autophagy, as demonstrated by increased expression of the microtubule‐associated proteins 1A/1B light chain 3 (LC3A/B) ortholog LC3 GABARAP and GATE‐16 family 1 (lgg‐1) and the TFEB ortholog helix loop helix 30 (hlh‐30). The mRNA expression of both lgg‐1 and hlh‐30 is upregulated upon voltage‐dependent anion channel 1 (VDAC1) overexpression, a protein that in turn is phosphorylated by sgk‐1 and subsequently targeted for degradation. Surprisingly, these mutants displayed a shortened lifespan. The authors showed that VDAC1 concomitantly activates mitochondrial permeability to initiate mitophagy, highlighting the potential deleterious effects of elevated levels of autophagy in vivo that can disrupt mitochondrial homeostasis (Zhou et al., 2019).

Oxidative stress has been identified as an inducer of mTORC2 activity in myofibroblasts (Bernard et al., 2020). Interestingly, Aspernig et al. showed that the mTORC2‐SGK‐1 axis is involved in maintaining mitochondrial homeostasis, whereas mTORC2 inhibits autophagy through activation of AKT and inactivation of FOXO (Aspernig et al., 2019). From these observations, we propose that a useful strategy to benefit from elevated autophagy flux while counteracting the harmful effects of mTORC2‐SGK‐1 inhibition would be the preservation of specific targets downstream of SGK‐1 that suppress mitochondrial permeability (Figure 2).

3.5. The foxos

The FOXO family of transcription factors includes several related proteins in mammals such as FOXO1, 3, 4, and 6 and orthologs across the animal kingdom. They contain a forkhead box DNA‐binding domain and nuclear localization and export signals that direct the nuclear shuttling and cytoplasmic export of these factors (Cheng, 2019). FOXOs have been associated with autophagy regulation in both transcription‐dependent and independent fashions. The factors can bind to the promoters of autophagy‐related genes through their DNA‐binding domain (Liu et al., 2018; Webb & Brunet, 2014). Cytoplasmic FOXO1 interacts with ATG7, a ubiquitin‐activating E1‐like enzyme required for autophagosome assembly (Xiong, 2015; Zhao et al., 2010). FOXO1 has been found to be acetylated by cyclic AMP (cAMP)‐response element‐binding protein (CREB)‐binding protein paralog p300 and phosphorylated by AKT. These modifications may facilitate its cytoplasmic retention and interaction with ATG7 (Shen et al., 2017). On the other hand, deacetylases like SIRT1/2 induce nuclear translocation of FOXO1/3 and transcriptional activation of target genes (Liu, Bi et al., 2015). SIRT1 was found to induce the transcription of RICTOR, phosphorylation of Ser473 of Akt and Ser253 of Foxo1 at S253, which results in decreased gluconeogenesis (Tobita et al., 2016; Wang et al., 2011). In addition to a role for mTORC2 as a regulator of FOXO through AKT‐induced phosphorylation and cytoplasmic localization, mTORC2 has been shown to directly interact with and suppress SIRT6, whereas inactivation through RICTOR knockdown lifts SIRT6 suppression and promotes FOXO1 deacetylation and nuclear translocation (Jung et al., 2019). These findings suggest that a fine‐tuned control of FOXO1 activity through post‐translational modification and subsequent changes in subcellular localization may lead to different roles in autophagy regulation (Cheng, 2019).

FOXO3 has been shown to drive autophagy in hematopoietic stem cells (HSCs) and to be a key player in the regulation of autophagy in muscle tissue. FOXO3a ^−/− knockout in HSCs leads to significant downregulation of the expression of autophagy‐related genes paralleled with an increase in LC3 protein levels that suggest impaired autophagic flux (Warr et al., 2013).

FOXO3 downstream of the mTORC2‐AKT axis is sufficient to drive autophagy in muscle cells (Mammucari et al., 2007; Zuleger et al., 2018). Two identified target effectors of FOXO3‐driven autophagy are LC3 and B‐cell lymphoma 2 (BCL2)‐interacting protein 3 (BNIP3). Conversely, mTORC2‐AKT phosphorylates and inactivates FOXO3 and autophagy independently of mTORC1 and is not prevented by rapamycin (Mammucari et al., 2007). SGK‐1 can also phosphorylate and inactivate FOXO3. Interestingly, phosphorylated FOXO3a has been reported to interact with LC3. Inhibiting SGK‐1 leads to reduced levels of p‐FOXO3 interacting with LC3 and increased LC3‐I/LC3‐II conversion, suggesting increased autophagic flux (Liu et al., 2017). In a rat model of burn‐induced muscle wasting, down‐regulating Phlpp1 led to elevated Akt and FoxO3 phosphorylation, which in turn suppressed detrimental autophagy and mitigated muscle wasting (Yu et al., 2019b; Zhao et al., 2007). Another mechanism by which mTORC2 regulates FOXO3 activity is through phosphorylation and inactivation of histone deacetylase (HDAC), resulting in increased acetylation of FOXO3 and reduced transcriptional activity (Masui et al., 2013).

Another study shed additional light on mechanism involved in autophagy regulation by FOXO3, involving glutamine metabolism as a regulator of mTORC1 localization. FOXO3 can induce the expression of glutamine synthetase, leading to rising glutamine levels that, in turn, can inhibit mTORC1 localization to the lysosomal membrane impairing the ability of mTORC1 to function as an autophagy negative regulator (van der Vos et al., 2012) (Figure 3).

3.6. AMPK

In endothelial cells, silencing of RICTOR increased the expression of LC3‐II while it decreased protein levels of p62, two markers that are associated with enhanced autophagic flux, in parallel with AMP‐activated protein kinase (AMPK) activation. In addition, arginase 2 (ARG2) can inactivate autophagy through positive regulation of mTORC2 and subsequent inactivation of AMPK (Xiong et al., 2014). AMPK appears to participate in a regulatory feedback loop as activating AMPK with metformin, a drug employed in the treatment of type 2 diabetes, increased liver mTORC2 in vivo (Kazyken et al., 2019). Metformin‐mediated AMPK activation has been shown to induce autophagosome maturation and induce cell‐cycle arrest through combined mTORC1/2 repression (Jang et al., 2018; Wang et al., 2018).

Additionally, AMPK has been identified as an upstream regulator of the MEK/ERK pathway which, in turn, can disassemble and inhibit both mTOR complexes and induce autophagy through upregulation of Beclin‐1. Interestingly, the authors of this study suggested that whereas individually inhibiting mTORC1 or mTORC2 results in moderate activation of Beclin‐1 and protective autophagy, dual inhibition of mTORC1/2 and the subsequent robust activation of Beclin‐1 can result in destructive autophagy (Wang et al., 2009).