The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor‐infiltrating CD4 and CD8 T cells. The Peli1‐deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild‐type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1‐inhibitory proteins, TSC1 and TSC2. Peli1 mediates non‐degradative ubiquitination of TSC1, thereby promoting TSC1‐TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1‐mediated actions on T cell metabolism and antitumor immunity.     

Requirement for lysosomal localization of mTOR for its activation differs between leucine and other amino acids

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and metabolism. It controls many cell functions by integrating nutrient availability and growth factor signals. Amino acids, and in particular leucine, are among the main positive regulators of mTORC1 signaling. The current model for the regulation of mTORC1 by amino acids involves the movement of mTOR to the lysosome mediated by the Rag-GTPases. Here, we have examined the control of mTORC1 signaling and mTOR localization by amino acids and leucine in serum-fed cells, because both serum growth factors (or, e.g., insulin) and amino acids are required for full activation of mTORC1 signaling. We demonstrate that mTORC1 activity does not closely correlate with the lysosomal localization of mTOR. In particular, leucine controls mTORC1 activity without any detectable modification of the lysosomal localization of mTOR, indicating that the signal(s) exerted by leucine is likely distinct from those exerted by other amino acids. In addition, knock-down of the Rag-GTPases attenuated the inhibitory effect of amino acid- or leucine-starvation on the phosphorylation of mTORC1 targets. Furthermore, data from cells where Rag expression has been knocked down revealed that leucine can promote mTORC1 signaling independently of the lysosomal localization of mTOR. Our data complement existing models for the regulation of mTORC1 by amino acids and provide new insights into this important topic.     

AKAP13 couples GPCR signaling to mTORC1 inhibition

The mammalian target of rapamycin complex 1 (mTORC1) senses multiple stimuli to regulate anabolic and catabolic processes. mTORC1 is typically hyperactivated in multiple human diseases such as cancer and type 2 diabetes. Extensive research has focused on signaling pathways that can activate mTORC1 such as growth factors and amino acids. However, less is known about signaling cues that can directly inhibit mTORC1 activity. Here, we identify A-kinase anchoring protein 13 (AKAP13) as an mTORC1 binding protein, and a crucial regulator of mTORC1 inhibition by G-protein coupled receptor (GPCR) signaling. GPCRs paired to Gα_s proteins increase cyclic adenosine 3’5’ monophosphate (cAMP) to activate protein kinase A (PKA). Mechanistically, AKAP13 acts as a scaffold for PKA and mTORC1, where PKA inhibits mTORC1 through the phosphorylation of Raptor on Ser 791. Importantly, AKAP13 mediates mTORC1-induced cell proliferation, cell size, and colony formation. AKAP13 expression correlates with mTORC1 activation and overall lung adenocarcinoma patient survival, as well as lung cancer tumor growth in vivo. Our study identifies AKAP13 as an important player in mTORC1 inhibition by GPCRs, and targeting this pathway may be beneficial for human diseases with hyperactivated mTORC1.     

mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.     

SH3BP4 is a negative regulator of amino acid-Rag GTPase-mTORC1 signaling

Amino acids stimulate cell growth and suppress autophagy through activation of mTORC1. The activation of mTORC1 by amino acids is mediated by Rag guanosine triphosphatase (GTPase) heterodimers on the lysosome. The molecular mechanism by which amino acids regulate the Rag GTPase heterodimers remains to be elucidated. Here, we identify SH3 domain-binding protein 4 (SH3BP4) as a binding protein and a negative regulator of Rag GTPase complex. SH3BP4 binds to the inactive Rag GTPase complex through its Src homology 3 (SH3) domain under conditions of amino acid starvation and inhibits the formation of active Rag GTPase complex. As a consequence, the binding abrogates the interaction of mTORC1 with Rag GTPase complex and the recruitment of mTORC1 to the lysosome, thus inhibiting amino acid-induced mTORC1 activation and cell growth and promoting autophagy. These results demonstrate that SH3BP4 is a negative regulator of the Rag GTPase complex and amino acid-dependent mTORC1 signaling.     

Glutaminolysis Activates Rag-mTORC1 Signaling

Amino acids control cell growth via activation of the?highly conserved kinase TORC1. Glutamine is a particularly important amino acid in cell growth control and metabolism. However, the role of glutamine in TORC1 activation remains poorly defined. Glutamine is metabolized through glutaminolysis to?produce α-ketoglutarate. We demonstrate that glutamine in combination with leucine activates mammalian TORC1 (mTORC1) by enhancing glutaminolysis and α-ketoglutarate production. Inhibition of glutaminolysis prevented GTP loading of RagB and lysosomal translocation and subsequent activation of mTORC1. Constitutively active Rag heterodimer activated mTORC1 in the absence of glutaminolysis. Conversely, enhanced glutaminolysis or?a cell-permeable α-ketoglutarate analog stimulated lysosomal translocation and activation of mTORC1. Finally, cell growth and autophagy, two processes controlled by mTORC1, were regulated by glutaminolysis. Thus, mTORC1 senses and is activated by?glutamine and leucine via glutaminolysis and α-ketoglutarate production upstream of Rag. This may provide an explanation for glutamine addiction in cancer cells.     

Amino acid regulation of TOR complex 1

TOR complex 1 (TORC1), an oligomer of the mTOR (mammalian target of rapamycin) protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GbetaL, controls cell growth in all eukaryotes in response to nutrient availability and in metazoans to insulin and growth factors, energy status, and stress conditions. This review focuses on the biochemical mechanisms that regulate mTORC1 kinase activity, with special emphasis on mTORC1 regulation by amino acids. The dominant positive regulator of mTORC1 is the GTP-charged form of the ras-like GTPase Rheb. Insulin, growth factors, and a variety of cellular stressors regulate mTORC1 by controlling Rheb GTP charging through modulating the activity of the tuberous sclerosis complex, the Rheb GTPase activating protein. In contrast, amino acids, especially leucine, regulate mTORC1 by controlling the ability of Rheb-GTP to activate mTORC1. Rheb binds directly to mTOR, an interaction that appears to be essential for mTORC1 activation. In addition, Rheb-GTP stimulates phospholipase D1 to generate phosphatidic acid, a positive effector of mTORC1 activation, and binds to the mTOR inhibitor FKBP38, to displace it from mTOR. The contribution of Rheb's regulation of PL-D1 and FKBP38 to mTORC1 activation, relative to Rheb's direct binding to mTOR, remains to be fully defined. The rag GTPases, functioning as obligatory heterodimers, are also required for amino acid regulation of mTORC1. As with amino acid deficiency, however, the inhibitory effect of rag depletion on mTORC1 can be overcome by Rheb overexpression, whereas Rheb depletion obviates rag's ability to activate mTORC1. The rag heterodimer interacts directly with mTORC1 and may direct mTORC1 to the Rheb-containing vesicular compartment in response to amino acid sufficiency, enabling Rheb-GTP activation of mTORC1. The type III phosphatidylinositol kinase also participates in amino acid-dependent mTORC1 activation, although the site of action of its product, 3'OH-phosphatidylinositol, in this process is unclear.     

MAPK signaling regulates c‐MYC for melanoma cell adaptation to asparagine restriction

Amino acid restriction is among promising potential cancer treatment strategies. However, cancer cells employ a multitude of mechanisms to mount resistance to amino acid restriction, which impede the latter’s clinical development. Here we show that MAPK signaling activation in asparagine‐restricted melanoma cells impairs GSK3‐β‐mediated c‐MYC degradation. In turn, elevated c‐MYC supports ATF4 translational induction by enhancing the expression of the amino acid transporter SLC7A5, increasing the uptake of essential amino acids, and the subsequent maintenance of mTORC1 activity in asparagine‐restricted melanoma cells. Blocking the MAPK‐c‐MYC‐SLC7A5 signaling axis cooperates with asparagine restriction to effectively suppress melanoma cell proliferation. This work reveals a previously unknown axis of cancer cell adaptation to asparagine restriction and informs mechanisms that may be targeted for enhanced therapeutic efficacy of asparagine limiting strategies.     

Proteomic analyses reveal GNG12 regulates cell growth and casein synthesis by activating the Leu-mediated mTORC1 signaling pathway

In cow mammary epithelial cells (CMECs), cell growth and casein synthesis are regulated by amino acids (AAs), and lysosomes are important organelles in this regulatory process, but the mechanisms remain unclear. Herein, lysosomal membrane proteins (LMPs) in CMECs in the presence (Leu+) and absence (Leu-) of leucine were quantitatively analysed using Sequential Windowed Acquisition of All Theoretical Fragment Ion (SWATH) mass spectrometry. In identified LMPs, Guanine nucleotide-binding protein subunit gamma-12 (GNG12) was a markedly up-regulated protein in Leu+ group. CMECs were treated with Leu+ or Leu−, expression and lysosomal localization of GNG12 were decreased in response to Leu absence. Overexpressing or inhibiting GNG12 demonstrated that cell growth, casein synthesis and activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway were all up-regulated by GNG12. Cell growth, casein synthesis and mTORC1 signaling pathway were decreased in response to Leu absence, but these decreases were partially restored by GNG12 overexpression, and those effects were partially reversed by inhibiting GNG12. Co-immunoprecipitation analysis showed that GNG12 activates the mTORC1 pathway via interaction with Ragulator. Taken together, these results suggest that GNG12 is a positive regulator of the Leu-mediated mTORC1 signaling pathway in CMECs that promotes cell growth and casein synthesis.     

Key mediators of intracellular amino acids signaling to mTORC1 activation

Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

     

mTORC2: The other mTOR in autophagy regulation

The mechanistic target of rapamycin (mTOR) has gathered significant attention as a ubiquitously expressed multimeric kinase with key implications for cell growth, proliferation, and survival. This kinase forms the central core of two distinct complexes, mTORC1 and mTORC2, which share the ability of integrating environmental, nutritional, and hormonal cues but which regulate separate molecular pathways that result in different cellular responses. Particularly, mTORC1 has been described as a major negative regulator of endosomal biogenesis and autophagy, a catabolic process that degrades intracellular components and organelles within the lysosomes and is thought to play a key role in human health and disease. In contrast, the role of mTORC2 in the regulation of autophagy has been considerably less studied despite mounting evidence this complex may regulate autophagy in a different and perhaps complementary manner to that of mTORC1. Genetic ablation of unique subunits is currently being utilized to study the differential effects of the two mTOR complexes. RICTOR is the best‐described subunit specific to mTORC2 and as such has become a useful tool for investigating the specific actions of this complex. The development of complex‐specific inhibitors for mTORC2 is also an area of intense interest. Studies to date have demonstrated that mTORC1/2 complexes each signal to a variety of exclusive downstream molecules with distinct biological roles. Pinpointing the particular effects of these downstream effectors is crucial toward the development of novel therapies aimed at accurately modulating autophagy in the context of human aging and disease.     

Multiple amino acid sensing inputs to mTORC1

The evolutionarily conserved target of rapamycin complex 1 (TORC1) is a master regulator of cell growth and metabolism. In mammals, growth factors and cellular energy stimulate mTORC1 activity through inhibition of the TSC complex (TSC1-TSC2-TBC1D7), a negative regulator of mTORC1. Amino acids signal to mTORC1 independently of the TSC complex. Here, we review recently identified regulators that link amino acid sufficiency to mTORC1 activity and how mutations affecting these regulators cause human disease.     

Class III PI-3-kinase activates phospholipase D in an amino acid-sensing mTORC1 pathway

The rapamycin-sensitive mammalian target of rapamycin (mTOR) complex, mTORC1, regulates cell growth in response to mitogenic signals and amino acid availability. Phospholipase D (PLD) and its product, phosphatidic acid, have been established as mediators of mitogenic activation of mTORC1. In this study, we identify a novel role for PLD1 in an amino acid-sensing pathway. We find that amino acids activate PLD1 and that PLD1 is indispensable for amino acid activation of mTORC1. Activation of PLD1 by amino acids requires the class III phosphatidylinositol 3-kinase hVps34, which stimulates PLD1 activity through a functional interaction between phosphatidylinositol 3-phosphate and the Phox homology (PX) domain of PLD1. Furthermore, amino acids stimulate PLD1 translocation to the lysosomal region where mTORC1 activation occurs in an hVps34-dependent manner, and this translocation is necessary for mTORC1 activation. The PX domain is required for PLD1 translocation, mTORC1 activation, and cell size regulation. Finally, we show that the hVps34-PLD1 pathway acts independently of, and in parallel to, the Rag pathway in regulating amino acid activation of mTORC1.     

Regulation of mTORC1 by the Rab and Arf GTPases

The mammalian target of rapamycin (mTOR) is a key cell growth regulator, which forms two distinct functional complexes (mTORC1 and mTORC2). mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. mTORC1 is regulated by a wide range of extra- and intracellular signals, including growth factors, nutrients, and energy levels. Precise regulation of mTORC1 is important for normal cellular physiology and development, and dysregulation of mTORC1 contributes to hypertrophy and tumorigenesis. In this study, we screened Drosophila small GTPases for their function in TORC1 regulation and found that TORC1 activity is regulated by members of the Rab and Arf family GTPases, which are key regulators of intracellular vesicle trafficking. In mammalian cells, uncontrolled activation of Rab5 and Arf1 strongly inhibit mTORC1 activity. Interestingly, the effect of Rab5 and Arf1 on mTORC1 is specific to amino acid stimulation, whereas glucose-induced mTORC1 activation is not blocked by Rab5 or Arf1. Similarly, active Rab5 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds and activates mTORC1. Our data demonstrate a key role of Rab and Arf family small GTPases and intracellular trafficking in mTORC1 activation, particularly in response to amino acids.     

Growth factor signaling to mTORC1 by amino acid–laden macropinosomes

The rapid activation of the mechanistic target of rapamycin complex-1 (mTORC1) by growth factors is increased by extracellular amino acids through yet-undefined mechanisms of amino acid transfer into endolysosomes. Because the endocytic process of macropinocytosis concentrates extracellular solutes into endolysosomes and is increased in cells stimulated by growth factors or tumor-promoting phorbol esters, we analyzed its role in amino acid–dependent activation of mTORC1. Here, we show that growth factor-dependent activation of mTORC1 by amino acids, but not glucose, requires macropinocytosis. In murine bone marrow–derived macrophages and murine embryonic fibroblasts stimulated with their cognate growth factors or with phorbol myristate acetate, activation of mTORC1 required an Akt-independent vesicular pathway of amino acid delivery into endolysosomes, mediated by the actin cytoskeleton. Macropinocytosis delivered small, fluorescent fluid-phase solutes into endolysosomes sufficiently fast to explain growth factor–mediated signaling by amino acids. Therefore, the amino acid–laden macropinosome is an essential and discrete unit of growth factor receptor signaling to mTORC1.     

The role of S6K1 in ER-positive breast cancer

The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases, which also includes Akt and many others. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1). S6K1 is sensitive to a wide range of signaling inputs, including growth factors, amino acids, energy levels and hypoxia. S6K1 relays these signals to regulate a growing list of substrates and interacting proteins in control of oncogenic processes, such as cell growth and proliferation, cell survival and apoptosis and cell migration and invasion. Several lines of evidence suggest an important role for S6K1 in estrogen receptor (ER)-positive breast cancer. S6K1 directly phosphorylates and activates ERα. Furthermore, S6K1 expression is estrogenically regulated. Therefore, hyperactivation of mTORC1/S6K1 signaling may be closely related to ER-positive status in breast cancer and may be utilized as a marker for prognosis and a therapeutic target.     

Alkaline intracellular pH (pHi) activates AMPK–mTORC2 signaling to promote cell survival during growth factor limitation

The mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) signaling controls cell metabolism, promotes cell survival, and contributes to tumorigenesis, yet its upstream regulation remains poorly defined. Although considerable evidence supports the prevailing view that amino acids activate mTOR complex 1 but not mTORC2, several studies reported paradoxical activation of mTORC2 signaling by amino acids. We noted that after amino acid starvation of cells in culture, addition of an amino acid solution increased mTORC2 signaling. Interestingly, we found the pH of the amino acid solution to be alkaline, ∼pH 10. These observations led us to discover and demonstrate here that alkaline intracellular pH (pHi) represents a previously unknown activator of mTORC2. Using a fluorescent pH-sensitive dye (cSNARF1-AM) coupled with live-cell imaging, we demonstrate that culturing cells in media at an alkaline pH induces a rapid rise in the pHi, which increases mTORC2 catalytic activity and downstream signaling to the pro-growth and pro-survival kinase Akt. Alkaline pHi also activates AMPK, a canonical sensor of energetic stress. Functionally, alkaline pHi activates AMPK-mTOR signaling, which attenuates apoptosis caused by growth factor withdrawal. Collectively, these findings reveal that alkaline pHi increases mTORC2- and AMPK-mediated signaling to promote cell survival during conditions of growth factor limitation, analogous to the demonstrated ability of energetic stress to activate AMPK–mTORC2 and promote cell survival. As an elevated pHi represents an underappreciated hallmark of cancer cells, we propose that the alkaline pHi stress sensing by AMPK–mTORC2 may contribute to tumorigenesis by enabling cancer cells at the core of a growing tumor to evade apoptosis and survive.     

Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

Metabolic reprogramming of non‐cancer cells residing in a tumor microenvironment, as a result of the adaptations to cancer‐derived metabolic and non‐metabolic factors, is an emerging aspect of cancer–host interaction. We show that in normal and cancer‐associated fibroblasts, breast cancer‐secreted extracellular vesicles suppress mTOR signaling upon amino acid stimulation to globally reduce mRNA translation. This is through delivery of cancer‐derived miR‐105 and miR‐204, which target RAGC, a component of Rag GTPases that regulate mTORC1 signaling. Following amino acid starvation and subsequent re‐feeding, ¹³C‐arginine labeling of de novo synthesized proteins shows selective translation of proteins that cluster to specific cellular functional pathways. The repertoire of these newly synthesized proteins is altered in fibroblasts treated with cancer‐derived extracellular vesicles, in addition to the overall suppressed protein synthesis. In human breast tumors, RAGC protein levels are inversely correlated with miR‐105 in the stroma. Our results suggest that through educating fibroblasts to reduce and re‐prioritize mRNA translation, cancer cells rewire the metabolic fluxes of amino acid pool and dynamically regulate stroma‐produced proteins during periodic nutrient fluctuations.