Ryan C. Russell

Amino acid signalling upstream of mTOR

Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that is part of mTOR complex 1 (mTORC1), a master regulator that couples amino acid availability to cell growth and autophagy. Multiple cues modulate mTORC1 activity, such as growth factors, stress, energy status and amino acids. Although amino acids are key environmental stimuli, exactly how they are sensed and how they activate mTORC1 is not fully understood. Recently, a model has emerged whereby mTORC1 activation occurs at the lysosome and is mediated through an amino acid sensing cascade involving RAG GTPases, Ragulator and vacuolar H+-ATPase (v-ATPase).

Differential Regulation of Distinct Vps34 Complexes by AMPK in Nutrient Stress and Autophagy

Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the nonautophagy Vps34 complex by phosphorylating T163/S165 in Vps34 and therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the proautophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy-essential gene present only in the proautophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy.

Autophagy regulation by nutrient signaling

The ability of cells to respond to changes in nutrient availability is essential for the maintenance of metabolic homeostasis and viability. One of the key cellular responses to nutrient withdrawal is the upregulation of autophagy. Recently, there has been a rapid expansion in our knowledge of the molecular mechanisms involved in the regulation of mammalian autophagy induction in response to depletion of key nutrients. Intracellular amino acids, ATP, and oxygen levels are intimately tied to the cellular balance of anabolic and catabolic processes. Signaling from key nutrient-sensitive kinases mTORC1 and AMP-activated protein kinase (AMPK) is essential for the nutrient sensing of the autophagy pathway. Recent advances have shown that the nutrient status of the cell is largely passed on to the autophagic machinery through the coordinated regulation of the ULK and VPS34 kinase complexes. Identification of extensive crosstalk and feedback loops converging on the regulation of ULK and VPS34 can be attributed to the importance of these kinases in autophagy induction and maintaining cellular homeostasis.

Differential regulation of mTORC1 by leucine and glutamine

Getting specific about amino acid sensing The protein kinase complex mTORC1 regulates growth and metabolism, and its activity is controlled in response to the abundance of cellular amino acids. Jewell et al. report that control of mTORC1 in response to glutamine does not require the Rag guanosine triphosphatase (GTPase) implicated in the sensing of other amino acids such as leucine (see the Perspective by Abraham). For sensing of glutamine, another GTPase, Arf1, was required. Distinct mechanisms thus appear to couple various amino acids to signaling by the mTORC1 complex. Science, this issue p. 194; see also p. 128 Distinct mechanisms sense amino acids leucine and glutamine at the lysosome. [Also see Perspective by Abraham] The mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates environmental and intracellular signals to regulate cell growth. Amino acids stimulate mTORC1 activation at the lysosome in a manner thought to be dependent on the Rag small guanosine triphosphatases (GTPases), the Ragulator complex, and the vacuolar H+–adenosine triphosphatase (v-ATPase). We report that leucine and glutamine stimulate mTORC1 by Rag GTPase-dependent and -independent mechanisms, respectively. Glutamine promoted mTORC1 translocation to the lysosome in RagA and RagB knockout cells and required the v-ATPase but not the Ragulator. Furthermore, we identified the adenosine diphosphate ribosylation factor–1 GTPase to be required for mTORC1 activation and lysosomal localization by glutamine. Our results uncover a signaling cascade to mTORC1 activation independent of the Rag GTPases and suggest that mTORC1 is differentially regulated by specific amino acids.

YAP mediates crosstalk between the Hippo and PI(3)K–TOR pathways by suppressing PTEN via miR-29

Organ development is a complex process governed by the interplay of several signalling pathways that have critical functions in the regulation of cell growth and proliferation. Over the past years, the Hippo pathway has emerged as a key regulator of organ size. Perturbation of this pathway has been shown to play important roles in tumorigenesis. YAP, the main downstream target of the mammalian Hippo pathway, promotes organ growth, yet the underlying molecular mechanism of this regulation remains unclear. Here we provide evidence that YAP activates the mammalian target of rapamycin (mTOR), a major regulator of cell growth. We have identified the tumour suppressor PTEN, an upstream negative regulator of mTOR, as a critical mediator of YAP in mTOR regulation. We demonstrate that YAP downregulates PTEN by inducing miR-29 to inhibit PTEN translation. Last, we show that PI(3)K–mTOR is a pathway modulated by YAP to regulate cell size, tissue growth and hyperplasia. Our studies reveal a functional link between Hippo and PI(3)K–mTOR, providing a molecular basis for the coordination of these two pathways in organ size regulation.

An emerging role for TOR signaling in mammalian tissue and stem cell physiology

The mammalian target of rapamycin (mTOR) is a kinase that responds to a myriad of signals, ranging from nutrient availability and energy status, to cellular stressors, oxygen sensors and growth factors. The finely tuned response of mTOR to these stimuli results in alterations to cell metabolism and cell growth. Recent studies of conditional knockouts of mTOR pathway components in mice have affirmed the role of mTOR signaling in energy balance, both at the cell and whole organism levels. Such studies have also highlighted a role for mTOR in stem cell homeostasis and lifespan determination. Here, we discuss the molecular mechanisms of TOR signaling and review recent in vitro and in vivo studies of mTOR tissue-specific activities in mammals.

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.

Organ Size Control by Hippo and TOR Pathways

The determination of final organ size is a highly coordinated and complex process that relies on the precise regulation of cell number and/or cell size. Perturbation of organ size control contributes to many human diseases, including hypertrophy, degenerative diseases, and cancer. Hippo and TOR are among the key signaling pathways involved in the regulation of organ size through their respective functions in the regulation of cell number and cell size. Here, we review the general mechanisms that regulate organ growth, describe how Hippo and TOR control key aspects of growth, and discuss recent findings that highlight a possible coordination between Hippo and TOR in organ size regulation.

Defects of Vps15 in skeletal muscles lead to autophagic vacuolar myopathy and lysosomal disease

The complex of Vacuolar Protein Sorting 34 and 15 (Vps34 and Vps15) has Class III phosphatidylinositol 3‐kinase activity and putative roles in nutrient sensing, mammalian Target Of Rapamycin (mTOR) activation by amino acids, cell growth, vesicular trafficking and autophagy. Contrary to expectations, here we show that Vps15‐deficient mouse tissues are competent for LC3‐positive autophagosome formation and maintain mTOR activation. However, an impaired lysosomal function in mutant cells is traced by accumulation of adaptor protein p62, LC3 and Lamp2 positive vesicles, which can be reverted to normal levels after ectopic overexpression of Vps15. Mice lacking Vps15 in skeletal muscles, develop a severe myopathy. Distinct from the autophagy deficient Atg7−/− mutants, pathognomonic morphological hallmarks of autophagic vacuolar myopathy (AVM) are observed in Vps15−/− mutants, including elevated creatine kinase plasma levels, accumulation of autophagosomes, glycogen and sarcolemmal features within the fibres. Importantly, Vps34/Vps15 overexpression in myoblasts of Danon AVM disease patients alleviates the glycogen accumulation. Thus, the activity of the Vps34/Vps15 complex is critical in disease conditions such as AVMs, and possibly a variety of other lysosomal storage diseases.

Rag GTPases are cardioprotective by regulating lysosomal function

The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection.