Dysfunction of Avo3, an essential component of target of rapamycin complex 2, induces ubiquitin-proteasome-dependent downregulation of Avo2 in Saccharomyces cerevisiae.

The ubiquitin-proteasome system (UPS) plays a key role in maintaining cellular protein homeostasis and participates in modulating various cellular functions. Target of rapamycin (TOR), a highly conserved Ser/Thr kinase found across species from yeasts to humans, forms two multi-protein complexes, TORC1 and TORC2, to orchestrate cellular processes crucial for optimal growth, survival, and stress responses. While UPS-mediated regulation of mammalian TOR complexes has been documented, the ubiquitination of yeast TOR complexes remains largely unexplored. Here we report a functional interplay between the UPS and TORC2 in Saccharomyces cerevisiae. Using avo3-2ts, a temperature-sensitive mutant of the essential TORC2 component Avo3 exhibiting TORC2 defects at restrictive temperatures, we obtained evidence for UPS-dependent protein degradation and downregulation of the TORC2 component Avo2. Our results established the involvement of the E3 ubiquitin ligase Ubr1 and its catalytic activity in mediating Avo2 degradation in cells with defective Avo3. Coimmunoprecipitation revealed the interaction between Avo2 and Ubr1, indicating Avo2 as a potential substrate of Ubr1. Furthermore, depleting Ubr1 rescued the growth of avo3-2ts cells at restrictive temperatures, suggesting an essential role of Avo2 in sustaining cell viability under heat stress and/or TORC2 dysfunction. This study uncovers a role of UPS in yeast TORC2 regulation, highlighting the impact of protein degradation control on cellular signaling.

Casein kinase 1 controls components of a TORC2 signaling network in budding yeast.

Tor kinases play diverse and essential roles in control of nutrient signaling and cell growth. Tor kinases are assembled into two large multiprotein complexes referred to as Tor Complex 1 and Tor Complex 2 (TORC1 and TORC2). In budding yeast, TORC2 controls a signaling network that relays signals regarding carbon source that strongly influence growth rate and cell size. However, the mechanisms that control TORC2 signaling are poorly understood. Activation of TORC2 requires Mss4, a phosphoinositol kinase that initiates assembly of a multi-protein complex at the plasma membrane that recruits and activates downstream targets of TORC2. Localization of Mss4 to the plasma membrane is controlled by phosphorylation and previous work suggested that yeast homologs of casein kinase 1γ, referred to as Yck1 and Yck2, control phosphorylation of Mss4. Here, we generated a new analog-sensitive allele of YCK2 and used it to test whether Yck1/2 influence signaling in the TORC2 network. We found that multiple components of the TORC2 network are strongly influenced by Yck1/2 signaling.

Intestinal Mg2+ accumulation induced by cnnm mutations decreases the body size by suppressing TORC2 signaling in Caenorhabditis elegans.

Mg2+ is a vital ion involved in diverse cellular functions by forming complexes with ATP. Intracellular Mg2+ levels are tightly regulated by the coordinated actions of multiple Mg2+ transporters, such as the Mg2+ efflux transporter, cyclin M (CNNM). Caenorhabditis elegans (C. elegans) worms with mutations in both cnnm-1 and cnnm-3 exhibit excessive Mg2+ accumulation in intestinal cells, leading to various phenotypic abnormalities. In this study, we investigated the mechanism underlying the reduction in body size in cnnm-1; cnnm-3 mutant worms. RNA interference (RNAi) of gtl-1, which encodes a Mg2+-intake channel in intestinal cells, restored the worm body size, confirming that this phenotype is due to excessive Mg2+ accumulation. Moreover, RNAi experiments targeting body size-related genes and analyses of mutant worms revealed that the suppression of the target of rapamycin complex 2 (TORC2) signaling pathway was involved in body size reduction, resulting in downregulated DAF-7 expression in head ASI neurons. As the DAF-7 signaling pathway suppresses dauer formation under stress, cnnm-1; cnnm-3 mutant worms exhibited a greater tendency to form dauer upon induction. Collectively, our results revealed that excessive accumulation of Mg2+ repressed the TORC2 signaling pathway in C. elegans worms and suggest the novel role of the DAF-7 signaling pathway in the regulation of their body size.

The Greatwall-Endosulfine-PP2A/B55 pathway controls entry into quiescence by promoting translation of Elongator-tuneable transcripts.

Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. Here, we report that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications essential for sustaining the translation programme during entry into quiescence. This process promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, some of these mRNAs encode inhibitors of TORC1, activators of TORC2, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a novel mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between the tRNA epitranscriptome and biased codon usage.

GRD-1/PTR-11, the C. elegans hedgehog/patched-like morphogen-receptor pair, modulates developmental rate.

Both hedgehog (Hh) and target of rapamycin complex 2 (TORC2) are central, evolutionarily conserved signaling pathways that regulate development and metabolism. In C. elegans, loss of the essential TORC2 component RICTOR (rict-1) causes delayed development, shortened lifespan, reduced brood, small size and increased fat. Here, we report that knockdown of both the hedgehog-related morphogen grd-1 and its patched-related receptor ptr-11 rescues delayed development in TORC2 loss-of-function mutants, and grd-1 and ptr-11 overexpression delays wild-type development to a similar level to that in TORC2 loss-of-function animals. These findings potentially indicate an unexpected role for grd-1 and ptr-11 in slowing developmental rate downstream of a nutrient-sensing pathway. Furthermore, we implicate the chronic stress transcription factor pqm-1 as a key transcriptional effector in this slowing of whole-organism growth by grd-1 and ptr-11. We propose that TORC2, grd-1 and ptr-11 may act linearly or converge on pqm-1 to delay organismal development.

Aft1 Nuclear Localization and Transcriptional Response to Iron Starvation Rely upon TORC2/Ypk1 Signaling and Sphingolipid Biosynthesis.

Iron scarcity provokes a cellular response consisting of the strong expression of high-affinity systems to optimize iron uptake and mobilization. Aft1 is a primary transcription factor involved in iron homeostasis and controls the expression of high-affinity iron uptake genes in Saccharomyces cerevisiae. Aft1 responds to iron deprivation by translocating from the cytoplasm to the nucleus. Here, we demonstrate that the AGC kinase Ypk1, as well as its upstream regulator TOR Complex 2 (TORC2), are required for proper Aft1 nuclear localization following iron deprivation. We exclude a role for TOR Complex 1 (TORC1) and its downstream effector Sch9, suggesting this response is specific for the TORC2 arm of the TOR pathway. Remarkably, we demonstrate that Aft1 nuclear localization and a robust transcriptional response to iron starvation also require biosynthesis of sphingolipids, including complex sphingolipids such as inositol phosphorylceramide (IPC) and upstream precursors, e.g., long-chain bases (LCBs) and ceramides. Furthermore, we observe the deficiency of Aft1 nuclear localization and impaired transcriptional response in the absence of iron when TORC2-Ypk1 is impaired is partially suppressed by exogenous addition of the LCB dihydrosphingosine (DHS). This latter result is consistent with prior studies linking sphingolipid biosynthesis to TORC2-Ypk1 signaling. Taken together, these results reveal a novel role for sphingolipids, controlled by TORC2-Ypk1, for proper localization and activity of Aft1 in response to iron scarcity.

MoVast2 combined with MoVast1 regulates lipid homeostasis and autophagy in Magnaporthe oryzae.

Macroautophagy/autophagy is an evolutionarily conserved biological process among eukaryotes that degrades unwanted materials such as protein aggregates, damaged mitochondria and even viruses to maintain cell survival. Our previous studies have demonstrated that MoVast1 acts as an autophagy regulator regulating autophagy, membrane tension, and sterol homeostasis in rice blast fungus. However, the detailed regulatory relationships between autophagy and VASt domain proteins remain unsolved. Here, we identified another VASt domain-containing protein, MoVast2, and further uncovered the regulatory mechanism of MoVast2 in M. oryzae. MoVast2 interacted with MoVast1 and MoAtg8, and colocalized at the PAS and deletion of MoVAST2 results in inappropriate autophagy progress. Through TOR activity analysis, sterols and sphingolipid content detection, we found high sterol accumulation in the ΔMovast2 mutant, whereas this mutant showed low sphingolipids and low activity of both TORC1 and TORC2. In addition, MoVast2 colocalized with MoVast1. The localization of MoVast2 in the MoVAST1 deletion mutant was normal; however, deletion of MoVAST2 leads to mislocalization of MoVast1. Notably, the wide-target lipidomic analyses revealed significant changes in sterols and sphingolipids, the major PM components, in the ΔMovast2 mutant, which was involved in lipid metabolism and autophagic pathways. These findings confirmed that the functions of MoVast1 were regulated by MoVast2, revealing that MoVast2 combined with MoVast1 maintained lipid homeostasis and autophagy balance by regulating TOR activity in M. oryzae.

Rapamycin and Torin2 inhibit Candida auris TOR: Insights through growth profiling, docking, and MD simulations.

The fungus Candida auris is a pathogen of utmost concern due to its rapid emergence across the globe, acquired antifungal drug tolerance, thermotolerance, and ability to survive in hospital settings and preserved foods. Recent incidences of comorbidity of corona patients with its infection in hospital settings highlighted the importance of understanding the pathobiology and drug tolerance of this fungus on priority. The Target of rapamycin (TOR) is a central regulator of growth across eukaryotes with an illustrated role in fungal pathology. The role of the TOR signalling pathway in the growth of C. auris is yet to be described. In-silico, analysis revealed the presence of highly conserved Tor kinase, components of TORC, and key downstream components in C. auris. Rapamycin and Torin2, the specific inhibitors of Tor reduce the growth of C. auris. An inhibition of Tor leads to cell cycle arrest at the G1 phase with a defect in cytokinesis. Interestingly, with an insignificant difference in growth at 30 and 37 °C, a sharp decline in growth is seen with Torin2 at 37 °C. The heterogeneous response emphasizes the importance of physiology-based differential cellular response at different temperatures. In addition, the inhibition of Tor suppresses the biofilm formation. In silico studies through docking and simulations showed rapamycin and torin2 as specific inhibitors of C. auris Tor kinase (CauTor kinase) and hence can be exploited for a thorough understanding of the TOR signalling pathway in pathobiology and drug tolerance of C. auris. HIGHLIGHTSConservation of TOR signalling pathway in Candida aurisRapamycin and torin2 are specific inhibitors of Cau TorUnderstanding of the role of TOR signalling pathway through the use of inhibitors rapamycin and torin2.Heterogenous response of C. auris to torin2 at different physiological conditions.Communicated by Ramaswamy H. Sarma.

Roles of phosphatidylserine and phospholipase C in the activation of TOR complex 2 signaling in Saccharomyces cerevisiae.

Target of rapamycin (TOR) forms two distinct complexes, TORC1 and TORC2, to exert its essential functions in cellular growth and homeostasis. TORC1 signaling is regulated in response to nutrients such as amino acids and glucose; however, the mechanisms underlying the activation of TORC2 signaling are still poorly understood compared to those for TORC1 signaling. In the budding yeast Saccharomyces cerevisiae, TORC2 targets the protein kinases Ypk1 and Ypk2 (hereafter Ypk1/2), and Pkc1 for phosphorylation. Plasma membrane stress is known to activate TORC2-Ypk1/2 signaling. We have previously reported that methylglyoxal (MG), a metabolite derived from glycolysis, activates TORC2-Pkc1 signaling. In this study, we found that MG activates the TORC2-Ypk1/2 and TORC2-Pkc1 signaling, and that phosphatidylserine is involved in the activation of both signaling pathways. We also demonstrated that the Rho family GTPase Cdc42 contributes to the plasma membrane stress-induced activation of TORC2-Ypk1/2 signaling. Furthermore, we revealed that phosphatidylinositol-specific phospholipase C, Plc1, contributes to the activation of both TORC2-Ypk1/2 and TORC2-Pkc1 signaling.

Coordinated regulation of TORC2 signaling by MCC/eisosome-associated proteins, Pil1 and tetraspan membrane proteins during the stress response.

MCCs are linear invaginations of the yeast plasma membrane that form stable membrane microdomains. Although over 20 proteins are localized in the MCCs, it is not well understood how these proteins coordinately maintain normal MCC function. Pil1 is a core eisosome protein and is responsible for MCC-invaginated structures. In addition, six-tetraspan membrane proteins (6-Tsp) are localized in the MCCs and classified into two families, the Sur7 family and Nce102 family. To understand the coordinated function of these MCC proteins, single and multiple deletion mutants of Pil1 and 6-Tsp were generated and their MCC structure and growth under various stresses were investigated. Genetic interaction analysis revealed that the Sur7 family and Nce102 function in stress tolerance and normal eisosome assembly, respectively, by cooperating with Pil1. To further understand the role of MCCs/eisosomes in stress tolerance, we screened for suppressor mutants using the SDS-sensitive phenotype of pil1Δ 6-tspΔ cells. This revealed that SDS sensitivity is caused by hyperactivation of Tor kinase complex 2 (TORC2)-Ypk1 signaling. Interestingly, inhibition of sphingolipid metabolism, a well-known downstream pathway of TORC2-Ypk1 signaling, did not rescue the SDS-sensitivity of pil1Δ 6-tspΔ cells. These results suggest that Pil1 and 6-Tsp cooperatively regulate TORC2 signaling during the stress response.

TOR and MAP kinase pathways synergistically regulate autophagy in response to nutrient depletion in fission yeast.

General autophagy is an evolutionarily conserved process in eukaryotes, by which intracellular materials are transported into and degraded inside lysosomes or vacuoles, with the main goal of recycling those materials during periods of starvation. The molecular bases of autophagy have been widely described in Saccharomyces cerevisiae, and the specific roles of Atg proteins in the process were first characterized in this model system. Important contributions have been made in Schizosaccharomyces pombe highlighting the evolutionary similarity and, at the same time, diversity of Atg components in autophagy. However, little is known regarding signals, pathways and role of autophagy in this distant yeast. Here, we undertake a global approach to investigate the signals, the pathways and the consequences of autophagy activation. We demonstrate that not only nitrogen but several nutritional deprivations including lack of carbon, sulfur, phosphorus or leucine sources, trigger autophagy, and that the TORC1, TORC2 and MAP kinase Sty1 pathways control the onset of autophagy. Furthermore, we identify an unexpected phenotype of autophagy-defective mutants, namely their inability to survive in the absence of leucine when biosynthesis of this amino acid is impaired.Abbreviations: ATG: autophagy-related; cAMP: cyclic adenosine monophosphate; cDNA: complementary deoxyribonucleic acid; GFP: green fluorescence protein; Gluc: glucose; Leu: leucine; MAP: mitogen-activated protein; MM: minimal medium; PI: propidium iodine; PKA: protein kinase A; RNA: ribonucleic acid; RT-qPCR: real time quantitative polymerase chain reaction; S. cerevisiae: Saccharomyces cerevisiae; S. pombe: Schizosaccharomyces pombe; TCA: trichloroacetic acid; TOR: target of rapamycin; TORC1: target of rapamycin complex 1; TORC2: target of rapamycin complex 2; YE5S: yeast extract 5 amino acid supplemented.

Fission Yeast TORC2 Signaling Pathway Ensures Cell Proliferation under Glucose-Limited, Nitrogen-Replete Conditions.

Target of rapamycin (TOR) kinases form two distinct complexes, TORC1 and TORC2, which are evolutionarily conserved among eukaryotes. These complexes control intracellular biochemical processes in response to changes in extracellular nutrient conditions. Previous studies using the fission yeast, Schizosaccharomyces pombe, showed that the TORC2 signaling pathway, which is essential for cell proliferation under glucose-limited conditions, ensures cell-surface localization of a high-affinity hexose transporter, Ght5, by downregulating its endocytosis. The TORC2 signaling pathway retains Ght5 on the cell surface, depending on the presence of nitrogen sources in medium. Ght5 is transported to vacuoles upon nitrogen starvation. In this review, we discuss the molecular mechanisms underlying this regulation to cope with nutritional stress, a response which may be conserved from yeasts to mammals.

Expanding TOR Complex 2 Signaling: Emerging Regulators and New Connections.

Almost three decades after its seminal discovery, our understanding of the remarkable TOR pathway continues to expand. As a TOR complex, TORC2 lies at the nexus of many signaling pathways and directs a diverse array of fundamental processes such as cell survival, proliferation, and metabolism by integrating environmental and intracellular cues. The dysregulation of TORC2 activity disrupts cellular homeostasis and leads to many pathophysiological conditions. With continued efforts at mapping the signaling landscape, the pace of discovery in TORC2 regulation has been accelerated in recent years. Consequently, emerging evidence has expanded the repertoire of upstream regulators and has revealed unexpected diversity in the modes of TORC2 regulation. Multiple environmental cues and plasma membrane proteins that fine-tune TORC2 activity are unfolding. Furthermore, TORC2 signaling is intricately intertwined with other major signaling pathways. Therefore, feedback and crosstalk regulation also extensively modulate TORC2. In this context, we provide a comprehensive overview of revolutionary concepts regarding emerging regulators of TORC2 and discuss evidence of feedback and crosstalk regulation that shed new light on TORC2 biology.

A TOR (target of rapamycin) and nutritional phosphoproteome of fission yeast reveals novel targets in networks conserved in humans.

Fluctuations in TOR, AMPK and MAP-kinase signalling maintain cellular homeostasis and coordinate growth and division with environmental context. We have applied quantitative, SILAC mass spectrometry to map TOR and nutrient-controlled signalling in the fission yeast Schizosaccharomyces pombe. Phosphorylation levels at more than 1000 sites were altered following nitrogen stress or Torin1 inhibition of the TORC1 and TORC2 networks that comprise TOR signalling. One hundred and thirty of these sites were regulated by both perturbations, and the majority of these (119) new targets have not previously been linked to either nutritional or TOR control in either yeasts or humans. Elimination of AMPK inhibition of TORC1, by removal of AMPKα (ssp2::ura4+), identified phosphosites where nitrogen stress-induced changes were independent of TOR control. Using a yeast strain with an ATP analogue-sensitized Cdc2 kinase, we excluded sites that were changed as an indirect consequence of mitotic control modulation by nitrogen stress or TOR signalling. Nutritional control of gene expression was reflected in multiple targets in RNA metabolism, while significant modulation of actin cytoskeletal components points to adaptations in morphogenesis and cell integrity networks. Reduced phosphorylation of the MAPKK Byr1, at a site whose human equivalent controls docking between MEK and ERK, prevented sexual differentiation when resources were sparse but not eliminated.

Crosstalk between the mTOR and DNA Damage Response Pathways in Fission Yeast.

Cells have developed response systems to constantly monitor environmental changes and accordingly adjust growth, differentiation, and cellular stress programs. The evolutionarily conserved, nutrient-responsive, mechanistic target of rapamycin signaling (mTOR) pathway coordinates basic anabolic and catabolic cellular processes such as gene transcription, protein translation, autophagy, and metabolism, and is directly implicated in cellular and organismal aging as well as age-related diseases. mTOR mediates these processes in response to a broad range of inputs such as oxygen, amino acids, hormones, and energy levels, as well as stresses, including DNA damage. Here, we briefly summarize data relating to the interplays of the mTOR pathway with DNA damage response pathways in fission yeast, a favorite model in cell biology, and how these interactions shape cell decisions, growth, and cell-cycle progression. We, especially, comment on the roles of caffeine-mediated DNA-damage override. Understanding the biology of nutrient response, DNA damage and related pharmacological treatments can lead to the design of interventions towards improved cellular and organismal fitness, health, and survival.

The Target of Rapamycin Signalling Pathway in Ageing and Lifespan Regulation.

Ageing is a complex trait controlled by genes and the environment. The highly conserved mechanistic target of rapamycin signalling pathway (mTOR) is a major regulator of lifespan in all eukaryotes and is thought to be mediating some of the effects of dietary restriction. mTOR is a rheostat of energy sensing diverse inputs such as amino acids, oxygen, hormones, and stress and regulates lifespan by tuning cellular functions such as gene expression, ribosome biogenesis, proteostasis, and mitochondrial metabolism. Deregulation of the mTOR signalling pathway is implicated in multiple age-related diseases such as cancer, neurodegeneration, and auto-immunity. In this review, we briefly summarise some of the workings of mTOR in lifespan and ageing through the processes of transcription, translation, autophagy, and metabolism. A good understanding of the pathway's outputs and connectivity is paramount towards our ability for genetic and pharmacological interventions for healthy ageing and amelioration of age-related disease.

TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

Berberine Inhibits Gluconeogenesis in Skeletal Muscles and Adipose Tissues in Streptozotocin-induced Diabetic Rats via LKB1-AMPK-TORC2 Signaling Pathway.

The effect and potential molecular mechanisms of berberine on gluconeogenesis in skeletal muscles and adipose tissues were investigated. After adaptive feeding for one week, 8 rats were randomly selected as the normal group and fed on a standard diet. The remaining 32 rats were fed on a high-fat diet and given an intravenous injection of streptozotocin (STZ) for 2 weeks to induce the diabetic models. The diabetic rat models were confirmed by oral glucose tolerance test (OGTT) and randomly divided into 4 groups (n=8 each), which were all fed on a high-fat diet. Berberine (3 g/kg per day) or metformin (183 mg/kg per day) was intragastrically administered to the diabetic rats for 12 weeks, serving as berberine group and metformin group respectively. 5-aminoimidazole-4-carboxamide1-ß-D-ribofuranoside [AICAR, an agonist of AMP-activated protein kinase (AMPK), 0.5 mg/kg per day] was subcutaneously injected to the diabetic rats for 12 weeks, serving as AICAR group. The remaining 8 diabetic rats served as the model group, which was given a 0.5% carboxyl methylcellulose solution by oral gavage. Fasting serum insulin (FINS), OGTT as well as lipid parameters were tested by commercial kit. The protein levels of liver kinase B1 (LKB1), AMPK, phosphorylated AMP-activated protein kinase (p-AMPK), transducer of regulated CREB activity 2 (TORC2), phosphorylated transducer of regulated CREB activity 2 (p-TORC2), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6Pase) in skeletal muscles and adipose tissues were examined by Western blotting. The results showed that berberine significantly decreased the body weight, plasma glucose, insulin levels, and homeostatic model assessment for insulin resistance (HOMA-IR) of diabetic rats compared with those in the model group. Meanwhile, the serum total triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels were markedly decreased and high-density lipoprotein cholesterol (HDL-C) level was significantly increased after the treatment with berberine. In addition, we found that berberine significantly increased the expression of p-AMPK and LKB1, while decreasing the p-TORC2 levels in skeletal muscles and adipose tissues. Moreover, the expression of PEPCK and G6Pase was significantly down-regulated after the treatment with berberine compared to the model group. It was suggested that the mechanism by which berberine inhibited peripheral tissue gluconeogenesis may be attributed to the activation of the LKB1-AMPK-TORC2 signaling pathway.

Nuclear Functions of TOR: Impact on Transcription and the Epigenome.

The target of rapamycin (TOR) protein kinase is at the core of growth factor- and nutrient-dependent signaling pathways that are well-known for their regulation of metabolism, growth, and proliferation. However, TOR is also involved in the regulation of gene expression, genomic and epigenomic stability. TOR affects nuclear functions indirectly through its activity in the cytoplasm, but also directly through active nuclear TOR pools. The mechanisms by which TOR regulates its nuclear functions are less well-understood compared with its cytoplasmic activities. TOR is an important pharmacological target for several diseases, including cancer, metabolic and neurological disorders. Thus, studies of the nuclear functions of TOR are important for our understanding of basic biological processes, as well as for clinical implications.

Tumor-suppressor Fbxw7 targets SIK2 for degradation to interfere with TORC2-AKT signaling in pancreatic cancer.

The tumor suppressor F-box/WD repeat-containing protein 7 (Fbxw7) is a substrate-recognition subunit of a ubiquitin ligase complex. We have previously proposed that Fbxw7 inhibited pancreatic cancer cell proliferation and invasion by targeting ß-catenin. To identify other targets of Fbxw7 involved in pancreatic carcinogenesis, we screened the human protein database for Fbxw7 target candidates using the conserved Fbxw7-recognizing sequences. Twenty-three candidates are identified, including five known Fbxw7 targets and two cancer-related genes (salt inducible kinase 2 [SIK2] and ZMIZ1). We identified SIK2 as an Fbxw7 target for degradation by binding to the "TPPPS" motif of SIK2 in pancreatic cancer cells. We also demonstrated that SIK2 promoted proliferation and mitotic progression of pancreatic cancer cells. Moreover, endogenous Fbxw7 downregulates SIK2 protein level for controlling cell cycle progression, possibly by interfering the SIK2/TORC2/AKT signaling pathway to modulate p21 expression. Collectively, these data demonstrate that Fbxw7 targets the cell cycle controller, SIK2, for degradation, thereby leading to the disruption of downstream TORC2/AKT signaling to inhibit pancreatic cancer cell proliferation and cell cycle progression.