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1.  Oxidative Stress Activates SIRT2 to Deacetylate and Stimulate Phosphoglycerate Mutase 
Cancer research  2014;74(13):3630-3642.
Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.
doi:10.1158/0008-5472.CAN-13-3615
PMCID: PMC4303242  PMID: 24786789
2.  Rag GTPases are cardioprotective by regulating lysosomal function 
Nature communications  2014;5:4241.
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.
doi:10.1038/ncomms5241
PMCID: PMC4100214  PMID: 24980141
3.  Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy 
Autophagy  2013;9(12):1983-1995.
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.
doi:10.4161/auto.26058
PMCID: PMC4028342  PMID: 24013218
MTOR; autophagy; PIK3C3; ATG14; BECN1
4.  AMPK connects energy stress to PIK3C3/VPS34 regulation 
Autophagy  2013;9(7):1110-1111.
The class III phosphatidylinositol (PtdIns)-3 kinase, PIK3C3/VPS34, forms multiple complexes and regulates a variety of cellular functions, especially in intracellular vesicle trafficking and autophagy. Even though PtdIns3P, the product of PIK3C3, is thought to be a critical membrane marker for the autophagosome, it is unclear how PIK3C3 is regulated in response to autophagy-inducing stimuli. A complexity of PIK3C3 biology is due in part to the existence of multiple complexes, of which the ATG14- or UVRAG-containing complexes play important roles in autophagy. We recently discovered differential regulation of distinct PIK3C3 complexes in response to energy starvation and showed a mechanism by which AMPK directly phosphorylates PIK3C3 and BECN1 to regulate non- and pro-autophagic PIK3C3 complexes, respectively.
doi:10.4161/auto.24877
PMCID: PMC3722323  PMID: 23669030
VPS34 complexes; AMPK; BECN1; ATG14; autophagy
5.  YAP as oncotarget in uveal melanoma 
Oncoscience  2014;1(7):480-481.
PMCID: PMC4278320  PMID: 25594048
6.  LDH-A Acetylation: Implication in Cancer 
Oncotarget  2013;4(6):802-803.
PMCID: PMC3757233  PMID: 23868819
7.  Mechanistic insights into the regulation of metabolic enzymes by acetylation 
The Journal of Cell Biology  2012;198(2):155-164.
The activity of metabolic enzymes is controlled by three principle levels: the amount of enzyme, the catalytic activity, and the accessibility of substrates. Reversible lysine acetylation is emerging as a major regulatory mechanism in metabolism that is involved in all three levels of controlling metabolic enzymes and is altered frequently in human diseases. Acetylation rivals other common posttranslational modifications in cell regulation not only in the number of substrates it modifies, but also the variety of regulatory mechanisms it facilitates.
doi:10.1083/jcb.201202056
PMCID: PMC3410420  PMID: 22826120
8.  Upstream regulators of the Hippo pathway 
Cell Cycle  2012;11(22):4097-4098.
doi:10.4161/cc.22322
PMCID: PMC3524194  PMID: 23075495
Hippo; YAP; GPCR; cancer; LPA; Rho
9.  mTOR in podocyte function 
Cell Cycle  2011;10(20):3415-3416.
doi:10.4161/cc.10.20.17686
PMCID: PMC3266173  PMID: 22067710
10.  The autophagy initiating kinase ULK1 is regulated via opposing phosphorylation by AMPK and mTOR 
Autophagy  2011;7(6):645-646.
The serine/threonine kinase ULK1 is a mammalian homolog of Atg1, part of the Atg1 kinase complex, which is the most upstream component of the core autophagy machinery conserved from yeast to mammals. In budding yeast, activity of the Atg1 kinase complex is inhibited by TORC1 (target of rapamycin complex 1), but how the counterpart ULK1 complex in mammalian cells is regulated has been unknown. Our laboratories recently discovered that AMPK associates with, and directly phosphorylates, ULK1 on several sites and this modification is required for ULK1 activation after glucose deprivation. In contrast, when nutrients are plentiful, the mTORC1 complex phosphorylates ULK1, preventing its association and activation by AMPK. These studies have revealed a molecular mechanism of ULK1 regulation by nutrient signals via the actions of AMPK and mTORC1.
doi:10.4161/auto.7.6.15123
PMCID: PMC3359466  PMID: 21460621
autophagy; ULK1; AMPK; mTOR; 14-3-3
11.  mTORC1 activation in podocytes is a critical step in the development of diabetic nephropathy in mice 
The Journal of Clinical Investigation  2011;121(6):2181-2196.
Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria and glomerulosclerosis in glomerular diseases including DN. However, molecular mechanisms of podocyte dysfunction in the development of DN are not well understood. Here we have shown that activity of mTOR complex 1 (mTORC1), a kinase that senses nutrient availability, was enhanced in the podocytes of diabetic animals. Further, podocyte-specific mTORC1 activation induced by ablation of an upstream negative regulator (PcKOTsc1) recapitulated many DN features, including podocyte loss, glomerular basement membrane thickening, mesangial expansion, and proteinuria in nondiabetic young and adult mice. Abnormal mTORC1 activation caused mislocalization of slit diaphragm proteins and induced an epithelial-mesenchymal transition–like phenotypic switch with enhanced ER stress in podocytes. Conversely, reduction of ER stress with a chemical chaperone significantly protected against both the podocyte phenotypic switch and podocyte loss in PcKOTsc1 mice. Finally, genetic reduction of podocyte-specific mTORC1 in diabetic animals suppressed the development of DN. These results indicate that mTORC1 activation in podocytes is a critical event in inducing DN and suggest that reduction of podocyte mTORC1 activity is a potential therapeutic strategy to prevent DN.
doi:10.1172/JCI44771
PMCID: PMC3104745  PMID: 21606597
12.  Critical role for hypothalamic mTOR activity in energy balance 
Cell metabolism  2009;9(4):362-374.
Summary
The mammalian target of Rapamycin (mTOR) promotes anabolic cellular processes in response to growth factors and metabolic cues. The TSC1 and TSC2 tumor suppressors are major upstream inhibitory regulators of mTOR signaling. Mice with Rip2/Cre-mediated deletion of Tsc1 (Rip-Tsc1cKO mice) developed hyperphagia and obesity, suggesting that hypothalamic disruption (for which Rip2/Cre is well known) of Tsc1 may dysregulate feeding circuits via mTOR activation. Indeed, Rip-Tsc1cKO mice displayed increased mTOR signaling and enlarged neuron cell size in a number of hypothalamic populations, including Pomc neurons. Furthermore, Tsc1 deletion with Pomc/Cre (Pomc-Tsc1cKO mice) resulted in dysregulation of Pomc neurons and hyperphagic obesity. Treatment with the mTOR inhibitor, rapamycin, ameliorated the hyperphagia, obesity, and the altered Pomc neuronal morphology in developing or adult Pomc-Tsc1cKO mice, and cessation of treatment reinstated these phenotypes. Thus, ongoing mTOR activation in Pomc neurons blocks the catabolic function of these neurons to promote nutrient intake and increased adiposity.
doi:10.1016/j.cmet.2009.03.005
PMCID: PMC2790375  PMID: 19356717
13.  Regulation of TORC1 by Rag GTPases in nutrient response 
Nature cell biology  2008;10(8):935-945.
TORC1 (Target of rapamycin complex 1) has a critical role in the regulation of cell growth and cell size. A wide range of signals, including amino acids, is known to activate TORC1. Here, we report the identification of Rag GTPases as novel activators of TORC1 in response to amino acid signals. Knockdown of Rag gene expression suppressed the stimulatory effect of amino acids on TORC1 in Drosophila S2 cells. Expression of constitutively active (GTP-bound) Rag in mammalian cells enhances TORC1 in the absence of amino acids while expression of dominant negative Rag blocks the stimulatory effects of amino acids on TORC1. Drosophila genetic studies also show that the Rag GTPases regulate cell growth, autophagy, and animal viability under starvation. Together, our studies establish a novel function of Rag GTPases in TORC1 activation in response to amino acid signals.
doi:10.1038/ncb1753
PMCID: PMC2711503  PMID: 18604198
14.  Autophagy regulation by nutrient signaling 
Cell Research  2013;24(1):42-57.
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.
doi:10.1038/cr.2013.166
PMCID: PMC3879708  PMID: 24343578
autophagy; ULK1; AMPK; VPS34; amino acids; oxygen; mTORC1
15.  LATS2 Suppresses Oncogenic Wnt Signaling by Disrupting β-catenin/BCL9 Interaction 
Cell reports  2013;5(6):1650-1663.
SUMMARY
Abnormal activation of Wnt/β-catenin-mediated transcription is associated with a variety of human cancers. Here we report that LATS2 inhibited oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. LATS2 directly interacted with β-catenin and to be present on Wnt target gene promoters. Mechanistically, LATS2 inhibited the interaction between BCL9 and β-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 was down-regulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an anti-microtubule drug, potently induced LATS2 to suppress tumor growth in vivo by targeting β-catenin/BCL9. Our results suggest that LATS2 is not only a key tumor suppressor in human cancer, but may also be an important target for anti-cancer therapy.
doi:10.1016/j.celrep.2013.11.037
PMCID: PMC3897473  PMID: 24360964
16.  Acetylation of Metabolic Enzymes Coordinates Carbon Source Utilization and Metabolic Flux 
Science (New York, N.Y.)  2010;327(5968):1004-1007.
Lysine acetylation regulates many eukaryotic cellular processes, but its function in prokaryotes is largely unknown. We demonstrated that central metabolism enzymes in Salmonella were acetylated extensively and differentially in response to different carbon sources, concomitantly with changes in cell growth and metabolic flux. The relative activities of key enzymes controlling the direction of glycolysis versus gluconeogenesis and the branching between citrate cycle and glyoxylate bypass were all regulated by acetylation. This modulation is mainly controlled by a pair of lysine acetyltransferase and deacetylase, whose expressions are coordinated with growth status. Reversible acetylation of metabolic enzymes ensure that cells respond environmental changes via promptly sensing cellular energy status and flexibly altering reaction rates or directions. It represents a metabolic regulatory mechanism conserved from bacteria to mammals.
doi:10.1126/science.1179687
PMCID: PMC4183141  PMID: 20167787
17.  Mitogenic and Oncogenic Stimulation of K433 Acetylation Promotes PKM2 Protein Kinase Activity and Nuclear Localization 
Molecular cell  2013;52(3):340-352.
SUMMARY
Alternative splicing of the PKM2 gene produces two isoforms, M1 and M2, which are preferentially expressed in adult and embryonic tissues, respectively. The M2 isoform is reexpressed in human cancer and has nonmetabolic functions in the nucleus as a protein kinase. Here, we report that PKM2 is acetylated by p300 acetyltransferase at K433, which is unique to PKM2 and directly contacts its allosteric activator, fructose 1,6-bisphosphate (FBP). Acetylation prevents PKM2 activation by interfering with FBP binding and promotes the nuclear accumulation and protein kinase activity of PKM2. Acetylationmimetic PKM2(K433) mutant promotes cell proliferation and tumorigenesis. K433 acetylation is decreased by serum starvation and cell-cell contact, increased by cell cycle stimulation, epidermal growth factor (EGF), and oncoprotein E7, and enriched in breast cancers. Hence, K433 acetylation links cell proliferation and transformation to the switch of PKM2 from a cytoplasmic metabolite kinase to a nuclear protein kinase.
doi:10.1016/j.molcel.2013.09.004
PMCID: PMC4183148  PMID: 24120661
18.  The regulation of Hippo pathway and implications for anticancer drug development 
Trends in pharmacological sciences  2013;34(10):581-589.
Research in the past decade has revealed key components of the Hippo tumour-suppressor pathway and its critical role in organ size regulation and tumorigenesis. Recent progress has identified a wide range of upstream factors that control the Hippo pathway, which include cell-cell contact, various diffusible signals, and cognate receptors. Dysregulation of the Hippo pathway, caused by gene mutation or aberrant expression, promotes cell proliferation and tumorigenesis. Here, we discuss the current state of Hippo pathway research, primarily focusing on upstream regulators and protein-protein interactions as potential therapeutic targets. Consideration of pharmacological intervention of the Hippo pathway may provide novel avenues for future therapeutic treatment of human diseases, particularly in cancer.
doi:10.1016/j.tips.2013.08.006
PMCID: PMC3929107  PMID: 24051213
Hippo pathway; YAP; phosphorylation; GPCR; cancer
19.  Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth 
Molecular cell  2013;51(4):506-518.
SUMMARY
Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.
doi:10.1016/j.molcel.2013.07.002
PMCID: PMC4180208  PMID: 23932781
21.  Nutrient Signaling to mTOR and Cell Growth 
Trends in biochemical sciences  2013;38(5):233-242.
The mammalian target of rapamycin (mTOR) is a conserved protein kinase involved in a multitude of cellular processes including cell growth. Increased mTOR activation is observed in multiple human cancers and inhibition of mTOR has proven efficacious in numerous clinical trials. mTOR comprises two complexes, termed mTORC1 and mTORC2. Both complexes respond to growth factors, whereas only mTORC1 is controlled by nutrients, such as glucose and amino acids. Since the discovery of mTOR, extensive studies have intricately detailed the molecular mechanisms by which mTORC1 is regulated. Somewhat paradoxically, amino acid induced mTORC1 activation—arguably the most essential stimulus leading to mTORC1 activation—is the least understood. Here we review the current knowledge of nutrient dependent regulation of mTORC1.
doi:10.1016/j.tibs.2013.01.004
PMCID: PMC3634910  PMID: 23465396
22.  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).
doi:10.1038/nrm3522
PMCID: PMC3988467  PMID: 23361334
23.  Lysine-5 Acetylation Negatively Regulates Lactate Dehydrogenase A and Is Decreased in Pancreatic Cancer 
Cancer cell  2013;23(4):10.1016/j.ccr.2013.02.005.
SUMMARY
Tumor cells commonly have increased glucose uptake and lactate accumulation. Lactate is produced from pyruvate by lactate dehydrogenase A (LDH-A), which is frequently overexpressed in tumor cells and is important for cell growth. Elevated transcription by c-Myc or HIF1α may contribute to increased LDH-A in some cancer types. Here, we show that LDH-A is acetylated at lysine 5 (K5) and that this acetylation inhibits LDH-A activity. Furthermore, the K5-acetylated LDH-A is recognized by the HSC70 chaperone and delivered to lysosomes for degradation. Replacement of endogenous LDH-A with an acetylation mimetic mutant decreases cell proliferation and migration. Importantly, K5 acetylation of LDH-A is reduced in human pancreatic cancers. Our study reveals a mechanism of LDH-A upregulation in pancreatic cancers.
doi:10.1016/j.ccr.2013.02.005
PMCID: PMC3885615  PMID: 23523103
24.  The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal 
Nature cell biology  2011;13(8):877-883.
Precise control of organ size is crucial during animal development and regeneration. In Drosophila and mammals, studies over the past decade have uncovered a critical role for the Hippo tumour-suppressor pathway in the regulation of organ size. Dysregulation of this pathway leads to massive overgrowth of tissue. The Hippo signalling pathway is highly conserved and limits organ size by phosphorylating and inhibiting the transcription co-activators YAP and TAZ in mammals and Yki in Drosophila, key regulators of proliferation and apoptosis. The Hippo pathway also has a critical role in the self-renewal and expansion of stem cells and tissue-specific progenitor cells, and has important functions in tissue regeneration. Emerging evidence shows that the Hippo pathway is regulated by cell polarity, cell adhesion and cell junction proteins. In this review we summarize current understanding of the composition and regulation of the Hippo pathway, and discuss how cell polarity and cell adhesion proteins inform the role of this pathway in organ size control and regeneration.
doi:10.1038/ncb2303
PMCID: PMC3987945  PMID: 21808241
25.  AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 
Nature cell biology  2011;13(2):132-141.
Autophagy is a process by which components of the cell are degraded to maintain essential activity and viability in response to nutrient limitation. Extensive genetic studies have shown that the yeast ATG1 kinase has an essential role in autophagy induction. Furthermore, autophagy is promoted by AMP activated protein kinase (AMPK), which is a key energy sensor and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by the mammalian target of rapamycin (mTOR), a central cell-growth regulator that integrates growth factor and nutrient signals. Here we demonstrate a molecular mechanism for regulation of the mammalian autophagy-initiating kinase Ulk1, a homologue of yeast ATG1. Under glucose starvation, AMPK promotes autophagy by directly activating Ulk1 through phosphorylation of Ser 317 and Ser 777. Under nutrient sufficiency, high mTOR activity prevents Ulk1 activation by phosphorylating Ulk1 Ser 757 and disrupting the interaction between Ulk1 and AMPK. This coordinated phosphorylation is important for Ulk1 in autophagy induction. Our study has revealed a signalling mechanism for Ulk1 regulation and autophagy induction in response to nutrient signalling.
doi:10.1038/ncb2152
PMCID: PMC3987946  PMID: 21258367

Results 1-25 (66)