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1.  ULK1 inhibits the kinase activity of mTORC1 and cell proliferation 
Autophagy  2011;7(10):1212-1221.
ULK1 (Unc51-like kinase, hATG1) is a Ser/Thr kinase that plays a key role in inducing autophagy in response to starvation. ULK1 is phosphorylated and negatively regulated by the mammalian target of rapamycin complex 1 (mTORC1). Previous studies have shown that ULK1 is not only a downstream effector of mTORC1 but also a negative regulator of mTORC1 signaling.1–3 Here, we investigated how ULK1 regulates mTORC1 signaling, and found that ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Deficiency or knockdown of ULK1 or its homolog ULK2 enhanced mTORC1 signaling, cell proliferation rates and accumulation of cell mass, whereas overexpression of ULK1 had the opposite effect. Knockdown of Atg13, the binding partner of ULK1 and ULK2, mimicked the effects of ULK1 or ULK2 deficiency or knockdown. Both insulin and leucine stimulated mTORC1 signaling to a greater extent when ULK1 or ULK2 was deficient or knocked down. In contrast, Atg5 deficiency did not have a significant effect on mTORC1 signaling and cell proliferation. The stimulatory effect of ULK1 knockdown on mTORC1 signaling occurred even in the absence of tuberous sclerosis complex 2 (TSC2), the negative regulator of mTORC1 signaling. In addition, ULK1 was found to bind raptor, induce its phosphorylation, and inhibit the kinase activity of mTORC1. These results demonstrate that ULK1 negatively regulates the kinase activity of mTORC1 and cell proliferation in a manner independent of Atg5 and TSC2. The inhibition of mTORC1 by ULK1 may be important to coordinately regulate cell growth and autophagy with optimized utilization of cellular energy.
doi:10.4161/auto.7.10.16660
PMCID: PMC3210307  PMID: 21795849
ULK1; ULK2; Atg5; raptor; mTOR
2.  ULK1 inhibits mTORC1 signaling, promotes multisite Raptor phosphorylation and hinders substrate binding 
Autophagy  2011;7(7):737-747.
Protein synthesis and autophagy work as two opposing processes to control cell growth in response to nutrient supply. The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway, which acts as a master regulator to control protein synthesis, has recently been shown to inhibit autophagy by phosphorylating and inactivating ULK1, an autophagy regulatory protein. ULK1 also inhibits phosphorylation of a mTORC1 substrate, S6K1, indicating that a complex signaling interplay exists between mTORC1 and ULK1. Here, we demonstrate that ULK1 induces multisite phosphorylation of Raptor in vivo and in vitro. Using phospho-specific antibodies we identify Ser855 and Ser859 as being strongly phosphorylated by ULK1, with moderate phosphorylation of Ser792 also observed. Interestingly, ULK1 overexpression also increases phosphorylation of Raptor Ser863 and the mTOR autophosphorylation site, Ser2481 in a mTORC1-dependent manner. Despite this evidence for heightened mTORC1 kinase activity following ULK1 overexpresssion, mTORC1-mediated phosphorylation of S6K1 and 4E-BP1 is significantly inhibited. ULK1 expression has no effect on protein-protein interactions between the components of mTORC1, but does reduce the ability of Raptor to bind to the substrate 4E-BP1. Furthermore, shRNA knockdown of ULK1 leads to increased phosphorylation of mTORC1 substrates and decreased phosphorylation of Raptor at Ser859 and Ser792. We propose a new mechanism whereby ULK1 contributes to mTORC1 inhibition through hindrance of substrate docking to Raptor. This is a novel negative feedback loop that occurs upon activation of autophagy to maintain mTORC1 inhibition when nutrient supplies are limiting.
doi:10.4161/auto.7.7.15491
PMCID: PMC3149699  PMID: 21460630
autophagy; mTORC1; raptor; mTOR; S6K1; ULK1; ULK2
3.  The Association of AMPK with ULK1 Regulates Autophagy 
PLoS ONE  2010;5(11):e15394.
Autophagy is a highly orchestrated intracellular bulk degradation process that is activated by various environmental stresses. The serine/threonine kinase ULK1, like its yeast homologue Atg1, is a key initiator of autophagy that is negatively regulated by the mTOR kinase. However, the molecular mechanism that controls the inhibitory effect of mTOR on ULK1-mediated autophagy is not fully understood. Here we identified AMPK, a central energy sensor, as a new ULK1-binding partner. We found that AMPK binds to the PS domain of ULK1 and this interaction is required for ULK1-mediated autophagy. Interestingly, activation of AMPK by AICAR induces 14-3-3 binding to the AMPK-ULK1-mTORC1 complex, which coincides with raptor Ser792 phosphorylation and mTOR inactivation. Consistently, AICAR induces autophagy in TSC2-deficient cells expressing wild-type raptor but not the mutant raptor that lacks the AMPK phosphorylation sites (Ser722 and Ser792). Taken together, these results suggest that AMPK association with ULK1 plays an important role in autophagy induction, at least in part, by phosphorylation of raptor to lift the inhibitory effect of mTOR on the ULK1 autophagic complex.
doi:10.1371/journal.pone.0015394
PMCID: PMC2972217  PMID: 21072212
4.  Nutrient-dependent mTORC1 Association with the ULK1–Atg13–FIP200 Complex Required for Autophagy 
Molecular Biology of the Cell  2009;20(7):1981-1991.
Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable ∼3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1–Atg13–FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1–Atg13–FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the ∼3-MDa ULK1–Atg13–FIP200 complex.
doi:10.1091/mbc.E08-12-1248
PMCID: PMC2663915  PMID: 19211835
5.  ULK-Atg13-FIP200 Complexes Mediate mTOR Signaling to the Autophagy Machinery 
Molecular Biology of the Cell  2009;20(7):1992-2003.
Autophagy, the starvation-induced degradation of bulky cytosolic components, is up-regulated in mammalian cells when nutrient supplies are limited. Although mammalian target of rapamycin (mTOR) is known as the key regulator of autophagy induction, the mechanism by which mTOR regulates autophagy has remained elusive. Here, we identify that mTOR phosphorylates a mammalian homologue of Atg13 and the mammalian Atg1 homologues ULK1 and ULK2. The mammalian Atg13 binds both ULK1 and ULK2 and mediates the interaction of the ULK proteins with FIP200. The binding of Atg13 stabilizes and activates ULK and facilitates the phosphorylation of FIP200 by ULK, whereas knockdown of Atg13 inhibits autophagosome formation. Inhibition of mTOR by rapamycin or leucine deprivation, the conditions that induce autophagy, leads to dephosphorylation of ULK1, ULK2, and Atg13 and activates ULK to phosphorylate FIP200. These findings demonstrate that the ULK-Atg13-FIP200 complexes are direct targets of mTOR and important regulators of autophagy in response to mTOR signaling.
doi:10.1091/mbc.E08-12-1249
PMCID: PMC2663920  PMID: 19225151
6.  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
7.  Deficiency in AMPK attenuates ethanol-induced cardiac contractile dysfunction through inhibition of autophagosome formation 
Cardiovascular Research  2012;94(3):480-491.
Aims
Binge drinking often triggers compromised myocardial contractile function while activating AMP-activated protein kinase (AMPK). Given the role of AMPK in the initiation of autophagy through the mammalian target of rapamycin complex 1 (mTORC1) and Unc51-like kinase (ULK1), this study was designed to examine the impact of AMPK deficiency on cardiac function and the mechanism involved with a focus on autophagy following an acute ethanol challenge.
Methods and results
Wild-type (WT) and transgenic mice overexpressing a kinase-dead (KD) α2 isoform (K45R mutation) of AMPK were challenged with ethanol. Glucose tolerance, echocardiography, Langendorff heart and cardiomyocyte contractile function, autophagy, and autophagic signalling including AMPK, acetyl-CoA carboxylase (ACC), mTOR, the mTORC1-associated protein Raptor, and ULK1 were examined. Ethanol exposure triggered glucose intolerance and compromised cardiac contraction accompanied by increased phosphorylation of AMPK and ACC as well as autophagosome accumulation (increased LC3II and p62), the effects of which were attenuated or mitigated by AMPK deficiency or inhibition. Ethanol dampened and stimulated, respectively, the phosphorylation of mTOR and Raptor, the effects of which were abolished by AMPK deficiency. ULK1 phosphorylation at Ser757 and Ser777 was down-regulated and up-regulated, respectively, by ethanol, the effect of which was nullified by AMPK deficiency or inhibition. Moreover, the ethanol challenge enhanced LC3 puncta in H9c2 cells and promoted cardiac contractile dysfunction, and these effects were ablated by the inhibition of autophagy or AMPK. Lysosomal inhibition failed to accentuate ethanol-induced increases in LC3II and p62.
Conclusion
In summary, these data suggest that ethanol exposure may trigger myocardial dysfunction through a mechanism associated with AMPK-mTORC1-ULK1-mediated autophagy.
doi:10.1093/cvr/cvs127
PMCID: PMC3353804  PMID: 22451512
Ethanol; AMPK deficiency; Autophagy; Cardiac function; ULK1
8.  Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction 
Autophagy  2011;7(12):1424-1433.
Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.
doi:10.4161/auto.7.12.18027
PMCID: PMC3327613
Atg13; autophagy; FIP200; Ulk1; Ulk2
9.  AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization 
Autophagy  2012;8(8):1197-1214.
Autophagy is activated in response to a variety of cellular stresses including metabolic stress. While elegant genetic studies in yeast have identified the core autophagy machinery, the signaling pathways that regulate this process are less understood. AMPK is an energy sensing kinase and several studies have suggested that AMPK is required for autophagy. The biochemical connections between AMPK and autophagy, however, have not been elucidated. In this report, we identify a biochemical connection between a critical regulator of autophagy, ULK1, and the energy sensing kinase, AMPK. ULK1 forms a complex with AMPK, and AMPK activation results in ULK1 phosphorylation. Moreover, we demonstrate that the immediate effect of AMPK-dependent phosphorylation of ULK1 results in enhanced binding of the adaptor protein YWHAZ/14-3-3ζ; and this binding alters ULK1 phosphorylation in vitro. Finally, we provide evidence that both AMPK and ULK1 regulate localization of a critical component of the phagophore, ATG9, and that some of the AMPK phosphorylation sites on ULK1 are important for regulating ATG9 localization. Taken together these data identify an ULK1-AMPK signaling cassette involved in regulation of the autophagy machinery.
doi:10.4161/auto.20586
PMCID: PMC3679237  PMID: 22932492
14-3-3 proteins; AMP-activated protein kinase; Atg9; autophagy; energy metabolism; metabolic stress; phosphorylation; Unc-51-like kinase 1
10.  The requirement of uncoordinated 51-like kinase 1 (ULK1) and ULK2 in the regulation of autophagy 
Autophagy  2011;7(7):689-695.
Autophagy is an evolutionarily conserved physiological process of self-digestion by a cell to adapt to various stresses, including starvation. Its molecular basis involves the concerted activation of proteins encoded by the family of autophagy-related (Atg) genes. The best characterized is the serine/threonine protein kinase Atg1 in yeast which appears to be essential at the early stage of autophagy. In mammals, five Atg1 homologues have been identified as uncoordinated (UNC) 51-like kinase 1 to 4 and STK36. ULK1 and ULK2 are the most closely related members of the family, sharing 78% homology within their protein kinase domains. However, the specific function of ULK1 and ULK2 in mammalian autophagy is not fully understood. Here, we demonstrate that ULK1 and ULK2 are functionally redundant protein kinases required to mediate autophagy under nutrient-deprived conditions in fibroblasts. In contrast, ULK1, but not ULK2, is critical to induce the autophagic response of cerebellar granule neurons (CGN) to low potassium concentration in serum-free conditions. Furthermore, we found that ULK1 has a cytoprotective function in neurons. Together, these results provide strong genetic evidence that ULK1 is an essential component of the autophagic signaling pathway. The ability of ULK2 to compensate for the loss of ULK1 function is cell-type specific.
doi:10.4161/auto.7.7.15450
PMCID: PMC3149696  PMID: 21460635
Atg1; ULK1; ULK2; mTOR; autophagy; apoptosis; neurons; MEFs
11.  Active-Site Inhibitors of mTOR Target Rapamycin-Resistant Outputs of mTORC1 and mTORC2 
PLoS Biology  2009;7(2):e1000038.
The mammalian target of rapamycin (mTOR) regulates cell growth and survival by integrating nutrient and hormonal signals. These signaling functions are distributed between at least two distinct mTOR protein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to the selective inhibitor rapamycin and activated by growth factor stimulation via the canonical phosphoinositide 3-kinase (PI3K)→Akt→mTOR pathway. Activated mTORC1 kinase up-regulates protein synthesis by phosphorylating key regulators of mRNA translation. By contrast, mTORC2 is resistant to rapamycin. Genetic studies have suggested that mTORC2 may phosphorylate Akt at S473, one of two phosphorylation sites required for Akt activation; this has been controversial, in part because RNA interference and gene knockouts produce distinct Akt phospho-isoforms. The central role of mTOR in controlling key cellular growth and survival pathways has sparked interest in discovering mTOR inhibitors that bind to the ATP site and therefore target both mTORC2 and mTORC1. We investigated mTOR signaling in cells and animals with two novel and specific mTOR kinase domain inhibitors (TORKinibs). Unlike rapamycin, these TORKinibs (PP242 and PP30) inhibit mTORC2, and we use them to show that pharmacological inhibition of mTOR blocks the phosphorylation of Akt at S473 and prevents its full activation. Furthermore, we show that TORKinibs inhibit proliferation of primary cells more completely than rapamycin. Surprisingly, we find that mTORC2 is not the basis for this enhanced activity, and we show that the TORKinib PP242 is a more effective mTORC1 inhibitor than rapamycin. Importantly, at the molecular level, PP242 inhibits cap-dependent translation under conditions in which rapamycin has no effect. Our findings identify new functional features of mTORC1 that are resistant to rapamycin but are effectively targeted by TORKinibs. These potent new pharmacological agents complement rapamycin in the study of mTOR and its role in normal physiology and human disease.
Author Summary
Growth factor pathways are required for normal development but are often inappropriately activated in many cancers. One growth-factor–sensitive pathway of increasing interest to cancer researchers relies on the mammalian target of rapamycin (mTOR), a kinase that (like all kinases) delivers phosphate groups from ATP to amino acid residues of downstream proteins. TOR proteins were first discovered in yeast as the cellular targets of rapamycin, a small, naturally occurring molecule derived from bacteria that is widely used as an immunosuppressant and more recently in some cancer therapies. The study of TOR proteins has relied heavily on the use of rapamycin, but rapamycin does not directly inhibit TOR kinase activity; rather, rapamycin influences TOR's enzymatic activities by binding to a domain far from the kinase's active site. Some mTOR functions are resistant to rapamycin, as a result of the kinase activity of one kind of multiprotein complex, the mTOR complex 2 (mTORC2), whereas rapamycin-sensitive functions of mTOR are due to the mTOR complex 1 (mTORC1). We have developed new inhibitors of mTOR that bind to the ATP-binding site of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2 without inhibiting other kinases. Unexpectedly, these inhibitors had profound effects on protein synthesis and cell proliferation due to their inhibition of mTORC1 rather than mTORC2. We found that the phosphorylation of a protein that controls protein synthesis, the mTORC1 substrate 4E binding protein (4EBP) is partially resistant to rapamycin but fully inhibited by our new inhibitors. The finding that 4EBP phosphorylation is resistant to rapamycin suggests that active-site inhibitors may be more effective than rapamycin in the treatment of cancer and may explain why rapamycin is so well tolerated when taken for immunosuppression.
Cells rely on the mammalian target of rapamycin kinase (mTOR) to sense growth factors. Inhibition of all forms of mTOR using newly developed inhibitors of its active site reveals new insights into the function of two mTOR-containing protein complexes and their potential as therapeutic targets.
doi:10.1371/journal.pbio.1000038
PMCID: PMC2637922  PMID: 19209957
12.  The ULK1 complex 
Autophagy  2013;9(2):124-137.
The Atg1/ULK1 complex plays a central role in starvation-induced autophagy, integrating signals from upstream sensors such as MTOR and AMPK and transducing them to the downstream autophagy pathway. Much progress has been made in the last few years in understanding the mechanisms by which the complex is regulated through protein-protein interactions and post-translational modifications, providing insights into how the cell modulates autophagy, particularly in response to nutrient status. However, how the ULK1 complex transduces upstream signals to the downstream central autophagy pathway is still unclear. Although the protein kinase activity of ULK1 is required for its autophagic function, its protein substrate(s) responsible for autophagy activation has not been identified. Furthermore, examples of potential ULK1-independent autophagy have emerged, indicating that under certain specific contexts, the ULK1 complex might be dispensable for autophagy activation. This raises the question of how the autophagic machinery is activated independent of the ULK1 complex and what are the biological functions of such noncanonical autophagy pathways.
doi:10.4161/auto.23323
PMCID: PMC3552878  PMID: 23295650
AMPK; Atg1; kinase; MTOR; regulation
13.  Distinct functions of Ulk1 and Ulk2 in the regulation of lipid metabolism in adipocytes 
Autophagy  2013;9(12):2103-2114.
ULK1 (unc-51 like kinase 1) is a serine/threonine protein kinase that plays a key role in regulating the induction of autophagy. Recent studies using autophagy-defective mouse models, such as atg5- or atg7-deficient mice, revealed an important function of autophagy in adipocyte differentiation. Suppression of adipogenesis in autophagy-defective conditions has made it difficult to study the roles of autophagy in metabolism of differentiated adipocytes. In this study, we established autophagy defective-differentiated 3T3-L1 adipocytes, and investigated the roles of Ulk1 and its close homolog Ulk2 in lipid and glucose metabolism using the established adipocytes. Through knockdown approaches, we determined that Ulk1 and Ulk2 are important for basal and MTORC1 inhibition-induced autophagy, basal lipolysis, and mitochondrial respiration. However, unlike other autophagy genes (Atg5, Atg13, Rb1cc1/Fip200, and Becn1) Ulk1 was dispensable for adipogenesis without affecting the expression of CCAAT/enhancer binding protein α (CEBPA) and peroxisome proliferation-activated receptor gamma (PPARG). Ulk1 knockdown reduced fatty acid oxidation and enhanced fatty acid uptake, the metabolic changes that could contribute to adipogenesis, whereas Ulk2 knockdown had opposing effects. We also found that the expression levels of insulin receptor (INSR), insulin receptor substrate 1 (IRS1), and glucose transporter 4 (SLC2A4/GLUT4) were increased in Ulk1-silenced adipocytes, which was accompanied by upregulation of insulin-stimulated glucose uptake. These results suggest that ULK1, albeit its important autophagic role, regulates lipid metabolism and glucose uptake in adipocytes distinctly from other autophagy proteins.
doi:10.4161/auto.26563
PMCID: PMC4028344  PMID: 24135897
ULK1; ULK2; mTORC1; adipogenesis; adipocytes; lipid metabolism
14.  Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy 
Science (New York, N.Y.)  2010;331(6016):456-461.
Adenosine monophosphate-activated protein kinase (AMPK) is a conserved sensor of intracellular energy activated in response to low nutrient availability and environmental stress. In a screen for conserved substrates of AMPK, we identified ULK1 and ULK2, mammalian orthologs of the yeast protein kinase Atg1, which is required for autophagy. Genetic analysis of AMPK or ULK1 in mammalian liver and C. elegans revealed a requirement for these kinases in autophagy. In mammals, loss of AMPK or ULK1 resulted in aberrant accumulation of the autophagy adaptor p62 and defective mitophagy. Reconstitution of ULK1-deficient cells with a mutant ULK1 that cannot be phosphorylated by AMPK revealed that such phosphorylation is required for mitochondrial homeostasis and cell survival following starvation. These findings uncover a conserved biochemical mechanism coupling nutrient status with autophagy and cell survival.
doi:10.1126/science.1196371
PMCID: PMC3030664  PMID: 21205641
15.  Hsp90-Cdc37 chaperone complex regulates Ulk1- and Atg13-mediated mitophagy 
Molecular cell  2011;43(4):572-585.
SUMMARY
Autophagy, the primary recycling pathway of cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the response to cellular stress. The Hsp90-Cdc37 chaperone complex coordinately regulates the activity of select kinases to orchestrate many facets of the stress response. Although both maintain mitochondrial integrity, the relationship between Hsp90-Cdc37 and autophagy has not been well characterized. Ulk1, one of the mammalian homologues of yeast Atg1, is a serine-threonine kinase required for mitophagy. Here we show that the interaction between Ulk1 and Hsp90-Cdc37 stabilizes and activates Ulk1, which in turn is required for the phosphorylation and release of Atg13 from Ulk1, and for the recruitment of Atg13 to damaged mitochondria. Hsp90-Cdc37, Ulk1 and Atg13 phosphorylation are all required for efficient mitochondrial clearance. These findings establish a direct pathway that integrates Ulk1- and Atg13- directed mitophagy with the stress response coordinated by Hsp90 and Cdc37.
doi:10.1016/j.molcel.2011.06.018
PMCID: PMC3485687  PMID: 21855797
16.  LKB1 and AMPK control of mTOR signalling and growth 
The AMP-activated serine/threonine protein kinase (AMPK) is a sensor of cellular energy status found in all eukaryotes that is activated under conditions of low intracellular ATP following stresses such as nutrient deprivation or hypoxia. In the past five years, work from a large number of laboratories has revealed that one of the major downstream signaling pathways regulated by AMPK is the mammalian target-of-rapamycin (mTOR pathway). Interestingly, like AMPK, the mTOR serine/threonine kinase plays key roles not only in growth control and cell proliferation but also in metabolism. Recent work has revealed that across eukaryotes mTOR orthologs are found in two biochemically distinct complexes and only one of those complexes (mTORC1 in mammals) is acutely sensitive to rapamycin and regulated by nutrients and AMPK. Many details of the molecular mechanism by which AMPK inhibits mTORC1 signaling have also been decoded in the past 5 years. AMPK directly phosphorylates at least two proteins to induce rapid suppression of mTORC1 activity, the TSC2 tumor suppressor and the critical mTORC1 binding subunit raptor. Here we explore the molecular connections between AMPK and mTOR signaling pathways and examine the physiological processes in which AMPK regulation of mTOR is critical for growth or metabolic control. The functional conservation of AMPK and TOR in all eukaryotes, and the sequence conservation around the AMPK phosphorylation sites in raptor across all eukaryotes examined suggest that this represents a fundamental cell growth module connecting nutrient status to the cell growth machinery. These findings have broad implications for the control of cell growth by nutrients in a number of cellular and organismal contexts.
doi:10.1111/j.1748-1716.2009.01972.x
PMCID: PMC2760308  PMID: 19245654
LKB1; AMPK; mTOR; raptor; TSC2; metabolism; checkpoint
17.  Upregulation of human autophagy-initiation kinase ULK1 by tumor suppressor p53 contributes to DNA-damage-induced cell death 
Cell Death and Differentiation  2011;18(10):1598-1607.
In yeast, activation of ATG1/ATG13 kinase complex initiates autophagy. This mechanism of autophagy initiation is conserved, as unc-51-like kinase 1 (ULK1) and unc-51-like kinase 2 (ULK2) are two mammalian functional homologues of ATG1 and form similar complex with mammalian ATG13. Here, we report that both ULK1 and ULK2 are transcriptional targets of tumor suppressor p53. In response to DNA damage, ULK1 and ULK2 are upregulated by p53. The upregulation of ULK1 (ULK2)/ATG13 complex by p53 is necessary for the sustained autophagy activity induced by DNA damage. In this context, elevated autophagy contributes to subsequent cell death. These findings suggest that ULK1 and ULK2 may mediate part of tumor suppression activity in mammalian cells and contribute to the efficacy of genotoxic chemotherapeutic drugs.
doi:10.1038/cdd.2011.33
PMCID: PMC3172118  PMID: 21475306
autophagy; p53; DNA damage; Ulk1
18.  ULK1 induces autophagy by phosphorylating Beclin-1 and activating Vps34 lipid kinase 
Nature cell biology  2013;15(7):10.1038/ncb2757.
Autophagy is the primary cellular catabolic program activated in response to nutrient starvation. Initiation of autophagy, particularly by amino acid withdrawal, requires the ULK kinases. Despite its pivotal role in autophagy initiation, little is known about the mechanisms by which ULK promotes autophagy. Here we describe a molecular mechanism linking ULK to the pro-autophagic lipid kinase VPS34. Upon amino acid starvation or mTOR inhibition the activated ULK1 phosphorylates Beclin-1 on S14, thereby, enhancing the activity of the ATG14L-containing VPS34 complexes. The Beclin-1 S14 phosphorylation by ULK is required for full autophagic induction in mammals and this requirement is conserved in C. elegans. Our study reveals a molecular link from ULK1 to activation of the autophagy specific VPS34 complex and autophagy induction.
doi:10.1038/ncb2757
PMCID: PMC3885611  PMID: 23685627
19.  Regulation of nutrient-sensitive autophagy by uncoordinated 51-like kinases 1 and 2 
Autophagy  2013;9(3):361-373.
Macroautophagy, commonly referred to as autophagy, is a protein degradation pathway that occurs constitutively in cells, but can also be induced by stressors such as nutrient starvation or protein aggregation. Autophagy has been implicated in multiple disease mechanisms including neurodegeneration and cancer, with both tumor suppressive and oncogenic roles. Uncoordinated 51-like kinase 1 (ULK1) is a critical autophagy protein near the apex of the hierarchal regulatory pathway that receives signals from the master nutrient sensors MTOR and AMP-activated protein kinase (AMPK). In mammals, ULK1 has a close homolog, ULK2, although their functional distinctions have been unclear. Here, we show that ULK1 and ULK2 both function to support autophagy activation following nutrient starvation. Increased autophagy following amino acid or glucose starvation was disrupted only upon combined loss of ULK1 and ULK2 in mouse embryonic fibroblasts. Generation of PtdIns3P and recruitment of WIPI2 or ZFYVE1/DFCP1 to the phagophore following amino acid starvation was blocked by combined Ulk1/2 double knockout. Autophagy activation following glucose starvation did not involve recruitment of either WIPI1 or WIPI2 to forming autophagosomes. Consistent with a PtdIns3P-independent mechanism, glucose-dependent autophagy was resistant to wortmannin. Our findings support functional redundancy between ULK1 and ULK2 for nutrient-dependent activation of autophagy and furthermore highlight the differential pathways that respond to amino acid and glucose deprivation.
doi:10.4161/auto.23066
PMCID: PMC3590256  PMID: 23291478
ULK1; ULK2; WIPI1; WIPI2; knockout; MEF; nutrient starvation
20.  The incredible ULKs 
Macroautophagy (commonly abbreviated as autophagy) is an evolutionary conserved lysosome-directed vesicular trafficking pathway in eukaryotic cells that mediates the lysosomal degradation of intracellular components. The cytoplasmic cargo is initially enclosed by a specific double membrane vesicle, termed the autophagosome. By this means, autophagy either helps to remove damaged organelles, long-lived proteins and protein aggregates, or serves as a recycling mechanism for molecular building blocks. Autophagy was once invented by unicellular organisms to compensate the fluctuating external supply of nutrients. In higher eukaryotes, it is strongly enhanced under various stress conditions, such as nutrient and growth factor deprivation or DNA damage. The serine/threonine kinase Atg1 was the first identified autophagy-related gene (ATG) product in yeast. The corresponding nematode homolog UNC-51, however, has additional neuronal functions. Vertebrate genomes finally encode five closely related kinases, of which UNC-51-like kinase 1 (Ulk1) and Ulk2 are both involved in the regulation of autophagy and further neuron-specific vesicular trafficking processes. This review will mainly focus on the vertebrate Ulk1/2-Atg13-FIP200 protein complex, its function in autophagy initiation, its evolutionary descent from the yeast Atg1-Atg13-Atg17 complex, as well as the additional non-autophagic functions of its components. Since the rapid nutrient- and stress-dependent cellular responses are mainly mediated by serine/threonine phosphorylation, it will summarize our current knowledge about the relevant upstream signaling pathways and the altering phosphorylation status within this complex during autophagy induction.
doi:10.1186/1478-811X-10-7
PMCID: PMC3330011  PMID: 22413737
Atg1; Atg13; Atg17; UNC-51; EPG-1; Ulk1; Ulk2; FIP200; Atg101; Autophagy; Serine/threonine phosphorylation
21.  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
22.  FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells 
The Journal of Cell Biology  2008;181(3):497-510.
Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51–like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.
doi:10.1083/jcb.200712064
PMCID: PMC2364687  PMID: 18443221
23.  Kinase-Inactivated ULK Proteins Inhibit Autophagy via Their Conserved C-Terminal Domains Using an Atg13-Independent Mechanism▿  
Molecular and Cellular Biology  2008;29(1):157-171.
The yeast Atg1 serine/threonine protein kinase and its mammalian homologs ULK1 and ULK2 play critical roles during the activation of autophagy. Previous studies have demonstrated that the conserved C-terminal domain (CTD) of ULK1 controls the regulatory function and localization of the protein. Here, we explored the role of kinase activity and intramolecular interactions to further understand ULK function. We demonstrate that the dominant-negative activity of kinase-dead mutants requires a 7-residue motif within the CTD. Our data lead to a model in which the functions of ULK1 and ULK2 are controlled by autophosphorylation and conformational changes involving exposure of the CTD. Additional mapping indicates that the CTD contains other distinct regions that direct membrane association and interaction with the putative human homologue of Atg13, which we have here characterized. Atg13 is required for autophagy and Atg9 trafficking during autophagy. However, Atg13 does not bind the 7-residue dominant-negative motif in the CTD of ULK proteins nor is the inhibitory activity of the CTDs rescued by Atg13 ectopic expression, suggesting that in mammalian cells, the CTD may interact with additional autophagy proteins.
doi:10.1128/MCB.01082-08
PMCID: PMC2612494  PMID: 18936157
24.  Analysis of a lung defect in autophagy-deficient mouse strains 
Autophagy  2013;10(1):45-56.
Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.
doi:10.4161/auto.26505
PMCID: PMC4028323  PMID: 24275123
Ulk1/2 DKO mice; Atg5 KO mice; perinatal mortality; glycogen; lung development
25.  Aspirin Inhibits mTOR Signaling, Activates AMP-Activated Protein Kinase, and Induces Autophagy in Colorectal Cancer Cells 
Gastroenterology  2012;142(7):1504-15.e3.
BACKGROUND & AIMS
Aspirin reduces the incidence of and mortality from colorectal cancer (CRC) by unknown mechanisms. Cancer cells have defects in signaling via the mechanistic target of rapamycin (mTOR), which regulates proliferation. We investigated whether aspirin affects adenosine monophosphate–activated protein kinase (AMPK) and mTOR signaling in CRC cells.
METHODS
The effects of aspirin on mTOR signaling, the ribosomal protein S6, S6 kinase 1 (S6K1), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) were examined in CRC cells by immunoblotting. Phosphorylation of AMPK was measured; the effects of loss of AMPKα on the aspirin-induced effects of mTOR were determined using small interfering RNA (siRNA) in CRC cells and in AMPKα1/α2−/− mouse embryonic fibroblasts. LC3 and ULK1 were used as markers of autophagy. We analyzed rectal mucosa samples from patients given 600 mg aspirin, once daily for 1 week.
RESULTS
Aspirin reduced mTOR signaling in CRC cells by inhibiting the mTOR effectors S6K1 and 4E-BP1. Aspirin changed nucleotide ratios and activated AMPK in CRC cells. mTOR was still inhibited by aspirin in CRC cells after siRNA knockdown of AMPKα, indicating AMPK-dependent and AMPK-independent mechanisms of aspirin-induced inhibition of mTOR. Aspirin induced autophagy, a feature of mTOR inhibition. Aspirin and metformin (an activator of AMPK) increased inhibition of mTOR and Akt, as well as autophagy in CRC cells. Rectal mucosal samples from patients given aspirin had reduced phosphorylation of S6K1 and S6.
CONCLUSIONS
Aspirin is an inhibitor of mTOR and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism. These could contribute to its protective effects against development of CRC.
doi:10.1053/j.gastro.2012.02.050
PMCID: PMC3682211  PMID: 22406476
Chemoprevention; Colon Cancer; Oncogene; Tumor Suppressor

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