A Non-canonical MEK/ERK Signaling Pathway Regulates Autophagy via Regulating Beclin 1
2009; Elsevier BV; Volume: 284; Issue: 32 Linguagem: Inglês
10.1074/jbc.m109.026013
ISSN1083-351X
AutoresJianrong Wang, Mary W. Whiteman, Huiqin Lian, Guangxin Wang, Amit Kumar Singh, Dongyang Huang, Ted Denmark,
Tópico(s)Islanding Detection in Power Systems
ResumoAutophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy. Autophagy-essential proteins are the molecular basis of protective or destructive autophagy machinery. However, little is known about the signaling mechanisms governing these proteins and the opposing consequences of autophagy in mammals. Here we report that a non-canonical MEK/ERK module, which is positioned downstream of AMP-activated protein kinase (AMPK) and upstream of tuberous sclerosis complex (TSC), regulates autophagy by regulating Beclin 1. Depletion of ERK partially inhibited autophagy, whereas specific inhibition on MEK completely inhibited autophagy. MEK could bypass ERK to promote autophagy. Basal MEK/ERK activity conferred basal Beclin 1 by preventing disassembly of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2. Activation of MEK/ERK by AMPK upon autophagy stimuli disassembled mTORC1 via binding to and activating TSC but disassembled mTORC2 independently of TSC. Inhibition of mTORC1 or mTORC2 by transiently or moderately activated MEK/ERK caused moderately enhanced Beclin 1 resulting in cytoprotective autophagy, whereas inhibition of both mTORC1 and mTORC2 by sustained MEK/ERK activation caused strongly pronounced Beclin 1 leading to cytodestructive autophagy. Our findings thus propose that the AMPK-MEK/ERK-TSC-mTOR pathway regulation of Beclin 1 represents different thresholds responsible for a protective or destructive autophagy. Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3140) Google Scholar), antiaging (2Longo V.D. Finch C.E. Science. 2003; 299: 1342-1346Crossref PubMed Scopus (489) Google Scholar, 3Meléndez A. Tallóczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Crossref PubMed Scopus (1009) Google Scholar, 4Vellai T. Takacs-Vellai K. Zhang Y. Kovacs A. Orosz L. Müller F. Nature. 2003; 426: 620Crossref PubMed Scopus (814) Google Scholar), development (1Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3140) Google Scholar, 5Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2362) Google Scholar), protection of the genome (6Karantza-Wadsworth V. Patel S. Kravchuk O. Chen G. Mathew R. Jin S. White E. Genes Dev. 2007; 21: 1621-1635Crossref PubMed Scopus (670) Google Scholar), and regulation of cell size (7Vellai T. Bicsák B. Tóth M.L. Takács-Vellai K. Kovács A.L. Autophagy. 2008; 4: 507-509Crossref PubMed Scopus (33) Google Scholar). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. Cattoretti G. Levine B. J. Clin. Investig. 2003; 112: 1809-1820Crossref PubMed Scopus (1837) Google Scholar, 9Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2134) Google Scholar, 10Yue Z. Jin S. Yang C. Levine A.J. Heintz N. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 15077-15082Crossref PubMed Scopus (1746) Google Scholar), cardiomyopathy (11Tanaka Y. Guhde G. Suter A. Eskelinen E.L. Hartmann D. Lüllmann-Rauch R. Janssen P.M. Blanz J. von Figura K. Saftig P. Nature. 2000; 406: 902-906Crossref PubMed Scopus (716) Google Scholar), and neurodegenerative disorders (12Yuan J. Lipinski M. Degterev A. Neuron. 2003; 40: 401-413Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar).Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg) 2The abbreviations used are: Atgautophagy-essential proteinAMPKAMP-activated protein kinaseTSCtuberous sclerosis complexmTORmammalian target of rapamycinLC3light chain 3Bcl2B-cell lymphoma-2PI3Kphosphatidylinositol 3-kinasePI3KC3class III phosphatidylinositol 3-kinaseAmbra 1activating molecule in Beclin 1-regulated autophagy protein 1MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseERKextracellular signal-regulated kinasemTORCmTOR complexGΒLG-protein Β-subunit-likeAICAR5-aminoimidazole-4-carboxamide ribosideGFPgreen fluorescent proteinsiRNAshort interfering RNAGSTglutathione S-transferase. 2The abbreviations used are: Atgautophagy-essential proteinAMPKAMP-activated protein kinaseTSCtuberous sclerosis complexmTORmammalian target of rapamycinLC3light chain 3Bcl2B-cell lymphoma-2PI3Kphosphatidylinositol 3-kinasePI3KC3class III phosphatidylinositol 3-kinaseAmbra 1activating molecule in Beclin 1-regulated autophagy protein 1MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseERKextracellular signal-regulated kinasemTORCmTOR complexGΒLG-protein Β-subunit-likeAICAR5-aminoimidazole-4-carboxamide ribosideGFPgreen fluorescent proteinsiRNAshort interfering RNAGSTglutathione S-transferase. are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (898) Google Scholar, 14Reggiori F. Tucker K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: 79-90Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar).In mammalian systems, autophagosomes fuse with lysosomes to generate autophagolysosomes, which undergo a maturation process by fusing with endocytic compartments and lysosomes (15Eskelinen E.L. Autophagy. 2005; 1: 1-10Crossref PubMed Scopus (497) Google Scholar). Because it is not known how the Atg1 homolog acts in mammals, a different mechanism may be involved in regulating autophagy. Beclin 1/Atg6, microtubule-associated protein 1 light chain 3 (LC3)/Atg8, Atg5, Atg12, and Atg13 are essential for autophagosome formation in mammalian species (5Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2362) Google Scholar, 16Chan E.Y. Longatti A. McKnight N.C. Tooze S.A. Mol. Cell. Biol. 2009; 29: 157-171Crossref PubMed Scopus (341) Google Scholar, 17Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar, 18Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2700) Google Scholar, 19Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-668Crossref PubMed Scopus (1146) Google Scholar, 20Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1892) Google Scholar). Atg7 and Atg3 are required in the conjugation reaction between Atg12 and Atg5 and in the lipidation of LC3. During the formation of autophagosomes in mammalian cells, LC3 is lipidated via a ubiquitylation-like system (17Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5363) Google Scholar, 21Tanida I. Ueno T. Kominami E. Int. J. Biochem. Cell Biol. 2004; 36: 2503-2518Crossref PubMed Scopus (1043) Google Scholar), generating a soluble form, LC3-I. LC3-I is further modified to a membrane-bound form, LC3-II, which is subsequently localized to autophagosomes and autolysosomes until being degraded by the lysosome.Beclin 1 was initially isolated as a B-cell lymphoma-2 (Bcl2)-interacting tumor suppressor in mammalian cells (22Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar). Overexpression of Bcl2 attenuates the formation of the kinase complex Beclin 1-class III phosphatidylinositol 3-kinase (PI3KC3) essential for the formation of autophagosomes (23Pattingre S. Tassa A. Qu X. Garuti R. Liang X.H. Mizushima N. Packer M. Schneider M.D. Levine B. Cell. 2005; 122: 927-939Abstract Full Text Full Text PDF PubMed Scopus (2896) Google Scholar). The UV radiation resistance-associated gene tumor suppressor and the activating molecule in Beclin 1-regulated autophagy protein 1 (Ambra 1) were identified as new Beclin 1-binding partners that also regulate autophagy by regulating the Beclin 1-PI3KC3 kinase complex. Association of Beclin 1 with PI3KC3 is negatively regulated by Bcl2 (22Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar) and positively regulated by UV radiation resistance-associated gene tumor suppressor and Ambra 1 (24Fimia G.M. Stoykova A. Romagnoli A. Giunta L. Di Bartolomeo S. Nardacci R. Corazzari M. Fuoco C. Ucar A. Schwartz P. Gruss P. Piacentini M. Chowdhury K. Cecconi F. Nature. 2007; 447: 1121-1125Crossref PubMed Scopus (773) Google Scholar, 25Liang C. Feng P. Ku B. Dotan I. Canaani D. Oh B.H. Jung J.U. Nat. Cell Biol. 2006; 8: 688-699Crossref PubMed Scopus (828) Google Scholar). Beclin 1 is homoallelically deleted in many human tumors. A decreased Beclin 1 level causes defective autophagy and breast cancer, but restoration of Beclin 1 induces autophagy and inhibits tumorigenicity of human breast cancer cells (18Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2700) Google Scholar). These reports evidence the dependence on Beclin 1 for a functional autophagy mechanism.Diverse signaling pathways have been reported in the regulation of autophagy in mammalian cells (26Meijer A.J. Codogno P. Int. J. Biochem. Cell Biol. 2004; 36: 2445-2462Crossref PubMed Scopus (538) Google Scholar, 27Shinojima N. Yokoyama T. Kondo Y. Kondo S. Autophagy. 2007; 3: 635-637Crossref PubMed Scopus (280) Google Scholar). In contrast to yeast, mammalian cells regulate autophagy via both class I and class III PI3K. Class I PI3K plays an inhibitory role, whereas class III PI3K kinase complex, which includes Beclin 1, plays a stimulatory role in autophagy by promoting the nucleation of autophagic vesicles (28Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (797) Google Scholar, 29Petiot A. Ogier-Denis E. Blommaart E.F. Meijer A.J. Codogno P. J. Biol. Chem. 2000; 275: 992-998Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). A recent study also indicates that hVps15 is required in regulation of class III PI3K in mammalian cells (30Yan Y. Flinn R.J. Wu H. Schnur R.S. Backer J.M. Biochem. J. 2009; 417: 747-755Crossref PubMed Scopus (75) Google Scholar). However, the signaling mechanisms controlling autophagy-essential proteins, in particular Beclin 1, and the opposing consequences of autophagy remain to be resolved.Our present studies identified and positioned a non-canonical MEK/ERK pathway downstream of AMPK and upstream of TSC and mTOR. This MEK/ERK module regulated autophagy via regulating the Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR pathway. Moderately enhanced Beclin 1 by transient or moderate activation of MEK/ERK and subsequent inhibition on mTORC1 and mTORC2 individually caused protective autophagy. Strongly pronounced Beclin 1 by sustained or strong activation of MEK/ERK followed by dual inhibition on mTORC1 and mTORC2 caused destructive autophagy. Our results thus reveal interesting Beclin 1 thresholds in regulating autophagy.DISCUSSIONThe results presented here demonstrate that a non-canonical MEK/ERK module regulates autophagy by regulating Beclin 1 level through the AMPK-MEK/ERK-TSC-mTOR signaling pathway (Fig. 9). Basal MEK/ERK activity conferred constitutive Beclin 1 that was insufficient to trigger the autophagic response. MEK/ERK activation by AMPK upon various autophagy stimuli inactivated mTOR by disassembling the functional complexes mTORC1 and/or mTORC2, resulting in the up-regulation of Beclin 1 to different thresholds leading to autophagy of opposing consequences, suggesting that Beclin 1 may play a central role in integrating signals downstream of mTOR. Our studies thus provide the first mechanistic link between a signaling pathway and autophagy-essential proteins in the mammalian system.Mechanism of the Non-canonical MEK/ERK Module Regulating AutophagyERKs are a widely conserved family of serine/threonine protein kinases implicated in many cellular programs such as cell proliferation, differentiation, and apoptosis. ERK can be activated by a wide variety of oncogenes and extracellular stimuli including mitogens, growth factors, cytokines, and chemokines. An increasing number of studies have suggested that ERK plays a role in modulating autophagy (27Shinojima N. Yokoyama T. Kondo Y. Kondo S. Autophagy. 2007; 3: 635-637Crossref PubMed Scopus (280) Google Scholar, 43Aoki H. Takada Y. Kondo S. Sawaya R. Aggarwal B.B. Kondo Y. Mol. Pharmacol. 2007; 72: 29-39Crossref PubMed Scopus (464) Google Scholar, 44Zhu J.H. Horbinski C. Guo F. Watkins S. Uchiyama Y. Chu C.T. Am. J. Pathol. 2007; 170: 75-86Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar, 54Corcelle E. Nebout M. Bekri S. Gauthier N. Hofman P. Poujeol P. Fénichel P. Mograbi B. Cancer Res. 2006; 66: 6861-6870Crossref PubMed Scopus (152) Google Scholar, 55Ogier-Denis E. Pattingre S. El Benna J. Codogno P. J. Biol. Chem. 2000; 275: 39090-39095Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 56Pattingre S. Bauvy C. Codogno P. J. Biol. Chem. 2003; 278: 16667-16674Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 57Subramaniam S. Unsicker K. Neuroscience. 2006; 138: 1055-1065Crossref PubMed Scopus (76) Google Scholar). However, the role of ERK in autophagy was primarily concluded from the pharmacological blockage of autophagy by compound PD98059, an MEK-specific inhibitor. Because this chemical indirectly blocks ERK via inhibiting its canonical upstream regulator, MEK, it is not surprising that ERK was exclusively credited in regulating autophagy because ERK has been believed to be the only known substrate of MEK.We demonstrated in this study that basal MEK/ERK activity maintains the activity of mTORC1 and mTORC2 by protecting them from being disassembled (Fig. 6E, lower panel) and thus maintains a basal Beclin 1 level incapable of triggering autophagic response (Figs. 1E, 3C, and 6E, upper panel). Although we found that ERK is involved in the regulation of autophagy because constitutive expression of active ERK caused autophagy response (Figs. 1C and 7A) whereas specific inhibition on ERK by RNA interference (Fig. 2C) or dominant negative ERK (Fig. 2D) partially inhibited autophagy, knockdown of the canonical effectors ERK, p90RSK, and Elk by RNA interference did not cripple autophagic response (Fig. 2, A and B). This unusual observation suggests that ERK may use non-canonical downstream effectors in the context of autophagic signaling. Coincidentally MEK could bypass ERK (Fig. 2D) and interact with a downstream effector other than ERK in the context of autophagy stimuli (Figs. 4D and 5B), indicating that ERK is not the sole downstream effector of MEK and that MEK plays a more important role than ERK in regulating autophagy. Interestingly MEK regulates Golgi fragmentation in mitosis independently of ERK1/2 (58Colanzi A. Deerinck T.J. Ellisman M.H. Malhotra V. J. Cell Biol. 2000; 149: 331-339Crossref PubMed Scopus (85) Google Scholar, 59Colanzi A. Suetterlin C. Malhotra V. Curr. Opin. Cell Biol. 2003; 15: 462-467Crossref PubMed Scopus (103) Google Scholar), and the nucleation of autophagy by Beclin 1-PI3KC3 kinase complex is executed at the trans-Golgi network in mammalian cells (60Kihara A. Kabeya Y. Ohsumi Y. Yoshimori T. EMBO Rep. 2001; 2: 330-335Crossref PubMed Scopus (721) Google Scholar), both of which support the unusual role and the non-canonical module of MEK/ERK in regulating Beclin 1 and autophagy.Recent studies show that ERK up-regulates starvation-caused autophagy by down-regulating Akt/mTOR/S6K (27Shinojima N. Yokoyama T. Kondo Y. Kondo S. Autophagy. 2007; 3: 635-637Crossref PubMed Scopus (280) Google Scholar), but the molecular link between this pathway and the basis of autophagy machinery remains largely unclear. We observed here that activation of MEK/ERK coincided with up-regulation of Beclin 1 and autophagic responses, and MEK/ERK was activated by AMPK but not Raf1 in response to autophagy stimuli. AMPK has been proposed as a physiological cellular energy sensor because AMP is the most sensitive indicator for a cellular energy status. The cellular ATP concentration is much higher than that of AMP, and an insignificant decrease in ATP levels can result in an extraordinary increase in AMP levels that are sensed by and stimulate AMPK (24Fimia G.M. Stoykova A. Romagnoli A. Giunta L. Di Bartolomeo S. Nardacci R. Corazzari M. Fuoco C. Ucar A. Schwartz P. Gruss P. Piacentini M. Chowdhury K. Cecconi F. Nature. 2007; 447: 1121-1125Crossref PubMed Scopus (773) Google Scholar). MEK/ERK activation by AMPK thus confers the sensitivity and accuracy of MEK/ERK in regulating autophagic activity. TSC is known to be regulated by cellular energy levels. Activation of AMPK by energy starvation results in direct phosphorylation of TSC (49Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2365) Google Scholar). Our study showed that AMPK activation of TSC depends on MEK/ERK. Furthermore, activated MEK interacted with TSC, and the physical interaction between activated MEK and TSC required the ERK D docking site sequence motif contained in both MEK and TSC, consistent with the notion that stable binding of substrates to mitogen-activated protein kinase (MAPK) kinase involves recognition of a docking site on the substrate (2Longo V.D. Finch C.E. Science. 2003; 299: 1342-1346Crossref PubMed Scopus (489) Google Scholar). This non-canonical MEK/ERK signaling pathway functions not only in human and rat cell lines but also in neonatal mice, indicating a physiological significance. Our findings thus position this unusual MEK/ERK module downstream of AMPK and upstream of TSC in response to autophagy stimuli.Our present study showed that mTOR regulates autophagy caused both by starvation and non-starvation stimuli that activate MEK/ERK, consistent with the report that AMPK regulates autophagy through mTOR also in non-starved conditions (61Høyer-Hansen M. Jäättelä M. Autophagy. 2007; 3: 381-383Crossref PubMed Scopus (202) Google Scholar), suggesting a possible universal mechanism in the regulation of autophagy through mTOR. Because constitutive expression of Beclin 1 is due to the basal activity of MEK/ERK and enhanced Beclin 1 is attributed to the enhanced MEK/ERK activation in both human and rat cells, we postulate that the dependence of Beclin 1 on MEK/ERK signals may also be a universal mechanism in regulating autophagy.Beclin 1 Plays a Central Role in the Mechanisms Determining a Protective or Destructive AutophagyOur previous study shows that autophagic activity depends on the Beclin 1 level (41Wang J. Autophagy. 2008; 4: 947-948Crossref PubMed Scopus (123) Google Scholar). The present study found that the non-canonical MEK/ERK module regulates mutually antagonistic processes by regulating Beclin 1 levels. Basal activity of MEK/ERK protected mTORC1 and mTORC2 from being disassembled, thus conferring the basal mTOR activity and basal Beclin 1 level, which are incapable of triggering autophagy. MEK/ERK activation by AMPK up-regulated Beclin 1 through down-regulation of mTOR activity. Transient or moderate activation of MEK/ERK caused inhibition of mTORC1 or mTORC2, resulting in a moderate Beclin 1 increase and an ultimate cytoprotective autophagy, but sustained or strong MEK/ERK activation completely inhibited both mTORC1 and mTORC2, resulting in a strongly pronounced Beclin 1 and an ultimate cytodestructive autophagy. The requirement of an enhanced Beclin 1 level by MEK/ERK activation for autophagy thus appears to represent interesting threshold effects. The degree of enhanced Beclin 1 level may determine the level of autophagic activity, and the degree of autophagy elicited may play a role in cell fate decisions with low levels serving a homeostatic role and high levels promoting cell death. Our findings thus establish the molecular connection between MEK/ERK and mTOR in the regulation of mammalian autophagy-essential proteins and the opposing consequences of autophagy.Mammalian cells undergo autophagy resulting from exposure to nutritional, chemical, or physical autophagy stimuli or extreme physiological conditions such as birth. Despite the ubiquitous and constitutive expression of Beclin 1 in mammalian cells, our data demonstrate that autophagy is not evident until Beclin 1 is elevated, and autophagic activity is not detectable at a constitutive Beclin 1 level under normal growth conditions in cancer cells. In the brain, autophagic activity is very low even under starvation conditions (20Mizushima N. Yamamoto A. Matsui M. Yoshimori T. Ohsumi Y. Mol. Biol. Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1892) Google Scholar). These data suggest that either there is no constitutive autophagy or autophagy can only occur at extremely low levels under normal growth conditions when Beclin 1 is maintained at a basal level. Therefore, we doubt that autophagy plays a housekeeping role in all types of mammalian cells. Constitutive or basal Beclin 1 may be implicated in other cellular processes. Our recent studies show that constitutive Beclin 1 inhibits activation of apoptosis and confers differentiation capability in leukemia HL-60 cells (37Wang J. Lian H. Zhao Y. Kauss M.A. Spindel S. J. Biol. Chem. 2008; 283: 25596-25605Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 41Wang J. Autophagy. 2008; 4: 947-948Crossref PubMed Scopus (123) Google Scholar). Because autophagy can be regulated by several proteins capable of binding Beclin 1, including Bcl2, UV radiation resistance-associated gene tumor suppressor, and Ambra 1 (22Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar, 24Fimia G.M. Stoykova A. Romagnoli A. Giunta L. Di Bartolomeo S. Nardacci R. Corazzari M. Fuoco C. Ucar A. Schwartz P. Gruss P. Piacentini M. Chowdhury K. Cecconi F. Nature. 2007; 447: 1121-1125Crossref PubMed Scopus (773) Google Scholar, 25Liang C. Feng P. Ku B. Dotan I. Canaani D. Oh B.H. Jung J.U. Nat. Cell Biol. 2006; 8: 688-699Crossref PubMed Scopus (828) Google Scholar), Beclin 1 may play a central role in integrating signals by various stimuli.Studies on Beclin 1 demonstrate its increasing importance in the mammalian system because mammalian species fail to survive in the absence of Beclin 1, develop cancers with low Beclin 1 levels, suppress tumorigenesis with normal Beclin 1 levels, secure a protective autophagy at moderately enhanced Beclin 1 levels, and suffer from a destructive autophagy when too much Beclin 1 is induced. Thus, our present study adds new understanding of Beclin 1 in the regulation of autophagy. Autophagy is an evolutionally conserved machinery involving the degradation and turnover of cytoplasmic material in lysosomes. Autophagy plays a role in cellular homeostasis (1Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3140) Google Scholar), antiaging (2Longo V.D. Finch C.E. Science. 2003; 299: 1342-1346Crossref PubMed Scopus (489) Google Scholar, 3Meléndez A. Tallóczy Z. Seaman M. Eskelinen E.L. Hall D.H. Levine B. Science. 2003; 301: 1387-1391Crossref PubMed Scopus (1009) Google Scholar, 4Vellai T. Takacs-Vellai K. Zhang Y. Kovacs A. Orosz L. Müller F. Nature. 2003; 426: 620Crossref PubMed Scopus (814) Google Scholar), development (1Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3140) Google Scholar, 5Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2362) Google Scholar), protection of the genome (6Karantza-Wadsworth V. Patel S. Kravchuk O. Chen G. Mathew R. Jin S. White E. Genes Dev. 2007; 21: 1621-1635Crossref PubMed Scopus (670) Google Scholar), and regulation of cell size (7Vellai T. Bicsák B. Tóth M.L. Takács-Vellai K. Kovács A.L. Autophagy. 2008; 4: 507-509Crossref PubMed Scopus (33) Google Scholar). Autophagy may act as a means of defense against bacterium and virus invasion and be linked to various diseases including cancer (8Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. Cattoretti G. Levine B. J. Clin. Investig. 2003; 112: 1809-1820Crossref PubMed Scopus (1837) Google Scholar, 9Shintani T. Klionsky D.J. Science. 2004; 306: 990-995Crossref PubMed Scopus (2134) Google Scholar, 10Yue Z. Jin S. Yang C. Levine A.J. Heintz N. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 15077-15082Crossref PubMed Scopus (1746) Google Scholar), cardiomyopathy (11Tanaka Y. Guhde G. Suter A. Eskelinen E.L. Hartmann D. Lüllmann-Rauch R. Janssen P.M. Blanz J. von Figura K. Saftig P. Nature. 2000; 406: 902-906Crossref PubMed Scopus (716) Google Scholar), and neurodegenerative disorders (12Yuan J. Lipinski M. Degterev A. Neuron. 2003; 40: 401-413Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Autophagy starts with the formation of an autophagosome, enclosed within a double membrane that engulfs part of the cytoplasm. During periods of autophagy stimuli, cells respond to either maintain the metabolism essential for survival or execute cell death. Autophagy-essential proteins (Atg) 2The abbreviations used are: Atgautophagy-essential proteinAMPKAMP-activated protein kinaseTSCtuberous sclerosis complexmTORmammalian target of rapamycinLC3light chain 3Bcl2B-cell lymphoma-2PI3Kphosphatidylinositol 3-kinasePI3KC3class III phosphatidylinositol 3-kinaseAmbra 1activating molecule in Beclin 1-regulated autophagy protein 1MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseERKextracellular signal-regulated kinasemTORCmTOR complexGΒLG-protein Β-subunit-likeAICAR5-aminoimidazole-4-carboxamide ribosideGFPgreen fluorescent proteinsiRNAshort interfering RNAGSTglutathione S-transferase. 2The abbreviations used are: Atgautophagy-essential proteinAMPKAMP-activated protein kinaseTSCtuberous sclerosis complexmTORmammalian target of rapamycinLC3light chain 3Bcl2B-cell lymphoma-2PI3Kphosphatidylinositol 3-kinasePI3KC3class III phosphatidylinositol 3-kinaseAmbra 1activating molecule in Beclin 1-regulated autophagy protein 1MEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseERKextracellular signal-regulated kinasemTORCmTOR complexGΒLG-protein Β-subunit-likeAICAR5-aminoimidazole-4-carboxamide ribosideGFPgreen fluorescent proteinsiRNAshort interfering RNAGSTglutathione S-transferase. are the molecular basis of autophagy machinery. About 30 Atg proteins in yeast and 10 in mammals have been identified. In yeast, the protein kinase target of rapamycin (TOR) mediates autophagy via Atg1-Atg13 kinase complex. Atg1 interacts with multiple components of the autophagic machinery through direct association, phosphorylation, and/or intracellular localization (13Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (898) Google Scholar, 14Reggiori F. Tucker K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: 79-90Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). autophagy-essential protein AMP-activated protein kinase tuberous sclerosis complex mammalian target of rapamycin light chain 3 B-cell lymphoma-2 phosphatidylinositol 3-kinase class III phosphatidylinositol 3-kinase activating molecu
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