AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation

Article

AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation Graphical Abstract

Authors Chunmei Chang, Hua Su, Danhong Zhang, ..., Han-Ming Shen, Jennifer Lippincott-Schwartz, Wei Liu

Correspondence [email protected]

In Brief The histone deacetylase Sirt1 is essential to starvation-induced autophagy, but it is currently unclear how Sirt1 is rapidly activated in the process. Chang et al. report that an AMPK-dependent phosphorylation and nuclear translocation of GAPDH supply a fast track for Sirt1 activation and subsequent autophagy initiation upon glucose withdraw.

Highlights d

GAPDH is essential for glucose starvation stimulated Sirt1 activation and autophagy

d

Phosphorylation at Ser122 by AMPK mediates GAPDH nuclear entry

d

GAPDH directly interacts with Sirt1 in the nucleus

d

Interaction with GAPDH displaces Sirt1 from DBC1

Chang et al., 2015, Molecular Cell 60, 1–11 December 17, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2015.10.037

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Molecular Cell

Article AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation Chunmei Chang,1 Hua Su,1 Danhong Zhang,1 Yusha Wang,1 Qiuhong Shen,1 Bo Liu,1 Rui Huang,1 Tianhua Zhou,1 Chao Peng,3 Catherine C.L. Wong,3 Han-Ming Shen,4 Jennifer Lippincott-Schwartz,5 and Wei Liu1,2,* 1Department of Biochemistry and Molecular Biology, Program in Molecular and Cell Biology, Zhejiang University School of Medicine, Hangzhou 310058, China 2Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China 3National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China 4Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore 5Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.10.037

SUMMARY

Eukaryotes initiate autophagy to cope with the lack of external nutrients, which requires the activation of the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase Sirtuin 1 (Sirt1). However, the mechanisms underlying the starvation-induced Sirt1 activation for autophagy initiation remain unclear. Here, we demonstrate that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a conventional glycolytic enzyme, is a critical mediator of AMP-activated protein kinase (AMPK)-driven Sirt1 activation. Under glucose starvation, but not amino acid starvation, cytoplasmic GAPDH is phosphorylated on Ser122 by activated AMPK. This causes GAPDH to redistribute into the nucleus. Inside the nucleus, GAPDH interacts directly with Sirt1, displacing Sirt1’s repressor and causing Sirt1 to become activated. Preventing this shift of GAPDH abolishes Sirt1 activation and autophagy, while enhancing it, through overexpression of nuclear-localized GAPDH, increases Sirt1 activation and autophagy. GAPDH is thus a pivotal and central regulator of autophagy under glucose deficiency, undergoing AMPK-dependent phosphorylation and nuclear translocation to activate Sirt1 deacetylase activity. INTRODUCTION Cells sense and adapt to changes in the environment through the initiation and execution of necessary and special processes. Autophagy is such a process conserved by eukaryotic cells to face poor conditions such as nutrient deficiency, although its original function was defined as the lysosomal digestion of intra-

cellular protein aggregates and damaged organelles (Klionsky, 2007; Mizushima and Klionsky, 2007). Despite the identification of numerous autophagy-related genes (Atgs), most of which contribute to the formation of autophagosomes, the signaling events for the initiation of autophagy remain indefinite. In addition to the verification of several protein complexes for phagophore generation and vesicle nucleation on membranes (Mizushima et al., 2011), and the ubiquitin-like conjugation system required for certain Atg proteins to execute their functions (Geng and Klionsky, 2008), emerging evidence suggests that the acetylation-deacetylation of a number of Atg proteins by specific acetyltransferases and deacetylases is essential for their activity in autophagy (Lee et al., 2008; Lee and Finkel, 2009; Lin et al., 2012; Yi et al., 2012). One such deacetylase is Sirtuin 1 (Sirt1), a highly conserved nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase involved in the regulation of numerous physiological processes including cell metabolism, survival, and differentiation, by deacetylating histone and non-histone proteins (Finkel et al., 2009). Sirt1 is activated during cell starvation and forms complexes with Atg5, Atg7, and Atg8 (LC3 in mammals) leading to the deacetylation of these key autophagy components (Lee et al., 2008). In addition, deacetylation of LC3 by Sirt1 in the nucleus has recently been identified as essential for the redistribution of LC3 to the cytoplasm and conjugation to autophagic membranes (Huang et al., 2015). These data explain previous findings showing that intracellular Sirt1 activity correlates well with the autophagy level in different cell systems (Hariharan et al., 2010; Jeong et al., 2013; Kume et al., 2010; Powell et al., 2011), and Sirt1 deficiency arrests while Sirt1 overexpression stimulates the formation of autophagosomes (Lee et al., 2008), suggesting a pivotal role of Sirt1 in autophagy triggered by energy-stress. The enzymatic activity of Sirt1 depends on NAD+, and it is principally activated by an elevated NAD+/NADH ratio (Revollo et al., 2004). Nevertheless, in addition to the post-translational modification of Sirt1 itself by environmental stimuli (Gerhart-Hines et al., 2011; Nasrin et al., 2009; Yang et al., 2007), Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc. 1

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

involved in multiple cellular processes based mainly on its subcellular localization (Tristan et al., 2011). Shift of GAPDH into the nucleus upon exposure of cell to environmental stressors has revealed its functions in DNA repair, RNA export, and cell death (Azam et al., 2008; Chuang and Ishitani, 1996; Nagy et al., 2000). Recently, it has been reported that GAPDH competes with mammalian target of rapamycin (mTOR) for binding to the mTOR activator Rheb and prevents mTOR activation under low-glucose conditions (Lee et al., 2009). Overexpression of wild-type but not nuclear localization signal (NLS)-deleted GAPDH may protect cells from apoptosis through promoting Atg12 expression (Colell et al., 2007). These findings suggest a potential role of GAPDH in autophagy that may involve a change in its localization in cell. In this study, we have investigated the dynamic subcellular localizations of GAPDH and analyzed its relationship with autophagy driven by nutrient depletion. By identifying GAPDH as a phosphorylation substrate for AMPK and the interaction between GAPDH and Sirt1 in the nucleus, we have revealed that AMPK-dependent phosphorylation and the nuclear translocation of GAPDH mediate rapid Sirt1 activation and autophagy initiation under glucose deprivation. RESULTS

Figure 1. Glucose Starvation Results in the Nuclear Translocation of GAPDH (A) Confocal images of the intracellular distribution of GAPDH and LC3 puncta formation in MEFs. MEFs were starved and then fed with nutrient-rich medium for 4 hr, followed by staining with anti-GAPDH and anti-LC3 antibodies. (B) MEFs after incubation in glucose-free or amino-acid-free medium were stained with anti-GAPDH and anti-LC3. (C and D) Western blots of subcellular fractions from MEFs treated as in (A) and (B). Lamin B and b-tubulin were used as markers for the nuclear and cytoplasmic fractions. NRM: nutrient-rich medium. Scale bars, 10 mm.

recent findings have suggested that a more immediate and dynamic form of Sirt1 regulation may be adopted by cells under metabolic stress when changes in NAD+ levels are nominal or absent (Gerhart-Hines et al., 2011; Nin et al., 2012). This rapid form of Sirt1 activation may involve the activation of AMP-activated protein kinase (AMPK) and Sirt1-interacting proteins localized in the nucleus (Kim et al., 2007; Kim et al., 2008; Nin et al., 2012; Zhao et al., 2008). However, in energy-stress-triggered autophagy, the potential messenger molecule that directs the AMPK signal to nuclear Sirt1 remains unknown. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved primarily cytoplasmic enzyme by which glyceraldehyde-3-phosphate is converted to D-glycerate 1,3-bisphospate for glycolysis. Besides its glycolytic activity, GAPDH is 2 Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc.

Glucose Starvation Results in the Nuclear Translocation of GAPDH To investigate the role of GAPDH in autophagy driven by nutrient deprivation, we began by observing the intracellular distribution of GAPDH during starvation in mouse embryonic fibroblasts (MEFs) using confocal microscopy. Compared to cells grown in nutrient medium, GAPDH in cells incubated in medium lacking both glucose and amino acids was dramatically redistributed from the cytoplasm to the nucleus, along with the formation of LC3-positive puncta (Figure 1A). Returning the starved cells to nutrient medium reversed the GAPDH redistribution, and the LC3-positive puncta disappeared (Figure 1A). Interestingly, when either glucose or amino acids was withdrawn from the medium, LC3 puncta formed, while only glucose starvation but not amino acid starvation resulted in the nuclear translocation of GAPDH (Figure 1B). This redistribution was confirmed by subcellular fractionation analysis (Figures 1C and 1D). These data suggested a correlation between the nuclear translocation of GAPDH and autophagosome formation during glucose deprivation and implied an involvement of AMPK activation rather than mTORC1 inhibition. AMPK Regulates the Nuclear Redistribution of GAPDH To verify the role of AMPK in the regulation of GAPDH redistribution, we first confirmed that it is indeed activated during glucose starvation by finding increased specific phosphorylation of AMPK (Figure 2A). We then showed that treatment with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR) also resulted in the redistribution of GAPDH to the nucleus and the formation of LC3 puncta (Figure 2B); also, the GAPDH redistribution and LC3 puncta triggered by glucose starvation were prevented by the AMPK inhibitor compound C (Figure 2C). Further, we found that, in AMPK-deleted cells, neither

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Figure 2. AMPK Regulates the Nuclear Redistribution of GAPDH (A) AMPK phosphorylation analyzed with antiAMPK and anti-phospho-AMPK antibodies. AMPK wild-type (WT) and a1/a2 double-knockout (DKO) MEFs were treated with AICAR or glucosestarved in the presence or absence of compound C (C.C). (B and C) Confocal images of MEFs treated with AICAR (B) or starved of glucose, in the presence of C.C (C). (D) AMPK DKO MEFs treated with AICAR or starved of glucose and stained with anti-GAPDH and anti-LC3. (E) MEFs treated with rapamycin and stained with anti-GAPDH and anti-LC3. (F) MEFs transiently expressing Rheb-GFP after glucose-starvation stained with anti-GAPDH. (G) Confocal images of the distribution of GAPDH and LC3-positive puncta in indicated TSC / MEFs starved of glucose. (H) Distribution of GAPDH in glucose-starved Atg7 / and Atg5 / MEFs. Scale bars, 10 mm. See also Figure S1.

glucose starvation nor AICAR treatment drove the movement of GAPDH and the formation of LC3 puncta (Figure 2D). Consistent with a previous report (Kwon et al., 2010), these data clearly showed that AMPK activation plays a pivotal role in the nuclear translocation of GAPDH. Activated AMPK can inhibit mTORC1 by directly phosphorylating the tumor suppressor TSC2 (tuberous sclerosis complex 2) and the critical mTORC1-binding subunit, raptor (Gwinn et al., 2008; Inoki et al., 2003). Although amino acid deprivation, which leads to the inhibition of mTORC1, showed no effect on GAPDH distribution (Figure 1B), to further exclude a role of mTOR inhibition in glucose-starvation-induced GAPDH nuclear translocation, we treated cells with the mTORC1 inhibitor rapamycin. Clearly, rapamycin stimulated the formation of LC3 puncta, but at the same time, GAPDH remained in the cytoplasm (Figure 2E). In addition, in cells in which mTORC1 was constitutively activated by overexpression of Rheb (Figure 2F) or with TSC deletion (Figure 2G), glucose starvation still caused the nuclear translocation of GAPDH, while LC3 puncta failed to form (Figure 2G). These results indicated the independence of nuclear translocation of GAPDH from mTORC1 inhibition. The nuclear translocation of GAPDH was further characterized in Atg5 / and Atg7 / MEFs that fail to form autophagosomes. Clearly, withdrawal of glucose caused redistribution of GAPDH from the cytoplasm to the nucleus in these cells (Figure 2H), suggesting that the movement of GAPDH is not dependent on the activity of Atg5 or Atg7. The nuclear translocation of GAPDH trig-

gered by AMPK activation is correlated with but not dependent on autophagosome formation. Recently, it has been reported that GAPDH shifts into the nucleus in nitric oxide (NO)-induced apoptosis, which involves S-nitrosylation of GAPDH and its association with Siah1 protein (Hara et al., 2005). To clarify whether the nuclear translocation during glucose starvation is mediated in the same way, we introduced mutations by changing Cys152 to Ser (GAPDHC152S) or Lys227 to Arg (GAPDHK227R) in GAPDH, which abolishes either the S-nitrosylation or the GAPDH-Siah1 interaction (Hara et al., 2005). We found that, when expressed in cells, GFP-GAPDHC152S and GFP-GAPDHC152S/K227R underwent nuclear translocation accompanied by increased intracellular LC3 puncta upon glucose starvation (Figure S1A). We also examined the redistribution of GAPDH by blocking intracellular NO production. N-(3-(aminomethyl)benzyl)acetamidine (1400W), an iNOS inhibitor, prevented lipopolysaccharide (LPS)-induced GAPDH nuclear translocation in RAW264.7 cells (Figure S1B). But suppression of NO production by 1400W had no effect on the nuclear accumulation of GAPDH resulting from glucose starvation (Figure S1B). Together, these results indicated that AMPK-activated GAPDH nuclear translocation does not rely on S-nitrosylation and its association with Siah1. Since the Cys152 site is also crucial to the function of GAPDH in glycolysis, these data suggested that glycolytic activity is not essential to the nuclear translocation of GAPDH. Nuclear Translocation of GAPDH Is Required for AMPK Activation-Evoked Autophagy To determine the function of the nuclear redistribution of GAPDH in autophagy triggered by AMPK activation, we first knocked down GAPDH and checked the autophagy level. Knockdown Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc. 3

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Figure 3. Nuclear Translocation of GAPDH Is Required for AMPK Activation-Evoked Autophagy (A) Confocal images of Cherry-LC3 puncta in HEK293 cells transfected with GAPDH shRNA for 72 hr, then starved of glucose or treated with AICAR. Scale bars, 10 mm. (B) Statistical analysis of the number of CherryLC3 puncta per cell in cells treated as in (A). Data are presented as mean ± SEM, n = 50. ***p < 0.001. (C) Western blots of LC3-PE formation in HEK293 cells transfected with GAPDH siRNA and starved of glucose or treated with AICAR in the presence of chloroquine (CQ). (D) Intracellular p62 levels in glucose-starved HEK293 cells transfected with GFP-GAPDH or GFP-NES-GAPDH after 48 hr culture with GAPDH siRNA. (E) TEM images of autophagic vacuoles in cells treated as in (D), except that CQ was added to the starvation medium. Arrows indicate formed autophagosomes and autolysosomes. Scale bars, 0.5 mm. (F) Statistical analysis of cytoplasmic occupancy of autophagic vacuoles in the cells shown in (E). Data are shown as mean ± SEM from 20 micrographs of duplicate experiments. ***p < 0.001. (G and H) LC3-PE and p62 levels in HEK293 cells expressing GAPDH-NLS-Myc incubated in nutrient medium or glucose-free medium with or without Baf A1. (I) Formation of LC3 puncta in MEFs transiently expressing GAPDH-NLS-Myc. Scale bars, 10 mm. (J and K) TEM images of autophagic vacuoles in GAPDH-NLS-Myc-expressing HEK293 cells. Statistical Data are shown as mean ± SEM from 20 micrographs of duplicate experiments. ***p < 0.001. Scale bars, 0.5 mm. See also Figure S2.

of GAPDH dramatically reduced the number of LC3 puncta and the generation of lipidated LC3 (LC3-PE) from LC3-I by either glucose starvation or AICAR treatment (Figures 3A–3C and S2A). The unchanged mRNA levels of LC3 excluded a possible influence of GAPDH knockdown on LC3 expression (Figure S2B). We then designed a knockdown and rescue experiment to further evaluate GAPDH and its nuclear translocation in AMPKinitiated autophagy. Clearly, knockdown of GAPDH inhibited the glucose-starvation-induced decrease of p62, an indicator of autophagic degradation, and prevented glucose-starvationinduced autophagosome/autolysosome formation as identified by electron microscopy (Figures 3D–3F and S2D). Wild-type GAPDH, but not NES-GAPDH, a mutant with extra nuclear export signals (NESs) in its N-terminal that trap the protein in the cytoplasm (Figure S2C), restored the effect of GAPDH knockdown (Figures 3D–3F and S2D). 4 Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc.

Further, we created a GAPDH mutant by adding a nuclear localization signal at the C-terminal (GAPDH-NLS), and this mutant predominantly localized in the nucleus (Figure 3I). We found that under fed conditions, expressing only GAPDHNLS resulted in an increase in LC3-PE (Figures 3G and S2E), degradation of p62 (Figures 3H and S2F), and the formation of autophagosomes (Figures 3I–3K), indicating an induction of autophagy. Taken together, these data suggested a critical role of GAPDH nuclear translocation in glucose-deprivation-induced autophagy, and once in the nucleus, GAPDH can trigger cell autophagy. Phosphorylation at Ser122 by AMPK Mediates GAPDH Nuclear Translocation In searching for the molecular mechanism by which AMPK regulates the redistribution of GAPDH for autophagy initiation, a simple proposal is that AMPK directly interacts with and phosphorylates GAPDH, since GAPDH contains two serine residues (Ser122 and Ser125) that match the predicted AMPK consensus

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Figure 4. Phosphorylation at Ser122 by AMPK Mediates GAPDH Nuclear Translocation Immunoprecipitation and western blot were carried out with antibodies against the indicated proteins. (A) GAPDH immunoprecipitated from MEFs treated as indicated were analyzed for phosphorylation. (B) Phosphorylation of GAPDH in AMPK WT or DKO MEFs with or without glucose starvation. (C) Co-immunoprecipitation of pAMPK or AMPK with GAPDH in MEFs treated as indicated. (D) In vitro kinase assays carried out with purified active AMPK, GST-GAPDH, and [g-32P]-ATP. Upper panel, autoradiography; lower panel, Coomassie blue staining. (E) Phosphorylation of Flag-tagged GAPDH or GAPDH mutants in MEFs with or without glucose starvation. (F) Phosphorylation of GAPDH-Flag or GAPDHS122A-Flag immunopurified from HEK293 cells and incubated with active AMPK. (G) Distribution of GAPDHS122D-Flag in MEFs or GAPDHS122A-Flag in glucose-starved MEFs. Scale bars, 10 mm. (H and I) Flag-tagged GAPDH or each of the GAPDH mutants was transfected into HEK293 cells after 48 hr incubation with GAPDH siRNA. After glucose starvation, the LC3-PE level (H) and the autophagic vacuoles (I) were analyzed by western blot and TEM. Arrows indicate formed autophagosomes and autolysosomes. Scale bars, 0.5 mm. Statistical data are shown as mean ± SEM from 20 micrographs of duplicate experiments. ***p < 0.001. See also Figure S3.

motif (Banko et al., 2011; Kim et al., 2013). After stimulating the cells with AICAR and glucose starvation, GAPDH was immunoprecipitated and checked for phosphorylation using a specific anti-phospho-serine/threonine antibody. As predicted, AICAR and glucose starvation resulted in a dramatic phosphorylation of GAPDH at serine/threonine but not tyrosine (Figure 4A), and this was abolished by either the AMPK inhibitor compound C (Figure 4A) or knockout of AMPK (Figure 4B). Co-immunoprecipitation experiments also detected an association of GAPDH and activated AMPK following AICAR treatment or glucose starvation (Figure 4C). Phosphorylation of GAPDH by AMPK was confirmed by an in vitro kinase assay using purified AMPK and recombinant GST-GAPDH, and the results indicated that GAPDH is a direct substrate of AMPK (Figure 4D). To identify the site(s) on GAPDH phosphorylated by AMPK, phosphorylated GAPDH was analyzed by mass spectrometry. The results demonstrated a phosphorylation site at Ser122 located in the predicted phosphorylation motif for AMPK (Figure S3A). GAPDH mutants were then constructed in which the Ser122 and Ser125 residues were changed to alanine via sitedirected mutagenesis. Flag-tagged GAPDH-SA mutants were transfected into HEK293 cells and assessed for phosphorylation. This revealed that serine-to-alanine replacement at Ser122 but not Ser125 abolished GAPDH phosphorylation upon glucose starvation (Figure 4E). Culture of the GAPDHS122A mutant

with AMPK also confirmed that Ser122 of GAPDH is the correct and may be the only phosphorylation site for AMPK (Figure 4F). We then investigated whether the phosphorylation of GAPDH at Ser122 is essential for its AMPK-evoked nuclear translocation and autophagy initiation. When expressed in cells, upon glucose starvation the GAPDHS122A mutant failed to move into the nucleus (Figures 4G and S3B), while the GAPDHS122D mutant in which the serine at Ser122 was replaced by aspartic acid mimicking phosphorylated GAPDH was localized predominantly in the nucleus even under fed conditions (Figures 4G and S3B). Consistently, in glucose-starved cells, GAPDHS122D, but not GAPDHS122A, restored the LC3-PE generation (Figures 4H and S3C) and autophagosome formation (Figure 4I) abrogated by GAPDH RNAi. Like that of GAPDH-NLS, GAPDHS122D expression was sufficient to stimulate LC3 puncta under fed conditions (Figure S3D). Together, these data suggested that phosphorylation of GAPDH at Ser122 by AMPK mediates the nuclear translocation and autophagy-initiation of GAPDH. GAPDH Activates Sirt1 in the Nucleus To investigate the function of GAPDH in the nucleus and identify its target for autophagy initiation, we first measured the expression of several autophagy-associated genes based on the reported function of GAPDH in gene transcription (Zheng et al., Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc. 5

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Figure 5. GAPDH Activates Sirt1 in the Nucleus Immunoprecipitation and western blot were carried out with antibodies against the indicated proteins. (A) Co-immunoprecipitation of Sirt1 with GAPDH from MEFs treated as indicated. (B) Co-immunoprecipitation of Sirt1 with GAPDH from glucose-starved AMPK WT and AMPK DKO MEFs. (C) Co-immunoprecipitation of Sirt1 with transiently expressed GAPDH-Myc, GAPDH-NLS-Myc, or GAPDHC152S-NLS-Myc from transfected MEFs. (D) MEF cell lysates were incubated with purified GST-GAPDH, and bound Sirt1 was detected by western blot. (E) Co-immunoprecipitation of Sirt1 with Myctagged GAPDH or GAPDH mutants from transfected MEFs. (F) Time course of glucose-starvation-stimulated activation of endogenous Sirt1 in MEFs, measured by using fluorophore-conjugated acetylated p53 peptide as a substrate. (G) Sirt1 activity in HEK293 cells treated as indicated. (H) Acetylation of GFP-LC3 in GFP-LC3-expressing HEK293 cells with AICAR treatment or glucose starvation. (I) GFP-LC3 acetylation in GFP-LC3-expressing HEK293 cells with or without GAPDH knockdown. The cells were starved of glucose or treated with AICAR. (J) Acetylation of GFP-LC3 in GFP-LC3-stable cells transfected with GAPDH-NLS-Myc and treated with TSA or EX-527. See also Figures S4 and S5 and Tables S1 and S2.

2003). None of the stimuli that caused the nuclear translocation of GAPDH led to detectable changes in the expression of either Atg12 (Figures S4A and S4B), which is regulated by GAPDH overexpression (Colell et al., 2007), or Beclin-1, which increases in autophagy stimulated by stress (Hariharan et al., 2011; Sun et al., 2013), or p62, which was decreased during GAPDH nuclear translocation (Figures 3H and S4A). We then employed a proteomic approach to identify nuclear interacting partners for GAPDH. The mass spectrometry results showed that the NAD+-dependent deacetylase Sirt1, a nuclear protein essential and sufficient for starvation-induced autophagy (Huang et al., 2015; Lee et al., 2008), was potentially one of the new binding proteins of GAPDH upon glucose deprivation (Table S1), and a particular robust partner of GAPDH-NLS under fed conditions (Table S2). To verify the potential Sirt1-GAPDH interaction, we performed co-immunoprecipitation experiments in cells. We found that glucose deprivation or AICAR treatment co-immunoprecipitated Sirt1 with GAPDH; this was inhibited by compound C and abolished by AMPK deletion (Figures 5A and 5B). Coimmunoprecipitation between Sirt1 and nuclear GAPDH-NLS was also detected in fed cells (Figure 5C). In addition, purified GAPDH pulled down endogenous Sirt1 from cell lysates (Figure 5D). Furthermore, in fed cells, stronger co-immunoprecipitation was detected between Sirt1 and GAPDHS122D, the nuclear-localized phosphorylated form of GAPDH, than that between Sirt1 and GAPDHS122A, the un-phosphorylated form 6 Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc.

localized only in the cytoplasm (Figure 5E). Nevertheless, Sirt1 was also co-immunoprecipitated with GAPDHS122A-NLS, the un-phosphorylated form localized in the nucleus (Figure 5E), indicating that phosphorylation of GAPDH at Ser122 is not indispensable for its interaction with Sirt1 in the nucleus. Together, these results suggested that once the phosphorylated GAPDH moves into the nucleus, it interacts directly with nuclear Sirt1. The effect of GAPDH association on Sirt1 activity was then investigated. With fluorescence-labeled acetylated p53 peptide as a substrate, we determined the cellular activity of Sirt1 as a deacetylase by in vitro assay. As expected, glucose starvation or GAPDH-NLS expression dramatically raised Sirt1 activity, and GAPDH RNAi or compound C clearly suppressed the activation (Figures 5F and 5G). Sirt1 activity was also assessed by examining the acetylation of intracellular LC3, a known substrate for Sirt1 (Huang et al., 2015; Lee et al., 2008). AICAR treatment or glucose starvation resulted in a decrease in LC3 acetylation (Figure 5H), while knockdown of GAPDH prevented the deacetylation of LC3 (Figure 5I). GAPDH-NLS expression-induced LC3 deacetylation was impaired by the specific Sirt1 inhibitor EX-527, but not by trichostatin A, an inhibitor of class I/II histone deacetylases (Figure 5J), indicating a specific activation of Sirt1. EX-527 treatment or knockdown of Sirt1 but not Sirt6 or Sirt7, two other nucleus-localized sirtuins, dramatically reduced LC3 puncta and LC3-PE production by either glucose starvation or GAPDH-NLS expression (Figures S5A–S5D). By comparison,

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Figure 6. Interaction with GAPDH Disassociates Sirt1 from Its Inhibitor DBC1 (A) NAD+ levels in MEFs treated as indicated. (B) Co-immunoprecipitation of DBC1 or GAPDH with Sirt1 from cells treated as indicated. The graph shows the quantification of three independent experiments. ***p < 0.001. (C and D) Co-immunoprecipitation of DBC1 with Sirt1 from HEK293 cells transiently expressing GAPDH-NLS-Myc or GAPDHC152S-NLS-Myc (C) or GAPDHS122D-Flag (D). (E) Sirt1-Flag immunoprecipitated by anti-Flag from transfected HEK293 cells and eluted by Flag peptide was cultured with purified GST-GAPDH. Then the Sirt1-Flag was immunoprecipitated by anti-Sirt1, and the associated DBC1 and GSTGAPDH were analyzed by western blot. (F) Activity of Sirt1-Flag after incubation with purified GAPDH as in (E) measured using fluorophore-conjugated acetylated p53 as a substrate. (G) GFP-LC3 puncta in HEK293 cells stably expressing GFP-LC3 and cultured with DBC1 siRNA for 72 hr. Scale bar, 10 mm. (H) MEFs cultured with GAPDH siRNA for 48 hr were transfected with wild-type GAPDH (WT), the GAPDH mutants, or Sirt1-Flag. Then the cells were starved of glucose for 24 hr, and cell viability was determined by FACS analysis using AnexinV and propidium iodide double staining. Results are reported as means ± SEM of three replicates. ***p < 0.001. See also Figure S6 and Table S3.

EX-527 showed scarcely any effect on the export of nuclear LC3, LC3 puncta formation and LC3 deacetylation triggered by amino acid starvation or rapamycin treatment (Figures S5E and S5F). These results verified a requirement of Sirt1 for the autophagic induction by nuclear GAPDH. Interaction with GAPDH Disassociates Sirt1 from Its Inhibitor DBC1 We then investigated the potential mechanism by which Sirt1 is activated by GAPDH. First, the intracellular NAD+ level was measured. We found that each of the stimuli that induce rapid activation of AMPK, GAPDH nuclear translocation, and LC3 puncta failed to raise the NAD+ level, while the cellular NAD+ concentration responded normally to the nicotinamide phosphoribosyltransferase inhibitor FK866 (Hasmann and Schemainda, 2003) or to long-term AICAR treatment (Canto´ et al., 2009) (Figure 6A). We therefore performed mass spectrometry to characterize the potential binding partners of Sirt1 before and after glucose deprivation (Table S3). Upon glucose starvation, the amount of GAPDH in the Sirt1 interactome increased as expected. Intriguingly, the amount of included DBC1 (deleted in breast cancer 1), a negative regulator of Sirt1 that stably complexes with Sirt1 and inhibits Sirt1 activity (Kim et al., 2008;

Zhao et al., 2008), was decreased by glucose starvation (Figure S6; Table S3), suggesting a disassociation of DBC1 from Sirt1. Consistently, we found that while activation of AMPK by AICAR or glucose depletion increased the co-immunoprecipitation of GAPDH with endogenous Sirt1, it evidently reduced the co-immunoprecipitation of DBC1 with Sirt1 (Figure 6B). We then checked the potential role of GAPDH in mediating the separation of Sirt1 from DBC1. Clearly, expression of the nuclear-localized GAPDH-NLS impaired the Sirt1-DBC1 interaction (Figure 6C). This effect of GAPDH was independent of its glycolytic activity and was augmented by its phosphorylation, because the disassociation of Sirt1 from DBC1 also occurred with the expression of GAPDHC152S-NLS (Figure 6C) and was further enhanced by GAPDHS122D (Figure 6D). We further performed an in vitro experiment to test this effect of GAPDH. Sirt1-Flag was immunoprecipitated from overexpressed HEK293 cells; after competitively eluting the beads from precipitates with substantial Flag peptide, purified GST-GAPDH was added and cultured. Then the Sirt1-Flag in the solution was further immunoprecipitated by Sirt1 antibody, and the associated DBC1 and GST-GAPDH were analyzed by western blot. Intriguingly, we found that the more GAPDH associated with Sirt1 by adding more GSTGAPDH, the more DBC1 disassociated from Sirt1 (Figure 6E). The activity of the precipitated Sirt1 was also checked using acetylated p53 peptide as a substrate and, as predicted, Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc. 7

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

Figure 7. Schematic Model for GAPDH-Mediated AMPK-Dependent Sirt1 Activation and Autophagy Initiation In response to glucose deprivation, activated AMPK phosphorylates GAPDH at Ser122 in the cytoplasm. The phosphorylated GAPDH moves into the nucleus and directly interacts with and activates Sirt1 by disassociating Sirt1 from its repressor DBC1; autophagy is initiated therefrom.

addition of GST-GAPDH dose-dependently elevated Sirt1 activity in vitro in the presence of NAD+ (Figure 6F). Furthermore, in HEK293 cells stably expressing GFP-LC3, knockdown of DBC1 induced the formation of LC3 puncta (Figure 6G). Together, these data suggested that interaction with GAPDH activates Sirt1 by dissociating Sirt1 from DBC1. Finally, to testify that the revealed AMPK-GAPDH-Sirt1 pathway is of importance for the physiological response of cell to glucose withdrawal, we checked cell viability under glucose starvation. We found that knockdown of GAPDH significantly aggravated the apoptotic cell death led by glucose starvation, while re-expression of the wild-type GAPDH, or overexpression of GAPDHS122D or Sirt1, but not GAPDHS122A, rescued the cell death by GAPDH knockdown (Figure 6H). These data suggested that AMPK activation-triggered GAPDH phosphorylation and subsequent Sirt1 activation and autophagy initiation play a crucial role in protecting cells from apoptosis induced by glucose withdrawal. DISCUSSION Autophagy can be induced by multiple stresses via different signaling mechanisms. The accomplishment of autophagosome formation for nonselective autophagy requires, on the one hand, the generation of the phagophore on membranes through the activation of class III phosphatidylinositol-3 kinase and, on the other hand, the activation of essential Atg proteins for the growth of isolation membranes, which involves the activation of Sirt1. Here, our findings revealed a previously unappreciated pathway for the activation of Sirt1 in response to cellular energy depletion. 8 Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc.

By serving as a downstream messenger of the energy sensor AMPK, GAPDH transfers the signal from cytoplasmic AMPK to nuclear Sirt1, allowing the activation of this pivotal deacetylase for the initiation of autophagy (Figure 7). Identification of GAPDH as a direct phosphorylation substrate for AMPK unveiled a previously unknown mechanism for AMPKtriggered Sirt1 activation and its role in the adaptation of cells to reduced nutrient availability. Activation of AMPK maintains the cellular energy store by switching on catabolic pathways and switching off anabolic pathways. Recent studies have indicated a pivotal role of Sirt1 in mediating the effect of AMPK. While activation of Sirt1 suppresses energy-demanding processes like cell differentiation (Fulco et al., 2008), Sirt1-mediated activation of PGC-1a and FOXOs enhances mitochondrial biogenesis (Canto´ et al., 2009). In both cases, AMPK activates Sirt1 by modulating the NAD+ level, suggesting a role of NAD+ metabolism in Sirt1 activation for transcriptional reprogramming. However, in fact, activation of AMPK maintains the energy store first by initiating post-translational events that involve key proteins that rapidly manipulate the metabolism of lipid, protein, and glucose (Hardie, 2007). The AMPK-GAPDH-Sirt1 pathway described here suggests that Sirt1 also participates in AMPK-evoked rapid posttranslational processes in which Sirt1 activation by AMPK is directly mediated by GAPDH and is independent of NAD+ elevation. In coordination with the AMPK-mTOR-ULK1 pathway, GAPDH-mediated Sirt1 activation ensures the rapid initiation of autophagy, supplying an alternative means by which AMPK enhances acute catabolic metabolism. Like other post-translational processes, this rapid response may quickly produce ATP at the onset of energy deficiency before the transcriptional reprogramming of the cell is activated. DBC1 is a native inhibitor of Sirt1 by interacting with the catalytic domain of Sirt1 (Kim et al., 2008; Zhao et al., 2008). Nevertheless, little is known about the regulation of this interaction. Recently, a study has shown that the pharmacological activation of protein kinase A and AMPK leading to Sirt1 activation is attributable to an inhibited DBC1-Sirt1 interaction (Nin et al., 2012). The GAPDH binding to Sirt1 and disassociation of Sirt1 from DBC1 suggested that the DBC1-Sirt1 interaction is a target for physiological regulation and defined GAPDH as a direct regulator. It is unlikely that disassociation of Sirt1 from DBC1 is due to an interaction between GAPDH and DBC1, since they did not co-immunoprecipitate in glucose-deprived or AICAR-treated cells (data not shown). In addition, binding of DBC1 to the catalytic domain of Sirt1 also rules out a mechanism by which a competitive association of GAPDH and DBC1 with Sirt1 could contribute GAPDH-mediated Sirt1 activation. Therefore, a reasonable interpretation is that association of GAPDH with the non-catalytic domain of Sirt1 causes a conformational change leading to its release from the DBC1-Sirt1 complex. It is worth noting that, at present, the effect of GAPDH phosphorylation by AMPK on the glycolytic activity of GAPDH is unclear. In rat cardiac muscle cells, GAPDH phosphorylation by insulin-stimulated Akt activation has been linked to elevated GAPDH activity (Baba et al., 2010). Although GAPDH is not a key rate-limiting enzyme in glycolysis like phosphofructokinase (PFK), PFK has recently been identified as a substrate of AMPK, and the phosphorylation of PFK by AMPK activates the

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

enzyme, contributing to the enhanced heart glycolysis during ischemia (Marsin et al., 2000). However, our data indicated that the glycolytic activity of GAPDH is not likely involved in the Sirt1 activation. In response to AMPK activation, the GAPDHC152S mutant that has little catalytic activity shifts to the nucleus, interacts normally with Sirt1, and disassociates DBC1 from Sirt1 like wild-type GAPDH. Considering that the acute GAPDH phosphorylation by activated AMPK occurred in the cytoplasm, and the GAPDHS122D mutant that mimics phosphorylated GAPDH localized to both the nucleus and the cytoplasm (Figure 4G), it would be interesting to clarify the influence of GAPDH phosphorylation on cell glycolysis. The nuclear translocation of GAPDH has been related to apoptotic cell death triggered by a number of cytotoxic stresses (Dastoor and Dreyer, 2001; Hara and Snyder, 2006; Kusner et al., 2004; Sawa et al., 1997; Schmitz, 2001). Our data indicate, on the contrary, a protective effect of GAPDH in the nucleus through initiating autophagy, suggesting that the action of this multifunctional protein can be influenced by different posttranslational modifications. While the S-nitrosylation of the protein leads to cytotoxicity by activating the acetyltransferase p300 (Hara et al., 2005; Sen et al., 2008), after phosphorylation by AMPK, GAPDH becomes anti-apoptotic through activation of the deacetylase Sirt1. Since the nuclear translocation of S-nitrosylated GAPDH and the pro-apoptotic effect usually occur with relatively prolonged stress (Hara et al., 2005; Li et al., 2012; Ortiz-Ortiz et al., 2010), the nuclear translocation of phosphorylated GAPDH may launch autophagy to supply energy or remove aberrant proteins or organelles, such as damaged mitochondria, at an early stage of cellular stress. When the stress is sustained, more GAPDH moves into the nucleus in an S-nitrosylated form in NO-producing cells or by other modifications to induce apoptosis. Intriguingly, it seems that the multiple functions of GAPDH are represented not only by its capacity to interact with different proteins, but also by acting on the same target in distinct forms, because it has been shown that S-nitrosylated GAPDH interacts with Sirt1 and inhibits its enzymatic activity through transnitrosylation (Kornberg et al., 2010).

Immunostaining and Confocal Microscopy Immunostaining of endogenous or tagged proteins was performed as described previously (Guo et al., 2012). Images were acquired on a scanning confocal microscope (LSM 510; Carl Zeiss) and analyzed with the LSM 510 software. See Supplemental Experimental Procedures for details. Transmission Electron Microscopy Transmission electron microscopy (TEM) was performed as described previously (Yla¨-Anttila et al., 2009). Detailed protocol is presented in the Supplemental Experimental Procedures. Immunoprecipitation and Western Blot Western blot was performed as described previously (Guo et al., 2012). Protein immunoprecipitation was performed using nondenaturing cell extracts, and immunocomplexes were separated by SDS-PAGE and analyzed by western blot. See Supplemental Experimental Procedures for details. Recombinant Protein Purification and In Vitro Kinase Assay Recombinant proteins were produced using procedures described in Supplemental Experimental Procedures. In vitro kinase assays were performed using purified GAPDH and AMPK a/b/g complex (Promega) in the presence of ATP or [g-32P] ATP and are specified in the Supplemental Experimental Procedures. HPLC/MS/MS in an LTQ Mass Spectrometer To prepare samples for mass spectrometric analysis of phosphorylation site(s) of GAPDH, purified GST-GAPDH proteins were cultured with active AMPK a/b/g complex in the presence of ATP and then separated by SDSPAGE and depicted with colloidal Coomassie blue staining. Bands of GAPDH were excised, digested with trypsin, and analyzed by LTQ-Orbitrap mass spectrometer. To characterize the binding proteins of GAPDH or Sirt1, the immunoprecipitates of GAPDH, GAPDH-NLS, or Sirt1 were analyzed by mass spectrometry. See Supplemental Experimental Procedures for details. Statistical Analysis All the statistical data are presented as mean ± SEM. The statistical significance of differences was determined using Student’s t test. p < 0.05 was considered to be statistically significant. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures, three tables, and Supplemental Experimental Procedures and can be found with this article online at http://dx. doi.org/10.1016/j.molcel.2015.10.037. AUTHOR CONTRIBUTIONS

EXPERIMENTAL PROCEDURES Plasmids, Antibodies, and Reagents Plasmids, antibodies, and reagents are described in Supplemental Experimental Procedures.

Cell Culture, Transfection, and Treatment Cells were cultured under standard conditions and transfected according to manufacturer’s instructions. Detailed descriptions of cell-culture conditions, transfection procedures, and chemical treatment are in Supplemental Experimental Procedures.

Autophagy Induction For starvation, cells were incubated in starvation medium (20 mM HEPES [pH 7.4], 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1% BSA) for 1.5 hr; for glucose starvation, cells were incubated in glucose-free DMEM (Life Technologies) containing 10% dialyzed fetal bovine serum (FBS) for 4 hr; and for amino acid starvation, cells were incubated in amino-acid-free medium containing 10% dialyzed FBS for 4 hr.

W.L. and C.C. designed the experiments. C.C., H.S., D.Z., Y.W., and Q.S. performed the experiments. C.P. and C.C.L.W. performed the mass spectrometry. W.L. and J.L.-S. wrote the manuscript. All authors discussed the results and commented on the manuscript. ACKNOWLEDGMENTS We are grateful to the Imaging Center of Zhejiang University School of Medicine for assistance with confocal microscopy and electron microscopy. We thank Tao Li for technical support in kinase assay. We thank Dr. Zongping Xia for providing the AMPKa1/a2 double-knockout MEFs. This study was supported by the National Natural Science Foundation of China (31171288, 31530040, and 31271431) and the National Basic Research Program of China (2011CB910100 and 2013CB910200). Received: June 3, 2015 Revised: September 10, 2015 Accepted: October 22, 2015 Published: November 25, 2015

Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc. 9

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

REFERENCES Azam, S., Jouvet, N., Jilani, A., Vongsamphanh, R., Yang, X., Yang, S., and Ramotar, D. (2008). Human glyceraldehyde-3-phosphate dehydrogenase plays a direct role in reactivating oxidized forms of the DNA repair enzyme APE1. J. Biol. Chem. 283, 30632–30641. Baba, T., Kobayashi, H., Kawasaki, H., Mineki, R., Naito, H., and Ohmori, D. (2010). Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle. FEBS Lett. 584, 2796–2800. Banko, M.R., Allen, J.J., Schaffer, B.E., Wilker, E.W., Tsou, P., White, J.L., Ville´n, J., Wang, B., Kim, S.R., Sakamoto, K., et al. (2011). Chemical genetic screen for AMPKa2 substrates uncovers a network of proteins involved in mitosis. Mol. Cell 44, 878–892. Canto´, C., Gerhart-Hines, Z., Feige, J.N., Lagouge, M., Noriega, L., Milne, J.C., Elliott, P.J., Puigserver, P., and Auwerx, J. (2009). AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature 458, 1056–1060. Chuang, D.M., and Ishitani, R. (1996). A role for GAPDH in apoptosis and neurodegeneration. Nat. Med. 2, 609–610. Colell, A., Ricci, J.E., Tait, S., Milasta, S., Maurer, U., Bouchier-Hayes, L., Fitzgerald, P., Guio-Carrion, A., Waterhouse, N.J., Li, C.W., et al. (2007). GAPDH and autophagy preserve survival after apoptotic cytochrome c release in the absence of caspase activation. Cell 129, 983–997. Dastoor, Z., and Dreyer, J.L. (2001). Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress. J. Cell Sci. 114, 1643–1653. Finkel, T., Deng, C.X., and Mostoslavsky, R. (2009). Recent progress in the biology and physiology of sirtuins. Nature 460, 587–591. Fulco, M., Cen, Y., Zhao, P., Hoffman, E.P., McBurney, M.W., Sauve, A.A., and Sartorelli, V. (2008). Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt. Dev. Cell 14, 661–673. Geng, J., and Klionsky, D.J. (2008). The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep. 9, 859–864. Gerhart-Hines, Z., Dominy, J.E., Jr., Bla¨ttler, S.M., Jedrychowski, M.P., Banks, A.S., Lim, J.H., Chim, H., Gygi, S.P., and Puigserver, P. (2011). The cAMP/PKA pathway rapidly activates SIRT1 to promote fatty acid oxidation independently of changes in NAD(+). Mol. Cell 44, 851–863. Guo, Y., Chang, C., Huang, R., Liu, B., Bao, L., and Liu, W. (2012). AP1 is essential for generation of autophagosomes from the trans-Golgi network. J. Cell Sci. 125, 1706–1715. Gwinn, D.M., Shackelford, D.B., Egan, D.F., Mihaylova, M.M., Mery, A., Vasquez, D.S., Turk, B.E., and Shaw, R.J. (2008). AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226. Hara, M.R., and Snyder, S.H. (2006). Nitric oxide-GAPDH-Siah: a novel cell death cascade. Cell. Mol. Neurobiol. 26, 527–538. Hara, M.R., Agrawal, N., Kim, S.F., Cascio, M.B., Fujimuro, M., Ozeki, Y., Takahashi, M., Cheah, J.H., Tankou, S.K., Hester, L.D., et al. (2005). S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat. Cell Biol. 7, 665–674. Hardie, D.G. (2007). AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat. Rev. Mol. Cell Biol. 8, 774–785. Hariharan, N., Maejima, Y., Nakae, J., Paik, J., Depinho, R.A., and Sadoshima, J. (2010). Deacetylation of FoxO by Sirt1 plays an essential role in mediating starvation-induced autophagy in cardiac myocytes. Circ. Res. 107, 1470– 1482. Hariharan, N., Zhai, P., and Sadoshima, J. (2011). Oxidative stress stimulates autophagic flux during ischemia/reperfusion. Antioxid. Redox Signal. 14, 2179–2190.

10 Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc.

Hasmann, M., and Schemainda, I. (2003). FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis. Cancer Res. 63, 7436–7442. Huang, R., Xu, Y., Wan, W., Shou, X., Qian, J., You, Z., Liu, B., Chang, C., Zhou, T., Lippincott-Schwartz, J., and Liu, W. (2015). Deacetylation of nuclear LC3 drives autophagy initiation under starvation. Mol. Cell 57, 456–466. Inoki, K., Zhu, T., and Guan, K.L. (2003). TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590. Jeong, J.K., Moon, M.H., Lee, Y.J., Seol, J.W., and Park, S.Y. (2013). Autophagy induced by the class III histone deacetylase Sirt1 prevents prion peptide neurotoxicity. Neurobiol. Aging 34, 146–156. Kim, E.J., Kho, J.H., Kang, M.R., and Um, S.J. (2007). Active regulator of SIRT1 cooperates with SIRT1 and facilitates suppression of p53 activity. Mol. Cell 28, 277–290. Kim, J.E., Chen, J., and Lou, Z. (2008). DBC1 is a negative regulator of SIRT1. Nature 451, 583–586. Kim, J., Kim, Y.C., Fang, C., Russell, R.C., Kim, J.H., Fan, W., Liu, R., Zhong, Q., and Guan, K.L. (2013). Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy. Cell 152, 290–303. Klionsky, D.J. (2007). Autophagy: from phenomenology to molecular understanding in less than a decade. Nat. Rev. Mol. Cell Biol. 8, 931–937. Kornberg, M.D., Sen, N., Hara, M.R., Juluri, K.R., Nguyen, J.V., Snowman, A.M., Law, L., Hester, L.D., and Snyder, S.H. (2010). GAPDH mediates nitrosylation of nuclear proteins. Nat. Cell Biol. 12, 1094–1100. Kume, S., Uzu, T., Horiike, K., Chin-Kanasaki, M., Isshiki, K., Araki, S., Sugimoto, T., Haneda, M., Kashiwagi, A., and Koya, D. (2010). Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney. J. Clin. Invest. 120, 1043–1055. Kusner, L.L., Sarthy, V.P., and Mohr, S. (2004). Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase: a role in high glucose-induced apoptosis in retinal Mu¨ller cells. Invest. Ophthalmol. Vis. Sci. 45, 1553–1561. Kwon, H.J., Rhim, J.H., Jang, I.S., Kim, G.E., Park, S.C., and Yeo, E.J. (2010). Activation of AMP-activated protein kinase stimulates the nuclear localization of glyceraldehyde 3-phosphate dehydrogenase in human diploid fibroblasts. Exp. Mol. Med. 42, 254–269. Lee, I.H., and Finkel, T. (2009). Regulation of autophagy by the p300 acetyltransferase. J. Biol. Chem. 284, 6322–6328. Lee, I.H., Cao, L., Mostoslavsky, R., Lombard, D.B., Liu, J., Bruns, N.E., Tsokos, M., Alt, F.W., and Finkel, T. (2008). A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proc. Natl. Acad. Sci. USA 105, 3374–3379. Lee, M.N., Ha, S.H., Kim, J., Koh, A., Lee, C.S., Kim, J.H., Jeon, H., Kim, D.H., Suh, P.G., and Ryu, S.H. (2009). Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of Rheb. Mol. Cell. Biol. 29, 3991–4001. Li, C., Feng, J.J., Wu, Y.P., and Zhang, G.Y. (2012). Cerebral ischemia-reperfusion induces GAPDH S-nitrosylation and nuclear translocation. Biochemistry (Mosc.) 77, 671–678. Lin, S.Y., Li, T.Y., Liu, Q., Zhang, C., Li, X., Chen, Y., Zhang, S.M., Lian, G., Liu, Q., Ruan, K., et al. (2012). GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 336, 477–481. Marsin, A.S., Bertrand, L., Rider, M.H., Deprez, J., Beauloye, C., Vincent, M.F., Van den Berghe, G., Carling, D., and Hue, L. (2000). Phosphorylation and activation of heart PFK-2 by AMPK has a role in the stimulation of glycolysis during ischaemia. Curr. Biol. 10, 1247–1255. Mizushima, N., and Klionsky, D.J. (2007). Protein turnover via autophagy: implications for metabolism. Annu. Rev. Nutr. 27, 19–40. Mizushima, N., Yoshimori, T., and Ohsumi, Y. (2011). The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27, 107–132. Nagy, E., Henics, T., Eckert, M., Miseta, A., Lightowlers, R.N., and Kellermayer, M. (2000). Identification of the NAD(+)-binding fold of

Please cite this article in press as: Chang et al., AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation, Molecular Cell (2015), http://dx.doi.org/10.1016/j.molcel.2015.10.037

glyceraldehyde-3-phosphate dehydrogenase as a novel RNA-binding domain. Biochem. Biophys. Res. Commun. 275, 253–260. Nasrin, N., Kaushik, V.K., Fortier, E., Wall, D., Pearson, K.J., de Cabo, R., and Bordone, L. (2009). JNK1 phosphorylates SIRT1 and promotes its enzymatic activity. PLoS ONE 4, e8414. Nin, V., Escande, C., Chini, C.C., Giri, S., Camacho-Pereira, J., Matalonga, J., Lou, Z., and Chini, E.N. (2012). Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMPactivated protein kinase. J. Biol. Chem. 287, 23489–23501. Ortiz-Ortiz, M.A., Mora´n, J.M., Ruiz-Mesa, L.M., Bravo-San Pedro, J.M., and Fuentes, J.M. (2010). Paraquat exposure induces nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the activation of the nitric oxide-GAPDH-Siah cell death cascade. Toxicol. Sci. 116, 614–622. Powell, M.J., Casimiro, M.C., Cordon-Cardo, C., He, X., Yeow, W.S., Wang, C., McCue, P.A., McBurney, M.W., and Pestell, R.G. (2011). Disruption of a Sirt1-dependent autophagy checkpoint in the prostate results in prostatic intraepithelial neoplasia lesion formation. Cancer Res. 71, 964–975. Revollo, J.R., Grimm, A.A., and Imai, S. (2004). The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. J. Biol. Chem. 279, 50754–50763. Sawa, A., Khan, A.A., Hester, L.D., and Snyder, S.H. (1997). Glyceraldehyde-3phosphate dehydrogenase: nuclear translocation participates in neuronal and nonneuronal cell death. Proc. Natl. Acad. Sci. USA 94, 11669–11674. Schmitz, H.D. (2001). Reversible nuclear translocation of glyceraldehyde3-phosphate dehydrogenase upon serum depletion. Eur. J. Cell Biol. 80, 419–427.

Sen, N., Hara, M.R., Kornberg, M.D., Cascio, M.B., Bae, B.I., Shahani, N., Thomas, B., Dawson, T.M., Dawson, V.L., Snyder, S.H., and Sawa, A. (2008). Nitric oxide-induced nuclear GAPDH activates p300/CBP and mediates apoptosis. Nat. Cell Biol. 10, 866–873. Sun, Q., Gao, W., Loughran, P., Shapiro, R., Fan, J., Billiar, T.R., and Scott, M.J. (2013). Caspase 1 activation is protective against hepatocyte cell death by up-regulating beclin 1 protein and mitochondrial autophagy in the setting of redox stress. J. Biol. Chem. 288, 15947–15958. Tristan, C., Shahani, N., Sedlak, T.W., and Sawa, A. (2011). The diverse functions of GAPDH: views from different subcellular compartments. Cell. Signal. 23, 317–323. Yang, Y., Fu, W., Chen, J., Olashaw, N., Zhang, X., Nicosia, S.V., Bhalla, K., and Bai, W. (2007). SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress. Nat. Cell Biol. 9, 1253–1262. Yi, C., Ma, M., Ran, L., Zheng, J., Tong, J., Zhu, J., Ma, C., Sun, Y., Zhang, S., Feng, W., et al. (2012). Function and molecular mechanism of acetylation in autophagy regulation. Science 336, 474–477. Yla¨-Anttila, P., Vihinen, H., Jokitalo, E., and Eskelinen, E.L. (2009). Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol. 452, 143–164. Zhao, W., Kruse, J.P., Tang, Y., Jung, S.Y., Qin, J., and Gu, W. (2008). Negative regulation of the deacetylase SIRT1 by DBC1. Nature 451, 587–590. Zheng, L., Roeder, R.G., and Luo, Y. (2003). S phase activation of the histone H2B promoter by OCA-S, a coactivator complex that contains GAPDH as a key component. Cell 114, 255–266.

Molecular Cell 60, 1–11, December 17, 2015 ª2015 Elsevier Inc. 11