Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy

Article

Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy Graphical Abstract

Authors Xu Qian, Xinjian Li, Qingsong Cai, ..., David X. Liu, Tao Jiang, Zhimin Lu

Correspondence [email protected]

In Brief Qian et al. demonstrate that glutamine deprivation and hypoxia inhibit mTOR and mTOR-dependent ARD1 S228 phosphorylation, which allows ARD1 to bind to PGK1 for PGK1 K388 acetylation. The acetylated PGK1 binds to and phosphorylates Beclin1 at S30, leading to activation of the VPS34-Beclin1 complex to initiate autophagosomal formation.

Highlights d

Glutamine deprivation and hypoxia result in ARD1-dependent PGK1 K388 acetylation

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PGK1 functioning as a protein kinase phosphorylates Beclin1 at S30

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Beclin1 S30 phosphorylation enhances VPS34-Beclin1ATG14L complex activity

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Beclin1 S30 phosphorylation is required for autophagy initiation and tumorigenesis

Qian et al., 2017, Molecular Cell 65, 1–15 March 2, 2017 ª 2017 Elsevier Inc. http://dx.doi.org/10.1016/j.molcel.2017.01.027

Please cite this article in press as: Qian et al., Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy, Molecular Cell (2017), http:// dx.doi.org/10.1016/j.molcel.2017.01.027

Molecular Cell

Article Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy Xu Qian,1 Xinjian Li,1 Qingsong Cai,1 Chuanbao Zhang,2 Qiujing Yu,3 Yuhui Jiang,1,3 Jong-Ho Lee,1 David Hawke,4 Yugang Wang,1 Yan Xia,1,3 Yanhua Zheng,1,3 Bing-Hua Jiang,5 David X. Liu,6 Tao Jiang,2 and Zhimin Lu1,7,8,9,* 1Brain

Tumor Center and Department of Neuro-Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA Neurosurgical Institute, Capital Medical University, Beijing 100050, China 3The Institute of Cell Metabolism and Diseases, Shanghai Key Laboratory of Pancreatic Cancer, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200080, China 4Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 5State Key Lab of Reproductive Medicine, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, Department of Pathology, Nanjing Medical University, Nanjing 210029, China 6Department of Pharmaceutical Sciences, Washington State University College of Pharmacy, Spokane, WA 99202, USA 7Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 8The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA 9Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2017.01.027 2Beijing

SUMMARY

Autophagy is crucial for maintaining cell homeostasis. However, the precise mechanism underlying autophagy initiation remains to be defined. Here, we demonstrate that glutamine deprivation and hypoxia result in inhibition of mTOR-mediated acetyl-transferase ARD1 S228 phosphorylation, leading to ARD1-dependent phosphoglycerate kinase 1 (PGK1) K388 acetylation and subsequent PGK1-mediated Beclin1 S30 phosphorylation. This phosphorylation enhances ATG14L-associated class III phosphatidylinositol 3-kinase VPS34 activity by increasing the binding of phosphatidylinositol to VPS34. ARD1dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumorigenesis. Furthermore, PGK1 K388 acetylation levels correlate with Beclin1 S30 phosphorylation levels and poor prognosis in glioblastoma patients. Our study unearths an important mechanism underlying cellular-stress-induced autophagy initiation in which the protein kinase activity of the metabolic enzyme PGK1 plays an instrumental role and reveals the significance of the mutual regulation of autophagy and cell metabolism in maintaining cell homeostasis. INTRODUCTION Autophagy is a process by which cells capture intracellular proteins, lipids, and organelles and deliver them to the lysosomal compartment, where they are degraded (Levine and Kroemer, 2008). The resulting breakdown products, such as amino acids,

nucleosides, carbohydrates, and fatty acids, provide substrates for both biosynthesis and energy generation, thus maintaining cellular metabolism (White, 2012). Autophagy works as a cellular housekeeper and is crucial for maintaining cell homeostasis. Accordingly, oncogenic proteins can inhibit autophagy, while many proteins with tumor-suppressing functions can stimulate an autophagic response (Galluzzi et al., 2015). However, many cancer cells upregulate autophagy, which is required to support metabolism, tumorigenesis, and resistance to therapy. Autophagy is robustly activated in tumor cells to promote survival upon stress stimulation, including starvation, growth factor deprivation, hypoxia, damaging stimuli, and proteasome inhibition (White, 2012). During the initiation of autophagy, autophagosome nucleation requires a complex that contains ATG6 or its mammalian homolog, Beclin1. Beclin1 recruits the class III phosphatidylinositol (PI) 3-kinase VPS34, which is in a complex with VPS15 and ATG14L, to generate phosphatidylinositol 3-phosphate (PI(3)P) by phosphorylating the 3-position of PI (Funderburk et al., 2010). PI(3)P recruits proteins with PI(3)P-binding domains to modulate intracellular trafficking and autophagosome formation (Kim et al., 2013). The process initiated with the Beclin1 complex gives rise to nascent autophagosome membranes, which encapsulate the cargo in a vesicle that subsequently fuses with a lysosome, generating an autolysosome (Rabinowitz and White, 2010). Beclin1 and VPS34 control autophagic activity, in which AMPactivated protein kinase (AMPK)- and UNC-51-like kinase-1 (ULK1)-dependent regulation of this complex plays an instrumental role (Funderburk et al., 2010; Kim et al., 2013; Russell et al., 2013); however, the precise mechanism by which Beclin1VPS34 complex activity is regulated remains to be further defined. In addition, although autophagy plays a critical role in metabolism regulation, whether metabolic enzymes are directly involved in regulating autophagy is not known. In the glycolytic pathway, there are two catalytic reactions that produce ATP: pyruvate kinase (PK) produces pyruvate and adenosine triphosphate (ATP) by transferring a phosphate group Molecular Cell 65, 1–15, March 2, 2017 ª 2017 Elsevier Inc. 1

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from phosphoenolpyruvate to adenosine diphosphate (ADP), and phosphoglycerate kinase 1 (PGK1) catalyzes the transfer of high-energy phosphate from the 1-position of 1,3-biphosphoglycerate (1,3-BPG) to ADP, which leads to the generation of 3-phosphoglycerate (3-PG) and ATP. Our work and that of others demonstrated that PKM2, acting as a protein kinase, phosphorylates histone H3, the spindle checkpoint protein Bub3, myosin light chain 2, Stat3, and ERK and regulates critical cellular functions, including metabolism, gene transcription, chromatid segregation, and cytokinesis (Jiang et al., 2014a, 2014b; Lu, 2012; Yang and Lu, 2015; Yang et al., 2012). As an ATP-generating enzyme, PGK1 expression is upregulated in many types of human cancer (Li et al., 2016a). We recently demonstrated that mitochondria-translocated PGK1 phosphorylates pyruvate dehydrogenase kinase 1 (PDHK1) at T338, which activates PDHK1 and inhibits mitochondrial pyruvate utilization to increase glycolysis production (Li et al., 2016a). However, it is not known whether PGK1 is involved in the regulation of other important cellular activities as a protein kinase. In this report, we showed that glutamine deprivation and hypoxia induced the binding of PGK1 to Beclin1 in an ARD1-dependent manner. Importantly, PGK1 directly phosphorylated Beclin1 at S30, leading to enhanced VPS34 activity and autophagy. RESULTS PGK1 Interacts with the VPS34-Beclin1 Complex Many cancer cells take up glutamine to support mitochondrial oxidative phosphorylation and provide metabolic intermediates (DeBerardinis et al., 2007). Glutamine deprivation, which occurs in tumors that are outgrowing the existing vasculature and ischemia, results in autophagy (Lin et al., 2012b). Autophagy can be enhanced by phosphorylating mouse Beclin1 at S91/ S94 (S93/S96 in humans) by AMPK in response to glucose starvation and at S14 (S15 in humans) by ULK1 after amino acid deprivation (Kim et al., 2013; Russell et al., 2013). As expected, glutamine (Gln) deprivation for short period of time (30 min) induced autophagy, as evidenced by an increased conversion of LC3B-I to LC3B-II (a phosphatidylethanolamine derivative of LC3B-I) and induced degradation of p62 (a receptor for cargo destined to be degraded by autophagy) in U87 human glioblastoma (GBM) cells (Figure S1A). Inhibition of lysosomal activity by chloroquine (CQ) increased expression of LC3B-II and p62 (Figure S1A). This autophagy was inhibited by Beclin1 small hairpin RNA (shRNA) expression and rescued by expressing RNAi-resistant (r) wild-type (WT) Beclin1, rBeclin1 S93/96A, and rBeclin1 S15A (Figure 1A), suggesting that AMPK- and ULK1-dependent Beclin1 phosphorylation plays a minor role in glutamine-deprivation-induced initiation of autophagy. This finding was supported by the results showing that short-term glutamine deprivation did not affect Beclin1 S93 or S15 phosphorylation (Figure 1B). In contrast, glucose deprivation, which activated AMPK, induced Beclin1 S93 phosphorylation, while amino acid deprivation, which inactivated mTOR and abrogated mTOR-mediated inhibition of ULK1 (Kim et al., 2013; Russell et al., 2013), resulted in moderate increase in Beclin1 S15 phosphorylation. This limited enhanced Beclin1 S15 phosphorylation may result from hypermethylation of ULK1/2 pro-

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moter and low expression of ULK1/2 in glioma cells (Shukla et al., 2014). It is known that an increased AMP/ATP ratio activates AMPK (Mihaylova and Shaw, 2011). A time course of glutamine deprivation experiment showed that the ATP level was decreased dramatically at a late stage of stress (12 hr), but not at an early stage (2 hr) (Figure S1B). Corresponding to the changes of ATP levels, AMPKa T172 and AMPK substrate ACC S79 phosphorylation was increased only after prolonged stress (Figure S1C). These results indicate that Beclin1 S93 and S15 phosphorylation is not involved in glutamine deprivation-induced autophagy initiation. To determine the mechanism underlying glutamine-deprivation-induced autophagy, we performed mass spectrometry analysis of the immunoprecipitated Beclin1 complex and found that PGK1 was associated with Beclin1 (Figure S1D). This association was validated by co-immunoprecipitation analyses, which showed that glutamine deprivation rapidly resulted in an enhanced interaction between PGK1 and Beclin1 exogenously (Figure 1C, top) or endogenously expressed in U87 cells (Figure 1C, bottom), BxPC-3 human pancreatic adenocarcinoma cells, and MDA-MB-231 human breast cancer cells (Figure S1E). Beclin1 is in complex with VPS34 and ATG14L to generate PI(3)P in autophagy (Funderburk et al., 2010). Immunoprecipitation with an anti-PGK1 antibody showed that glutamine deprivation also enhanced the binding of PGK1 to VPS34 and ATG14L (Figure 1D). These results indicate that PGK1 interacts with the VPS34-Beclin1-ATG14L complex and may play a role in glutamine-deprivation-induced autophagy in tumor cells. Acetylation of PGK1 Is Required for Its Binding to Beclin1 and Autophagy upon Glutamine Deprivation Posttranslational modifications of proteins often result in the alteration of their structures and subsequent protein-protein interactions (Lu and Hunter, 2009). Mass spectrometry analysis of immunoprecipitated PGK1 showed that it was acetylated at K388 upon glutamine deprivation (Figure 2A). Immunoblotting analyses with a specific anti-acetylated PGK1 K388 antibody (Figures S2A and S2B) showed that glutamine deprivation induced PGK1 K388 acetylation and that this acetylation was abrogated by the mutation of K388 into arginine (R) (Figure 2B). Co-immunoprecipitation analyses of U87 (Figure 2C) and U251 (Figure S2C) cells showed that PGK1 K388R, in contrast to its WT counterpart, lost its ability to bind to Beclin1. We next examined the role of PGK1 K388 acetylation in autophagy regulation. Reconstituted expression of rPGK1 K388R, but not WT rPGK1, in endogenous PGK1-depleted U87 cells significantly inhibited the production of PI(3)P without altering the complex formation of VPS34, Beclin1, and ATG14L (Figure 2D). As expected, glutamine deprivation resulted in increased numbers of PI(3)P puncta in U87 cells, as detected by the EGFP-FYVE domain fusion protein that binds PI(3)P with high specificity (Figure S2D). This increase was greatly reduced by rPGK1 K388R expression (Figure 2E). Furthermore, rPGK1 K388R expression largely inhibited the glutamine-deprivation-induced p62 degradation and conversion of LC3B-I to LC3B-II in U87 (Figure 2F), U251, BxPC-3, and MDA-MB-231 (Figure S2E) cells and autophagosome formation, as reflected by the LC3 puncta appearance in U87 cells (Figures S2F and 2G). In contrast to rPGK1 K388R

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Figure 1. PGK1 Interacts with the VPS34-Beclin1 Complex Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (A) Beclin1-depleted U87 cells with reconstituted expression of the indicated proteins (left) were pretreated with or without 100 mM chloroquine (CQ) for 30 min and cultured in the presence or absence of glutamine (Gln)-deprived medium for 2 hr (right). (B) U87 cells transfected with FLAG-Beclin1 were cultured with or without Gln, amino acids (AA), or glucose (Glc) for the indicated times. The band intensities were quantified. Data represent the mean ± SD of triplicate samples. (C and D) U87 cells were cultured with or without Gln for the indicated times (C) or 2 hr (D). Immunoprecipitation of FLAG-Beclin1 (C, top) or endogenous PGK1 (C, bottom; and D) was performed. See also Figure S1.

expression, expression of rPGK1 K388Q, an acetylation-mimic mutant, increased binding of PGK1 to Beclin1 (Figure S2G), p62 degradation, and the conversion of LC3B-I to LC3B-II even in glutamine-sufficient condition (Figure S2H). Of note, the expression of rPGK1 K388R, which had glycolytic activity comparable to its WT counterpart (Figure S2I), did not affect the glycolysis of U87 cells, as evidenced by unaltered glucose uptake and lactate production (Figure S2J). These results indicate that PGK1 K388

acetylation, which does not affect glycolysis, is specifically required for glutamine-deprivation-induced binding of PGK1 to Beclin1, VPS34 activation, and autophagy. ARD1 Acetylates PGK1 at K388 in an mTOR-InhibitionDependent Manner To determine the mechanism underlying glutamine deprivation-induced PGK1 K388 acetylation, we performed mass

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Figure 2. Acetylation of PGK1 Is Required for Its Binding to Beclin1 and Autophagy upon Glutamine Deprivation (A) PGK1 was immunoprecipitated from U87 cells cultured in Gln-deprived medium for 2 hr and analyzed by mass spectrometry. Mass spectrometric analysis was performed of a tryptic fragment at m/z 639.82 (mass error, 0.05 ppm) matched to the +4 charged peptide 383-WNTEDKVSHVSTGGGASLELLEGK-406; the results suggested that K388 was acetylated. The Mascot score was 12, and the expectation value was 3.6e-3. (B and C) U87 cells stably expressing with the indicated proteins were cultured in the presence or absence of Gln-deprived medium for 2 hr. A streptavidin pull-down assay (B) or immunoprecipitation analysis (C) was performed. (D–G) PGK1-depleted U87 cells with reconstituted expression of WT FLAG-rPGK1 or FLAG-rPGK1 K388R were cultured in the presence or absence of Gln-deprived medium for 2 hr. (D) The ATG14L-containing VPS34 complex was immunoprecipitated for an in vitro kinase assay. Autoradiography was performed. (E) EGFP-FYVE was transiently expressed in these cells. Representative images of EGFP-FYVE puncta are shown. (F) Immunoblotting analyses were performed. (G) GFP-LC3 was transiently expressed in these cells. Representative images of GFP-LC3 puncta are shown. (B–D and F) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (E and G) Data represent the mean ± SD of 10 different images. N.S., not significant. **p < 0.001. See also Figure S2.

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Figure 3. ARD1 Acetylates PGK1 at K388 in an mTOR-Inhibition-Dependent Manner Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (A) U87 cells were cultured in the presence or absence of Gln-deprived medium for 2 hr. (B) An in vitro acetylation assay was performed by mixing purified ARD1 and purified PGK1 in the presence or absence of acetyl-CoA (Ac-CoA). (C and E) U87 cells expressing SFB-PGK1(C) or FLAG-PGK1 (E) with or without ARD1 depletion and reconstituted expression of WT HA-rARD1 or HA-rARD1 R82/ G85A were cultured in the presence or absence of Gln-deprived medium for 2 hr. A streptavidin pull-down (C) or immunoprecipitation (E) assay was performed. S.E., short exposure; L.E., long exposure. (D) Purified His-PGK1 immobilized on Ni-NTA agarose beads was incubated with or without FLAG-ARD1 and Ac-CoA, followed by incubation with purified GST or GST-Beclin1. (F) Purified GST-ARD1 proteins were incubated with or without purified FLAG-mTOR or FLAG-mTOR KD in the presence of [g-32P] ATP. Autoradiography was performed. pSer, anti-phosphoserine antibody. (G) Purified GST-ARD1 proteins, immobilized on glutathione agarose beads, were incubated with or without purified FLAG-mTOR in the presence or absence of 20 nM Torin1. The beads were then washed and incubated with purified His-PGK1 for a GST pull-down assay. (H) U87 cells, with or without ARD1 depletion and reconstituted expression of the indicated rARD1 proteins, were cultured in the presence or absence of Gln-deprived medium for 2 hr. See also Figure S3.

spectrometry analysis of proteins associated with immunoprecipitated PGK1 and found that acetyl-transferase ARD1 was in the immunocomplex (Figure S3A). A glutathione S-transferase (GST) pull-down assay showed that purified GST-ARD1 interacted with purified His-PGK1, indicating that these two proteins interact with each other directly (Figure S3B). This finding was further validated by a co-immunoprecipitation analysis with an anti-PGK1 antibody, showing that glutamine deprivation enhanced the interaction between endogenous ARD1 and PGK1 in U87 (Figure 3A, top), BxPC-3, and MDA-MB-231 (Figure S3C) cells. A similar finding was observed using reciprocal immunoprecipitation with an anti-ARD1 antibody (Figure 3A, middle). Immunodepletion of ARD1-associated PGK1 with an anti-ARD1 antibody showed that 20% of total cellular PGK1 in U87 cells

was in complex with ARD1 upon glutamine deprivation stimulation (Figure S3D). To determine whether ARD1 acetylates PGK1, we performed an in vitro acetylation assay by incubating purified recombinant GSTARD1 with WT His-PGK1 or His-PGK1 K388R and found that ARD1 acetylated WT PGK1, but not PGK1 K388R (Figure 3B). In addition, glutamine-deprivation-induced PGK1 K388 acetylation was inhibited by ARD1 depletion, and this inhibition was abrogated by reconstituted expression of RNAi-resistant WT rARD1, but not the catalytically inactive rARD1 R82/G85A mutant, in U87 (Figure 3C) and U251 (Figure S3E) cells. These results indicate that ARD1 acetylates PGK1 K388 both in vitro and in vivo. In line with the finding that PGK1 K388 acetylation is required for the glutamine-deprivation-induced interaction between PGK1

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and Beclin1 and autophagy, an in vitro binding assay with incubation of purified GST-Beclin1 and His-PGK1 showed that these two proteins directly bound to each other in a PGK1 acetylationdependent manner (Figure 3D). Depletion of ARD1 inhibited the glutamine-deprivation-induced interaction between PGK1 and Beclin1 in U87 cells (Figure 3E) and p62 degradation and conversion of LC3B-I to LC3B-II in U87 (Figure S3F) and U251 (Figure S3G) cells; this inhibition was alleviated by reconstituted expression of WT rARD1, but not by expression of rARD1 R82/ G85A mutant. These results indicate that ARD1-dependent PGK1 acetylation is required for glutamine-deprivation-induced binding of PGK1 to Beclin1 and autophagy. We next determined the mechanism underlying ARD1-dependent PGK1 acetylation. It is known that glutamine deprivation suppresses mTOR activity (Jeon et al., 2015). As expected, glutamine deprivation and mTOR inhibitor Torin1 treatment reduced mTOR-mediated p70S6K T389 phosphorylation (Figure S3H). Mass spectrometry analysis revealed that ARD1 is potentially phosphorylated by mTOR at S182 and S228 (Hsu et al., 2011). An in vitro phosphorylation assay showed that WT mTOR, but not its kinase-dead mutant (mTOR KD), phosphorylated ARD1; this phosphorylation was abrogated by mutation of S228 but not S182 into alanine (A), as detected by autoradiography and immunoblotting analysis with an anti-phospho-serine antibody (Figure 3F). Of note, glutamine deprivation or Torin1 treatment of U87 cells reduced the total phospho-serine level of ARD1; ARD1 S228A mutant, unlike its WT counterpart, was largely resistant to be phosphorylated in a glutamine-sufficient condition (Figure S3I). These results indicate that ARD1 S228 is a primarily phosphorylated residue by mTOR upon glutamine deprivation. Torin1 treatment or glutamine deprivation of U87 cells resulted in enhanced interaction between PGK1 and ARD1, as detected by co-immunoprecipitation analyses (Figure S3J). An in vitro binding assay showed that purified WT GST-ARD1 bound to purified His-PGK1; this binding was inhibited by inclusion of mTOR for phosphorylation of ARD1, and this inhibition was abrogated by the mutation of ARD1 S228 or inclusion of mTOR inhibitor Torin1 (Figure 3G). Consistent with this result, mTOR inhibited ARD1-dependent PGK1 acetylation in vitro (Figure S3K), and expression of ARD1 S228A or treatment of U87 cells with Torin1 induced PGK1 K388 acetylation (Figure S3L), binding of PGK1 to Beclin1 (Figure S3M), and conversion of LC3B-I to LC3B-II (Figure 3H). These results indicate that glutamine deprivation inhibited mTOR-mediated ARD1 S228 phosphorylation, resulting in ARD1-dependent PGK1 acetylation, binding of PGK1 to Beclin1, and autophagy. PGK1 Phosphorylates Beclin1 at S30 The PGK1-catalyzed conversion of 1,3-BPG to 3-PG and ATP is a reversible reaction, such that PGK1 can also utilize ATP as a phosphate donor (Li et al., 2016a). To determine whether PGK1 acts as a protein kinase to phosphorylate Beclin1, we performed an in vitro phosphorylation assay by mixing purified GST-Beclin1 in the presence of [g-32P]ATP with bacterially purified and FLAG-ARD1-acetylated WT PGK1 or PGK1 T378P kinase-dead mutant. Figure 4A shows that WT PGK1, but not PGK1 T378P or Beclin1-binding deficient PGK1 K388R, phos-

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phorylated Beclin1. Liquid-chromatography-coupled Orbitrap tandem mass spectrometric analysis demonstrated that PGK1 phosphorylated Beclin1 at S30 (Figure 4B), which is an evolutionarily conserved residue in different species (Figure S4A). This phosphorylation was also detected using autoradiography with [g-32P]ATP and a specific anti-phospho-Beclin1 S30 antibody (Figure S4B) and was abrogated by the mutation of S30 into Ala (Figure 4C). In addition, glutamine deprivation rapidly induced Beclin1 S30 phosphorylation (Figure 4D). This enhanced phosphorylation was abrogated by PGK1 depletion and restored by reconstituted expression of rPGK1, but not of rPGK1 K388R, in U87 (Figure 4E), BxPC-3, and MDA-MB-231 (Figure S4C) cells. In line with the mTOR-inhibition-dependent regulation of ARD1 and PGK1, inhibition of mTOR by Torin1 or expression of ARD1 S228A induced Beclin1 S30 phosphorylation in the absence of glutamine deprivation (Figure 4F). In addition, amino acid starvation, which inactivated mTOR, increased ARD1dependent PGK1 K388 acetylation, the association between PGK1 and Beclin1, and subsequent PGK1-depdendent Beclin1 S30 phosphorylation (Figure S4D). These results indicate that mTOR-inhibition-dependent PGK1 acetylation results in the binding of PGK1 to Beclin1 and phosphorylation of Beclin1 S30 by PGK1. To gain insight into Beclin1 phosphorylation by PGK1, we performed a computer-based protein-peptide docking analysis. Due to the intrinsic disorder and lack of an available N-terminal structure of Beclin1 (Lee et al., 2016), we modeled Beclin1 peptide (27-LDTSFKI-33) with PGK1 (PDB: 2XE7) using CABS-dock server. We revealed a 4.6 A˚ distance between the –OH group of Beclin1 S30 and the high-energy –PO3 group of ATP and found that the –OH group of S30 and the –PO3 group of ATP are located in different arms of PGK1 (Figure S4E). This finding supports the feasibility that PGK1 transfers the –PO3 group from ATP to Beclin1 S30, resulting in Beclin1 S30 phosphorylation. To determine whether ULK1-mediated Beclin1 S15 phosphorylation and AMPK-dependent S93/S96 phosphorylation regulates Beclin1 S30 phosphorylation, we mutated S15/S93/S96 into Ala. Beclin1 S15/S93/S96A expression did not affect the interaction between PGK1 and Beclin1 and PGK1-mediated Beclin1 S30 phosphorylation in response to glutamine deprivation (Figure 4G), indicating that glutamine deprivation-induced Beclin1 S30 phosphorylation is independent of Beclin1 S15 or S93/S96 phosphorylation. PGK1-Phosphorylated Beclin1 Promotes GlutamineDeprivation-Induced VPS34 Activation and Autophagy To determine the functional consequence of PGK1-mediated Beclin1 phosphorylation, we first examined the effect of this phosphorylation on VPS34 activity in the ATG14L complex. Figure 5A shows that glutamine deprivation-induced PI(3)P production was much lower in U87 cells with reconstituted expression of rBeclin1 S30A than in those with reconstituted expression of its WT counterpart. However, this PGK1-enhanced VPS34 activity was not due to the alteration of VPS34-Beclin1-ATG14L complex formation, because PGK1 depletion did not affect the interactions among VPS34, Beclin1, and ATG14L (Figure S5A). This finding was further supported by immunoprecipitation analyses, which showed that immunoprecipitated ATG14L bound to

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Figure 4. PGK1 Phosphorylates Beclin1 at S30 (A and C) The indicated His-PGK1 proteins immobilized on Ni-NTA agarose beads were incubated with FLAG-ARD1 and Ac-CoA, followed by incubation with purified GST-Beclin1 (A and C) or GST-Beclin1 S30A (C), in the presence of [g-32P] ATP. Autoradiography was performed. (B) Purified GST-Beclin1 was phosphorylated by purified His-PGK1 in vitro and analyzed by mass spectrometry. Mass spectrometric analysis was performed of a tryptic fragment at m/z 1001.489 (mass error, 0.05 ppm) matched to the +4 charged peptide 21-CSQPLKLDTSFKILDR-36; the results suggested that S30 was phosphorylated. The Mascot score was 43, and the expectation value was 1.8e-3. (D) FLAG-Beclin1-expressing U87 cells were cultured with or without Gln for the indicated times. (E) U87 cells, with or without PGK1 depletion and reconstituted expression of WT HA-rPGK1 or HA-rPGK1 K388R, were cultured in the presence or absence of Gln-deprived medium for 2 hr. (F) ARD1-depleted U87 cells with reconstituted expression of WT FLAG-rARD1 or FLAG-rARD1 S228A were treated with or without Torin1 (20 nM) for 30 min before 2hr Gln deprivation. (G) WT FLAG-Beclin1- or FLAG-Beclin1 S15/S93/S96A-expressing U87 cells were cultured in the presence or absence of Gln-deprived medium for 2 hr. (A and C–G) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. See also Figure S4.

a comparable amount of WT rBeclin1 and rBeclin1 S30A in the presence or absence of glutamine deprivation (Figure 5B). To determine whether Beclin1 phosphorylation affects the binding of VPS34 to its substrate PI, we expressed hemagglutinin (HA)-tagged WT rBeclin1, rBeclin1 S30A, S93/S96A, or S15A mutant in Beclin1-depleted U87 cells and incubated immunoprecipitated ATG14L-associated VPS34 with 3H-labeled PI. The ability of VPS34, in complex with Beclin1 S30A, to bind to PI was lower than that of VPS34 in complex with WT rBeclin1, rBeclin1 S93/S96A, or rBeclin1 S15A (Figure 5C), suggesting a

negative regulation of VPS34 by Beclin1 S30A. In line with this finding, the number of glutamine-deprivation-increased PI(3)P puncta was lower in U87 cells expressing Beclin1 shRNA; this reduction was largely abrogated by reconstituted expression of WT rBeclin1, but not rBeclin1 S30A (Figure 5D). To determine the mechanism underlying Beclin1 S30 phosphorylation-dependent regulation of VPS34 complex activity, we purified ATG14L-containing VPS34 complex (Figure 5E, left) and digested the complex with trypsin. Immunoblotting with an anti-VPS34 antibody showed that glutamine deprivation resulted

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Figure 5. PGK1-Phosphorylated Beclin1 Promotes Glutamine-Deprivation-Induced VPS34 Activation and Autophagy (A) Beclin1-depleted U87 cells with reconstituted expression of WT FLAG-rBeclin1 or FLAG-rBeclin1 S30A transiently expressing V5-VPS34 and HA-ATG14L were cultured in the presence or absence of Gln-deprived medium for 2 hr. The HA-ATG14L-containing VPS34 complex was immunoprecipitated, and its activity was analyzed using an in vitro kinase assay. Autoradiography was performed. (B) WT HA-rBeclin1- or HA-rBeclin1 S30A-expressing U87 cells were co-expressed with HA-VPS34 and FLAG-ATG14L in the presence or absence of Glndeprived medium for 2 hr. The FLAG-ATG14L-containing VPS34 complex was immunoprecipitated. (C) Beclin1-depleted U87 cells expressing indicated HA-rBeclin1 proteins were cultured in the presence or absence of Gln-deprived medium for 2 hr. The FLAGATG14L-containing VPS34 complex was immunoprecipitated and incubated with 3H-PI. (D, F, and G) U87 cells, with or without Beclin1 depletion and reconstituted expression of WT FLAG-rBeclin1 or FLAG-rBeclin1 S30A, were cultured in the presence or absence of Gln-deprived medium for 2 hr. (D) EGFP-FYVE was transiently expressed in these cells. Representative images of EGFP-FYVE puncta are (legend continued on next page)

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in two additional digestion products with 63 kDa and 75 kDa molecular weights, which were detected in cells expressing WT Beclin1, but not Beclin1 S30A mutant (Figure 5E, right). These results strongly suggest that glutamine deprivation induces a Beclin1 S30 phosphorylation-dependent conformational change of VPS34, which promotes the binding of the VPS34 complex to its substrate PI and enhances the VPS34 complex activity. As expected, rBeclin1 S30A expression largely inhibited the glutamine deprivation-induced p62 degradation and conversion of LC3B-I to LC3B-II in U87 (Figure 5F), U251, BxPC-3, and MDA-MB-231 (Figure S5B) cells and reduced the number of LC3 puncta in U87 cells (Figure 5G). In line with that glutamine deprivation did not affect Beclin1 S15 or S93 phosphorylation, rBeclin1 S15/S93/S96A mutant did not inhibit glutamine deprivation-induced p62 degradation or conversion of LC3B-I to LC3B-II in U87 cells (Figure S5C). Thus, these results indicate that Beclin1 S30 phosphorylation by PGK1 is required for glutamine-deprivation-induced VPS34 activation and autophagy. In addition to regulation of autophagy initiation by forming complex with ATG14L and VPS34, Beclin1 interacts with VPS38/UV radiation resistance-associated gene (UVRAG) to regulate autophagosome maturation, vacuolar protein sorting, cytokinesis, and receptor degradation (Itakura et al., 2008; Kihara et al., 2001; Kim et al., 2015). Immunoprecipitation of FLAG-ATG14L or FLAG-UVRAG showed that S30-phosphorylated Beclin1 associated with ATG14L, but not with UVRAG (Figures S5D and S5E), suggesting that Beclin1 S30 phosphorylation was not involved in UVRAG-associated cellular activity. Beclin1 was reported to regulate endocytic trafficking of epidermal growth factor (EGF) receptor, and Beclin1 depletion enhanced EGF-stimulated and endosome maturation-dependent AKT and ERK activation (Rohatgi et al., 2015). We showed that EGF treatment did not affect Beclin1 S30 phosphorylation (Figure S5F). In addition, Beclin1-depletion-enhanced EGF-induced phosphorylation of AKT and ERK was suppressed by reconstituted expression of both WT rBeclin1 and rBeclin1 S30A (Figure S5G), suggesting that PGK1-mediated Beclin1 S30 phosphorylation specifically regulates autophagy initiation, but not vesicular trafficking. PGK1-Phosphorylated Beclin1 Promotes HypoxiaInduced Autophagy To determine whether Beclin1 S30 phosphorylation by PGK1 is unique to glutamine and amino acid deprivation, we cultured U87 cells under glucose deprivation and hypoxia stimulation. Glucose deprivation did not obviously affect PGK1-dependent Beclin1 S30 phosphorylation (Figure S6A). In contrast, hypoxia induced Beclin1 S30 phosphorylation at an early stage (6 hr) of autophagy, whereas Beclin1 S93 phosphorylation was increased after 48 hr of stimulation (Figure 6A). Of note, Beclin1 S30A mutant was phosphorylated at S93 to a level that was comparable to that of its WT counterpart (Figure 6B), whereas Beclin1 S93/

S96A mutation did not affect Beclin1 S30 phosphorylation upon hypoxic stimulation (Figure 6C), indicating that Beclin1 S30 and S93/S96 phosphorylation were independent of each other. Hypoxia inhibits mTOR activity (DeYoung et al., 2008). Consistent with the finding that mTOR regulates ARD1 phosphorylation and subsequent PGK1 K388 acetylation, hypoxia reduced ARD1 phosphorylation, and Torin1 treatment suppresses this phosphorylation in normoxic conditions (Figure S6B). In addition, ARD1 depletion or expression of inactive rARD1 R82/G85A mutant blocked hypoxia-induced PGK1 K388 acetylation and Beclin1 S30 phosphorylation in U87 (Figure 6D), U251 (Figure S6C), MDA-MB-231, and BxPC-3 cells (Figure S6D), while ARD1 S228A mutant stimulated PGK1 K388 acetylation and Beclin1 S30 phosphorylation in normoxic conditions. Similarly, PGK1 depletion inhibited hypoxia-induced Beclin1 S30 phosphorylation in U87 (Figure 6E), U251 (Figure S6E), MDA-MB-231, and BxPC-3 cells (Figure S6F), and this inhibition was abrogated by reconstituted expression of WT rPGK1, but not rPGK1 K388R mutant. Analyses of hypoxia-induced autophagy showed that hypoxia stimulation resulted in increased numbers of PI(3)P puncta, and this increase was abrogated by reconstituted expression of rBeclin1 S30A, but not its WT counterpart, in U87 cells with depleted endogenous Beclin1 (Figure 6F). In addition, rBeclin1 S30A expression significantly inhibited hypoxia-induced p62 degradation and conversion of LC3B-I to LC3B-II in U87 (Figure 6G) and U251 (Figure S6G) cells and reduced the number of LC3 puncta in U87 cells (Figure 6H). These results indicate that hypoxia induces PGK1-mediated Beclin1 phosphorylation, which is required for autophagy under this condition. Reactive oxygen species (ROS) is known to play a role in autophagy activation (Scherz-Shouval and Elazar, 2011). Both glutamine deprivation and hypoxia promote ROS production (Li et al., 2016a; Reid et al., 2013). Treating cells with N-acetyl-L-cysteine (NAC), a scavenger of free radicals, did not affect either glutamine deprivation- or hypoxia-induced PGK1 K388 acetylation or Beclin1 S30 phosphorylation (Figures S6H and S6I), suggesting that these modifications are ROS production-independent. It was known that hypoxia-upregulated BNIP3 and BNIP3L compete with Beclin1 to bind to BCL2 and release Beclin1 from BCL2 complex (Bellot et al., 2009). In line with this report, we showed that hypoxia increased the expression levels of BNIP3 and BNIP3L in U87 cells (Figure S6J) and released both WT Beclin1 and Beclin1 S30A from its binding to BCL2 (Figure S6K), suggesting that Beclin1 S30 phosphorylation is not involved in the interaction between Beclin1 and BCL2. Of note, depletion of both BNIP3 and BNIP3L moderately reduced hypoxia-induced Beclin1 S30 phosphorylation (Figure S6L), suggesting that Beclin1 is in distinct cellular complexes, including the Beclin1-VPS34-ATG14L and Beclin1-BCL2 complexes, and release of Beclin1 from BCL2 only partially contributes to Beclin1 S30 phosphorylation in the VPS34-ATG14L complex.

shown. (F) The immunoblotting intensity was quantified. (G) GFP-LC3 was transiently expressed in these cells. Representative images of GFP-LC3 puncta are shown. (E) WT Beclin1 or Beclin1 S30A was co-expressed with VPS34, VPS15, and ATG14L in 293T cells in the presence or absence of Gln-deprived medium for 2 hr. ATG14L-containing VPS34 complex was purified (left), digested by trypsin, and immunoblotted with an antibody against VPS34 (right). (A–C, E, and F) Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. (C and F) Data represent the mean ± SD of triplicate samples. (D and E) Data represent the mean ± SD of 10 different image. N.S., not significant. **p < 0.001. See also Figure S5.

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Figure 6. PGK1-Phosphorylated Beclin1 Promotes Hypoxia-Induced Autophagy (A) U87 cells expressing FLAG-Beclin1 were treated with hypoxia for the indicated times. (B, C, and G) U87 cells expressing the indicated FLAG-rBeclin1 proteins were treated with or without hypoxia for 48 hr (B) or 24 hr (C and G). (D) U87 cells, with or without ARD1 depletion and reconstituted expression of the indicated HA-rARD1 proteins, were incubated with or without hypoxia for 24 hr. (E) U87 cells, with or without PGK1 depletion and reconstituted expression of WT HA-rPGK1 or HA-rPGK1 K388R, were co-expressed with WT FLAG-rBeclin1 or FLAG-rBeclin1 S30A and treated with or without hypoxia for 24 hr. (F and H) U87 cells, with or without Beclin1 depletion and reconstituted expression of WT FLAG-rBeclin1 or FLAG-rBeclin1 S30A, were treated with or without hypoxia for 24 hr. (F) EGFP-FYVE was transiently expressed in these cells. Representative images of EGFP-FYVE puncta are shown. (H) GFP-LC3 was transiently expressed in these cells. Representative images of GFP-LC3 puncta are shown. (A–E and G) Immunoprecipitation and immunoblotting analyses were performed using the indicated antibodies. (F, H) Data represent the mean ± SD of 10 different images; **p < 0.001. See also Figure S6.

PGK1-Phosphorylated Beclin1 Promotes Cell Proliferation and Brain Tumorigenesis and Indicates a Poor Prognosis in GBM Patients Autophagy is activated in tumor cells to support cell proliferation and survival upon stress stimulation (White, 2012). As expected, depletion of PGK1 inhibited proliferation of U87 cells under

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hypoxic conditions (Figure 7A, left), and this inhibition was rescued by reconstituted expression of WT rPGK1, but not rPGK1 K388R. A similar inhibitory effect on cell proliferation was also detected by Beclin1 depletion, which was abrogated by reconstituted expression of WT rBeclin1, but not rBeclin1 S30A (Figure 7A, right). In contrast, under normoxic conditions,

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(legend on next page)

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PGK1-depletion-inhibited U87 cell proliferation was rescued by reconstituted expression of both WT rPGK1 and rPGK1 K388R (Figure S7A, left), which was consistent with the findings that PGK1 K388R mutant did not affect PGK1 glycolytic enzyme activity (Figure S2I) or glycolysis (Figure S2J). In addition, cell proliferation was not affected by depletion of Beclin1 or reconstituted expression of WT rBeclin1 or rBeclin1 S30A under normoxic conditions (Figure S7A, right). The effect of inhibited PGK1-mediated Beclin1 S30 phosphorylation on cell proliferation photocopied the effect of depletion of an essential autophagy-related gene, ATG5; ATG5 depletion, which did not affect cell proliferation under normoxic conditions, increased conversion of LC3B-I to LC3B-II and degradation of p62 (Figure S7B, left) and inhibited hypoxia-induced cell proliferation (Figure S7B, right). These results indicate that ARD1-mediated PGK1 acetylation and PGK1-phosphorylated Beclin1 specifically promote cell proliferation under hypoxic conditions. To determine the role of PGK1-mediated Beclin1 phosphorylation in brain tumor development, we intracranially injected athymic nude mice with U87 cells with or without depletion of PGK1 or Beclin1 and reconstituted expression of their WT counterparts, rPGK1 K388R, or rBeclin1 S30A. Consistent with the cell proliferation results, depletion of PGK1 or Beclin1 largely inhibited tumor growth, which was restored by reconstituted expression of WT rPGK1 or rBeclin1 (Figures 7B and 7C). In contrast, expression of rBeclin1 S30A or rPGK1 K388R exhibited significant tumor growth inhibition (Figures 7B and 7C) with suppressed autophagy, as evidenced by enhanced p62 and reduced Ki-67 expression levels (Figures 7D and S7C). To dynamically monitor the effect of regulation of PGK1 and Beclin1 on tumor growth, we subcutaneously injected U87 cells with reconstituted expression of WT rPGK1, rPGK1 K388R, WT rBeclin1, or rBeclin1 S30A into athymic nude mice. In contrast to expression of their WT counterparts, expression of rPGK1 K388R or rBeclin1 S30A significantly inhibited tumor growth (Figures S7D–S7F) and Ki-67 staining (Figure S7G). These results strongly suggest that PGK1-phosphorylated Beclin1 and subsequent autophagy activation plays an instrument role in brain tumor growth. We next analyzed 74 human primary GBM specimens with specificity-validated antibodies (Figures S7H and S7I). We showed that the acetylation levels of PGK1 K388 were positively correlated with phosphorylation levels of Beclin1 S30, while both of them were inversely correlated with p70S6K T389 phosphorylation levels (Figure 7E). Quantification of the staining showed that these correlations were significant (Figure S7J).

We compared the survival duration of the 74 patients, all of whom had received standard adjuvant radiotherapy after surgical resection of GBM followed by treatment with an alkylating agent (temozolomide in most cases), with tumor acetylation levels of PGK1 K388 and phosphorylation levels of Beclin1 S30. The median survival duration was 114.8 and 103.5 weeks for patients whose tumors had low PGK1 K388 acetylation and Beclin1 S30 phosphorylation levels, respectively, and 46.5 and 50.2 weeks for those whose tumors had high levels of PGK1 K388 acetylation and Beclin1 S30 phosphorylation, respectively (Figure 7F). These results support a role for PGK1-phosphorylated Beclin1 and subsequent autophagy activation in the clinical behavior of human GBM and reveal correlations among PGK1 K388 acetylation, Beclin1 S30 phosphorylation, and the clinical aggressiveness of GBM. DISCUSSION Many cancer cells require glutamine to support mitochondrial oxidative phosphorylation and provide metabolic intermediates, while glutamine deprivation and hypoxia occur in tumors that are outgrowing the existing vasculature and ischemia (Lin et al., 2012b). Glutamine deprivation and hypoxia induce autophagy to maintain cell homeostasis, including cellular metabolism. However, the precise mechanism underlying the regulation of autophagy initiation under these cellular stresses and the role of metabolic enzymes in this regulation remain to be defined. We demonstrated here that glutamine deprivation and hypoxia resulted in inhibition of mTOR-mediated ARD1 S228 phosphorylation, leading to an association of ARD1 and PGK1 and subsequent PGK1 K388 acetylation. Acetylated PGK1 directly interacted with Beclin1. Importantly, PGK1 acting as a protein kinase phosphorylated Beclin1 at S30. This phosphorylation, which did not affect Beclin1-VPS34-ATG14L complex formation, altered VPS34 conformation and dramatically enhanced the ability of VPS34 to bind to PI, thereby significantly increasing VPS34 activity toward PI(3)P production. Consequently, glutaminedeprivation- and hypoxia-induced ARD1-dependent PGK1 acetylation and PGK1-mediated Beclin1 S30 phosphorylation are required for the initiation of autophagy, which is instrumental for brain tumor development (Figure 7G). It is known that Beclin1 is phosphorylated at S93/S96 by AMPK and at S15 by ULK1 in response to glucose starvation and amino acid deprivation, respectively (Kim et al., 2013; Russell et al., 2013). However, glutamine deprivation and hypoxia at an early phase does not affect Beclin1 phosphorylation at these

Figure 7. PGK1-Dependent Beclin1 S30 Phosphorylation Promotes Brain Tumorigenesis and Is Associated with Poor Prognosis in GBM Patients (A) 1 3 105 U87 cells, with or without PGK1 or Beclin1 depletion and reconstituted expression of the indicated proteins, were cultured in hypoxia condition for 72 hr. These cells were collected and counted. Data represent the mean ± SD of quadruplicate samples. **p < 0.001. (B and C) U87 cells, with or without PGK1 or Beclin1 depletion and reconstituted expression of the indicated proteins, were intracranially injected into athymic nude mice (n = 7 per group). (B) Representative H&E stained coronal brain sections are shown. B, brain; T, tumor. Scale bar, 200 mm. (C) Tumor volumes were calculated. Data represent the mean ± SD of seven mice. **p < 0.001. (D and E) Immunohistochemical staining of mouse brain tumor tissues (D) or human GBM tissues (E). Representative images are shown. Scale bar, 200 mm. (F) Kaplan-Meier plots of the overall survival rates of GBM patients in the groups of low (staining score, 0–4) and high (staining score, 5–8) expression of PGK1 K388 acetylation (top) or Beclin1 S30 phosphorylation (bottom). p values were calculated using the log-rank test. (G) Schematic model of PGK1-regulated autophagy activation. See also Figure S7.

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residues. In contrast, both glutamine deprivation and hypoxia induced rapid PGK1 K388 acetylation and Beclin1 S30 phosphorylation, indicating that these posttranslational modifications are important in the initiation of autophagy in an AMPK-independent manner. Notably, prolonged glutamine deprivation or hypoxia stimulation, which resulted in reduction of cellular ATP levels and activation of AMPK, resulted in AMPK-dependent Beclin1 S93 phosphorylation. These results suggest that AMPK-dependent Beclin1 S93/S96 phosphorylation, in conjunction with PGK1regulated Beclin1 S30 phosphorylation, plays a role in sustained autophagy and that cells initiate and maintain autophagy in response to extracellular stresses using distinct mechanisms of signaling regulation. ULK1/2 are required in the autophagy response to amino acid deprivation, but not for autophagy induced by glucose deprivation or glucose metabolism inhibition (Cheong et al., 2011). GBM cells, which expressed low levels of ULK1/2, initiated autophagy in a manner that was dependent on PGK1-mediated Beclin1 S30 phosphorylation, but not on ULK1/2-regulated Beclin1 S15 phosphorylation, in response to glutamine deprivation. In addition, we showed that Beclin1 S30 phosphorylation was required for glutamine-deprivation-induced autophagy in BxPC-3 pancreatic ductal adenocarcinoma and MDA-MB-231 breast cancer cells expressing ULK1/2 (Pike et al., 2013; Wong et al., 2015), further supporting an essential role of PGK1mediated Beclin1 S30 phosphorylation in glutamine deprivation-induced autophagy. Pyruvate kinase and PGK1 catalyze two glycolytic reactions that produce ATP. We and others showed that PKM2 possesses protein kinase activity and phosphorylates multiple protein substrates (Yang and Lu, 2015). We recently revealed that PGK1 phosphorylates and activates PDHK1, leading to inhibition of mitochondrial pyruvate utilization and an increase in glycolysis. This finding provides initial evidence demonstrating that PGK1 functions as a protein kinase in a cellular signaling-dependent manner (Li et al., 2016a, 2016d). In addition, we unveiled that ketohexokinase-A (fructokinase-A) acts as a protein kinase, phosphorylating and activating phosphoribosyl pyrophosphate synthetase 1 (PRPS1) to promote de novo nucleic acid synthesis and hepatocellular carcinoma formation (Li et al., 2016b, 2016c). These findings, together with current studies, indicate that the non-metabolic functions of metabolic enzymes executed by their protein kinase activity play critical roles in the regulation of diverse cellular activities, including autophagy initiation. Hyperactivation of mTOR signaling has been implicated in human carcinogenesis, and different mTOR-inhibitory agents have been developed for cancer treatment. However, it was reported that the lack of clinical effects of mTOR inhibitors in some cancer treatments might be partially due to upregulated tumor-protective autophagy (Jiang et al., 2015). We showed here that mTOR inhibition resulted in PGK1-dependent autophagy in tumor cells, which in turn promoted survival of these cells in response to extracellular stresses. These findings suggest that approaches inhibiting both mTOR activity and PGK1regulated autophagy likely increase cancer treatment efficacy and overcome the cancer cell resistance to mTOR inhibitors that is potentially induced by activation of PGK1-mediated autophagy.

In summary, our findings highlight the dual roles of PGK1, which acts as both a glycolytic enzyme and a protein kinase, in cell metabolism, autophagy, and proliferation, and greatly affect our understanding of protein enzymes that have multiple distinct functions in the control of cellular activities. The demonstration of the mutual regulation of autophagy and cell metabolism integrated by the protein kinase activity of PGK1 revealed that PGK1 has a significant role in maintaining cell homeostasis and is an attractive molecular target for cancer treatment. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

d

d

KEY RESOURCES TABLE CONTACT FOR REAGENT AND RESOURCE SHARING EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mice B Human Subjects B Cell Lines METHOD DETAILS B Materials B Transfection B DNA Construction and Mutagenesis B Purification of Recombinant Proteins B Immunoprecipitation and Immunoblotting Analysis B Mass Spectrometry Analysis B Streptavidin and GST Pull-Down Assay B Measurement of PGK1 Activity B Measurement of ATP B In Vitro Acetylation Assay B In Vitro Kinase Assay B Protein-peptide Docking B VPS34 Lipid Kinase Assay B Phosphatidylinositol Binding Ability Assay B Trypsin Digestion of VPS34 Complex B Autophagy Assay B Measurement of Glucose Consumption and Lactate Production B Cell Proliferation Assay B Immunohistochemical Staining QUANTIFICATION AND STATISTICAL ANALYSIS

SUPPLEMENTAL INFORMATION Supplemental Information includes seven figures and two tables and can be found with this article online at http://dx.doi.org/10.1016/j.molcel.2017.01.027. AUTHOR CONTRIBUTIONS Z.L. and X.Q. conceived and designed the study. X.Q., X.L., Q.C., C.Z., Q.Y., Y.J., J.-H.L., D.H., Y.W., Y.X., and Y.Z. performed the experiments. B.-H.J., D.X.L., and T.J. provided reagents and technical support. Z.L. wrote the paper, with comments from all authors. ACKNOWLEDGMENTS We thank Bih-Fang Pan at the University of Texas MD Anderson Cancer Center for technical assistance and Ann Sutton for her critical reading of this

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manuscript. This work was supported by National Cancer Institute grants 2R01 CA109035 (Z.L.) and 1R01 CA169603 (Z.L.), Brain Cancer SPORE grant 2P50 CA127001, National Institute of Neurological Disorders and Stroke grant 1R01 NS089754 (Z.L.), The University of Texas MD Anderson Cancer Center support grant CA016672, James S. McDonnell Foundation 21st Century Science Initiative in Brain Cancer research award 220020318 (Z.L.), a Sister Institution Network Fund from The University of Texas MD Anderson Cancer Center (Z.L.), UTMDACC Institutional Research Grant (Y.X.) and National Natural Science Foundation of China 81672710 (Q.Y.), 81320108019 (B.-H.J.), 81572499 (Y.Z.), and 81572700 (Y.X.). Z.L. is a Ruby E. Rutherford Distinguished Professor. Received: June 9, 2016 Revised: October 28, 2016 Accepted: January 17, 2017 Published: February 23, 2017 REFERENCES Baskaran, S., Carlson, L.A., Stjepanovic, G., Young, L.N., Kim, D.J., Grob, P., Stanley, R.E., Nogales, E., and Hurley, J.H. (2014). Architecture and dynamics of the autophagic phosphatidylinositol 3-kinase complex. eLife 3, e05115. Bellot, G., Garcia-Medina, R., Gounon, P., Chiche, J., Roux, D., Pouysse´gur, J., and Mazure, N.M. (2009). Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol. Cell. Biol. 29, 2570–2581. Chen, C.H., and Sarbassov dos, D. (2011). The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2. J. Biol. Chem. 286, 40386–40394. Cheong, H., Lindsten, T., Wu, J., Lu, C., and Thompson, C.B. (2011). Ammonia-induced autophagy is independent of ULK1/ULK2 kinases. Proc. Natl. Acad. Sci. USA 108, 11121–11126. DeBerardinis, R.J., Mancuso, A., Daikhin, E., Nissim, I., Yudkoff, M., Wehrli, S., and Thompson, C.B. (2007). Beyond aerobic glycolysis: transformed cells can engage in glutamine metabolism that exceeds the requirement for protein and nucleotide synthesis. Proc. Natl. Acad. Sci. USA 104, 19345–19350. DeYoung, M.P., Horak, P., Sofer, A., Sgroi, D., and Ellisen, L.W. (2008). Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling. Genes Dev. 22, 239–251. Fang, D., Hawke, D., Zheng, Y., Xia, Y., Meisenhelder, J., Nika, H., Mills, G.B., Kobayashi, R., Hunter, T., and Lu, Z. (2007). Phosphorylation of beta-catenin by AKT promotes beta-catenin transcriptional activity. J. Biol. Chem. 282, 11221–11229. Funderburk, S.F., Wang, Q.J., and Yue, Z. (2010). The Beclin 1-VPS34 complex–at the crossroads of autophagy and beyond. Trends Cell Biol. 20, 355–362. Galluzzi, L., Pietrocola, F., Bravo-San Pedro, J.M., Amaravadi, R.K., Baehrecke, E.H., Cecconi, F., Codogno, P., Debnath, J., Gewirtz, D.A., Karantza, V., et al. (2015). Autophagy in malignant transformation and cancer progression. EMBO J. 34, 856–880.

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Mihaylova, M.M., and Shaw, R.J. (2011). The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat. Cell Biol. 13, 1016–1023. Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612. Pike, L.R., Singleton, D.C., Buffa, F., Abramczyk, O., Phadwal, K., Li, J.L., Simon, A.K., Murray, J.T., and Harris, A.L. (2013). Transcriptional up-regulation of ULK1 by ATF4 contributes to cancer cell survival. Biochem. J. 449, 389–400. Piya, S., White, E.J., Klein, S.R., Jiang, H., McDonnell, T.J., Gomez-Manzano, C., and Fueyo, J. (2011). The E1B19K oncoprotein complexes with Beclin 1 to regulate autophagy in adenovirus-infected cells. PLoS ONE 6, e29467. Rabinowitz, J.D., and White, E. (2010). Autophagy and metabolism. Science 330, 1344–1348. Reid, M.A., Wang, W.I., Rosales, K.R., Welliver, M.X., Pan, M., and Kong, M. (2013). The B55a subunit of PP2A drives a p53-dependent metabolic adaptation to glutamine deprivation. Mol. Cell 50, 200–211. Rohatgi, R.A., Janusis, J., Leonard, D., Bellve´, K.D., Fogarty, K.E., Baehrecke, E.H., Corvera, S., and Shaw, L.M. (2015). Beclin 1 regulates growth factor receptor signaling in breast cancer. Oncogene 34, 5352–5362. Russell, R.C., Tian, Y., Yuan, H., Park, H.W., Chang, Y.Y., Kim, J., Kim, H., Neufeld, T.P., Dillin, A., and Guan, K.L. (2013). ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase. Nat. Cell Biol. 15, 741–750. Scherz-Shouval, R., and Elazar, Z. (2011). Regulation of autophagy by ROS: physiology and pathology. Trends Biochem. Sci. 36, 30–38.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Rabbit polyclonal anti-PGK1 K388 acetylation

This paper

N/A

Rabbit polyclonal anti-Beclin1 S30 phosphorylation

This paper

N/A

Rabbit polyclonal anti-PGK1

Signalway Antibody

Cat# 32524

Rabbit polyclonal anti-HA tag

Signalway Antibody

Cat# 35534

Rabbit polyclonal anti-ERK1/2 phosphorylation

Signalway Antibody

Cat# 12082

Rabbit polyclonal anti-PGK1

Abcam

Cat# ab38007; RRID:AB_2161220

Antibodies

Rabbit polyclonal anti-p70S6K T389 phosphorylation

Abcam

Cat# ab126818

Rabbit polyclonal anti-V5 tag

Abcam

Cat# ab15828; RRID:AB_443253

Rabbit polyclonal anti-Ki-67

Millipore

Cat# AB9260; RRID:AB_2142366

Mouse monoclonal anti-phosphoserine

BD Biosciences

Cat# 612546; RRID:AB_399841

Rabbit monoclonal anti-Beclin1

Cell Signaling Technology

Cat# 3495S; RRID:AB_1903911

Rabbit monoclonal anti-Beclin1 S93 phosphorylation

Cell Signaling Technology

Cat# 14717S

Rabbit polyclonal anti-Beclin1 S15 phosphorylation

Cell Signaling Technology

Cat# 13825

Rabbit monoclonal anti-VPS34

Cell Signaling Technology

Cat# 4263S; RRID:AB_2299765

Rabbit monoclonal anti-LC3B

Cell Signaling Technology

Cat# 3868S; RRID:AB_2137707

Rabbit polyclonal anti-ARD1

Cell Signaling Technology

Cat# 9046S

Mouse monoclonal anti-p70S6K T389 phosphorylation

Cell Signaling Technology

Cat# 9206S; RRID:AB_331790

Rabbit polyclonal anti-p70S6K

Cell Signaling Technology

Cat# 9202S; RRID:AB_10695156

Rabbit monoclonal anti-AKT S473 phosphorylation

Cell Signaling Technology

Cat# 4060S; RRID:AB_2315049

Rabbit monoclonal anti-AKT

Cell Signaling Technology

Cat# 4685S; RRID:AB_10698888

Rabbit monoclonal anti-AMPKa T172 phosphorylation

Cell Signaling Technology

Cat# 2535S; RRID:AB_331250

Rabbit monoclonal anti- AMPKa

Cell Signaling Technology

Cat# 5831S; RRID:AB_10622186

Rabbit monoclonal anti-ACC S79 phosphorylation

Cell Signaling Technology

Cat# 11818S

Rabbit monoclonal anti-ACC

Cell Signaling Technology

Cat# 3676S; RRID:AB_10694239

Rabbit polyclonal anti-BNIP3

Cell Signaling Technology

Cat# 13795S

Rabbit monoclonal anti-BNIP3L

Cell Signaling Technology

Cat# 12396S

Mouse monoclonal anti-BCL2

Cell Signaling Technology

Cat# 2872S; RRID:AB_10693462

Rabbit polyclonal anti-p62

Santa Cruz Biotechnology

Cat# sc-25575; RRID:AB_2302590

Mouse monoclonal anti-GST tag

Santa Cruz Biotechnology

Cat# sc-138; RRID:AB_627677

Mouse monoclonal anti-a-tubulin

Santa Cruz Biotechnology

Cat# sc-5286; RRID:AB_628411

Rabbit polyclonal anti-ERK1/2

Santa Cruz Biotechnology

Cat# sc-94; RRID:AB_2140110

Normal rabbit IgG

Santa Cruz Biotechnology

Cat# sc-2027; RRID:AB_737197

Normal rabbit IgG

Santa Cruz Biotechnology

Cat# sc-2025; RRID:AB_737182

Rabbit polyclonal anti-ATG14

MBL International

Cat# PD026; RRID:AB_1953054

Mouse monoclonal anti-Flag

Sigma-Aldrich

Cat# F3165; RRID:AB_259529

Mouse monoclonal anti-His

Sigma-Aldrich

Cat# H1029; RRID:AB_260015

Anti-DYKDDDDK Tag (L5) Affinity Gel

BioLegend

Cat# 651503; RRID:AB_10962692

Torin1

LC Laboratories

Cat# T-7887

L-a-Phosphatidylinositol (PI) ammonium salt solution

Sigma-Aldrich

Cat# P2517

Acetyl coenzyme A (Ac-CoA)

Sigma-Aldrich

Cat# A2056

Biological Samples See Table S1 for information of GBM patients Chemicals, Peptides, and Recombinant Proteins

(Continued on next page)

e1 Molecular Cell 65, 1–15.e1–e6, March 2, 2017

Please cite this article in press as: Qian et al., Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy, Molecular Cell (2017), http:// dx.doi.org/10.1016/j.molcel.2017.01.027

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

Chloroquine

Sigma-Aldrich

Cat# C6628

Glyceraldehyde 3-phosphate

Sigma-Aldrich

Cat# G5251

ATP

Sigma-Aldrich

Cat# A6419

NADH

Sigma-Aldrich

Cat# N4505

N-acetyl-L-cysteine (NAC)

Sigma-Aldrich

Cat# A9165

EGF

Sigma-Aldrich

Cat# E9644

Recombinant active glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

Sigma-Aldrich

Cat# G2267

Chymotrypsin

Sigma-Aldrich

Cat# C8946

[g-32P]ATP

MP Biochemicals

Cat# 38101X

[3H]-Phosphatidylinositol

American Radiolabeled Chemicals

Cat# ART0184

Critical Commercial Assays ATP Colorimetric/Fluorometric Assay Kit

BioVision

Cat# K354-100

Glucose (GO) Assay Kit

Sigma-Aldrich

Cat# GAGO20

L-Lactate Assay Kit II

Eton Bioscience

Cat# 120005

VECTASTAIN Elite ABC HRP Kit

Vector Laboratories

Cat# PK-6101

Liquid DAB+ Substrate Chromogen System

Dako

Cat# K3468

U87

ATCC

Cat# HTB-14

HEK293T/17

ATCC

Cat# CRL-11268

Experimental Models: Cell Lines

BxPC-3

ATCC

Cat# CRL-1687

MDA-MB-231

ATCC

Cat# HTB-26

U251

Sigma

Cat# 09063001

ERO/MD Anderson Cancer Center

N/A

pcDNA3.1/hygro(+)-Flag-PGK1

(Li et al., 2016a)

N/A

pcDNA3.1/hygro(+)-Flag-PGK1 K388R

This study

N/A

pcDNA3.1/hygro(+)-Flag-PGK1 K388Q

This study

N/A

pcDNA3.1/hygro(+)-Flag-PGK1 T378P

(Li et al., 2016a)

N/A

pcDNA3-HA-PGK1

This study

N/A

pcDNA3-HA-PGK1 K388R

This study

N/A

pcDNA6/His-V5-PGK1

(Li et al., 2016a)

N/A

Experimental Models: Organisms/Strains Nu/Nu nude mice Recombinant DNA

pColdI-PGK1 (His-PGK1)

This study

N/A

pColdI-PGK1 K388R

This study

N/A

pColdI-PGK1 T378P

This study

N/A

pcDNA6/SFB-PGK1

(Li et al., 2016a)

N/A

pcDNA6/SFB-PGK1 K388R

This study

N/A

pGEX-4T-1-PGK1 (GST-PGK1)

This study

N/A

pcDNA3.1/hygro(+)-Flag-Beclin1

This study

N/A

pcDNA3.1/hygro(+)-Flag-Beclin1 S30A

This study

N/A

pcDNA3.1/hygro(+)-Flag-Beclin1 S15A

This study

N/A

pcDNA3.1/hygro(+)-Flag-Beclin1 S93/96A

This study

N/A

pcDNA3.1/hygro(+)-Flag-Beclin1 S15/93/96A

This study

N/A

pcDNA3-HA-Beclin1

This study

N/A

pcDNA3-HA-Beclin1 S30A

This study

N/A (Continued on next page)

Molecular Cell 65, 1–15.e1–e6, March 2, 2017 e2

Please cite this article in press as: Qian et al., Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy, Molecular Cell (2017), http:// dx.doi.org/10.1016/j.molcel.2017.01.027

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

pcDNA6/SFB-Beclin1 S30A

This study

N/A

pGEX-4T-1 Beclin1

This study

N/A

pGEX-4T-1 Beclin1 S30A

This study

N/A

pcDNA3-HA-ARD1 S182A

This study

N/A

pcDNA3-HA-ARD1 S228A

This study

N/A

pcDNA3-HA-ARD1 R82/G85A

This study

N/A

pGEX-4T-1 ARD1 S182A

This study

N/A

pGEX-4T-1 ARD1 S228A

This study

N/A

pcDNA3.1/hygro(+)-Flag-ATG14L

This study

N/A

pcDNA3-HA-ATG14L

This study

N/A

Flag-mTOR WT

This study

N/A

Flag-mTOR KD

This study

N/A

myc-mTOR WT

(Chen and Sarbassov dos, 2011)

N/A

myc-mTOR KD

(Chen and Sarbassov dos, 2011)

N/A

myc-Beclin1

(Russell et al., 2013)

N/A

Flag-Beclin1

(Piya et al., 2011)

N/A

EGFP-2 3 FYVE

(Piya et al., 2011)

N/A

GFP-LC3

(Piya et al., 2011)

N/A

HA-ARD1

(Kuo et al., 2010)

N/A

GST-ARD1

(Kuo et al., 2010)

N/A

HA-VPS34

(Ma et al., 2014)

N/A

mCherry-ATG14L

(Ma et al., 2014)

N/A

pCAG-twinSTREP-FLAG-VPS15

(Baskaran et al., 2014)

N/A

pCAG-twinSTREP-FLAG-VPS34

(Baskaran et al., 2014)

N/A

pCAG-twinSTREP-FLAG-Beclin1

(Baskaran et al., 2014)

N/A

GST-ATG14L

(Baskaran et al., 2014)

N/A

Flag-UVRAG

(He et al., 2013)

N/A

(Kurcinski et al., 2015)

http://biocomp.chem.uw.edu.pl/ CABSdock

Discovery Studio Visualizer

Accelrys

http://accelrys.com/

PyMOL v1.8.4.0

PyMOL

https://www.pymol.org/

UCSF Chimera

(Pettersen et al., 2004)

https://www.cgl.ucsf.edu/chimera/

Sequence-Based Reagents See Table S2 for list of primers Software and Algorithms CABS-dock server

Other

CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for reagents should be directed to and will be fulfilled by the corresponding author Zhimin Lu ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Mice For in vivo brain tumor growth experiments, 1 3 106 cells suspended in 100 mL of DMEM medium were subcutaneously injected into right flank of each 4- to 6-week-old female athymic nude mouse. Six mice were used for each group. Tumor dimensions were measured using a caliper every 3 days from day 10 after injection. Tumor volume was calculated by 0.5 3 L 3 W2 (L, length; W, width). Mice were sacrificed at day 28. Xenograft tumors were resected, weighed, and pictured. For intracranial injection, 1 3 106 cells were

e3 Molecular Cell 65, 1–15.e1–e6, March 2, 2017

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suspended in 5 ml of DMEM and intracranially injected into each 4- to 6-week-old female athymic nude mouse as described before (Yang et al., 2011). Seven mice were included for each group. 28 days after injection, mice were sacrificed. Brains were harvested, fixed in 4% formaldehyde, and embedded in paraffin. Tumor formation was determined by H&E staining. Tumor volume was calculated by 0.5 3 L 3 W2. The use of animals was approved by the Institutional Review Board of The University of Texas MD Anderson Cancer Center. Human Subjects The use of human glioblastoma samples and the clinical parameters was approved by the Institutional Review Board at Capital Medical University in Beijing, China. Informed consent was obtained from all subjects involved in this study or from parents of those who were younger than 18 years old. The human GBM samples and clinical information were from the Chinese Glioma Genome Atlas (CGGA, http://www.cgga.org.cn) and listed in Table S1. Cell Lines U87 and U251 human GBM cells, BxPC-3 pancreatic adenocarcinoma cells, MDA-MB-231 breast cancer cells, and 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum (Hyclone, Logan, UT). U87 and U251 cell lines in the experiments were authenticated using short tandem repeat profiling in The University of Texas MD Anderson Cancer Center. Glutamine or glucose starvation was carried out by culturing cells in the medium without glutamine (GIBCO, #11054) or glucose (GIBCO, #11966) supplemented with dialyzed FBS. Amino acid starvation was carried out by changing the medium to EBSS (GIBCO, #14105). For hypoxia treatment, cells were cultured under hypoxic (1% oxygen) condition. METHOD DETAILS Materials Rabbit polyclonal antibodies against PGK1 K388 acetylation and Beclin1 S30 phosphorylation were custom produced by Signalway Antibody (College Park, MD). Rabbit polyclonal antibodies against PGK1, HA and p-ERK1/2 were also obtained from Signalway Antibody. Rabbit polyclonal antibodies recognizing PGK1, p70S6K pT389 and V5 were obtained from Abcam (Cambridge, MA). Rabbit polyclonal antibody recognizing Ki-67 was purchased from Millipore (Billerica, MA). Mouse monoclonal antibody against phosphoserine was obtained from BD Biosciences (San Jose, CA). Antibodies recognizing Beclin1, Beclin1 pS93, Beclin1 pS15, VPS34, LC3B, ARD1, p70S6K pT389, p70S6K, AKT pS473, AKT, AMPKa pT172, AMPKa, ACC pS79, ACC, BNIP3, BNIP3L and BCL2 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against p62/SQSTM1, GST, a-tubulin, ERK1/2, normal rabbit IgG, and normal mouse IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibody against ATG14L was obtained from MBL International (Woburn, MA). Torin1 was purchased from LC Laboratories (Woburn, MA). Active glyceraldehyde 3-phosphate dehydrogenase (GAPDH) recombinant protein, mouse monoclonal anti-Flag and anti–His antibodies, phosphatidylinositol (PI), acetyl coenzyme A (Ac-CoA), chloroquine, EGF, Triton X-100, glyceraldehyde 3-phosphate, ATP, NADH, and N-acetyl-L-cysteine (NAC) were purchased from Sigma (St. Louis, MO). Anti-DYKDDDDK (Flag) tag affinity gel was purchased from BioLegend (San Diego, CA). Streptavidin beads were purchased from Thermo Fisher Scientific (Pittsburgh, PA). [g-32P]ATP was purchased from MP Biochemicals (Santa Ana, CA). [3H]-Phosphatidylinositol was obtained from American Radiolabeled Chemicals (St. Louis, MO). Transfection Cells were plated at a density of 4 3 105 per 60-mm dish or 1 3 105 per well of a 6-well plate 18 hr before transfection. The transfection procedure was performed as previously described (Xia et al., 2007). DNA Construction and Mutagenesis Polymerase chain reaction (PCR)-amplified human PGK1, Beclin1, ARD1, VPS34, and ATG14L were cloned into pcDNA3.1/hygro(+)Flag, pcDNA3-HA, pcDNA6/His-V5, pcDNA6/SFB, pColdI, or pGEX-4T-1 vector. pcDNA3.1/hygro(+)-Flag PGK1 K388R, PGK1 K388Q, PGK1 T378P, Beclin1 S30A, Beclin1 S15A, Beclin1 S93/96A, Beclin1 S15/93/96A; pcDNA3-HA PGK1 K388R, Beclin1 S30A, ARD1 S182A, ARD1 S228A, ARD1 R82/G85A; pcDNA6/SFB PGK1 K388R, Beclin1 S30A, pColdI PGK1 K388R, PGK1 T378P; and pGEX-4T-1 Beclin1 S30A, ARD1 S182A, and ARD1 S228A were made using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). shRNA-resistant (r) PGK1, Beclin1, and ARD1 were made using a non-sense mutation in shRNA-targeting sites, as described previously (Yang et al., 2011). Primers were list in Table S2. pGIPZ control was generated with the control oligonucleotide GCTTCTAACACCGGAGGTCTT. pGIPZ PGK1 shRNA was generated with GGATGTCTATGTCAATGATGC. pGIPZ Beclin1 shRNA, ARD1 shRNA, ATG5 shRNA, and siRNA pools against BNIP3 and BNIP3L were purchased from Thermo Fisher Scientific (Pittsburgh, PA). Purification of Recombinant Proteins His-PGK1 WT, His-PGK1 K388R, His-PGK1 T378P, GST-ARD1, GST-Beclin1, and GST-Beclin1 S30A were expressed in bacteria and purified as described previously (Xia et al., 2007).

Molecular Cell 65, 1–15.e1–e6, March 2, 2017 e4

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Immunoprecipitation and Immunoblotting Analysis The extraction of proteins using a modified buffer from cultured cells was followed by immunoprecipitation and immunoblotting using corresponding antibodies as described previously (Lu et al., 1998). Mass Spectrometry Analysis Immunoprecipitated PGK1 protein from glutamine-starved cells or the in vitro PGK1-phosphorylated Beclin1 sample was digested and analyzed by LC-MS/MS on an Obitrap-Ellite mass spectrometer (Thermo Fisher Scientific, Waltham, MA), as described previously (Jiang et al., 2014a). Streptavidin and GST Pull-Down Assay Streptavidin or glutathione agarose beads were incubated with cell lysates or purified proteins overnight. The beads were then washed with lysis buffer for 5 times. Measurement of PGK1 Activity Purified recombinant WT or mutant PGK1 (1 ng) proteins were incubated in 100 mL of reaction buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl2, 5 mM ATP, 0.2 mM NADH, 10 mM glycerol-3-phosphate, and 10 U of GAPDH) at 25 C in a 96-well plate and read at 339 nm in kinetic mode for 5 min. Measurement of ATP 1 3 106 cells were washed and lysed in ATP assay buffer. Samples were deproteinized using 10 kDa spin columns (Millipore, Billerica, MA). ATP levels were then determined using a BioVision ATP assay kit (BioVision, Milpitas, CA). In Vitro Acetylation Assay An in vitro acetylation reaction was performed as described previously (Lin et al., 2012a). In brief, purified recombinant PGK1 and ARD1 were incubated in 30 mL of reaction buffer (20 mM Tris-HCl [pH 8.0], 20% glycerol, 100 mM KCl, 1 mM DTT, 0.2 mM EDTA, 10 mM TSA, 10 mM nicotinamide, and 100 mM acetyl-CoA) at 30 C for 1 hr. The reaction was terminated by adding SDS-PAGE loading buffer. The samples were then subjected to immunoblotting analyses. In Vitro Kinase Assay The kinase reactions were performed as described previously (Fang et al., 2007). In brief, after the in vitro acetylation reaction, the acetylated PGK1 (200 ng) was washed and incubated with Beclin1 (100 ng) in 25 mL of kinase buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 50 mM MgCl2, 1 mM Na3VO4, 1 mM DTT, 5% glycerol, 0.5 mM ATP, and 10 mCi [g-32P]ATP) at 25 C for 1 hr. The reaction was terminated by adding SDS-PAGE loading buffer and heated at 100 C for 5 min. The reaction mixture was then subjected to an SDS-PAGE analysis. Protein-peptide Docking The docking between human PGK1 (PDB: 2XE7) and Beclin1 peptide 27-LDTSFKI-33 was performed by using CABS-dock server (http://biocomp.chem.uw.edu.pl/CABSdock/). The docking results were analyzed and adjusted with Discovery Studio Visualizer and PyMOL and displayed with UCSF Chimera (http://www.cgl.ucsf.edu/chimera). VPS34 Lipid Kinase Assay ATG14L-associated VPS34 complex was immunoprecipitated on beads and washed three times with lysis buffer, followed by three washes with the reaction buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, and 1 mM EDTA). The beads were then suspended in 60 mL of reaction buffer containing 10 mM MnCl2 and 10 mL of 2 mg/ml phosphatidylinositol. The reaction was started by adding ATP (10 mL of 440 mM ATP containing 10 mCi of [g-32P] ATP) and incubated at ambient temperature for 30 min. The reaction was quenched by adding 20 mL of 8 M HCl and extracted with 160 mL chloroform/methanol (1:1). Extracted phospholipid products were separated on TLC using a coated silica gel and a solvent composed of chloroform/methanol/H2O/NH4OH (v/v/v/v, 9:7:1.7:0.3). The TLC plates were dried and exposed by autoradiography to visualize PI(3)P production. Phosphatidylinositol Binding Ability Assay The ATG14L-associated VPS34 complex was immunoprecipitated on beads and washed three times using VPS34 lipid kinase reaction buffer. The beads were then incubated in 60 mL of reaction buffer containing 10 mM MnCl2, 10 mL of 440 mM ATP, and 10 mL of 2 mg/ml phosphatidylinositol (containing 1 mCi of [3H]-phosphatidylinositol) at ambient temperature for 30 min. The beads were then washed three times, and VPS34 complex-associated radioactivity was detected via liquid scintillation counting. Trypsin Digestion of VPS34 Complex ATG14L-containing VPS34 complex was purified according to previous publication (Baskaran et al., 2014). This complex was then digested with 5 ng of chymotrypsin (Sigma) for 5 min as described before (Zheng et al., 2011). The digested fragments were separated by SDS-PAGE followed by immunoblotting with an anti-VPS34 antibody.

e5 Molecular Cell 65, 1–15.e1–e6, March 2, 2017

Please cite this article in press as: Qian et al., Phosphoglycerate Kinase 1 Phosphorylates Beclin1 to Induce Autophagy, Molecular Cell (2017), http:// dx.doi.org/10.1016/j.molcel.2017.01.027

Autophagy Assay Cells grown on gelatinized coverslips were transfected with GFP-LC3 or EGFP-FYVE construct. 48 hr later, the medium was changed to complete or Gln-deprived medium. The fluorescence of GFP-LC3 or EGFP-FYVE puncta was observed under a fluorescence microscope. The numbers of puncta were counted in 10 independent visual fields at 400 3 magnification. Measurement of Glucose Consumption and Lactate Production Cells were seeded, and the medium was changed 6 hr later with non-serum DMEM. Cells were then incubated for another 20 hr. The culture medium was collected to measure the glucose and lactate concentrations. The glucose level was determined using a glucose assay kit (Sigma). The lactate level was determined using a lactate assay kit (Eton Bioscience, Inc., San Diego, CA). Glucose consumption and lactate production were normalized to cell numbers. Cell Proliferation Assay 1 3 105 cells were seeded in a 6-well plate and maintained in DMEM medium with 10% bovine calf serum during hypoxia. The cells were trypsined and counted. Data represent the means ± SD of three independent experiments. Immunohistochemical Staining Sections of paraffin-embedded human glioblastoma tissues were stained with indicated antibodies. The tissue sections were quantitatively scored according to the percentage of positive cells and staining intensity as described previously (Yang et al., 2011). The following proportion scores were assigned to the sections: 0 if 0% of tumor cells exhibited positive staining, 1 for 0%–1% positive cells, 2 for 2%–10% positive cells, 3 for 11%–30% positive cells, 4 for 31%–70% positive cells, and 5 for 71%–100% positive cells. In addition, the staining was scored on a scale of 0-3: 0, negative; 1, weak; 2, moderate; and 3, strong. The proportion and intensity scores were then added to obtain a total score ranging from 0-8 as described before (Ji et al., 2009). Scores were compared with overall survival duration, defined as the time from the date of diagnosis to death or last known follow-up examination. QUANTIFICATION AND STATISTICAL ANALYSIS All data represent the mean ± SD of at least three independent experiments. Sample number (n) indicates the number of independent biological samples in each experiment. Sample numbers and experimental repeats are indicated in figure legends. A statistical analysis was conducted with the two-tailed unpaired Student’s t test unless specifically indicated. Differences in means were considered statistically significant at p < 0.05. Significance levels are: *p < 0.05; **p < 0.001; N.S., not significant. Analyses were performed using the Microsoft Excel.

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