Protein kinase activity of the glycolytic enzyme PGK1 regulates autophagy to promote tumorigenesis

Macroautophagy/autophagy is a cellular defense response to stress conditions and is crucial for cell homeostasis maintenance. However, the precise mechanism underlying autophagy initiation, especially in response to glutamine deprivation and hypoxia, is yet to be explored. We recently discovered that PGK1 (phosphoglycerate kinase 1), a glycolytic enzyme, functions as a protein kinase, phosphorylating BECN1/Beclin 1 to initiate autophagy. Under glutamine deprivation or hypoxia stimulation, PGK1 is acetylated at K388 by NAA10/ARD1 in an MTOR-inhibition-dependent manner, leading to the interaction between PGK1 and BECN1 and the subsequent phosphorylation of BECN1 at S30 by PGK1. This phosphorylation enhances ATG14-associated PIK3C3/VPS34-BECN1-PIK3R4/VPS15 complex activity, thereby increasing phosphatidylinositol-3-phosphate (PtdIns3P) generation in the initiation stage of autophagy. Furthermore, NAA10-dependent PGK1 acetylation and PGK1-dependent BECN1 phosphorylation are required for glutamine deprivation- and hypoxia-induced autophagy and brain tumor formation. Our work reveals the important dual roles of PGK1 as a glycolytic enzyme and a protein kinase in the mutual regulation of cell metabolism and autophagy in maintaining cell homeostasis.     

Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40

Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Here, we identify PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt-mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.     

Beclin 1-mediated autophagy in hepatocellular carcinoma cells: implication in anticancer efficiency of oroxylin A via inhibition of mTOR signaling

Autophagy is a tightly-regulated catabolic process that involves the degradation of intracellular components via lysosomes. Although the pivotal role of autophagy in cell growth, development, and homeostasis has been well understood, its function in cancer prevention and intervention remains to be delineated. The aim of this study was to investigate the function and mechanism of autophagy induced by oroxylin A, a natural mono-flavonoid extracted from Scutellariae radix. We found for the first time that oroxylin A induced Beclin 1-mediated autophagy in human hepatocellular carcinoma HepG2 cells. Time-lapse video microscopy and western blotting studies showed that treatment of cells with 80 μM oroxylin A resulted in the conversion of water soluble MAP-LC3 (LC3-I) to the lipidated and autophagosome-associated form (LC3-II) after 12hours; then autophagosome-lysosome fusion and lysosome degradation after 24 hours was required in oroxylin A-mediated cell death. This induction was associated with the suppressing of PI3K-PTEN-Akt-mTOR signaling pathway by oroxylin A. Our results also showed that autophagy took place before noticeable apoptosis can be observed. It was further demonstrated that oroxylin A-triggered autophagy contributed to cell death using over-expression of autophagy-related gene (Atg5 and Atg7) and inhibition of autophagy by siBeclin 1 and 3-methyladenine (3-MA). In vivo study, oroxylin A inhibited xenograft tumor growth and induced obvious autophagy in tumors. Taken together, we conclude that oroxylin A exhibits autophagy-mediated antitumor activity in a dose and time-dependent manner in vivo and in vitro. These findings define and support a novel function of autophagy in promoting death of hepatocellular carcinoma cells.     

Inhibition of mTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1

AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser(757) and inhibited known targets of mTORC1 (p-mTOR Ser(2448), p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser(2481), p-AKT Ser(473)). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser(757) and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.     

Bim inhibits autophagy by recruiting beclin 1 to microtubules

Bim is a proapoptotic BH3-only Bcl-2 family member.?In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in?vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.     

Phosphorylation of PRAS40 on Thr246 by PKB/AKT facilitates efficient phosphorylation of Ser183 by mTORC1

Type 2 diabetes is associated with alterations in protein kinase B (PKB/Akt) and mammalian target of rapamycin complex 1 (mTORC1) signalling. The proline-rich Akt substrate of 40-kDa (PRAS40) is a component of mTORC1, which has a regulatory function at the intersection of the PKB/Akt and mTORC1 signalling pathway. Phosphorylation of PRAS40-Thr246 by PKB/Akt, and PRAS40-Ser183 and PRAS40-Ser221 by mTORC1 results in dissociation from mTORC1, and its binding to 14-3-3 proteins. Although all phosphorylation sites within PRAS40 have been implicated in 14-3-3 binding, substitution of Thr246 by Ala alone is sufficient to abolish 14-3-3 binding under conditions of intact mTORC1 signalling. This suggests that phosphorylation of PRAS40-Thr246 may facilitate efficient phosphorylation of PRAS40 on its mTORC1-dependent sites. In the present study, we investigated the mechanism of PRAS40-Ser183 phosphorylation in response to insulin. Insulin promoted PRAS40-Ser183 phosphorylation after a euglycaemic–hyperinsulinaemic clamp in human skeletal muscle. The insulin-induced PRAS40-Ser183 phosphorylation was further evidenced in vivo in rat skeletal and cardiac muscle, and in vitro in A14 fibroblasts, 3T3L1 adipocytes and L6 myotubes. Inhibition of mTORC1 by rapamycin or amino acid deprivation partially abrogated insulin-mediated PRAS40-Ser183 phosphorylation in cultured cell lines. However, lowering insulin-induced PRAS40-Thr246 phosphorylation using wortmannin or palmitate in cell lines, or by feeding rats a high-fat diet, completely abolished insulin-mediated PRAS40-Ser183 phosphorylation. In addition, replacement of Thr246 by Ala reduced insulin-mediated PRAS40-Ser183 phosphorylation. We conclude that PRAS40-Ser183 is a component of insulin action, and that efficient phosphorylation of PRAS40-Ser183 by mTORC1 requires the phosphorylation of PRAS40-Thr246 by PKB/Akt.     

PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis

TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.     

Beclin 1-independent autophagy induced by a Bcl-XL/Bcl-2 targeting compound,Z18

Inhibitors of Bcl-X_L/Bcl-2 can induce autophagy by releasing the autophagic protein Beclin 1 from its complexes with these proteins. Here we report a novel compound targeting the BH3 binding groove of Bcl-X_L/Bcl-2, Z18, which efficiently induces autophagy-associated cell death in HeLa cells, without apparent apoptosis. Unexpectedly, the inhibition of Beclin 1 and phosphatidylinositol 3-kinase have no obvious effect on Z18-induced autophagy in HeLa cells, implying that it is a non-canonical Beclin 1-independent autophagy. Meanwhile, the accumulation of autophagosomes is positively correlated with Z18-induced cell death and the full flux of autophagy is not necessary.     

PKC delta and tissue transglutaminase are novel inhibitors of autophagy in pancreatic cancer cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKC delta) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKC delta/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKC delta/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.     

A novel role for autophagy in neurodevelopment

We recently showed that Ambra 1, a WD40-containing approximately 130 KDa protein, is a novel activating molecule in Beclin 1-regulated autophagy and plays a role in the development of the nervous system. Ambra 1 binds to Beclin 1 and favors Beclin 1/Vps34 interaction. At variance with these factors, Ambra 1 is highly conserved among vertebrates only, and its expression is mostly confined to the neuroepithelium during early neurogenesis. Ambra 1 functional inactivation in mouse led to lethality in utero (starting from embryonic day 14.5), characterized by severe neural tube defects associated with autophagy impairment, unbalanced cell proliferation, accumulation of ubiquitinated proteins, and excessive apoptosis. We also demonstrated that hyperproliferation was the earliest detectable abnormality in the developing neuroepithelium, followed by a wave of caspase-dependent cell death. These findings provided in vivo evidence supporting the existence of a complex interplay between autophagy, cell proliferation and cell death during neural development in mammals. In this article, we review our findings in the contexts of autophagy and neurodevelopment and consider some of the issues raised.     

PKCδ and Tissue Transglutaminase are Novel Inhibitors of Autophagy in Pancreatic Cancer Cells

Apoptosis (type I) and autophagy (type II) are both highly regulated forms of programmed cell death and play crucial roles in physiological processes such as the development, homeostasis and selective, moderate to massive elimination of cells, if needed. Accumulating evidence suggests that cancer cells, including pancreatic cancer cells, in general tend to have reduced autophagy relative to their normal counterparts and premalignant lesions, supporting the contention that defective autophagy provides resistance to metabolic stress such as hypoxia, acidity and chemotherapeutics, promotes tumor cell survival and plays a role in the process of tumorigenesis. However, the mechanisms underlying the reduced capability of undergoing autophagy in pancreatic cancer remain elusive. In a recent study, we demonstrated a novel mechanism for regulation of autophagy in pancreatic ductal carcinoma cells. We found that protein kinase C-delta (PKCδ) constitutively suppresses autophagy through induction of tissue transglutaminase (TG2). Inhibition of PKCδ/TG2 signaling resulted in significant autophagic cell death that was mediated by Beclin 1. Elevated expression of TG2 in pancreatic cancer cells has been implicated in the development of drug resistance, metastatic phenotype and poor patient prognosis. In conclusion, our data suggest a novel role of PKCδ/TG2 in regulation of autophagy, and that TG2 may serve as an excellent therapeutic target in pancreatic cancer cells.

Addendum to:

Tissue Transglutaminase Inhibits Autophagy in Pancreatic Cancer Cells

U. Akar, B. Ozpolat, K. Mehta, J. Fok, Y. Kondo and G. Lopez-Berestein

Mol Cancer Res 2007; 5:241-9     

Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy

Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The anti-apoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Here, we show that wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and that cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death.     

Autophagy in tumorigenesis and energy metabolism: friend by day, foe by night

Autophagy is the mechanism by which cells consume parts of themselves to survive starvation and stress. This self-cannibalization limits cell death and tissue inflammation, recycles energy and biosynthetic substrates and removes damaged proteins and organelles, accumulation of which is toxic. In normal tissues, autophagy-mediated damage mitigation may suppress tumorigenesis, while in advanced tumors macromolecular recycling may support survival by buffering metabolic demand under stress. As a result, autophagy-activation in normal cells may suppress tumorigenesis, while autophagy inhibition may be beneficial for the therapy of established tumors. The mechanisms by which autophagy supports cancer cell metabolism are slowly emerging. As cancer is being increasingly recognized as a metabolic disease, how autophagy-mediated catabolism impacts cellular and mammalian metabolism and tumor growth is of great interest. Most cancer therapeutics induce autophagy, either directly by modulating signaling pathways that control autophagy in the case of many targeted therapies, or indirectly in the case of cytotoxic therapy. However, the functional consequence of autophagy induction in the context of cancer therapy is not yet clear. A better understanding of how autophagy modulates cell metabolism under various cellular stresses and the consequences of this on tumorigenesis will help develop better therapeutic strategies against cancer prevention and treatment.     

UVRAG: a new player in autophagy and tumor cell growth

Autophagy has a well-documented role in the maintenance of homeostasis and the response to stressful environments and it is often deregulated in various human diseases including cancer. The regulation of the Beclin 1-PI3KC3 complex lipid kinase activity is a critical element in the autophagy signaling pathway. Previous studies(1) have demonstrated that Beclin 1-PI3KC3-mediated autophagy is negatively regulated by a proto-oncogene Bcl-2. We have recently identified a novel coiled-coil UVRAG tumor suppressor candidate, which positively engages in Beclin 1-dependent autophagy. UVRAG interacts with Beclin 1, leading to activation of autophagy and thereof inhibition of tumorigenesis. This finding adds a new player to the emerging picture of the autophagy network, under-scoring the importance of the coordinated activity between Bcl-2 and UVRAG in the regulation of Beclin 1-PI3KC3-mediated autophagy and tumor cell control.     

The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser(183)). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser(183)) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.     

A tumor-associated mutation of FYVE-CENT prevents its interaction with Beclin 1 and interferes with cytokinesis

The tumor suppressor activity of Beclin 1 (BECN1), a subunit of class III phosphatidylinositol 3-kinase complex, has been attributed to its regulation of apoptosis and autophagy. Here, we identify FYVE-CENT (ZFYVE26), a phosphatidylinositol 3-phosphate binding protein important for cytokinesis, as a novel interacting protein of Beclin 1. A mutation in FYVE-CENT (R1945Q) associated with breast cancer abolished the interaction between FYVE-CENT and Beclin 1, and reduced the localization of these proteins at the intercellular bridge during cytokinesis. Breast cancer cells containing the FYVE-CENT R1945Q mutation displayed a significant increase in cytokinetic profiles and bi-multinuclear phenotype. Both Beclin 1 and FYVE-CENT were found to be downregulated in advanced breast cancers. These findings suggest a positive feedback loop for recruitment of FYVE-CENT and Beclin 1 to the intercellular bridge during cytokinesis, and reveal a novel potential tumor suppressor mechanism for Beclin 1.     

Beclin 1 cleavage by caspase-3 inactivates autophagy and promotes apoptosis

Autophagy and apoptosis are both highly regulated biological processes that play essential roles in tissue homeostasis, development and diseases. Autophagy is also described as a mechanism of death pathways, however, the precise mechanism of how autophagy links to cell death remains to be fully understood. Beclin 1 is a dual regulator for both autophagy and apoptosis. In this study we found that Beclin 1 was a substrate of caspase-3 with two cleavage sites at positions 124 and 149, respectively. Furthermore, the autophagosome formation occurred, followed by the appearance of morphological hallmarks of apoptosis after staurosporine treatment. The cleavage products of Beclin 1 reduced autophagy and promoted apoptosis in HeLa cells and the cells in which Beclin 1 was stably knocked down by specific shRNA. In addition, the cleavage of Beclin 1 resulted in abrogating the interaction between Bcl-2 with Beclin 1, which could be blocked by z-VAD-fmk. Thus, our results suggest that the cleavage of Beclin 1 by caspase-3 may contribute to inactivate autophagy leading towards augmented apoptosis.     

The role of Beclin 1 in IR-induced crosstalk between autophagy and G2/M cell cycle arrest

ObjectivesBeclin 1 is a well-established core mammalian autophagy protein. Autophagy has been demonstrated to play roles in cellular responses to DNA damage, such as cell cycle regulation and apoptosis. In the present study, we investigated the exact mechanism by which Beclin 1 acts as a bridge between autophagy and cell cycle, when cells are exposed to ionizing radiation (IR).Materials and methodsWestern blotting and coimmunoprecipitation were performed to investigate protein expression levels and interactions. Immunofluorescence was used to monitor the localization and distribution of the indicated proteins. The levels of apoptosis and cell cycle changes were evaluated by flow cytometry. Double thymidine deoxyribonucleoside (TdR) blocking was conducted to differentiate G2 from mitotic delay. In vitro kinase assays using ATM kinase were performed to elucidate the specific phosphorylation site in Beclin 1.ResultsIn this study, we show that Beclin 1 knockdown reduces IR-induced autophagy. IR enhanced Beclin 1/PIK3CIII complex activity as demonstrated by the results of coimmunoprecipitation and immunofluorescence assays. An investigation to assess the possible relationship between autophagy and G2/M arrest showed that, similar to the autophagy inhibitor 3MA, Beclin 1 knockdown delayed IR-induced G2/M arrest. Furthermore, the interactions between Beclin 1 and several G2/M checkpoint-related proteins, namely, PLK1 and CDC25C, were observed to increase. In addition, we observed that both 3MA and Beclin 1 inhibition decreased IR-induced apoptosis. Regarding the potential mechanism associated with this phenomenon, we showed that IR induced the interaction between Beclin 1 and Tip60 as well as their redistribution. Furthermore, we demonstrated that Beclin 1 T57 may be a targeted phosphorylation site for ATM.ConclusionsIn the present study, we demonstrate the crucial and intricate roles of Beclin 1 in IR-induced autophagy, G2/M cell cycle arrest, and apoptosis. Additionally, Tip60 and ATM were identified as important molecular regulators of Beclin 1. Our findings show the precise mechanism of crosstalk between IR-induced autophagy and G2/M cell cycle arrest.     

The role of autophagy in spinal cord injury

Previous studies have indicated that autophagy has an important function, not only in many neurodegenerative diseases, but also in traumatic and ischemic brain injury. However, no study has previously shown the contribution of autophagy to neural tissue damage after spinal cord injury. We recently investigated that the alterations in Beclin 1 expression and the involvement of autophagy and autophagic cell death after spinal cord injury using a spinal cord hemisection model in mice. The results showed that the expression of Beclin 1 dramatically increased in the damaged neural tissue and induced autophagic cell death after a spinal cord injury. These observations suggested that the increased expression of Beclin1 activates autophagy, while mediating a novel cell death mechanism at the lesion site in response to spinal cord injury. Here we discuss several unsolved issues and review the evidence in related articles regarding the role of autophagy and its contribution to the mechanism of cell death in spinal cord injury.