Transcription factors and cognate signalling cascades in the regulation of autophagy.

1

Biol. Rev. (2015), pp. 000–000. doi: 10.1111/brv.12177

Transcription factors and cognate signalling cascades in the regulation of autophagy Vemika Chandra, Ella Bhagyaraj, Raman Parkesh and Pawan Gupta∗ CSIR-Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India

ABSTRACT Autophagy is a process that maintains the equilibrium between biosynthesis and the recycling of cellular constituents; it is critical for avoiding the pathophysiology that results from imbalance in cellular homeostasis. Recent reports indicate the need for the design of high-throughput screening assays to identify targets and small molecules for autophagy modulation. For such screening, however, a better understanding of the regulation of autophagy is essential. In addition to regulation by various signalling cascades, regulation of gene expression by transcription factors is also critical. This review focuses on the various transcription factors as well as the corresponding signalling molecules that act together to translate the stimuli to effector molecules that up- or downregulate autophagy. This review rationalizes the importance of these transcription factors functioning in tandem with cognate signalling molecules and their interfaces as possible therapeutic targets for more specific pharmacological interventions. Key words: autophagy, transcription factors, nuclear receptors, gene regulation, signalling molecules. CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Transcriptional regulators of autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Transcription factor EB (TFEB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Transcription factor binding to immunoglobulin heavy constant mu enhancer 3 (TFE3) . . . . . . (3) Zinc finger with KRAB and SCAN domains 3 (ZKSCAN3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Forkhead box-containing protein, O subfamily (FOXO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) E2 transcription factor (E2F) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6) Unscheduled meiotic gene expression protein 6 (UME6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (7) Activating transcription factor (ATF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (8) Hypoxia-inducible factor-1 (HIF-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (9) Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-𝜅B) . . . . . . . . . . . . . . . . . . . . . . . . . . (10)p53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (11)Vitamin D3 receptor (VDR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (12)Peroxisome proliferator-activated receptor gamma (PPAR𝛾) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (13)Retinoic acid receptor (RAR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (14)CCAAT/enhancer-binding protein 𝛽 (C/EBP𝛽) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (15)Sterol regulatory element-binding protein 2 (SREBP-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (16)Androgen receptor (AR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (17)Nuclear receptor subfamily 4, group A, member 1 (NR4A1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (18)Glucocorticoid receptor (GR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Critical signalling cascades of autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Mammalian target of rapamycin (mTOR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) AMP-activated protein kinase (AMPK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Eukaryotic initiation factor 2 (eIF2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 3 4 5 5 8 8 8 9 9 9 10 10 10 10 11 11 11 11 11 11 12 13

* Address for correspondence (Tel: +91-172-666-5221; Fax: +91-172-269-0585; E-mail: [email protected]). Biological Reviews (2015) 000–000 © 2015 Cambridge Philosophical Society

Vemika Chandra and others

2

(4) PI3K signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) Mitogen-activated protein kinase (MAPK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (a) p38𝛼 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (b) JNKs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (c) ERK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (6) B-cell CLL/lymphoma 2 (BCL2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Post-Translational regulation of autophagy-associated proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Pharmacological modulation of the transcriptional regulators of autophagy, their cognate signalling cascades and the interface between them . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION Homeostasis is a sine qua non feature of cell viability and provides the imperative balance between biosynthesis and the recycling of cellular macromolecules. Macroautophagy, an evolutionarily conserved ‘self-eating’ vesicular transport process, is rather a recycling than a degradative pathway (Levine & Klionsky, 2004; Shintani & Klionsky, 2004; Klionsky, 2005; Esclatine, Chaumorcel & Codogno, 2009). Diverse stimuli that can modulate signalling pathways and potentially induce autophagy include nutrient depletion, growth hormone depletion, hypoxia, activated oncogenes, endoplasmic reticulum (ER) stress, microbial invasion, and drugs targeting mammalian target of rapamycin (mTOR) and insulin signalling pathways (Mortimore & Poso, 1987; Cuervo et al., 2005; Mizushima, 2005; Bao et al., 2010). These stimuli correlate well with autophagy-associated physiological processes such as cell differentiation and development, tumour suppression, adaptation to starvation, innate and adaptive immunity, and cell death (Gozuacik & Kimchi, 2004; Levine & Klionsky, 2004; Ogata et al., 2006; Yorimitsu et al., 2006; Deretic & Levine, 2009; Young et al., 2009). Autophagy dysfunction has been implicated in a growing number of human diseases and comorbidities, ranging from infectious diseases to cancer and neurodegeneration (Mathew, Karantza-Wadsworth & White, 2007; Nixon, Yang & Lee, 2008; Lee, 2009; Orvedahl & Levine, 2009; Deretic, 2010; Nixon, 2013). In brief, autophagy involves formation of intracytoplasmic vacuoles called autophagosomes, the hallmark of this dynamic process (Seglen & Bohley, 1992; Fengsrud et al., 2000; Kovacs et al., 2000). Post-induction, autophagy-related (ATG) proteins are recruited to a phagophore assembly site referred to as the pre-autophagosomal structure (PAS) (Ichimura et al., 2000; Suzuki et al., 2001; Kim et al., 2002; Klionsky et al., 2003). In cooperation with specific soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein receptors (SNAREs) and tethering factors, ATG proteins orchestrate the fusion of membranes derived from the Golgi complex, endosomes, and plasma membrane

13 13 13 14 14 14 15 16 18 18 18

for the generation of the phagophore (Chen & Scheller, 2001; Moreau et al., 2011; Nair & Klionsky, 2011). The PAS of yeast is probably distinct from the precursor structure of autophagosomes seen in mammalian cells (Mizushima, Ohsumi & Yoshimori, 2002). Starvation allows phosphatidylinositol-3-phosphate [PtdIns(3)P] bound to double FYVE domain-containing protein 1 (DFCP1) to translocate to specific sites at the ER and Golgi membranes. The structure formed at the ER where the autophagic proteins aggregate is called the omegasome, and it leads to the formation of autophagosomes (Cheung et al., 2001; Ridley et al., 2001; Axe et al., 2008; Polson et al., 2010; Lu et al., 2011). The autophagosomes fuse with lysosomes (or vacuoles in plants and yeast) to form autolysosomes, and their contents are broken down by hydrolases in the lumen (Liang et al., 2008; Funderburk, Wang & Yue, 2010; Boya, Reggiori & Codogno, 2013) (Fig. 1). The discovery of ATG proteins and their ability to affect autophagosome formation has led to an emphasis on the study of their regulation by several kinases and the associated post-translational modifications (PTMs) (McEwan & Dikic, 2011; Mizushima, Yoshimori & Ohsumi, 2011). Additional targets of interest, however, are emerging within the network of transcription factors that regulate expression of the ATG genes. These proteins transduce physiological signals and can themselves be modified in the process of relaying upstream stimuli to downstream effectors. Herein, we present a comprehensive review of current developments in autophagy and in particular, the transcription factors and cognate signalling cascades that modulate it. We first examine the transcription factors, then the corresponding signalling cascades, followed by the PTMs of autophagy-related proteins. Finally, we highlight the amenability of these transcription factors to pharmacological targeting by specific agonists and antagonists and encourage the cognitive exploration of the interface of this network, which may be manipulated for fine-tuning of cellular processes. We advocate looking beyond targeting of the major signalling cascades, with consideration of off-target effects.

Biological Reviews (2015) 000–000 © 2015 Cambridge Philosophical Society

Transcription and signalling proteins in autophagy

3

Fig. 1. Steps induced in autophagy. Stimuli such as starvation, hypoxia, and nutrient depletion induce nucleation of the phagophore membrane. The whole process is regulated by autophagy-related (ATG) gene products. ATG proteins comprise the core machinery, which assembles at the phagophore assembly site known as the pre-autophagososmal structure (PAS) in a sequential and concerted order. In a ULK1-mediated process, class III PI3K VPS34 generates PtdIns(3)P at the phagophore; PtdIns(3)P is required for the recruitment of WIPI proteins and DFCP1. The elongation and expansion steps in autophagosome formation involve the ATG12–ATG5 and LC3-PE or LC3-II conjugation systems. After the phagophore membrane encloses its cargo to form the double-membraned autophagosome, the autophagosome fuses with a lysosome to form an autolysosome. The sequestered cytosolic components are degraded by the action of hydrolases present in the lumen of the lysosome, and recycled. ATG5, autophagy related 5; ATG12, autophagy related 12; ATG16L, autophagy related 16-like 1; DFCP1, double FYVE domain-containing protein 1; LC3-PE/LC3-II, LC3-phosphatidylethanolamine; PI3K, phosphoinositide-3-kinase; PtdIns(3)P, phosphatidylinositol-3-phosphate; ULK1, Unc-51 like autophagy activated kinase 1; VPS34, vacuolar protein sorting 34; WIPI 2, WD repeat domain, phosphoinositide interacting protein 2.

II. TRANSCRIPTIONAL REGULATORS OF AUTOPHAGY The homeostatic control system is highly complex and exceptionally modulatory in nature; it controls the internal environment of the cell by numerous compensatory regulatory mechanisms involving various transcription factors. These proteins do not remain in one state but instead keep switching between states to maintain equilibrium. The ‘on’ and ‘off’ states of these molecules allow the temporal and spatial regulation of effector proteins of autophagy. Some of the transcription factors exclusively upregulate the expression of the target genes, others repress, and a few act both as an activator and a suppressor as byzantine regulators, see Table 1 and Figs 2 and 3. Any violation or perturbation of these

transcriptional regulatory circuits leads to pathophysiological conditions. The factors described here regulate various steps in autophagosome induction, formation, and maturation. (1) Transcription factor EB (TFEB) TFEB is a basic helix–loop–helix (HLH) leucine zipper multitasking transcription factor that regulates lysosomal biogenesis, autophagy, and endocytosis (Sardiello et al., 2009; Pena-Llopis et al., 2011; Settembre et al., 2011, 2012). Ectopic expression of TFEB results in increased expression of autophagy genes, similar to that seen under starvation conditions (Settembre et al., 2011). It regulates the expression of genes involved at various steps in the autophagy process, including UV radiation resistance-associated gene (UVRAG), WD repeat domain phosphoinositide interacting



4 (WIPI ), microtubule-associated proteins 1A/1B light chain 3B (MAPLC3B), sequestosome 1 (SQSTM1/p62), vacuolar protein sorting 11 homolog (VPS11), vacuolar protein sorting protein 18 (VPS18), and autophagy related 9 homolog B (ATG9B) (Settembre et al., 2011). TFEB also regulates gene expression downstream of mammalian target of rapamycin complex 1 (mTORC1) by interacting with the v-ATPase/mTORC1 complex on the lysosomal surface and sensing lysosomal state. Activity of TFEB

requires its localization to the nucleus in a complex, context-dependent mechanism that involves PTM. (2) Transcription factor binding to immunoglobulin heavy constant mu enhancer 3 (TFE3) TFE3, like TFEB, also harbours a basic helix–loop– helix leucine zipper DNA-binding domain, which binds MUE3-type E-box sequences in gene promoters. It belongs to the microphthalamia-associated

Table 1. List of transcription factors with cognate signalling proteins and associated post-translational modification that positively or negatively regulate autophagy

Transcription factor TFEB

Organism Human

Impact on autophagy Upregulates

TFE3

Human

Upregulates

ZKSCAN3

Human

Downregulates

FOXO

Human, mouse

Upregulates

E2F

Human

Upregulates

UME6 ATF4

Yeast Human

Downregulates Upregulates

ATF5 HIF-1

Human Human, mouse

Downregulates Upregulates

NF-𝜅B

Human

p53

Human, mouse

Upregulates/ downregulates Nuclear p53: upregulates

Targets UVRAG, WIPI, MAPLC3B, SQSTM1, VPS11, VPS18, ATG9B

ATG16L1, ATG9B, GABARAP-L1, WIPI, UVRAG MAPLC3B, ULK1, ATG18b, DFCP1, Diras3, Rela, Tak1, Cdkn2a, Stx5, Sec22b, Bloc1s1, Rilp, Rptor, Akt1, Lamtor2, Ywhaz, Impa2, Ubqln2, CTSA ATG8, GABARAP-L1, VPS34, BECLIN1, glutamine synthtetase, BNIP3

MAPLC3B, ULK1, DRAM, ATG5, BNIP3, BECLIN 1 ATG8 HRK, PUMA, NOXA, MAPLC3B, ULK1 mTOR BNIP3

Human

Upregulates/ downregulates

References

ERK, mTORC1

Settembre et al. (2011)

Phosphorylation – S142↓, S211↓ mTOR

Martina et al. (2014)

—

Chauhan et al. (2013)

PI3K/AKT, SGK, SIRT1

Lin et al. (2013), van der Vos et al. (2012), Sengupta et al. (2009), Zhao et al. (2008), Mammucari et al. (2007) and Zhao et al. (2007)

Phosphorylation of FOXO3a – T32 ↓, S315 ↓, S253 ↓ Deacetylation of FOXO1 ↑ —

— — — —

SKP2, BECLIN 1

—

DRAM, PTEN, TSC (1/2), AEN/ISG20L1, Sestrins, ATG8

HDAC1, PIAS4

Cytoplasmic p53: downregulates PPAR𝛾

Cognate signalling molecules/PTMs

Cathepsin L, HIF-1𝛼


Deacetylation – K382 (h) and K379 (m) ↑ Acetylation and SUMOylation – K120 and K386 ↑ —

Polager et al. (2008), Tracy et al. (2007) and Weinmann et al. (2001) Bartholomew et al. (2012) Pike et al. (2012), (2013) Sheng et al. (2011) Bellot et al. (2009) and Zhang et al. (2008) Copetti et al. (2009) and Chen et al. (2008) Eby et al. (2010), Budanov & Karin (2008), Tasdemir et al. (2008), Crighton et al. (2006) and Stambolic et al. (2001) Mahmood et al. (2011) and Zhou et al. (2009)


5

Table 1. Continued Transcription factor

Organism

Impact on autophagy

C/EBP𝛽

Human

Upregulates

SREBP-2

Mouse

Upregulates

Targets ULK1, GABARAP-L1, BNIP3, LC3, ATG4 Maplc3, Atg4B, Atg4D

Cognate signalling molecules/PTMs

References

—

Ma et al. (2011)

—

Seo et al. (2011)

AEN/ISG20L1, apoptosis enhancing nuclease/interferon stimulated exonuclease gene 20 kDa-like 1; AKT, protein kinase B; ATF4, activating transcription factor 4; ATF5, activating transcription factor 5; ATG ‘#’, autophagy related ‘#’ (#: 4B/4D/5/8/9B/16L1/18B); Bloc1S1, biogenesis of lysosomal organelles complex-1, subunit 1; BNIP3, BCL2/adenovirus E1B 19 kDa interacting protein 3; Cdkn2a, cyclin-dependent kinase inhibitor 2A; C/EBP𝛽, CCAAT/enhancer binding protein beta; CTSA, cathepsin A; DFCP1, double FYVE domain-containing protein 1; Diras3, distinct subgroup of the Ras family member 3; DRAM, DNA-damage regulated autophagy modulator 1; E2F1, E2 transcription factor 1; ERK, extracellular-signal-regulated kinase; FOXO, forkhead box O; GABARAP-L1, GABA(A) receptor-associated protein like 1; HDAC1, histone deacetylase 1; HIF-1, hypoxia inducible factor 1; HRK, harakiri, BCL2 interacting protein; IMPA2, inositol(myo)-1(or 4)-monophosphatase 2; IP3R, inositol trisphosphate receptor; LC3/MAPLC3, microtubule-associated protein 1 light chain 3; LAMTOR2, late endosomal/lysosomal adaptor, MAPK and mTOR activator; mTORC1, mammalian target of rapamycin complex 1; NF𝜅B, nuclear factor kappa-light-chain-enhancer of activated B cells; np53, nuclear p53; NOXA or PMAIP1, Phorbol-12-myristate-13-acetate-induced protein 1; PI3K, phosphatidylinositide 3-kinases; PIAS4, protein inhibitor of activated STAT 4; PPAR𝛾, peroxisome proliferator-activated receptor gamma; PTEN, phosphatase and tensin homolog; PUMA, p53 upregulated modulator of apoptosis; Rela, v-rel avian reticuloendotheliosis viral oncogene homolog A; RILP, Rab interacting lysosomal protein; RPTOR, regulatory associated protein of mTOR, complex 1; SEC22b, SEC22 vesicle trafficking protein homolog B; SGK, serum/glucocorticoid regulated kinase; SIRT1, sirtuin 1; SKP2, S-phase kinase-associated protein 2; SREBP-2, sterol regulatory element binding protein 2; Stx5, syntaxin 5; SQSTM1, sequestosome 1; Tak1, transforming growth factor beta activated kinase-1; TFE3, transcription factor binding to IGHM (immunoglobulin heavy constant mu) enhancer 3; TFEB, transcription factor EB; TSC1/2, tuberous sclerosis 1/2; UBQLN2, ubiquilin 2; ULK1, Unc-51 like autophagy activated kinase 1; UME6, unscheduled meiotic gene expression protein 6; UVRAG, UV radiation-resistance associated gene; VPS ‘#’, vacuolar protein sorting ‘#’ (#: 11/18/34); WIPI, WD repeat domain, phosphoinositide interacting protein; YWHAZ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta; ZKSCAN3, zinc finger with KRAB and SCAN domains 3. ↑ and ↓ indicate upregulation and downregulation of autophagy process, respectively. h and m indicate protein modification in human and mouse, respectively.

transcription factor (MiTF) and TFE (MiTF/TFE) family, of which TFEB is also a member. TFE3 regulates the expression of genes that are important for the formation of autophagosomes [autophagy related 16-like 1 (ATG16L1), ATG9B, GABA(A) receptor-associated protein-like 1 (GABARAP-L1), and WIPI ] and their fusion with lysosomes (UVRAG) (Martina et al., 2014). It plays a critical role in the cellular response to nutrient depletion, and does so in the same way as TFEB, in that it translocates to the nucleus and binds to coordinated lysosomal expression and regulation (CLEAR) elements present in the promoter region of lysosomal genes to promote lysosome biogenesis. (3) Zinc finger with KRAB and SCAN domains 3 (ZKSCAN3) ZKSCAN3 is a zinc-finger protein containing a DNA binding domain for its transcriptional activity. It was first identified as a contributor to the malignancy of colorectal cancer and later in prostate cancer cell progression (Yang et al., 2008a; Zhang et al., 2012). Other functions of ZKSCAN3 include apoptosis, cellular proliferation, and neoplastic transformation. Cyclin D2, vascular endothelial growth factor (VEGF ), and integrin beta 4 are its direct downstream gene targets (Yang et al., 2008b, 2011a). Recently, ZKSCAN3 has been identified as a master transcriptional repressor of autophagy (Chauhan et al., 2013). It is negatively regulated by starvation and acts in opposition to TFEB. Specifically, it represses autophagy and lysosome biogenesis

by downregulating essential genes involved at multiple steps, including initiation of autophagosome biogenesis, maturation of autophagosomes to autolysosomes, and repression of lysosomal hydrolases and lysosome H+ transporting ATPase subunits. Given the opposite responses of ZKSCAN3 and TFEB to starvation and their opposing roles in regulation of autophagy, it is tempting to speculate that together they act to fine-tune the autophagy process. (4) Forkhead box-containing protein, O subfamily (FOXO) FOXO subfamily of transcription factors, particularly FOXO1 and 3, are pivotal for relaying stress stimuli to elicit autophagic responses (Mammucari et al., 2007; Zhao et al., 2007, 2010). Their essentiality in stress-induced autophagy and their ability independently to regulate multiple ATG genes, including ATG8/MAPLC3B, GABARAP-L1, ATG12, ATG4B, vacuolar protein sorting 34 (VPS34), BECLIN 1, glutamine synthetase, and B-cell CLL/lymphoma 2 (BCL2)/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), have given the associated responses the moniker of FOXOphagy, which involves direct induction of autophagy or repression of the mTOR pathway (Zhao et al., 2007; Demontis & Perrimon, 2010; van der Vos et al., 2012; Lin et al., 2013). The induction of glutamine synthetase expression by FOXO transcription factors results in elevated levels of glutamine, which in part impedes



6

Fig. 2. Association and regulation of core machinery proteins and transcriptional regulators in autophagy. In response to stimuli, autophagy is initiated by the activation of ULK1 complex (ULK1/2–ATG13–FIP200). This complex in turn activates VPS34, a class-III PI3K which is essential for the nucleation of the phagophore membrane. VPS34 forms a complex with BECLIN 1, VPS15, and ATG14. The PI3K complex, ULK1, and ATG9 recruit ATG2 to the PAS, where ATG2 interacts with ATG18 followed by conjugation of the ATG12–ATG5 complex and ATG16. Autophagosomes are formed with the addition of the LC3–ATG4–ATG3–ATG7 lipidation machinery. The ATG12–ATG5–ATG16 complex dissociates from the membrane and leaves behind LC3. The various transcription factors are colour-coded to indicate their effect on autophagy. Green represents autophagy inducers, red represents autophagy inhibitors, and yellow represents those transcription factors that have been reported for both functions, depending on cell type and state. These transcription factors are depicted at the autophagy steps they are known to or may regulate. AMPK, AMP-activated protein kinase 𝛼; ATF4, activating transcription factor 4; ATF5, activating transcription factor 5; ATG ‘#’, autophagy related ‘#’ (#: 2/3/4/5/7/9/12/13/14/16/18); BCL2, B-cell CLL/lymphoma 2; C/EBP𝛽, CCAAT/enhancer binding protein beta; E2F1, E2 transcription factor 1; FIP200, focal adhesion kinase family interacting protein of 200 kD; FOXO, forkhead box O; HIF-1, hypoxia inducible factor 1; LC3/MAPLC3, microtubule-associated protein 1 light chain 3; LC3-PE/LC3-II, LC3-phosphatidylethanolamine; mTORC1, mammalian target of rapamycin complex 1; NF𝜅B, nuclear factor kappa-light-chain-enhancer of activated B cells; np53, nuclear p53; PPAR𝛾, peroxisome proliferator-activated receptor gamma; RXR, retinoid X receptor; SREBP-2, sterol regulatory element binding protein 2; TFEB, transcription factor EB; ULK1, Unc-51 like autophagy activated kinase 1; UME6, unscheduled meiotic gene expression protein 6; VDR, vitamin D receptor; VPS15, vacuolar protein sorting 15; VPS34, vacuolar protein sorting 34; ZKSCAN3, zinc finger with KRAB and SCAN domains 3.

the localization of the mTORC1 complex to the lysososome and, as such, induces autophagy (van der Vos et al., 2012). In rat cardiomyocytes, both FoxO1 and FoxO3 have been reported to bind to the promoters of Gabarap-l1 and Atg12. However, preferential

binding to autophagy gene regulatory elements has been observed: Atg12 gene expression is preferentially activated by FoxO1, whereas FoxO3 preferentially activates expression of microtubule-associated protein 1 light chain 3 (Map1lc3), which is crucial for



7

Fig. 3. Transcriptional regulation of autophagy-related genes and associated signalling molecules. Stimuli such as nutrient depletion, starvation, and hypoxia increase the cell’s AMP:ATP ratio and activate AMPK. Activated AMPK inhibits mTORC1 activity, which causes activation of the ULK1 complex essential for nucleation of the phagophore membrane. Starvation also activates JNK1, which phosphorylates BCL2 and releases BECLIN 1 for formation of the VPS34 complex. Transcription factors regulating expression of various autophagy genes represent only a minor fraction of a larger genomic pool of autophagy regulators. Colour coding is as in Fig. 2. For abbreviations see Fig. 2 legend and as abbreviated below. AEN/ISG20L1, apoptosis enhancing nuclease/interferon stimulated exonuclease gene 20 kDa-like 1; AKT, protein kinase B; ATG ‘#’, autophagy related ‘#’ (#: 4/4B/5/8/9B/12/18B); BNIP3, BCL2/adenovirus E1B 19 kDa interacting protein 3; CaMK𝛽, Ca2+ /calmodulin-dependent protein kinase 𝛽; CatL, cathepsin L; DRAM, DNA-damage regulated autophagy modulator 1; GABARAP-L1, GABA(A) receptor-associated protein like 1; GDP, guanosine diphosphate; GTP, guanosine triphosphate; IP3R, inositol trisphosphate receptor; JNK, c-Jun N-terminal kinases; PI3K, phosphatidylinositide 3-kinases; PTEN, phosphatase and tensin homolog; RHEB, Ras homolog enriched in brain, ROS, reactive oxygen species; SKP2, S-phase kinase-associated protein 2; SQSTM1, sequestosome 1; TCR, T-cell receptor; TSC1/2, tuberous sclerosis 1/2; UVRAG, UV radiation-resistance associated gene; VPS ‘#’, vacuolar protein sorting ‘#’ (#; 11/18/34); WIPI, WD repeat domain, phosphoinositide interacting protein.

completion of autophagosome formation (Sengupta, Molkentin & Yutzey, 2009). In mouse neurons, FoxO activation in cooperation with c-Jun N-terminal kinase (JNK) activation leads to the expression of Bim, a proapoptotic Bcl-2 Homology domain 3 (BH3)-only protein that mediates cell death. JNK deficiency, however, prevents induction of Bim, and an autophagic survival response mediated by increased expression of Atg8/Lc3b, Atg12, and Bnip3 ensues (Xu et al., 2011).

5′ adenosine monophosphate-activated protein kinase (AMPK) induction in autophagy activates FoxO3a and leads to upregulation of autophagy-related proteins Lc3b-II, Gabarap-l1, and Beclin 1 in murine skeletal muscles (Sanchez et al., 2012). In hepatic and neuronal cells, FoxO selectively modulates lysosomal pathways, but in muscle cells, it coordinates both proteosomal and lysosomal pathways (Mammucari et al., 2007; Zhao et al., 2007,



8 2008). Autophagy plays a key role in protecting adult haematopoietic stem cells (HSCs) from metabolic stress through FOXO3a-mediated, pro-autophagic gene expression (Warr et al., 2013). Nuclear FOXO1, 3, and 4 can act both as transcriptional activators or repressors of many of the target genes, and the associated transcriptional responses have been implicated in several other cellular processes in addition to autophagy (Sandri, 2012). Another study showed the concerted modulation of autophagy by FOXO1 and FOXO3 (Zhou et al., 2012). Recently, in a mouse model of Huntington’s disease, it was reported that deficiency of another transcription factor, X-box-binding protein 1 (Xbp1), results in augmented FoxO1 expression and consequently upregulation of autophagy in neurons (Vidal et al., 2012). This leads to enhanced clearance of mutant huntingtin protein (mHtt) formed during the pathogenesis of the disorder. Interaction of FOXO1 and truncated XBP1 (XBP1u) is critical in the regulation of turnover of FOXO1 protein. Extracellular signal-regulated kinase 2/1 (ERK2/1)-mediated phosphorylation of XBP1u is important for its enhanced interaction with FOXO1 and the latter’s consequent degradation through 20S proteasome (Zhao et al., 2013). This summarizes that the FOXO family which is crucial for both genomic and non-genomic regulation of autophagy could also be an amenable regulatory point. (5) E2 transcription factor (E2F) E2F is well known for its ability to regulate cell cycle progression via its inhibitory interaction with the retinoblastoma tumour suppressor gene product (Rb). In addition, it is a regulator of apoptosis, transcription, DNA replication, and DNA damage (Dimova & Dyson, 2005; Polager & Ginsberg, 2008). E2F1, a member of the E2F family, promotes apoptosis and DNA-damage-induced autophagy (Stevens & La Thangue, 2004; Tracy et al., 2007; Polager, Ofir & Ginsberg, 2008). It upregulates the expression of LC3, Unc-51 like autophagy activated kinase 1 (ULK1) and damage-regulated autophage modulator (DRAM ) by binding directly to their gene promoters; it also upregulates ATG5, but indirectly (Polager et al., 2008). Another interesting set of data has revealed that E2F1 can induce autophagy by upregulating LC3 and ATG5, even in the absence of its transcriptional activity domain (TAD) (Garcia-Garcia et al., 2012). E2F1 also binds to the BECLIN 1 gene promoter, although the effect of its binding is not yet defined (Weinmann et al., 2001). Furthermore, a pro-apoptotic gene BNIP3 has been identified as an E2F-regulated gene that is required for hypoxia-induced autophagy (Tracy et al., 2007). Nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-𝜅B) competes with E2F1 for binding to the BNIP3 promoter to repress BNIP3 expression (Shaw et al., 2008). E2F induces expression of p53-related proteins, but its role both for oncogenes and tumour

suppressors is paradoxical (Polager et al., 2008). The intricate biology of E2F function suggests that E2F may affect vital cellular decisions leading to the complex regulation of autophagy (Fullgrabe, Klionsky & Joseph, 2014). (6) Unscheduled meiotic gene expression protein 6 (UME6) UME6 is a multifunctional transcriptional regulatory protein. It regulates initiation and progression of meiosis in yeast (Strich et al., 1994) in addition to the transcription of genes responding to metabolites and DNA repair (Jackson & Lopes, 1996; Kratzer & Schuller, 1997; Sweet, Jang & Sancar, 1997). It negatively regulates autophagy through interaction with the corepressor switch-independent 3 (SIN3), histone deacetylase reduced potassium dependency 3 (RPD3), and meiotic activator inducer of meiosis 1 (IME1) (Steber & Esposito, 1995; Williams et al., 2002). Recent studies have demonstrated its role in repressing ATG8 transcription, the promoter of which contains an upstream regulatory sequence (URS1) site. UME6 recruits the histone deacetylase complex containing RPD3 and SIN3 to the ATG8 promoter, deacetylating histone 3 (H3) and histone 4 (H4) and thereby repressing transcription. During meiosis and upon nitrogen starvation, however, UME6 is phosphorylated and degraded, which relieves its transcriptional repression on its target genes (Bartholomew et al., 2012). (7) Activating transcription factor (ATF) ATF4 and ATF5 belongs to the basic leucine zipper ATF/cyclic-AMP response element binding protein (CREB) transcription factor superfamily (Ameri & Harris, 2008). ATF4 plays a critical role in orchestrating the expression of genes involved in stress conditions such as oxidative stress, ER stress, hypoxia, and amino acid deprivation, as well as in integrated stress responses, including DNA damage, apoptosis, differentiation, metastasis and angiogenesis. It is well known that responses to stress conditions such as hypoxia mediate cell survival through induction of autophagy. This induction is via activation of ATF4, which regulates expression of the BH3-only protein harakiri (HRK), p53 upregulated modulator of apoptosis (PUMA) and NADPH oxidase activator 1 (NOXA) (Pike et al., 2012). ATF4 also facilitates autophagy by genomic upregulation of LC3B via direct binding to a cyclic AMP response element binding site in the LC3B promoter (Rzymski et al., 2010). Recently, it was also established that ATF4 transcriptionally upregulates ULK1 under severe hypoxia and ER stress by directly binding to its promoter (Pike et al., 2013). Another ATF family member, ATF5, is an anti-apoptotic factor. It is upregulated by breakpoint cluster region protein-Abelson



9

murine leukemia viral oncogene homolog 1 (BCR-ABL) through phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB/AKT)/FOXO4 signalling and inhibits autophagy through promoter-specific transcriptional upregulation of mTOR (Sheng et al., 2011).

ubiquitin ligase complex induces proteasome-mediated degradation of p27 Kip1, an activator of the autophagy pathway (Chen et al., 2008). NF-𝜅B regulates BECLIN 1 gene expression (and phagophore nucleation) through direct binding of its p65 subunit to the NF-𝜅B binding site of BECLIN 1 in activated Jurkat T cells (Copetti et al., 2009). Furthermore, growth of p65-deficient (p65-/-) mouse embryonic fibroblast cells (mEFs) in basal and starvation media demonstrated that p65 is necessary for starvation-mediated expression of classic NF-𝜅B target genes but not for pro-autophagic genes (Comb et al., 2011). Interestingly, a recent study reported the significance of p62/SQSTM1-mediated selective autophagy in NF-𝜅B p65 degradation. Toll-like receptor 2 (TLR2)-dependent signals as generated by pathogen recognition and activation of innate immunity stimulate accumulation of ubiquitinated NF-𝜅B p65 in the cytoplasm, which recruits the ubiquitin-binding protein p62/SQSTM1. Autophagosomes then recognize the NF-𝜅B p65 and subsequently degrade it via the lysosomal pathway. TLR2 signals induce sustained phosphorylation of ERK1/2, which facilitates autophagosome maturation (Chang et al., 2013). It thus appears that NF-𝜅B and autophagy may in part reciprocally regulate each other.

(8) Hypoxia-inducible factor-1 (HIF-1) HIF-1 is an oxygen-dependent transcription factor that primarily ensures cell survival under hypoxic conditions until normal oxygen levels are restored (Schofield & Ratcliffe, 2004). It has been demonstrated that hypoxia selectively induces mitochondrial but not ER autophagy through HIF-1-mediated expression of BNIP3/ BNIP3L, which disrupts the interaction of BECLIN 1 with anti-apoptotic protein BCL2 via their Bcl-2 Homology 1 (BH-1) domains, reducing mitochondrial mass and respiration (Zhang et al., 2008; Bellot et al., 2009). Another study on human tumour cells reports that hypoxia can induce HIF-1-independent autophagy in the absence of nutrient deprivation via activation of AMPK signalling (Papandreou et al., 2008). Furthermore, ATG5 is downstream of AMPK signalling, and its deficiency leads to an alteration in the metabolic response to hypoxia. ATG5 is essential for autophagosome formation and is indispensible in hypoxia-induced autophagy, whether HIF dependent or independent. (9) Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-𝜿B) NF-𝜅B encompasses a family of transcription factors that perform an essential role in regulating the expression of a wide range of genes involved in numerous developmental, inflammatory, immune response, and metabolic pathways (Kumar et al., 2004; Vallabhapurapu & Karin, 2009; Hayden & Ghosh, 2012). Several studies have highlighted its intricate role in the direct and indirect regulation of autophagy in response to inducers of NF-𝜅B signalling (Djavaheri-Mergny et al., 2007; Copetti et al., 2009; Comb et al., 2011; Trocoli & Djavaheri-Mergny, 2011). Tumour necrosis factor 𝛼 (TNF𝛼)-mediated activation of NF-𝜅B leads to mTORC1 activation and, consequently, repression of autophagy in Ewing sarcoma cells (Djavaheri-Mergny et al., 2006). Inhibitor of NF-𝜅B kinase (IKK) family members, such as IKK𝜀 and TANK-binding kinase 1 (TBK1), have been reported to have prosurvival (i.e. autophagy-stimulating) roles that are both NF-𝜅B dependent and independent (Baldwin, 2012). NF-𝜅B regulates autophagy by interacting with other transcription factors and by controlling the expression of autophagy regulatory genes (Trocoli & Djavaheri-Mergny, 2011). For instance, S-phase kinase-associated protein 2 (SKP2) is an NF-𝜅B target gene whose incorporation into the Skp1/Cul1/F-box (SCF)

(10) p53 Renowned as ‘The Guardian of the Genome’ (Lane, 1992) fine-tunes autophagy by virtue of its cellular localization, such that transcription-dependent and -independent mechanisms both contribute to autophagic homeostasis. p53 has now been unequivocally accepted as a bimodal regulator of autophagy: nuclear p53 acts as a transcription factor that induces autophagy by upregulating target genes such as DRAM , phosphatase and tensin homolog (PTEN ), tuberous sclerosis complex 1/2 (TSC1/2), apoptosis enhancing nuclease/interferon stimulated exonuclease gene 20 kDa-like 1 (AEN/ISG20L1), and sestrins, whereas cytoplasmic p53 suppresses autophagy post-trancriptionally or by modulation of yet insufficiently characterized signalling networks (Stambolic et al., 2001; Feng et al., 2005; Crighton et al., 2006; Budanov & Karin, 2008; Tasdemir et al., 2008; Eby et al., 2010). Under normal conditions, basal p53 promotes cell survival, but genotoxic stress or oncogenic activation induces autophagy through p53 activation, suggesting that basal p53 may orchestrate the balance between regulatory signals during stress and normal conditions. However, AMPK activation and upregulation of target genes PTEN , DRAM or TSC, which are inhibitors of mTORC1, illustrates the dependence of cell type and stimulus on the transcription independent and dependent functions of p53, respectively (Feng et al., 2005). In human colorectal cancer cells (HCT116) under chronic starvation conditions, p53 downregulates LC3 mRNA post-transcriptionally (Polager & Ginsberg, 2009), presumably corresponding



10 to the autophagy-suppressing role of cytoplasmic p53. It is clear that attempts to target p53 for pharmacological modulation of autophagy will be context dependent and should be governed by differences in nuclear versus cytoplasmic abundance of the protein and its isoforms among different cell types. (11) Vitamin D3 receptor (VDR) VDR, also known as highly versatile nuclear receptor, is a 1,25-dihydroxyvitamin D3-activated member of the nuclear receptor superfamily. It plays a regulatory role in bone development, calcium homeostasis, and bone mineralization, as well as control of cell growth and differentiation, apoptosis, immunity, and autophagy (Norman, 2008; Bikle, 2010; Hewison, 2011). Vitamin D stimulation induces slow calcium release from the ER, which leads to autophagosome accumulation. This accumulation is the result of activation of calcium/calmodulin-dependent kinase kinase-𝛽 (CaMKK-𝛽), which then later activates AMPK, a substrate of CaMKK-𝛽 (Hoyer-Hansen et al., 2007). Vitamin D can also repress mTORC1, resulting in induction of autophagy (Wang et al., 2008). Two other important proteins of core autophagic machinery, BECLIN 1 and ATG5, are also regulated by vitamin D in human myeloid leukaemia cells and human primary monocytes/macrophage cells (Wang et al., 2008; Yuk et al., 2009). The modulation of ATG5 and BECLIN 1 is mediated through cathelicidin, an antimicrobial peptide crucial for autophagosome–lysosome fusion during mycobacterial infection. In addition to its direct action in autophagy modulation, vitamin D has been linked with the regulation of other molecules such as NF-𝜅B and proinflammatory cytokines during infection and inflammation, all of which play an important role in inflammation and immune homeostasis (Martineau et al., 2007). The indirect modulation of these regulators compels us to explore VDR-mediated signalling further in autophagy. (12) Peroxisome proliferator-activated receptor gamma (PPAR𝜸) PPAR𝛾 belongs to the nuclear receptor superfamily that modulates an array of genes involved in lipid metabolism, glucose homeostasis, and inflammation (Picard & Auwerx, 2002; Watkins et al., 2002; Kim & Ahn, 2004; Bouhlel et al., 2007; Chawla, 2010). Transcriptional modulation by PPAR𝛾 involves heterodimer formation with retinoid-X-receptor (RXR) and binding to the PPAR response element (PPRE) (Gearing et al., 1993; Youssef & Badr, 2011). It has been reported that PPAR𝛾 induces expression of cathepsin L (CATL), a cysteine protease, in human monocyte-derived macrophages (hMDMs) (Mahmood et al., 2011). This induction of CATL concurrently induces apoptosis via degradation of BCL2 but not

BCL2-associated X protein (BAX), while suppressing autophagy via reduction of BECLIN 1 and LC3 protein levels. Consistent with this reported suppression of autophagy in macrophages, PPAR𝛾 and testicular receptor 4 (TR4) augments Mycobacterium tuberculosis survival in macrophages (Mahajan et al., 2012). In hepatocarcinoma cells, PPAR𝛾 activation has been shown to be imperative for its anti-cancerous activity in part by suppressing autophagy, but this effect has been postulated to be indirect and probably involves phosphorylation by different kinases (Vara et al., 2013). PPAR𝛾 has also been shown to suppress prostatic carcinogenesis in part by suppressing autophagy (Jiang et al., 2010b). However, primary mammary epithelial cells transduced with active PPAR𝛾 showed induction of BNIP3 and HIF𝛼 expression, where the latter is essential for activated PPAR𝛾-induced autophagy (Zhou et al., 2009). In hepatocellular carcinoma (HCC) cells, PPAR𝛾 contributes to the increase in autophagic vacuoles, illustrating the importance of PPAR𝛾 in autophagy flux promoted by cannabinoids, a process required for the antitumoral activity of cannabinoids (Vara et al., 2011). It seems that PPAR𝛾 modulation of autophagy is differential and depends on cell origin. (13) Retinoic acid receptor (RAR) Retinoid ligands to RARs have been shown to bisect the compensatory crosstalk between macroautophagy and chaperone-mediated autophagy (CMA), activating the former while repressing the latter (Anguiano et al., 2013). Retinoid-mediated modulation of CMA has been identified through RAR𝛼. The mechanism behind the complex RAR𝛼 signalling by retinoids in CMA is unclear, but lysosomal-associated membrane protein 2A (LAMP2A) has been shown to be a major downstream target gene involved in the process. Retinoic acid induces autophagy by upregulating BECLIN 1 while repressing the mTORC1 pathway (Trocoli et al., 2011). Given the essentiality of CMA in ageing, age-related and neurodegenerative disorders, and the opposing effects of retinoids, there is a need for development of new pharmacological agents capable of selectively activating CMA with no effect on macroautophagy. (14) CCAAT/enhancer-binding protein 𝜷 (C/EBP𝜷) C/EBP𝛽 is a basic leucine zipper transcription factor that is an important genomic regulator of immune homeostasis, metabolism, cell differentiation, and inflammation. In murine liver cells, C/EBP𝛽 orchestrates circadian autophagy rhythm by inducing rhythmic expression of various genes involved in autophagy, such as ULK1, GABARAP-L1, BNIP3, and LC3 (Ma, Panda & Lin, 2011). It also induces expression of ATG4B during adipocyte differentiation. The resultant induction of autophagy is required for the degradation of



11

anti-adipogenic factors Kruppel-like factor (KLF) 2 and KLF3 (Guo et al., 2013).

L929 cells. NR4A1 is clearly a general modulator of autophagy-related cell death emanating from IGF1R or caspase inhibition.

(15) Sterol regulatory element-binding protein 2 (SREBP-2)

(18) Glucocorticoid receptor (GR)

The SREBP gene encodes two protein isoforms: SREBP-1 and SREBP-2. SREBP-2 is important for maintaining sterol homeostasis (Gregor & Hotamisligil, 2007). Genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) screening using specific antibodies in various tissue types from mice under nutrient-deprived conditions has identified the presence of the SREBP motif in genes involved in autophagy (Seo et al., 2011). In addition, the positive role of SREBP-2 in promoting autophagy by direct gene regulation of LC3, ATG4B, and ATG4D was demonstrated with knockdown experiments in nutrient-deprived conditions.

Immunomodulatory glucocorticoid hormone inhibits inositol 1,4,5-trisphosphate (IP3 )-mediated calcium signalling and cell proliferation in T-cells (Baus et al., 1996), which is dependent on signals mediated by Src kinases Fyn and Lck, components of the T-cell receptor complex (Zamoyska et al., 2003; Palacios & Weiss, 2004; Harr et al., 2010). Fyn expression boosts IP3 receptor type 1 (IP3 R1) phosphorylation at tyrosine 353, which is followed by release of calcium from the ER (Cui et al., 2004). Fyn also regulates mTORC1 activity, again linking Fyn to autophagy regulation via glucocorticoids (Harr et al., 2010; Decuypere et al., 2013).

(16) Androgen receptor (AR)

III. CRITICAL SIGNALLING CASCADES OF AUTOPHAGY

ARs are steroid hormone receptors that are activated upon binding to androgen, an endogenous ligand. AR signalling is vital in both the development of the prostate and the progression of prostate cancer (Heinlein & Chang, 2004), but the regulatory role of AR in the autophagic process has been equivocally reported and not adequately investigated. Some recent studies have reported androgens to stimulate reactive oxygen species (ROS) generation both in genomic and non-genomic pathways, inducing autophagy-mediated mobilization of intracellular lipid depots and consequently promoting prostate cancer cell growth in LNCaP and VcaP cells (Shi et al., 2013). Another report, however, suggests that AR loss-of-function in LNCaP or CWRrv1 human prostate cancer cells leads to increased autophagy, possibly through modulation of p62/SQSTM1 protein. Furthermore, decreased autophagy has been demonstrated upon subsequent ectopic expression of AR in AR-deficient PC3 human prostate cancer cells, revealing a negative role of AR in autophagy regulation (Jiang et al., 2012). (17) Nuclear receptor subfamily 4, group A, member 1 (NR4A1) NR4A1, also known as Nur77, belongs to the orphan nuclear receptor superfamily. NR4A1 has recently been reported to promote cell death via autophagy. It interacts with BCL2 and p53. NR4A1 links the signals from the G protein-coupled receptor called neurokinin 1 receptor (NK1R) and the tyrosine kinase receptor called insulin-like growth factor 1 receptor (IGF1R), and in the process, it contributes to autophagy-dependent non-apoptotic cell death (Bouzas-Rodriguez et al., 2012). Moreover, loss-of-function of NR4A1 activity by a dominant negative approach prevents autophagy in

Autophagy is governed by various environmental cues. As described above, a variety of transcription factors directly regulate the genes responsible for induction or abrogation of autophagy. We now turn to the cognate signalling cascades of proteins involved in the process (Fig. 3). These proteins with transcription factors act in tandem to translate stimuli to effector proteins for autophagy modulation. (1) Mammalian target of rapamycin (mTOR) mTOR [also called rapamycin and FKBP12 target-1 (RAFT1), FKBP12-rapamycin associated protein (FRAP), and rapamycin target-1 (RAPT1)] orchestrates a wide variety of cellular functions, including transcription, upregulation of protein synthesis, mitochondria and ribosome biogenesis, cytoskeletal reorganization, cell growth and proliferation, modulation of the immune system, and autophagy (Hay & Sonenberg, 2004; Caron et al., 2010; Laplante & Sabatini, 2012). mTOR consists of two structurally and functionally discrete complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Whereas mTORC1 comprises the mTOR catalytic subunit, regulatory associated protein of mTOR (RAPTOR), G protein 𝛽-subunit-like protein/mLST8 (G𝛽L), proline-rich AKT substrate of 40 kDa (PRAS40), and DEP domain containing mTOR-interacting protein (DEPTOR), mTORC2 consists of the mTOR catalytic subunit, rapamycin-insensitive companion of mTOR (RICTOR), G𝛽L, SAPK-interacting protein 1 (SIN1), DEPTOR, and protein observed with rictor (PRR5/PROTOR) (Jacinto et al., 2004; Sarbassov et al., 2004; Sengupta, Peterson & Sabatini, 2010; Laplante & Sabatini, 2012).



12 Under normal conditions, mTORC1 associates with the ULK1–autophagy-related 13–focal adhesion kinase family-interacting protein of 200 kDa (ULK1– ATG13–FIP200) complex through RAPTOR. mTORC1 phosphorylates and inactivates ULK1 and ATG13, resulting in the inhibition of autophagy. Phosphorylation not only disturbs the ATG13 interaction at the early stages of autophagosome formation, but also the ULK1 interaction with AMPK (Ganley et al., 2009; Hosokawa et al., 2009). During starvation or upon rapamycin treatment, mTORC1 dissociates from the ULK1–ATG13–FIP200 complex and the inhibitory effect of mTORC1 is abrogated. This induces dephosphorylation of both ULK1 and ATG13; consequently, ULK1 mediates autophosphorylation and transphosphorylation of autophagy proteins ATG13 and FIP200, which triggers autophagy in mammalian cells. This phosphorylated ULK1 complex colocalizes with ATG16L, which then translocates to the autophagosome membrane and specifies the site of LC3’s lipidation and translocation to the phagophore membrane. ULK1 also phosphorylates ATG9, allowing recruitment of ATG18 and ATG8 to the phagophore membrane and binding of ATG9 to ATG18, which is essential for membrane elongation (Papinski et al., 2014). Recently, FIP200 was demonstrated to interact directly with ATG16L, a component of the ATG12–ATG5–ATG16L complex, providing the mechanism by which the ULK1 complex colocalizes with ATG16L (Gammoh et al., 2013). Because ATG16L interacting with ATG12–ATG5 conjugation machinery on the isolation membrane is tightly coupled with ATG4–ATG7–ATG3–ATG8 conjugation machinery, the colocalization of the ULK1 and ATG16L complexes provide a likely mechanism by which ULK1 is mediating formation of the autophagic isolation membrane via regulation of the conjugation systems that form ATG12–ATG5 and ATG8–phosphatidylethanolamine (PE) conjugates (Jung et al., 2010; Boya et al., 2013). mTORC1 regulates nucleo-cytoplasmic localization of TFEB and prevents autophagy (Settembre et al., 2012). Interestingly, inhibition of mTORC1 activity by rapamycin disturbs the transcriptional loop between C/EBP𝛼 and PPAR𝛾 in adipogenesis by targeting PPAR𝛾 transactivation directly (Kim & Chen, 2004). Although the role of autophagy was not mentioned, these results raise speculation about the intricate relationship between adipogenesis and autophagy. Another important molecule, class III PI3K VPS34, is a critical component of the BECLIN 1 complex and is essential for the generation of PtdIns(3)P at the phagophore membrane for the formation of autophagosomes. Under modulated amino acid levels, VPS34 upregulates mTORC1 activity (Nobukuni et al., 2005). Another member of the BECLIN 1 protein complex, activating molecule in BECN1-regulated

autophagy protein 1 (AMBRA1), is a substrate of mTORC1. By forming a complex with TNF receptorassociated factor 6 (TRAF6), AMBRA1 promotes lysine 63-linked ubiquitination of ULK1, thereby regulating ULK1’s stability in addition to its activity (Nazio et al., 2013). AMBRA1 is downregulated by phosphorylation at serine 52, which inhibits its role in ULK1 modification.

(2) AMP-activated protein kinase (AMPK) AMPK is an evolutionarily conserved, heterotrimeric serine/threonine kinase (Hardie, Carling & Sim, 1989; Hardie & Carling, 1997; Hardie et al., 2003). It is activated primarily by stress signals that deplete cellular ATP levels (i.e. by a rise in the AMP:ATP ratio) in conditions such as low glucose, hypoxia, ischaemia, heat shock, or oxidative stress, which globally promote catabolic processes to maintain energy homeostasis and induce autophagy (Corton, Gillespie & Hardie, 1994; Wang et al., 2001; Kahn et al., 2005). This cellular energy homeostasis is maintained by cross-talk between AMPK and mTORC1. AMPK inactivates mTORC1 through AMPK-mediated phosphorylation of the TSC2 and the RAPTOR subunit of mTORC1 (Gwinn et al., 2008). Inactivation of mTORC1 leads to inhibition of its signalling through abrogation of phosphorylation of ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1), which consequently promotes autophagy in response to the increased cellular AMP:ATP ratio (Connolly et al., 2006). Conversely, TSC2 activity is suppressed upon phosphorylation by AKT (Inoki et al., 2002). AMPK activity also affects two important regulators of autophagy initiation and progression, the ULK1 and VPS34 complexes (Kim et al., 2011, 2013). Upon glucose starvation, AMPK interacts with and activates ULK1 by phosphorylating serine 317 and serine 777. At normal cellular glucose levels, however, mTORC1 disrupts the interaction of ULK1 and AMPK by phosphorylating ULK1 at serine 757. Inhibition of mTORC1 alone by amino acid starvation or rapamycin treatment can activate ULK1 in an AMPK-independent manner (Kim et al., 2011). AMPK-mediated phosphorylation of ULK1 is essential for mitochondrial homeostasis and cell survival (Egan et al., 2011). In addition, AMPK differentially regulates VPS34 complexes that modulate intracellular vesicle trafficking and autophagy. AMPK inhibits VPS34 that is devoid of ATG14L and UVRAG by phosphorylating it at threonine 163 and serine 165. Conversely, AMPK activates the VPS34 proautophagy complex containing ATG14L by phosphorylating BECLIN 1 at serine 91 and serine 94. The autophagy-specific subunit ATG14L prevents the inhibitory phosphorylation of VPS34 and directs the complex to the ER for activation of autophagy. Thus, ATG14L acts as a switch for the



13

intricate molecular mechanism underlying AMPK regulation of pro-autophagic and non-autophagic VPS34 complexes (Kim et al., 2013).

2008). PKB/AKT1 is inactivated when it is phosphorylated by TSC2. TSC2 operates as a GTPase-activating protein (GAP) for ras homolog enriched in brain (Rheb), a GTP-binding protein. GTP-bound Rheb activates mTORC1, relaying signals that result in cellular growth and translation as well as autophagy inhibition (Inoki et al., 2003; Sridharan, Jain & Basu, 2011). Hyperactivation of the tumour suppressor phosphoinositide phosphatase and tensin homologue (PTEN) inhibits PI3K activity by converting PtdIns(3,4,5)P3 to PtdIns(4,5)P2 , followed by induction of autophagy (Song, Salmena & Pandolfi, 2012). PtdIns(3,4,5)P3 is an activator of PDK1 and PKB/AKT1. In contrast to class I PI3Ks, class III PI3K VPS34 is important for upregulation of autophagy. It mediates vesicle nucleation and hydrolase sorting through the vacuolar protein sorting (VPS) pathway. It phosphorylates PtdIns and thus increases PtdIns(3)P levels; the PtdIns(3)P binds to ATG18, facilitating recruitment of the ATG18–ATG2 complex to the autophagic membrane and thereby activating autophagy. PI3K VPS34 also directly interacts with BECLIN 1 to form a multi-protein structure and also facilitates recruitment of phospholipid interacting proteins, WIPI proteins and DFCP1 to the early autophagosome structure (Obara & Ohsumi, 2008).

(3) Eukaryotic initiation factor 2 (eIF2) The eIF2 family of proteins are evolutionarily conserved serine/threonine kinases that regulate stressmediated translational arrest and selectively enhance translation of genes involved in autophagy regulation (Harding, Zhang & Ron, 1999; Wek, Jiang & Anthony, 2006; B’Chir et al., 2013). Four diverse kinases that target the alpha subunit of eIF2 (eIF2𝛼) are known: general control non-derepressible 2 (GCN2), protein kinase regulated by RNA (PKR), (PKR)-like ER kinase (PERK), and heme-regulated inhibitor (HRI). These kinases are activated by amino-acid starvation, viral infection, ER stress, and heme depletion, respectively (Wek et al., 2006). In yeast, during cellular stress, only GCN2 is activated. GCN2 phosphorylates eIF2𝛼, which leads to global translation arrest but preferentially induces translation of specific mRNAs encoding basic leucine zipper transactivators such as GCN4 and ATF4, which regulate autophagy (Hinnebusch, 1997; Talloczy et al., 2002). eIF2 phosphorylation is also important for viral infection and ER stress-mediated autophagy (Talloczy et al., 2002). The latter can be interrupted by phosphorylation mutants of eIF2 or can be restricted by dominant-negative inhibition of PERK (Kouroku et al., 2007). It has been implied that phosphorylated eIF2 induces translation of transcription factors that augment ATG12 expression and ATG12–ATG5–ATG16 complex formation, which decisively induces autophagy. (4) PI3K signalling Mammalian PI3Ks are mainly divided into classes I, II, and III. PI3Ks are lipid kinases that catalyse the phosphorylation of phosphoinositide lipids at 3′ -hydroxyl groups, generating diverse secondary messengers (Datta, Brunet & Greenberg, 1999; Engelman, Luo & Cantley, 2006). Regulation of autophagy by these kinases is class-specific: class I PI3K is a negative modulator and classes II and III are positive modulators. Class I PI3K is activated by growth factors or insulin, resulting in formation of PtdIns(3)P, phosphatidylinositol 3,4-bisphosphate, [PtdIns(3,4)P2 ], and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3 ], which consequently mobilizes and activates pleckstrin homology (PH) domain proteins such as serine/threonine kinases, 3-phosphoinositide-dependent kinase 1 (PDK1 /PDPK1), and PKB (also known as AKT1) at the plasma membrane (Fresno Vara et al., 2004; Bayascas et al., 2008; Wu et al., 2009). PDK1 activates kinases such as PKB/AKT1, but the activity is lost when mutations in PDK1 inhibit its interaction with phosphoinositides, a key event for AKT1 activation (Bayascas et al.,

(5) Mitogen-activated protein kinase (MAPK) MAPKs are a family of serine/threonine protein kinases involved in various cellular responses. Among MAPK family members, the roles of p38, ERK, and JNK in autophagy have been well explained. Whereas ERK is generally activated by growth factor-stimulated signals, JNK and p38 are activated by chemical and physical stresses, including inflammatory cytokines, oxidative stress, and DNA-damaging agents (Roux & Blenis, 2004). (a) p38𝛼 The p38 MAPK family includes four splice variants, p38𝛼, -𝛽, -𝛾, and -𝛿, and is involved in amino acid signalling. Besides its roles in the regulation of apoptosis, the cell cycle and differentiation, inflammation, and tumour suppression, it also modulates autophagy in response to various stimuli in a cell-type-dependent manner. In yeast during osmotic and ER stress, its homolog Hog1 MAPK positively regulates autophagy by stabilizing Atg8 (Bicknell, Tourtellotte & Niwa, 2010). In cultured rat hepatocytes and perfused rat liver, however, an influx of amino acids caused by elevated Na+ levels activates p38 and inhibits autophagy in an mTOR-independent manner (vom Dahl et al., 2001; Kanazawa et al., 2004; Codogno & Meijer, 2005). In colorectal cancer cells, autophagy is induced by inactivation of p38 signalling (Comes et al., 2007). Conversely, in murine glycolytic and oxidative skeletal muscles,



14 endotoxin-induced p38 activation increases expression of autophagy genes Atg6, Atg7 and Atg12 during the initiation of cachectic muscle wasting (McClung et al., 2010). p38𝛼 activity is undetectable during starvation, and only basal phosphorylation of p38𝛼 is observed in nutrient-rich conditions. In HEK293 cells, p38𝛼 has been shown to be a negative regulator of autophagy that competes with ATG9 for p38-interacting protein (p38IP) and abrogates the trafficking of ATG9 to the autophagosome (Webber & Tooze, 2010). More recently, it has also been demonstrated that interferon 𝛾 (IFN𝛾)-induced autophagy requires Janus kinase 1/2 (JAK 1/2), PI3K, and p38 MAPK, but not signal transducer and activator of transcription 1 (STAT1), which was previously reported to enlist immunity-related GTPase family M member 1 (Irgm1), that plays a role in the innate immune response by regulating autophagy formation in response to intracellular pathogens (Matsuzawa et al., 2012). (b) JNKs The JNK family includes JNK1, -2, and -3. Expression of JNK1 and -2 is ubiquitous, whereas JNK3 is predominantly expressed in brain (Bogoyevitch, 2006). JNKs are activated by diverse stimuli, including inflammatory cytokines, starvation, surface receptor stimulation, growth factors, genotoxic agents, and other environmental stresses (Wagner & Nebreda, 2009). JNK1 promotes autophagy in response to starvation by phosphorylating anti-apoptotic protein BCL2 and releasing its association with BECLIN 1 (Wei et al., 2008). Activation of JNK by ceramide can also regulate BECLIN 1 gene expression in human cancer cell lines CNE2 and Hep3B through c-Jun phosphorylation (Li et al., 2009). JNK can promote autophagy by phosphorylating p53. Targeted deletion of JNK1, -2 and -3 increases autophagy in neurons in an mTORC1-independent manner through FOXO activation, which causes expression of autophagy-related genes ATG8, ATG12 and BNIP3 and results in autophagy-mediated neuroprotection (Xu et al., 2011). (c) ERK ERK1 (p44) and ERK2 (p42) are the two splice variants of ERK. After activation by upstream tyrosine kinase receptors (RTKs), the protein superfamily of small GTPase rat sarcoma (RAS) binds to and activates rapidly accelerated fibrosarcoma (RAF), leading to MAPK signalling activation. RAF phosphorylates and activates MAPK kinase (MEK), which activates the serine/threonine kinase ERK1/2 by phosphorylating serine 217 and 221 (Ogier-Denis et al., 2000; McKay & Morrison, 2007). ERK1 and ERK2 are activated upon nutrient starvation conditions, and thus downregulate starvation-induced autophagy when inhibited. This was demonstrated in the human colon cancer cell line

HT-29 using the MAPK pathway inhibitor PD-98059, which abrogated starvation-induced autophagy. ERK phosphorylation and the concomitant activation of G𝛼-interacting protein (GAIP), a regulator of G-protein signalling (RGS) protein, determine starvation-induced autophagy. Interestingly, inhibition of autophagy by stimulus with amino acids correlates with ERK1/2 MAPK inhibition and reduced GAIP phosphorylation (Ogier-Denis et al., 2000). Inhibition of ERK1/2 signalling leads to a decrease in TNF𝛼-induced autophagy in MCF-7 breast cancer cells (Sivaprasad & Basu, 2008). ERK elevates autophagy induction by TNF𝛼 in L929 mouse fibroblast cells via p53 activation (Cheng et al., 2008). It has also been shown that differential activation of MEK/ERK can stimulate autophagy by disassembly of mTORC1 and mTORC2 complexes and regulation of BECLIN 1 expression to different threshold levels via its non-canonical pathway (AMPK–MEK/ERK– TSC–mTOR). Transient activation of MEK/ERK leads to cytoprotective autophagy, whereas prolonged activation leads to cytodestructive autophagy (Wang et al., 2009). (6) B-cell CLL/lymphoma 2 (BCL2) The BCL2 proteins were historically known for their anti-apoptotic functions via interactions with family members BCL2-antagonist/killer (BAK) and BAX, but they are now far more appreciated for their anti-autophagy roles. BECLIN 1 interaction with BCL2 toggles between these key molecular events (Levine, Sinha & Kroemer, 2008; Sinha & Levine, 2008; Marquez & Xu, 2012; Tzifi et al., 2012). BECLIN 1, as a member of a core complex of class III PI3K VPS34 and other proteins [UVRAG, BAX-interacting factor-1 (BIF-1), etc.], is required for recruitment of the autophagic proteins to preautophagosomes (Itakura et al., 2008; Noble et al., 2008). Under normal conditions, BCL2 binds to and sequesters BECLIN 1 away from this complex with no basal autophagy. Cellular stress conditions such as starvation, however, activate JNK1 but not JNK2, and induce BCL2 phosphorylation and its consequent separation from BECLIN 1, thus leading to autophagy initiation (Pattingre et al., 2005; Wei et al., 2008; He & Levine, 2010). The role of BCL2 as an inhibitor of both cell survival and cell death mechanisms plausibly involves multisite phosphorylation of BCL2. Wei et al. (2008) reported that although brief intervals of nutrient deprivation led to phosphorylation at BCL2 sites threonine 60, serine 70 and serine 87 and the dissociation of BECLIN 1 from BCL2 with no effect on BAX, prolonged nutrient deprivation releases BAX. This implies that autophagy is activated by subtle and stepwise regulation of BCL2, whereas stimulation of apoptosis requires higher levels of BCL2 multi-site phosphorylation that results in slow dissociation of BCL2 from BECLIN 1 (Wei et al., 2008). BCL2 is also post-translationally modified by parkin, an E3 ubiquitin ligase (Chen et al., 2010).



15

Parkin mediates non-degradative mono-ubiquitination of BCL2 by binding directly to BCL-2’s C-terminus, altering its turnover. The increased level of BCL2 allows it to bind to BECLIN 1 more efficiently, thus inhibiting autophagy. The genomic regulation of BCL2 by other transcription factors such as testicular receptor 4 (TR4), GATA binding protein 4 (GATA4), and NF-𝜅B highlights the importance of investigating these factors for their ability to modulate autophagy (Catz & Johnson, 2001; Kobayashi et al., 2006; Kim et al., 2007).

acetylation is dependent on p300 acetyltransferase. Lack of SIRT1 inhibits autophagosome formation during starvation and results in elevated p62/SQSTM1 levels (Lee et al., 2008). Other PTMs are also important for autophagy. One study highlights the involvement of stress-induced, acetylated heat shock protein 70 (hsp70)-dependent control of VPS34 SUMOylation (Yang et al., 2013). Binding of acetylated hsp70 with the BECLIN 1–VPS34 complex induces recruitment of the E3 small ubiquitin-like modifier (SUMO) ligase KRAB–ZFP-associated protein 1 (KAP1), resulting in the SUMOylation of VPS34 and thereby enhancing its stability and lipid kinase activity in addition to promoting binding with BECLIN 1. In yeast, a screen for ATG1 kinase substrates by consensus peptide array identified ATG9, ATG8, and ATG18 (Papinski et al., 2014). ATG1 phosphorylates ATG9, which then interacts with ATG18 and recruits ATG8 and other autophagic factors to the PAS. In mammals, depletion of WIPI2 (a mammalian homolog of ATG18) does not affect the localization of ATG9 to DFCP1-positive omegasomes, but these omegasomes are unable to mature into LC3-positive autophagosomes (Orsi et al., 2012). LC3, a signature autophagic protein, is specifically phosphorylated at different sites by protein kinase A and protein kinase C in different cellular systems, with the resultant phosphorylations inhibiting autophagy induction (Cherra et al., 2010; Jiang et al., 2010a). Recently, the nutrient-sensing kinase ULK1 was identified as being hyperphosphorylated in nutrient-rich conditions. Among the various kinases involved in the phosphorylation of ULK1 are mTORC1 and AMPK (Ganley et al., 2009; Mack et al., 2012). As mentioned above, mTORC1-dependent phosphorylation changes the localization of ULK1, resulting in dissociation of the ULK1 complex and hence inhibition of autophagy; AMPK activation under starvation conditions enhances ULK1 kinase activity and regulates localization. Acetylation of ULK1 at lysine 162 and lysine 606 by the lysine acetyltransferase TIP60 is another mechanism whereby nutrient sensing is linked with ULK1-mediated autophagy (Lin et al., 2012). Ubiquitination is critical in autophagosome-mediated recognition and recruitment of specific cargo proteins. In neurons, ULK1 is ubiquitinated in response to nerve growth factor, but the functional relevance to autophagy is not clear (Zhou et al., 2007; Wong et al., 2013). As alluded to previously, the transcription factors that regulate autophagy are also subject to PTMs. Under normal conditions, ERK phosphorylates TFEB at serine 142 and keeps it in the cytoplasm; this pattern is reversed under starvation. mTORC1-mediated phosphorylation of TFEB at its C-terminal serine-rich motif causes nuclear localization of TFEB (Pena-Llopis et al., 2011), whereas under starvation or if treated with mTORC1 inhibitors, TFEB is entirely nuclear and in its hypo-phosphorylated state (Settembre et al., 2012).

IV. POST-TRANSLATIONAL REGULATION OF AUTOPHAGY-ASSOCIATED PROTEINS Many of the proteins involved in these autophagyaffecting signalling cascades, as well as the transcription factors that regulate autophagy, can themselves be regulated by PTMs. Genomic modulation of autophagy and cognate signalling cascades is incoherent without reference to PTM and associated protein–protein interaction at the various steps of the process. Two ubiquitination-like covalent modification reactions take place during autophagy progression. First, ATG12 conjugates to ATG5 and acts as an E3 ligase for the second conjugation of ATG8/LC3 to the polar head of the lipid PE. Both reactions are essential for autophagosome formation. ATG7, which behaves as an ubiquitin E1-like enzyme, activates ATG12 through a thio-ester bond formation. After ATG12’s transfer to ATG10, an E2-like enzyme, it is directed to ATG5 for conjugation. The ATG12–ATG5 conjugate later associates noncovalently with ATG16L. This protein complex then homo-oligomerizes through the ATG16L C-terminal homo-oligomerization domain to form a multimeric protein complex (Matsushita et al., 2007). Conjugation of ATG8/LC3 to PE begins with activation of ATG8 by ATG7, followed by transfer of ATG8 to the E2-like ATG3 protein. Eventually, ATG3 catalyses the conjugation of PE to ATG8. The site of LC3 lipidation depends on membrane localization of the ATG16L complex, which functions as a scaffold for ATG8–PE conjugation (Hanada et al., 2007; Geng & Klionsky, 2008). ATG4, a cysteine protease that cleaves the C-terminus of ATG8, is indispensable for ATG8–PE coupling on the autophagosomal membrane. It also decouples PE from ATG8 to release other membrane proteins and induce recycling of ATG8 from the mature autophagosome. Protein family members ATG4A and ATG4B are regulated by ROS because of the presence of oxidation-prone cysteine residues in their active sites (Scherz-Shouval et al., 2007); oxidation mediated by local ROS generation near mitochondria results in loss of ATG4 activity. Other ATG proteins, including ATG5, ATG7, ATG8, and ATG12, interact directly with NAD-dependent protein deacetylase sirtuin-1 (SIRT1), resulting in their deacetylation under starvation. ATG



16 Another report suggests that TFEB phosphorylation at serine 211 by an mTORC1-dependent pathway promotes interaction of TFEB with members of the YWHA (14-3-3) family of proteins, which promotes retention of TFEB in the cytosol (Martina et al., 2012). The varied observations regarding compartmentalization of TFEB and its activity necessitate more experimental analysis of TFEB’s phosphorylation status. FOXO homologue functions are also controlled by various PTMs. PI3K/AKT is activated by growth factor and phosphorylates FOXO family members, which move from the nucleus to the cytoplasm, where they remain transcriptionally abrogated. FOXO3a is phosphorylated by serum- and glucocorticoid-inducible kinases (SGKs) at the same sites as those phosphorylated by AKT (Brunet et al., 2001). Further, AKT-independent IKK-mediated phosphorylation of FOXO3a also results in its cytoplasmic retention and inhibition of its function by virtue of ubiquitination-mediated proteolysis (Hu et al., 2004). Stress such as growth factor limitation or glucose deprivation relieves the AKT-induced inhibition and consequent cytoplasmic sequestration of FOXO, and the dephosphorylated FOXO transcription factors return to the nucleus, where they activate autophagy pathway gene expression (Sengupta et al., 2009). In cardiac myocytes, SIRT1-induced deacetylation of FOXO1 is required for starvation-induced autophagy, which then later upregulates autophagic flux through ras-related protein (RAB7) activation (Hariharan et al., 2010). Physiological abundance of p53 is maintained by E3 ubiquitin ligase mouse double minute 2 homolog (MDM2)-mediated ubiquitination and proteasomal degradation (Li et al., 2003; Nie, Sasaki & Maki, 2007). However, adverse stress conditions such as genotoxic, hypoxic, and oncogenic stress induce rapid, reversible PTM of p53 that result in its stabilization (Siliciano et al., 1997; Jimenez et al., 1999; Shieh, Taya & Prives, 1999; Ashcroft, Taya & Vousden, 2000; Appella & Anderson, 2001; Koumenis et al., 2001). IFN𝛾 induces autophagy through histone deacetylase 1 (HDAC1)-mediated deacetylation of human p53 at lysine 382 and murine p53 at lysine 379, which leads to increased nuclear p53 levels and interaction with the promoter of the BH3 domain-only protein Bcl2 modifying factor (BMF) and consequently leads to downregulation of BMF mRNA expression (Juan et al., 2000; Contreras et al., 2013). Suppression of BMF reduces the BECLIN 1 and BCL2 interaction and thus facilitates autophagy (Contreras et al., 2013). Recent findings, however, demonstrated that lysine 120-acetylation and lysine 386-SUMOylation of p53 lead to its increased cytoplasmic localization and promotes PUMA-independent autophagy (Naidu, Lakhter & Androphy, 2012). In this case, the lysine acetyltransferase TIP60 and p53 both interact with SUMO E3 ligase PIASy (PIAS4). SUMOylation of TIP60

increases its acetylase activity, augmenting p53 acetylation at lysine 120. This indicates that independent or synergistic effects of PTMs modulate autophagy in several ways.

V. PHARMACOLOGICAL MODULATION OF THE TRANSCRIPTIONAL REGULATORS OF AUTOPHAGY, THEIR COGNATE SIGNALLING CASCADES AND THE INTERFACE BETWEEN THEM Thus far, we have focused on the relevance of the transcription factors and signalling molecules that regulate autophagy. We aim now to highlight the intriguing tandem network that exists between them, as well as their potential for pharmacological manipulation. Major emphasis has been put on the signalling molecules and ATG proteins as therapeutic targets, but it is also important to consider potential off-target effects. Rapamycin, the first drug to be identified for the upregulation of autophagy, involves inactivation of mTOR. It is effective in ameliorating the toxicity from proteinopathy in neurodegenerative disorders like Huntington’s disease and spinocerebellar ataxias in mouse models of the respective disease conditions (Ravikumar et al., 2004). Later, small molecule derivatives of rapamycin such as CCI-779 were discovered and successfully tested experimentally in vivo as autophagy enhancers/inducers (Hochfeld, Lee & Rubinsztein, 2013). However, a major limitation of these experimentally verified drugs is that mTOR substrates control several other biological processes such as human malignancies, cellular energy homeostasis, metabolism, and T-cell function. This necessitated the identification of mTOR-independent autophagy modulators such as verapamil and loperamide, which block L-type Ca2+ channels and decrease cytosolic Ca2+ levels. This leads to induction of autophagy by abrogation of calpain activity, resulting in autophagy-dependent, enhanced clearance of mutant protein aggregates in Drosophila and zebrafish models of Huntington’s disease. An increase in cytosolic Ca2+ levels activates calpain, which in turn inhibits autophagy by cleaving ATG5 and BECLIN 1 proteins (Yousefi et al., 2006; Russo et al., 2011). In cancer, the role of autophagy is double-edged: it promotes tumour suppression but also functions as an adaptive mechanism for stress response and maintains tumour cell survival (Yang et al., 2011b). The pathological complexity of cancer contributes to a lack of specificity of drugs inhibiting autophagy. Chloroquine and hydrochloroquine are under clinical trials in combination with cytotoxic chemotherapy for targeted intervention of myelomas, but a deeper understanding of autophagy regulation in cancer is needed (Kimura et al., 2013). Table 2 lists some of the drugs that are used



17

Table 2. Plausible pharmacological modulators and their implication in autophagy and associated diseases on the basis of experimental evidence Experimental evidence on autophagy modulation

Experimental evidence on autophagy-associated disease

Rapamycin

Enhances autophagy by inhibition of mTOR pathway

Verapamil Loperamide Nimodipine

Enhances autophagy by blocking Ca2+ channel

Hydroxychloroquine + Imatinib

Inhibits autophagy

Chloroquine + bortezomib

Inhibits autophagy

Cannabinoids

Induces autophagy through ER stress and AMPK activation

Chloroquine + Src kinase inhibitors

Inhibits autophagy

Minoxidil

Enhances autophagy by opening K+ ATP channel

Huntington’s disease: increases mutant Htt, 𝛼Syn, and tau aggregate clearance by autophagy Huntington’s disease: increases mutant Htt and 𝛼Syn aggregate clearance by autophagy Reduces the number of residual leukaemia cells in chronic myelogenous leukaemia Synergistically promotes anti-tumour effect on multiple myelomas Induces anti-tumour effect on hepatocellular carcinoma and pancreatic cancer Inhibits tumour growth and enhances apoptosis in prostate cancer Huntington’s disease: increases mutant Htt and 𝛼Syn aggregate clearance by autophagy

Name

References Hochfeld et al. (2013) and Ravikumar et al. (2004) Williams et al. (2008) and Zhang et al. (2007) Trela et al. (2014)

Jia et al. (2012) Vara et al. (2011)

Wu et al. (2010) Williams et al. (2008)

List includes drugs already in use for the treatment of some pathological conditions other than autophagy-associated pathological conditions. These drugs are experimentally validated for their role in modulation of autophagy. The mentioned drugs are in the process of being repurposed for the regulation of autophagy in associated pathophysiologies and some are the subject of clinical trials. References document successful observations of the effect in vitro and in vivo. 𝛼Syn, alpha-synuclein; AMPK, AMP-activated protein kinase 𝛼; ATP, adenosine triphosphate; ER, endoplasmic reticulum; Htt: huntingtin; mTORC1, mammalian target of rapamycin complex 1; tau, microtubule associated protein tau.

for the treatment of disease conditions that are different from autophagy-associated pathological conditions. These drugs are in the process of being repurposed for their role in autophagy modulation on the basis of experimental evidence and some of these drugs are undergoing clinical trials (Zhang et al., 2007; Williams et al., 2008; Carew et al., 2010; Wu et al., 2010; Vara et al., 2011; Jia et al., 2012; Trela, Glowacki & Blasiak, 2014). Transcription factors, particularly those in the nuclear receptor family, are attractive targets for drug development because of their ability to bind to specific ligands that modulate their activity. This characteristic invokes the possibility of using combinatorial therapies involving ligands and modulators of autophagy to achieve more specific responses while eliminating the risks of side effects such as excitotoxicity. Recently, RAR has been identified as a negative regulator of CMA in addition to being a macroautophagy inducer (Anguiano et al., 2013). CMA is important in nutritional and oxidative stress, ageing, kidney disease, lysosomal storage disease, neurodegenerative disorders, and neuronal homeostasis (Bejarano & Cuervo, 2010). Antagonism

of RAR function could promote CMA while preventing ageing (Anguiano et al., 2013), and therefore structure-based, chemically designed molecules such as 𝛼-aminonitrile retinoids, boron-aminonitrile retinoids, and guanidine retinoids with possible antagonistic function have been investigated. There is need, however, for design of antagonists that can selectively upregulate CMA with no effect on macroautophagy. Further investigation of the ability of other transcription factors to modulate autophagy could reveal new targets, particularly among those factors that are known to modulate pathological conditions reported for autophagy disorders. For instance, the underlying mechanism behind the anti-M. tuberculosis role of liver-X-receptor 𝛼 (LXR𝛼) and Reverse-erb𝛼 should be investigated (Mahajan et al., 2012; Chandra et al., 2013). VDR should be examined for cathelicidin-independent pathways of autophagy modulation. High-throughput screening techniques and structure-based drug design have already helped identify several ligands/modulators that could be promising candidates.



18 Experimental advances have confirmed that transcription factors are substantially different from signalling molecules in terms of their associated off-target effects. The degeneracy and pleiotropy provided through signalling molecules increase the diversity in gene regulation, and although valuable for cellular homeostasis, these properties are not useful for a therapeutic target, for which specificity and selectivity are of paramount importance. There are instances in which transcription factors and other molecular components that regulate autophagy do so in a stimulation-dependent manner. As described above, TFEB localization and activity responds to phosphorylation by the MAPK ERK and mTORC1, and p53 activity is modulated by acetylation by TIP60 and SUMOylation by PIAS4. Similarly, PI3K/AKT-dependent phosphorylation of FOXO protein is critical in determining its cellular localization, which directly affects autophagy. These examples are indicative of the complex regulatory mechanisms by which transcription factors are involved in cross talk with their cognate signalling cascades. They also highlight the importance of investigating the potential advantage of targeting the factors that reside at the interface of these interactions. The increase in drug discovery and development has led to more demand for polypharmacological drugs: those with multiple targets in single or multiple disease pathways. We encourage further examination of transcription factors as novel therapeutic targets to alleviate disease conditions involving autophagy. Autophagy modulators should be designed according to cell type and specific condition, with an emphasis on the fine-tuners, i.e. ligand-activated transcriptional regulators at the pivotal juncture between selective autophagy pathways. Furthermore, combinatorial approaches involving genomics, proteomics, cheminformatics, systems biology, and polypharmacology can frame a constructive platform for integrating diverse information involving molecular mechanisms, genomic regulations, binding affinities, crystal structure, side effects, and drug targets.

upstream stimuli are relayed to downstream effectors by undergoing several discrete multiple sitespecific modifications. These modification sites hold promise for therapeutic interventions because of their different stimuli-specific roles. Noteworthy molecules include TFEB, FOXO, and p53, which are posttranslationally modified by mTORC1, PI3K/AKT, and PIAS4, respectively. The modifications not only alter protein localization (TFEB and FOXO), but also activity (p53), making them plausible targets for pharmacological manipulation. (3) The complexity of autophagy pathways and the inadequate understanding of transcriptional networks and the associated pathological conditions calls for a concerted effort towards identifying transcriptional regulators and checkpoints that can be exploited for pharmacological selectivity and specificity. (4) A comprehensive analysis of the efficiency and polypharmacological effects of known drugs for autophagy modulation in addition to their off-target effects is exigent. Known drugs should be optimized with further combinatorial approaches to obtain specificity towards multiple pathways that include regulation of transcription factors and cognate signalling cascades. A combinatorial and polypharmacological approach for novel drug design aimed at these interactomes holds promise.

VII. ACKNOWLEDGEMENTS We sincerely thank Dr Girish Sahni for help and efforts. We thank CSIR-IMTECH for providing facilities. This work was supported by the Department of Biotechnology-India project BT/01/IYBA/2009 and CSIR 12th Plan Network project Bugs to Drugs, Infectious Disease (BSC0211, BSC0210) and OSDD to P.G. We express regret for not citing the work of many our colleagues due to space constraints.

VIII. REFERENCES VI. CONCLUSIONS (1) Despite the progress made towards understanding the regulation of autophagy through various cytoplasmic molecules, transcriptional regulation of autophagy has been undervalued. This review highlights the importance of understanding the transcriptional regulators of the autophagy process, from initiation of autophagosome formation to its maturation. It highlights the newly discovered master regulators of autophagy such as TFEB and ZKSCAN3 in addition to critical transcription factors such as FOXO, E2F, and p53. (2) These transcriptional modulators function in tandem with associated signalling molecules, so that

Ameri, K. & Harris, A. L. (2008). Activating transcription factor 4. The International Journal of Biochemistry and Cell Biology 40, 14–21. Anguiano, J., Garner, T. P., Mahalingam, M., Das, B. C., Gavathiotis, E. & Cuervo, A. M. (2013). Chemical modulation of chaperone-mediated autophagy by retinoic acid derivatives. Nature Chemical Biology 9, 374–382. Appella, E. & Anderson, C. W. (2001). Post-translational modifications and activation of p53 by genotoxic stresses. European Journal of Biochemistry 268, 2764–2772. Ashcroft, M., Taya, Y. & Vousden, K. H. (2000). Stress signals utilize multiple pathways to stabilize p53. Molecular and Cellular Biology 20, 3224–3233. Axe, E. L., Walker, S. A., Manifava, M., Chandra, P., Roderick, H. L., Habermann, A., Griffiths, G. & Ktistakis, N. T. (2008). Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. The Journal of Cell Biology 182, 685–701. Baldwin, A. S. (2012). Regulation of cell death and autophagy by IKK and NF-kappaB: critical mechanisms in immune function and cancer. Immunological Reviews 246, 327–345.



19

Bao, X. H., Naomoto, Y., Hao, H. F., Watanabe, N., Sakurama, K., Noma, K., Motoki, T., Tomono, Y., Fukazawa, T., Shirakawa, Y., Yamatsuji, T., Matsuoka, J. & Takaoka, M. (2010). Autophagy: can it become a potential therapeutic target? International Journal of Molecular Medicine 25, 493–503. Bartholomew, C. R., Suzuki, T., Du, Z., Backues, S. K., Jin, M., Lynch-Day, M. A., Umekawa, M., Kamath, A., Zhao, M., Xie, Z., Inoki, K. & Klionsky, D. J. (2012). Ume6 transcription factor is part of a signaling cascade that regulates autophagy. Proceedings of the National Academy of Sciences of the United States of America 109, 11206–11210. Baus, E., Andris, F., Dubois, P. M., Urbain, J. & Leo, O. (1996). Dexamethasone inhibits the early steps of antigen receptor signaling in activated T lymphocytes. The Journal of Immunology 156, 4555–4561. Bayascas, J. R., Wullschleger, S., Sakamoto, K., Garcia-Martinez, J. M., Clacher, C., Komander, D., van Aalten, D. M., Boini, K. M., Lang, F., Lipina, C., Logie, L., Sutherland, C., Chudek, J. A., van Diepen, J. A., Voshol, P. J., Lucocq, J. M. & Alessi, D. R. (2008). Mutation of the PDK1 PH domain inhibits protein kinase B/Akt, leading to small size and insulin resistance. Molecular and Cellular Biology 28, 3258–3272. B’Chir, W., Maurin, A. C., Carraro, V., Averous, J., Jousse, C., Muranishi, Y., Parry, L., Stepien, G., Fafournoux, P. & Bruhat, A. (2013). The eIF2alpha/ATF4 pathway is essential for stress-induced autophagy gene expression. Nucleic Acids Research 41, 7683–7699. Bejarano, E. & Cuervo, A. M. (2010). Chaperone-mediated autophagy. Proceedings of the American Thoracic Society 7, 29–39. Bellot, G., Garcia-Medina, R., Gounon, P., Chiche, J., Roux, D., Pouyssegur, J. & Mazure, N. M. (2009). Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Molecular and Cellular Biology 29, 2570–2581. Bicknell, A. A., Tourtellotte, J. & Niwa, M. (2010). Late phase of the endoplasmic reticulum stress response pathway is regulated by Hog1 MAP kinase. The Journal of Biological Chemistry 285, 17545–17555. Bikle, D. D. (2010). Vitamin D: newly discovered actions require reconsideration of physiologic requirements. Trends in Endocrinology and Metabolism 21, 375–384. Bogoyevitch, M. A. (2006). The isoform-specific functions of the c-Jun N-terminal Kinases (JNKs): differences revealed by gene targeting. BioEssays 28, 923–934. Bouhlel, M. A., Derudas, B., Rigamonti, E., Dievart, R., Brozek, J., Haulon, S., Zawadzki, C., Jude, B., Torpier, G., Marx, N., Staels, B. & Chinetti-Gbaguidi, G. (2007). PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metabolism 6, 137–143. Bouzas-Rodriguez, J., Zarraga-Granados, G., Sanchez-Carbente Mdel, R., Rodriguez-Valentin, R., Gracida, X., Anell-Rendon, D., Covarrubias, L. & Castro-Obregon, S. (2012). The nuclear receptor NR4A1 induces a form of cell death dependent on autophagy in mammalian cells. PLoS ONE 7, e46422. Boya, P., Reggiori, F. & Codogno, P. (2013). Emerging regulation and functions of autophagy. Nature Cell Biology 15, 713–720. Brunet, A., Park, J., Tran, H., Hu, L. S., Hemmings, B. A. & Greenberg, M. E. (2001). Protein kinase SGK mediates survival signals by phosphorylating the forkhead transcription factor FKHRL1 (FOXO3a). Molecular and Cellular Biology 21, 952–965. Budanov, A. V. & Karin, M. (2008). p53 target genes sestrin1 and sestrin2 connect genotoxic stress and mTOR signaling. Cell 134, 451–460. Carew, J. S., Medina, E. C., Esquivel, J. A. II, Mahalingam, D., Swords, R., Kelly, K., Zhang, H., Huang, P., Mita, A. C., Mita, M. M., Giles, F. J. & Nawrocki, S. T. (2010). Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. Journal of Cellular and Molecular Medicine 14, 2448–2459. Caron, E., Ghosh, S., Matsuoka, Y., Ashton-Beaucage, D., Therrien, M., Lemieux, S., Perreault, C., Roux, P. P. & Kitano, H. (2010). A comprehensive map of the mTOR signaling network. Molecular Systems Biology 6, 453. Catz, S. D. & Johnson, J. L. (2001). Transcriptional regulation of bcl-2 by nuclear factor kappa B and its significance in prostate cancer. Oncogene 20, 7342–7351. Chandra, V., Mahajan, S., Saini, A., Dkhar, H. K., Nanduri, R., Raj, E. B., Kumar, A. & Gupta, P. (2013). Human IL10 gene repression by Rev-erbalpha ameliorates Mycobacterium tuberculosis clearance. The Journal of Biological Chemistry 288, 10692–10702. Chang, C. P., Su, Y. C., Hu, C. W. & Lei, H. Y. (2013). TLR2-dependent selective autophagy regulates NF-kappaB lysosomal degradation in hepatoma-derived M2 macrophage differentiation. Cell Death and Differentiation 20, 515–523. Chauhan, S., Goodwin, J. G., Manyam, G., Wang, J., Kamat, A. M. & Boyd, D. D. (2013). ZKSCAN3 is a master transcriptional repressor of autophagy. Molecular Cell 50, 16–28. Chawla, A. (2010). Control of macrophage activation and function by PPARs. Circulation Research 106, 1559–1569.

Chen, D., Gao, F., Li, B., Wang, H., Xu, Y., Zhu, C. & Wang, G. (2010). Parkin mono-ubiquitinates Bcl-2 and regulates autophagy. The Journal of Biological Chemistry 285, 38214–38223. Chen, Y. A. & Scheller, R. H. (2001). SNARE-mediated membrane fusion. Nature Reviews Molecular Cell Biology 2, 98–106. Chen, Q., Xie, W., Kuhn, D. J., Voorhees, P. M., Lopez-Girona, A., Mendy, D., Corral, L. G., Krenitsky, V. P., Xu, W., Moutouh-de Parseval, L., Webb, D. R., Mercurio, F., Nakayama, K. I., Nakayama, K. & Orlowski, R. Z. (2008). Targeting the p27 E3 ligase SCF(Skp2) results in p27- and Skp2-mediated cell-cycle arrest and activation of autophagy. Blood 111, 4690–4699. Cheng, Y., Qiu, F., Tashiro, S., Onodera, S. & Ikejima, T. (2008). ERK and JNK mediate TNFalpha-induced p53 activation in apoptotic and autophagic L929 cell death. Biochemical and Biophysical Research Communications 376, 483–488. Cherra, S. J. III, Kulich, S. M., Uechi, G., Balasubramani, M., Mountzouris, J., Day, B. W. & Chu, C. T. (2010). Regulation of the autophagy protein LC3 by phosphorylation. The Journal of Cell Biology 190, 533–539. Cheung, P. C., Trinkle-Mulcahy, L., Cohen, P. & Lucocq, J. M. (2001). Characterization of a novel phosphatidylinositol 3-phosphate-binding protein containing two FYVE fingers in tandem that is targeted to the Golgi. Biochemical Journal 355, 113–121. Codogno, P. & Meijer, A. J. (2005). Autophagy and signaling: their role in cell survival and cell death. Cell Death and Differentiation 12(Suppl. 2), 1509–1518. Comb, W. C., Cogswell, P., Sitcheran, R. & Baldwin, A. S. (2011). IKK-dependent, NF-kappaB-independent control of autophagic gene expression. Oncogene 30, 1727–1732. Comes, F., Matrone, A., Lastella, P., Nico, B., Susca, F. C., Bagnulo, R., Ingravallo, G., Modica, S., Lo Sasso, G., Moschetta, A., Guanti, G. & Simone, C. (2007). A novel cell type-specific role of p38alpha in the control of autophagy and cell death in colorectal cancer cells. Cell Death and Differentiation 14, 693–702. Connolly, E., Braunstein, S., Formenti, S. & Schneider, R. J. (2006). Hypoxia inhibits protein synthesis through a 4E-BP1 and elongation factor 2 kinase pathway controlled by mTOR and uncoupled in breast cancer cells. Molecular and Cellular Biology 26, 3955–3965. Contreras, A. U., Mebratu, Y., Delgado, M., Montano, G., Hu, C. A., Ryter, S. W., Choi, A. M., Lin, Y., Xiang, J., Chand, H. & Tesfaigzi, Y. (2013). Deacetylation of p53 induces autophagy by suppressing Bmf expression. The Journal of Cell Biology 201, 427–437. Copetti, T., Bertoli, C., Dalla, E., Demarchi, F. & Schneider, C. (2009). p65/RelA modulates BECN1 transcription and autophagy. Molecular and Cellular Biology 29, 2594–2608. Corton, J. M., Gillespie, J. G. & Hardie, D. G. (1994). Role of the AMP-activated protein kinase in the cellular stress response. Current Biology 4, 315–324. Crighton, D., Wilkinson, S., O’Prey, J., Syed, N., Smith, P., Harrison, P. R., Gasco, M., Garrone, O., Crook, T. & Ryan, K. M. (2006). DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell 126, 121–134. Cuervo, A. M., Bergamini, E., Brunk, U. T., Droge, W., Ffrench, M. & Terman, A. (2005). Autophagy and aging: the importance of maintaining “clean” cells. Autophagy 1, 131–140. Cui, J., Matkovich, S. J., deSouza, N., Li, S., Rosemblit, N. & Marks, A. R. (2004). Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353. The Journal of Biological Chemistry 279, 16311–16316. vom Dahl, S., Dombrowski, F., Schmitt, M., Schliess, F., Pfeifer, U. & Haussinger, D. (2001). Cell hydration controls autophagosome formation in rat liver in a microtubule-dependent way downstream from p38MAPK activation. Biochemical Journal 354, 31–36. Datta, S. R., Brunet, A. & Greenberg, M. E. (1999). Cellular survival: a play in three Akts. Genes and Development 13, 2905–2927. Decuypere, J. P., Kindt, D., Luyten, T., Welkenhuyzen, K., Missiaen, L., De Smedt, H., Bultynck, G. & Parys, J. B. (2013). mTOR-controlled autophagy requires intracellular Ca(2+) signaling. PLoS ONE 8, e61020. Demontis, F. & Perrimon, N. (2010). FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging. Cell 143, 813–825. Deretic, V. (2010). Autophagy in infection. Current Opinion in Cell Biology 22, 252–262. Deretic, V. & Levine, B. (2009). Autophagy, immunity, and microbial adaptations. Cell Host and Microbe 5, 527–549. Dimova, D. K. & Dyson, N. J. (2005). The E2F transcriptional network: old acquaintances with new faces. Oncogene 24, 2810–2826. Djavaheri-Mergny, M., Amelotti, M., Mathieu, J., Besancon, F., Bauvy, C. & Codogno, P. (2007). Regulation of autophagy by NFkappaB transcription factor and reactives oxygen species. Autophagy 3, 390–392. Djavaheri-Mergny, M., Amelotti, M., Mathieu, J., Besancon, F., Bauvy, C., Souquere, S., Pierron, G. & Codogno, P. (2006). NF-kappaB activation



20 represses tumor necrosis factor-alpha-induced autophagy. The Journal of Biological Chemistry 281, 30373–30382. Eby, K. G., Rosenbluth, J. M., Mays, D. J., Marshall, C. B., Barton, C. E., Sinha, S., Johnson, K. N., Tang, L. & Pietenpol, J. A. (2010). ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy. Molecular Cancer 9, 95. Egan, D. F., Shackelford, D. B., Mihaylova, M. M., Gelino, S., Kohnz, R. A., Mair, W., Vasquez, D. S., Joshi, A., Gwinn, D. M., Taylor, R., Asara, J. M., Fitzpatrick, J., Dillin, A., Viollet, B., Kundu, M., Hansen, M. & Shaw, R. J. (2011). Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456–461. Engelman, J. A., Luo, J. & Cantley, L. C. (2006). The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nature Reviews Genetics 7, 606–619. Esclatine, A., Chaumorcel, M. & Codogno, P. (2009). Macroautophagy signaling and regulation. Current Topics in Microbiology and Immunology 335, 33–70. Feng, Z., Zhang, H., Levine, A. J. & Jin, S. (2005). The coordinate regulation of the p53 and mTOR pathways in cells. Proceedings of the National Academy of Sciences of the United States of America 102, 8204–8209. Fengsrud, M., Erichsen, E. S., Berg, T. O., Raiborg, C. & Seglen, P. O. (2000). Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy. European Journal of Cell Biology 79, 871–882. Fresno Vara, J. A., Casado, E., de Castro, J., Cejas, P., Belda-Iniesta, C. & Gonzalez-Baron, M. (2004). PI3K/Akt signalling pathway and cancer. Cancer Treatment Reviews 30, 193–204. Fullgrabe, J., Klionsky, D. J. & Joseph, B. (2014). The return of the nucleus: transcriptional and epigenetic control of autophagy. Nature Reviews Molecular Cell Biology 15, 65–74. Funderburk, S. F., Wang, Q. J. & Yue, Z. (2010). The Beclin 1-VPS34 complex--at the crossroads of autophagy and beyond. Trends in Cell Biology 20, 355–362. Gammoh, N., Florey, O., Overholtzer, M. & Jiang, X. (2013). Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy. Nature Structural and Molecular Biology 20, 144–149. Ganley, I. G., Lam du, H., Wang, J., Ding, X., Chen, S. & Jiang, X. (2009). ULK1.ATG13.FIP200 complex mediates mTOR signaling and is essential for autophagy. The Journal of Biological Chemistry 284, 12297–12305. Garcia-Garcia, A., Rodriguez-Rocha, H., Tseng, M. T., Montes de Oca-Luna, R., Zhou, H. S., McMasters, K. M. & Gomez-Gutierrez, J. G. (2012). E2F-1 lacking the transcriptional activity domain induces autophagy. Cancer Biology and Therapy 13, 1091–1101. Gearing, K. L., Gottlicher, M., Teboul, M., Widmark, E. & Gustafsson, J. A. (1993). Interaction of the peroxisome-proliferator-activated receptor and retinoid X receptor. Proceedings of the National Academy of Sciences of the United States of America 90, 1440–1444. Geng, J. & Klionsky, D. J. (2008). The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Reports 9, 859–864. Gozuacik, D. & Kimchi, A. (2004). Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23, 2891–2906. Gregor, M. F. & Hotamisligil, G. S. (2007). Thematic review series: adipocyte biology. Adipocyte stress: the endoplasmic reticulum and metabolic disease. Journal of Lipid Research 48, 1905–1914. Guo, L., Huang, J. X., Liu, Y., Li, X., Zhou, S. R., Qian, S. W., Zhu, H., Huang, H. Y., Dang, Y. J. & Tang, Q. Q. (2013). Transactivation of Atg4b by C/EBPbeta promotes autophagy to facilitate adipogenesis. Molecular and Cellular Biology 33, 3180–3190. Gwinn, D. M., Shackelford, D. B., Egan, D. F., Mihaylova, M. M., Mery, A., Vasquez, D. S., Turk, B. E. & Shaw, R. J. (2008). AMPK phosphorylation of raptor mediates a metabolic checkpoint. Molecular Cell 30, 214–226. Hanada, T., Noda, N. N., Satomi, Y., Ichimura, Y., Fujioka, Y., Takao, T., Inagaki, F. & Ohsumi, Y. (2007). The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy. The Journal of Biological Chemistry 282, 37298–37302. Hardie, D. G. & Carling, D. (1997). The AMP-activated protein kinase--fuel gauge of the mammalian cell? European Journal of Biochemistry 246, 259–273. Hardie, D. G., Carling, D. & Sim, A. T. R. (1989). The AMP-activated protein kinase—a multisubstrate regulator of lipid metabolism. Trends in Biochemical Sciences 14, 20–23. Hardie, D. G., Scott, J. W., Pan, D. A. & Hudson, E. R. (2003). Management of cellular energy by the AMP-activated protein kinase system. Federation of European Biochemical Societies Letters 546, 113–120. Harding, H. P., Zhang, Y. & Ron, D. (1999). Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271–274. Hariharan, N., Maejima, Y., Nakae, J., Paik, J., Depinho, R. A. & Sadoshima, J. (2010). Deacetylation of FoxO by Sirt1 plays an essential role in mediating

starvation-induced autophagy in cardiac myocytes. Circulation Research 107, 1470–1482. Harr, M. W., McColl, K. S., Zhong, F., Molitoris, J. K. & Distelhorst, C. W. (2010). Glucocorticoids downregulate Fyn and inhibit IP(3)-mediated calcium signaling to promote autophagy in T lymphocytes. Autophagy 6, 912–921. Hay, N. & Sonenberg, N. (2004). Upstream and downstream of mTOR. Genes and Development 18, 1926–1945. Hayden, M. S. & Ghosh, S. (2012). NF-kappaB, the first quarter-century: remarkable progress and outstanding questions. Genes and Development 26, 203–234. He, C. & Levine, B. (2010). The Beclin 1 interactome. Current Opinion in Cell Biology 22, 140–149. Heinlein, C. A. & Chang, C. (2004). Androgen receptor in prostate cancer. Endocrine Reviews 25, 276–308. Hewison, M. (2011). Antibacterial effects of vitamin D. Nature Reviews Endocrinology 7, 337–345. Hinnebusch, A. G. (1997). Translational regulation of yeast GCN4. A window on factors that control initiator-trna binding to the ribosome. The Journal of Biological Chemistry 272, 21661–21664. Hochfeld, W. E., Lee, S. & Rubinsztein, D. C. (2013). Therapeutic induction of autophagy to modulate neurodegenerative disease progression. Acta Pharmacologica Sinica 34, 600–604. Hosokawa, N., Hara, T., Kaizuka, T., Kishi, C., Takamura, A., Miura, Y., Iemura, S., Natsume, T., Takehana, K., Yamada, N., Guan, J. L., Oshiro, N. & Mizushima, N. (2009). Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Molecular Biology of the Cell 20, 1981–1991. Hoyer-Hansen, M., Bastholm, L., Szyniarowski, P., Campanella, M., Szabadkai, G., Farkas, T., Bianchi, K., Fehrenbacher, N., Elling, F., Rizzuto, R., Mathiasen, I. S. & Jaattela, M. (2007). Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Molecular Cell 25, 193–205. Hu, M. C., Lee, D. F., Xia, W., Golfman, L. S., Ou-Yang, F., Yang, J. Y., Zou, Y., Bao, S., Hanada, N., Saso, H., Kobayashi, R. & Hung, M. C. (2004). IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 117, 225–237. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara, N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T. & Ohsumi, Y. (2000). A ubiquitin-like system mediates protein lipidation. Nature 408, 488–492. Inoki, K., Li, Y., Xu, T. & Guan, K. L. (2003). Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes and Development 17, 1829–1834. Inoki, K., Li, Y., Zhu, T., Wu, J. & Guan, K. L. (2002). TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biology 4, 648–657. Itakura, E., Kishi, C., Inoue, K. & Mizushima, N. (2008). Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Molecular Biology of the Cell 19, 5360–5372. Jacinto, E., Loewith, R., Schmidt, A., Lin, S., Ruegg, M. A., Hall, A. & Hall, M. N. (2004). Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nature Cell Biology 6, 1122–1128. Jackson, J. C. & Lopes, J. M. (1996). The yeast UME6 gene is required for both negative and positive transcriptional regulation of phospholipid biosynthetic gene expression. Nucleic Acids Research 24, 1322–1329. Jia, L., Gopinathan, G., Sukumar, J. T. & Gribben, J. G. (2012). Blocking autophagy prevents bortezomib-induced NF-kappaB activation by reducing I-kappaBalpha degradation in lymphoma cells. PLoS One 7, e32584. Jiang, H., Cheng, D., Liu, W., Peng, J. & Feng, J. (2010a). Protein kinase C inhibits autophagy and phosphorylates LC3. Biochemical and Biophysical Research Communications 395, 471–476. Jiang, M., Fernandez, S., Jerome, W. G., He, Y., Yu, X., Cai, H., Boone, B., Yi, Y., Magnuson, M. A., Roy-Burman, P., Matusik, R. J., Shappell, S. B. & Hayward, S. W. (2010b). Disruption of PPARgamma signaling results in mouse prostatic intraepithelial neoplasia involving active autophagy. Cell Death and Differentiation 17, 469–481. Jiang, Q., Yeh, S., Wang, X., Xu, D., Zhang, Q., Wen, X., Xia, S. & Chang, C. (2012). Targeting androgen receptor leads to suppression of prostate cancer via induction of autophagy. The Journal of Urology 188, 1361–1368. Jimenez, G. S., Khan, S. H., Stommel, J. M. & Wahl, G. M. (1999). p53 regulation by post-translational modification and nuclear retention in response to diverse stresses. Oncogene 18, 7656–7665. Juan, L. J., Shia, W. J., Chen, M. H., Yang, W. M., Seto, E., Lin, Y. S. & Wu, C. W. (2000). Histone deacetylases specifically down-regulate p53-dependent gene activation. The Journal of Biological Chemistry 275, 20436–20443.



21

Jung, C. H., Ro, S. H., Cao, J., Otto, N. M. & Kim, D. H. (2010). mTOR regulation of autophagy. Federation of European Biochemical Societies Letters 584, 1287–1295. Kahn, B. B., Alquier, T., Carling, D. & Hardie, D. G. (2005). AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metabolism 1, 15–25. Kanazawa, T., Taneike, I., Akaishi, R., Yoshizawa, F., Furuya, N., Fujimura, S. & Kadowaki, M. (2004). Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes. The Journal of Biological Chemistry 279, 8452–8459. Kim, H. I. & Ahn, Y. H. (2004). Role of peroxisome proliferator-activated receptor-gamma in the glucose-sensing apparatus of liver and beta-cells. Diabetes 53(Suppl. 1), S60–S65. Kim, J. E. & Chen, J. (2004). Regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 53, 2748–2756. Kim, J., Huang, W. P., Stromhaug, P. E. & Klionsky, D. J. (2002). Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation. The Journal of Biological Chemistry 277, 763–773. Kim, J., Kim, Y. C., Fang, C., Russell, R. C., Kim, J. H., Fan, W., Liu, R., Zhong, Q. & Guan, K. L. (2013). Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy. Cell 152, 290–303. Kim, J., Kundu, M., Viollet, B. & Guan, K. L. (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nature Cell Biology 13, 132–141. Kim, E., Ma, W. L., Lin, D. L., Inui, S., Chen, Y. L. & Chang, C. (2007). TR4 orphan nuclear receptor functions as an apoptosis modulator via regulation of Bcl-2 gene expression. Biochemical and Biophysical Research Communications 361, 323–328. Kimura, T., Takabatake, Y., Takahashi, A. & Isaka, Y. (2013). Chloroquine in cancer therapy: a double-edged sword of autophagy. Cancer Research 73, 3–7. Klionsky, D. J. (2005). The molecular machinery of autophagy: unanswered questions. Journal of Cell Science 118, 7–18. Klionsky, D. J., Cregg, J. M., Dunn, W. A. Jr., Emr, S. D., Sakai, Y., Sandoval, I. V., Sibirny, A., Subramani, S., Thumm, M., Veenhuis, M. & Ohsumi, Y. (2003). A unified nomenclature for yeast autophagy-related genes. Developmental Cell 5, 539–545. Kobayashi, S., Lackey, T., Huang, Y., Bisping, E., Pu, W. T., Boxer, L. M. & Liang, Q. (2006). Transcription factor gata4 regulates cardiac BCL2 gene expression in vitro and in vivo. Federation of American Societies for Experimental Biology Journal 20, 800–802. Koumenis, C., Alarcon, R., Hammond, E., Sutphin, P., Hoffman, W., Murphy, M., Derr, J., Taya, Y., Lowe, S. W., Kastan, M. & Giaccia, A. (2001). Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation. Molecular and Cellular Biology 21, 1297–1310. Kouroku, Y., Fujita, E., Tanida, I., Ueno, T., Isoai, A., Kumagai, H., Ogawa, S., Kaufman, R. J., Kominami, E. & Momoi, T. (2007). ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death and Differentiation 14, 230–239. Kovacs, A. L., Rez, G., Palfia, Z. & Kovacs, J. (2000). Autophagy in the epithelial cells of murine seminal vesicle in vitro. Formation of large sheets of nascent isolation membranes, sequestration of the nucleus and inhibition by wortmannin and 3-ethyladenine. Cell and Tissue Research 302, 253–261. Kratzer, S. & Schuller, H. J. (1997). Transcriptional control of the yeast acetyl-CoA synthetase gene, ACS1, by the positive regulators CAT8 and ADR1 and the pleiotropic repressor UME6. Molecular Microbiology 26, 631–641. Kumar, A., Takada, Y., Boriek, A. M. & Aggarwal, B. B. (2004). Nuclear factor-kappaB: its role in health and disease. Journal of Molecular Medicine (Berlin) 82, 434–448. Lane, D. P. (1992). Cancer. p53, guardian of the genome. Nature 358, 15–16. Laplante, M. & Sabatini, D. M. (2012). mTOR signaling in growth control and disease. Cell 149, 274–293. Lee, J. A. (2009). Autophagy in neurodegeneration: two sides of the same coin. BMB Reports 42, 324–330. Lee, I. H., Cao, L., Mostoslavsky, R., Lombard, D. B., Liu, J., Bruns, N. E., Tsokos, M., Alt, F. W. & Finkel, T. (2008). A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proceedings of the National Academy of Sciences of the United States of America 105, 3374–3379. Levine, B. & Klionsky, D. J. (2004). Development by self-digestion: molecular mechanisms and biological functions of autophagy. Developmental Cell 6, 463–477. Levine, B., Sinha, S. & Kroemer, G. (2008). Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy 4, 600–606.

Li, M., Brooks, C. L., Wu-Baer, F., Chen, D., Baer, R. & Gu, W. (2003). Monoversus polyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972–1975. Li, D. D., Wang, L. L., Deng, R., Tang, J., Shen, Y., Guo, J. F., Wang, Y., Xia, L. P., Feng, G. K., Liu, Q. Q., Huang, W. L., Zeng, Y. X. & Zhu, X. F. (2009). The pivotal role of c-Jun NH2-terminal kinase-mediated Beclin 1 expression during anticancer agents-induced autophagy in cancer cells. Oncogene 28, 886–898. Liang, C., Lee, J. S., Inn, K. S., Gack, M. U., Li, Q., Roberts, E. A., Vergne, I., Deretic, V., Feng, P., Akazawa, C. & Jung, J. U. (2008). Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nature Cell Biology 10, 776–787. Lin, S. Y., Li, T. Y., Liu, Q., Zhang, C., Li, X., Chen, Y., Zhang, S. M., Lian, G., Ruan, K., Wang, Z., Zhang, C. S., Chien, K. Y., Wu, J., Li, Q., Han, J. & Lin, S. C. (2012). GSK3-TIP60-ULK1 signaling pathway links growth factor deprivation to autophagy. Science 336, 477–481. Lin, A., Yao, J., Zhuang, L., Wang, D., Han, J., Lam, E. W. & Gan, B. (2013). The FoxO-BNIP3 axis exerts a unique regulation of mTORC1 and cell survival under energy stress. Oncogene 33, 3183–3194. Lu, Q., Yang, P., Huang, X., Hu, W., Guo, B., Wu, F., Lin, L., Kovacs, A. L., Yu, L. & Zhang, H. (2011). The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes. Developmental Cell 21, 343–357. Ma, D., Panda, S. & Lin, J. D. (2011). Temporal orchestration of circadian autophagy rhythm by C/EBPbeta. The EMBO Journal 30, 4642–4651. Mack, H. I., Zheng, B., Asara, J. M. & Thomas, S. M. (2012). AMPK-dependent phosphorylation of ULK1 regulates ATG9 localization. Autophagy 8, 1197–1214. Mahajan, S., Dkhar, H. K., Chandra, V., Dave, S., Nanduri, R., Janmeja, A. K., Agrewala, J. N. & Gupta, P. (2012). Mycobacterium tuberculosis modulates macrophage lipid-sensing nuclear receptors PPARgamma and TR4 for survival. The Journal of Immunology 188, 5593–5603. Mahmood, D. F., Jguirim-Souissi, I., Khadija el, H., Blondeau, N., Diderot, V., Amrani, S., Slimane, M. N., Syrovets, T., Simmet, T. & Rouis, M. (2011). Peroxisome proliferator-activated receptor gamma induces apoptosis and inhibits autophagy of human monocyte-derived macrophages via induction of cathepsin L: potential role in atherosclerosis. The Journal of Biological Chemistry 286, 28858–28866. Mammucari, C., Milan, G., Romanello, V., Masiero, E., Rudolf, R., Del Piccolo, P., Burden, S. J., Di Lisi, R., Sandri, C., Zhao, J., Goldberg, A. L., Schiaffino, S. & Sandri, M. (2007). FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metabolism 6, 458–471. Marquez, R. T. & Xu, L. (2012). Bcl-2: Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. American Journal of Cancer Research 2, 214–221. Martina, J. A., Chen, Y., Gucek, M. & Puertollano, R. (2012). MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 8, 903–914. Martina, J. A., Diab, H. I., Lishu, L., Jeong, A. L., Patange, S., Raben, N. & Puertollano, R. (2014). The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Science Signaling 7, ra9. Martineau, A. R., Wilkinson, K. A., Newton, S. M., Floto, R. A., Norman, A. W., Skolimowska, K., Davidson, R. N., Sorensen, O. E., Kampmann, B., Griffiths, C. J. & Wilkinson, R. J. (2007). IFN-gamma- and TNF-independent vitamin D-inducible human suppression of mycobacteria: the role of cathelicidin LL-37. The Journal of Immunology 178, 7190–7198. Mathew, R., Karantza-Wadsworth, V. & White, E. (2007). Role of autophagy in cancer. Nature Reviews Cancer 7, 961–967. Matsushita, M., Suzuki, N. N., Obara, K., Fujioka, Y., Ohsumi, Y. & Inagaki, F. (2007). Structure of Atg5.Atg16, a complex essential for autophagy. The Journal of Biological Chemistry 282, 6763–6772. Matsuzawa, T., Kim, B. H., Shenoy, A. R., Kamitani, S., Miyake, M. & Macmicking, J. D. (2012). IFN-gamma elicits macrophage autophagy via the p38 MAPK signaling pathway. The Journal of Immunology 189, 813–818. McClung, J. M., Judge, A. R., Powers, S. K. & Yan, Z. (2010). p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting. American Journal of Physiology - Cell Physiology 298, C542–C549. McEwan, D. G. & Dikic, I. (2011). The Three Musketeers of Autophagy: phosphorylation, ubiquitylation and acetylation. Trends in Cell Biology 21, 195–201. McKay, M. M. & Morrison, D. K. (2007). Integrating signals from RTKs to ERK/MAPK. Oncogene 26, 3113–3121. Mizushima, N. (2005). The pleiotropic role of autophagy: from protein metabolism to bactericide. Cell Death and Differentiation 12 (Suppl. 2), 1535–1541. Mizushima, N., Ohsumi, Y. & Yoshimori, T. (2002). Autophagosome formation in mammalian cells. Cell Structure and Function 27, 421–429.



22 Mizushima, N., Yoshimori, T. & Ohsumi, Y. (2011). The role of Atg proteins in autophagosome formation. Annual Review of Cell and Developmental Biology 27, 107–132. Moreau, K., Ravikumar, B., Renna, M., Puri, C. & Rubinsztein, D. C. (2011). Autophagosome precursor maturation requires homotypic fusion. Cell 146, 303–317. Mortimore, G. E. & Poso, A. R. (1987). Intracellular protein catabolism and its control during nutrient deprivation and supply. Annual Review of Nutrition 7, 539–564. Naidu, S. R., Lakhter, A. J. & Androphy, E. J. (2012). PIASy-mediated Tip60 sumoylation regulates p53-induced autophagy. Cell Cycle 11, 2717–2728. Nair, U. & Klionsky, D. J. (2011). Autophagosome biogenesis requires SNAREs. Autophagy 7, 1570–1572. Nazio, F., Strappazzon, F., Antonioli, M., Bielli, P., Cianfanelli, V., Bordi, M., Gretzmeier, C., Dengjel, J., Piacentini, M., Fimia, G. M. & Cecconi, F. (2013). mTOR inhibits autophagy by controlling ULK1 ubiquitylation, self-association and function through AMBRA1 and TRAF6. Nature Cell Biology 15, 406–416. Nie, L., Sasaki, M. & Maki, C. G. (2007). Regulation of p53 nuclear export through sequential changes in conformation and ubiquitination. The Journal of Biological Chemistry 282, 14616–14625. Nixon, R. A. (2013). The role of autophagy in neurodegenerative disease. Nature Medicine 19, 983–997. Nixon, R. A., Yang, D. S. & Lee, J. H. (2008). Neurodegenerative lysosomal disorders: a continuum from development to late age. Autophagy 4, 590–599. Noble, C. G., Dong, J. M., Manser, E. & Song, H. (2008). Bcl-xL and UVRAG cause a monomer-dimer switch in Beclin1. The Journal of Biological Chemistry 283, 26274–26282. Nobukuni, T., Joaquin, M., Roccio, M., Dann, S. G., Kim, S. Y., Gulati, P., Byfield, M. P., Backer, J. M., Natt, F., Bos, J. L., Zwartkruis, F. J. & Thomas, G. (2005). Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proceedings of the National Academy of Sciences of the United States of America 102, 14238–14243. Norman, A. W. (2008). From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health. The American Journal of Clinical Nutrition 88, 491S–499S. Obara, K. & Ohsumi, Y. (2008). Dynamics and function of PtdIns(3)P in autophagy. Autophagy 4, 952–954. Ogata, M., Hino, S., Saito, A., Morikawa, K., Kondo, S., Kanemoto, S., Murakami, T., Taniguchi, M., Tanii, I., Yoshinaga, K., Shiosaka, S., Hammarback, J. A., Urano, F. & Imaizumi, K. (2006). Autophagy is activated for cell survival after endoplasmic reticulum stress. Molecular and Cellular Biology 26, 9220–9231. Ogier-Denis, E., Pattingre, S., El Benna, J. & Codogno, P. (2000). Erk1/2-dependent phosphorylation of Galpha-interacting protein stimulates its GTPase accelerating activity and autophagy in human colon cancer cells. The Journal of Biological Chemistry 275, 39090–39095. Orsi, A., Razi, M., Dooley, H. C., Robinson, D., Weston, A. E., Collinson, L. M. & Tooze, S. A. (2012). Dynamic and transient interactions of Atg9 with autophagosomes, but not membrane integration, are required for autophagy. Molecular Biology of the Cell 23, 1860–1873. Orvedahl, A. & Levine, B. (2009). Eating the enemy within: autophagy in infectious diseases. Cell Death and Differentiation 16, 57–69. Palacios, E. H. & Weiss, A. (2004). Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23, 7990–8000. Papandreou, I., Lim, A. L., Laderoute, K. & Denko, N. C. (2008). Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L. Cell Death and Differentiation 15, 1572–1581. Papinski, D., Schuschnig, M., Reiter, W., Wilhelm, L., Barnes, C. A., Maiolica, A., Hansmann, I., Pfaffenwimmer, T., Kijanska, M., Stoffel, I., Lee, S. S., Brezovich, A., Lou, J. H., Turk, B. E., Aebersold, R., Ammerer, G., Peter, M. & Kraft, C. (2014). Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase. Molecular Cell 53, 471–483. Pattingre, S., Tassa, A., Qu, X., Garuti, R., Liang, X. H., Mizushima, N., Packer, M., Schneider, M. D. & Levine, B. (2005). Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122, 927–939. Pena-Llopis, S., Vega-Rubin-de-Celis, S., Schwartz, J. C., Wolff, N. C., Tran, T. A., Zou, L., Xie, X. J., Corey, D. R. & Brugarolas, J. (2011). Regulation of TFEB and V-ATPases by mTORC1. The EMBO Journal 30, 3242–3258. Picard, F. & Auwerx, J. (2002). PPAR(gamma) and glucose homeostasis. Annual Review of Nutrition 22, 167–197. Pike, L. R., Phadwal, K., Simon, A. K. & Harris, A. L. (2012). ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. Molecular Biology Reports 39, 10811–10822. Pike, L. R., Singleton, D. C., Buffa, F., Abramczyk, O., Phadwal, K., Li, J. L., Simon, A. K., Murray, J. T. & Harris, A. L. (2013). Transcriptional up-regulation of ULK1 by ATF4 contributes to cancer cell survival. Biochemical Journal 449, 389–400.

Polager, S. & Ginsberg, D. (2008). E2F – at the crossroads of life and death. Trends in Cell Biology 18, 528–535. Polager, S. & Ginsberg, D. (2009). p53 and E2f: partners in life and death. Nature Reviews Cancer 9, 738–748. Polager, S., Ofir, M. & Ginsberg, D. (2008). E2F1 regulates autophagy and the transcription of autophagy genes. Oncogene 27, 4860–4864. Polson, H. E., de Lartigue, J., Rigden, D. J., Reedijk, M., Urbe, S., Clague, M. J. & Tooze, S. A. (2010). Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation. Autophagy 6, 506–522. Ravikumar, B., Vacher, C., Berger, Z., Davies, J. E., Luo, S., Oroz, L. G., Scaravilli, F., Easton, D. F., Duden, R., O’Kane, C. J. & Rubinsztein, D. C. (2004). Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nature Genetics 36, 585–595. Ridley, S. H., Ktistakis, N., Davidson, K., Anderson, K. E., Manifava, M., Ellson, C. D., Lipp, P., Bootman, M., Coadwell, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P., Cooper, M. A., Thuring, J. W., Lim, Z. Y., Holmes, A. B., Stephens, L. R. & Hawkins, P. T. (2001). FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments. Journal of Cell Science 114, 3991–4000. Roux, P. P. & Blenis, J. (2004). ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiology and Molecular Biology Reviews 68, 320–344. Russo, R., Berliocchi, L., Adornetto, A., Varano, G. P., Cavaliere, F., Nucci, C., Rotiroti, D., Morrone, L. A., Bagetta, G. & Corasaniti, M. T. (2011). Calpain-mediated cleavage of Beclin-1 and autophagy deregulation following retinal ischemic injury in vivo. Cell Death and Disease 2, e144. Rzymski, T., Milani, M., Pike, L., Buffa, F., Mellor, H. R., Winchester, L., Pires, I., Hammond, E., Ragoussis, I. & Harris, A. L. (2010). Regulation of autophagy by ATF4 in response to severe hypoxia. Oncogene 29, 4424–4435. Sanchez, A. M., Csibi, A., Raibon, A., Cornille, K., Gay, S., Bernardi, H. & Candau, R. (2012). AMPK promotes skeletal muscle autophagy through activation of forkhead FoxO3a and interaction with Ulk1. Journal of Cellular Biochemistry 113, 695–710. Sandri, M. (2012). FOXOphagy path to inducing stress resistance and cell survival. Nature Cell Biology 14, 786–788. Sarbassov, D. D., Ali, S. M., Kim, D. H., Guertin, D. A., Latek, R. R., Erdjument-Bromage, H., Tempst, P. & Sabatini, D. M. (2004). Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Current Biology 14, 1296–1302. Sardiello, M., Palmieri, M., di Ronza, A., Medina, D. L., Valenza, M., Gennarino, V. A., Di Malta, C., Donaudy, F., Embrione, V., Polishchuk, R. S., Banfi, S., Parenti, G., Cattaneo, E. & Ballabio, A. (2009). A gene network regulating lysosomal biogenesis and function. Science 325, 473–477. Scherz-Shouval, R., Shvets, E., Fass, E., Shorer, H., Gil, L. & Elazar, Z. (2007). Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. The EMBO Journal 26, 1749–1760. Schofield, C. J. & Ratcliffe, P. J. (2004). Oxygen sensing by HIF hydroxylases. Nature Reviews Molecular Cell Biology 5, 343–354. Seglen, P. O. & Bohley, P. (1992). Autophagy and other vacuolar protein degradation mechanisms. Experientia 48, 158–172. Sengupta, A., Molkentin, J. D. & Yutzey, K. E. (2009). FoxO transcription factors promote autophagy in cardiomyocytes. The Journal of Biological Chemistry 284, 28319–28331. Sengupta, S., Peterson, T. R. & Sabatini, D. M. (2010). Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Molecular Cell 40, 310–322. Seo, Y. K., Jeon, T. I., Chong, H. K., Biesinger, J., Xie, X. & Osborne, T. F. (2011). Genome-wide localization of SREBP-2 in hepatic chromatin predicts a role in autophagy. Cell Metabolism 13, 367–375. Settembre, C., Di Malta, C., Polito, V. A., Garcia Arencibia, M., Vetrini, F., Erdin, S., Erdin, S. U., Huynh, T., Medina, D., Colella, P., Sardiello, M., Rubinsztein, D. C. & Ballabio, A. (2011). TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433. Settembre, C., Zoncu, R., Medina, D. L., Vetrini, F., Erdin, S., Huynh, T., Ferron, M., Karsenty, G., Vellard, M. C., Facchinetti, V., Sabatini, D. M. & Ballabio, A. (2012). A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. The EMBO Journal 31, 1095–1108. Shaw, J., Yurkova, N., Zhang, T., Gang, H., Aguilar, F., Weidman, D., Scramstad, C., Weisman, H. & Kirshenbaum, L. A. (2008). Antagonism of E2F-1 regulated Bnip3 transcription by NF-kappaB is essential for basal cell survival. Proceedings of the National Academy of Sciences of the United States of America 105, 20734–20739.



23

Sheng, Z., Ma, L., Sun, J. E., Zhu, L. J. & Green, M. R. (2011). BCR-ABL suppresses autophagy through ATF5-mediated regulation of mTOR transcription. Blood 118, 2840–2848. Shi, Y., Han, J. J., Tennakoon, J. B., Mehta, F. F., Merchant, F. A., Burns, A. R., Howe, M. K., McDonnell, D. P. & Frigo, D. E. (2013). Androgens promote prostate cancer cell growth through induction of autophagy. Molecular Endocrinology 27, 280–295. Shieh, S. Y., Taya, Y. & Prives, C. (1999). DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization. The EMBO Journal 18, 1815–1823. Shintani, T. & Klionsky, D. J. (2004). Autophagy in health and disease: a double-edged sword. Science 306, 990–995. Siliciano, J. D., Canman, C. E., Taya, Y., Sakaguchi, K., Appella, E. & Kastan, M. B. (1997). DNA damage induces phosphorylation of the amino terminus of p53. Genes and Development 11, 3471–3481. Sinha, S. & Levine, B. (2008). The autophagy effector Beclin 1: a novel BH3-only protein. Oncogene 27 (Suppl. 1), S137–S148. Sivaprasad, U. & Basu, A. (2008). Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells. Journal of Cellular and Molecular Medicine 12, 1265–1271. Song, M. S., Salmena, L. & Pandolfi, P. P. (2012). The functions and regulation of the PTEN tumour suppressor. Nature Reviews Molecular Cell Biology 13, 283–296. Sridharan, S., Jain, K. & Basu, A. (2011). Regulation of autophagy by kinases. Cancers 3, 2630–2654. Stambolic, V., MacPherson, D., Sas, D., Lin, Y., Snow, B., Jang, Y., Benchimol, S. & Mak, T. W. (2001). Regulation of PTEN transcription by p53. Molecular Cell 8, 317–325. Steber, C. M. & Esposito, R. E. (1995). UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression. Proceedings of the National Academy of Sciences of the United States of America 92, 12490–12494. Stevens, C. & La Thangue, N. B. (2004). The emerging role of E2F-1 in the DNA damage response and checkpoint control. DNA Repair (Amsterdam) 3, 1071–1079. Strich, R., Surosky, R. T., Steber, C., Dubois, E., Messenguy, F. & Esposito, R. E. (1994). UME6 is a key regulator of nitrogen repression and meiotic development. Genes and Development 8, 796–810. Suzuki, K., Kirisako, T., Kamada, Y., Mizushima, N., Noda, T. & Ohsumi, Y. (2001). The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation. The EMBO Journal 20, 5971–5981. Sweet, D. H., Jang, Y. K. & Sancar, G. B. (1997). Role of UME6 in transcriptional regulation of a DNA repair gene in Saccharomyces cerevisiae. Molecular and Cellular Biology 17, 6223–6235. Talloczy, Z., Jiang, W., Virgin, H. W.t., Leib, D. A., Scheuner, D., Kaufman, R. J., Eskelinen, E. L. & Levine, B. (2002). Regulation of starvationand virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proceedings of the National Academy of Sciences of the United States of America 99, 190–195. Tasdemir, E., Chiara Maiuri, M., Morselli, E., Criollo, A., D’Amelio, M., Djavaheri-Mergny, M., Cecconi, F., Tavernarakis, N. & Kroemer, G. (2008). A dual role of p53 in the control of autophagy. Autophagy 4, 810–814. Tracy, K., Dibling, B. C., Spike, B. T., Knabb, J. R., Schumacker, P. & Macleod, K. F. (2007). BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy. Molecular and Cellular Biology 27, 6229–6242. Trela, E., Glowacki, S. & Blasiak, J. (2014). Therapy of chronic myeloid leukemia: twilight of the Imatinib era? International Scholarly Research Notices Oncology 2014, 596483. Trocoli, A. & Djavaheri-Mergny, M. (2011). The complex interplay between autophagy and NF-kappaB signaling pathways in cancer cells. American Journal of Cancer Research 1, 629–649. Trocoli, A., Mathieu, J., Priault, M., Reiffers, J., Souquere, S., Pierron, G., Besancon, F. & Djavaheri-Mergny, M. (2011). ATRA-induced upregulation of Beclin 1 prolongs the life span of differentiated acute promyelocytic leukemia cells. Autophagy 7, 1108–1114. Tzifi, F., Economopoulou, C., Gourgiotis, D., Ardavanis, A., Papageorgiou, S. & Scorilas, A. (2012). The role of BCL2 family of apoptosis regulator proteins in acute and chronic leukemias. Advances in Hematology 2012, 524308. Vallabhapurapu, S. & Karin, M. (2009). Regulation and function of NF-kappaB transcription factors in the immune system. Annual Review of Immunology 27, 693–733. Vara, D., Morell, C., Rodriguez-Henche, N. & Diaz-Laviada, I. (2013). Involvement of PPARgamma in the antitumoral action of cannabinoids on hepatocellular carcinoma. Cell Death and Disease 4, e618.

Vara, D., Salazar, M., Olea-Herrero, N., Guzman, M., Velasco, G. & Diaz-Laviada, I. (2011). Anti-tumoral action of cannabinoids on hepatocellular carcinoma: role of AMPK-dependent activation of autophagy. Cell Death and Differentiation 18, 1099–1111. van der Vos, K. E., Eliasson, P., Proikas-Cezanne, T., Vervoort, S. J., van Boxtel, R., Putker, M., van Zutphen, I. J., Mauthe, M., Zellmer, S., Pals, C., Verhagen, L. P., Groot Koerkamp, M. J., Braat, A. K., Dansen, T. B., Holstege, F. C., Gebhardt, R., Burgering, B. M. & Coffer, P. J. (2012). Modulation of glutamine metabolism by the PI(3)K-PKB-FOXO network regulates autophagy. Nature Cell Biology 14, 829–837. Vidal, R. L., Figueroa, A., Court, F. A., Thielen, P., Molina, C., Wirth, C., Caballero, B., Kiffin, R., Segura-Aguilar, J., Cuervo, A. M., Glimcher, L. H. & Hetz, C. (2012). Targeting the UPR transcription factor XBP1 protects against Huntington’s disease through the regulation of FoxO1 and autophagy. Human Molecular Genetics 21, 2245–2262. Wagner, E. F. & Nebreda, A. R. (2009). Signal integration by JNK and p38 MAPK pathways in cancer development. Nature Reviews Cancer 9, 537–549. Wang, J., Lian, H., Zhao, Y., Kauss, M. A. & Spindel, S. (2008). Vitamin D3 induces autophagy of human myeloid leukemia cells. The Journal of Biological Chemistry 283, 25596–25605. Wang, J., Whiteman, M. W., Lian, H., Wang, G., Singh, A., Huang, D. & Denmark, T. (2009). A non-canonical MEK/ERK signaling pathway regulates autophagy via regulating Beclin 1. The Journal of Biological Chemistry 284, 21412–21424. Wang, Z., Wilson, W. A., Fujino, M. A. & Roach, P. J. (2001). Antagonistic controls of autophagy and glycogen accumulation by Snf1p, the yeast homolog of AMP-activated protein kinase, and the cyclin-dependent kinase Pho85p. Molecular and Cellular Biology 21, 5742–5752. Warr, M. R., Binnewies, M., Flach, J., Reynaud, D., Garg, T., Malhotra, R., Debnath, J. & Passegue, E. (2013). FOXO3A directs a protective autophagy program in haematopoietic stem cells. Nature 494, 323–327. Watkins, S. M., Reifsnyder, P. R., Pan, H. J., German, J. B. & Leiter, E. H. (2002). Lipid metabolome-wide effects of the PPARgamma agonist rosiglitazone. Journal of Lipid Research 43, 1809–1817. Webber, J. L. & Tooze, S. A. (2010). Coordinated regulation of autophagy by p38alpha MAPK through mAtg9 and p38IP. The EMBO Journal 29, 27–40. Wei, Y., Pattingre, S., Sinha, S., Bassik, M. & Levine, B. (2008). JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Molecular Cell 30, 678–688. Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q. & Farnham, P. J. (2001). Use of chromatin immunoprecipitation to clone novel E2F target promoters. Molecular and Cellular Biology 21, 6820–6832. Wek, R. C., Jiang, H. Y. & Anthony, T. G. (2006). Coping with stress: eIF2 kinases and translational control. Biochemical Society Transactions 34, 7–11. Williams, R. M., Primig, M., Washburn, B. K., Winzeler, E. A., Bellis, M., Sarrauste de Menthiere, C., Davis, R. W. & Esposito, R. E. (2002). The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast. Proceedings of the National Academy of Sciences of the United States of America 99, 13431–13436. Williams, A., Sarkar, S., Cuddon, P., Ttofi, E. K., Saiki, S., Siddiqi, F. H., Jahreiss, L., Fleming, A., Pask, D., Goldsmith, P., O’Kane, C. J., Floto, R. A. & Rubinsztein, D. C. (2008). Novel targets for Huntington’s disease in an mTOR-independent autophagy pathway. Nature Chemical Biology 4, 295–305. Wong, P. M., Puente, C., Ganley, I. G. & Jiang, X. (2013). The ULK1 complex: sensing nutrient signals for autophagy activation. Autophagy 9, 124–137. Wu, Z., Chang, P. C., Yang, J. C., Chu, C. Y., Wang, L. Y., Chen, N. T., Ma, A. H., Desai, S. J., Lo, S. H., Evans, C. P., Lam, K. S. & Kung, H. J. (2010). Autophagy blockade sensitizes prostate cancer cells towards Src family kinase inhibitors. Genes and Cancer 1, 40–49. Wu, Y. T., Tan, H. L., Huang, Q., Ong, C. N. & Shen, H. M. (2009). Activation of the PI3K-Akt-mTOR signaling pathway promotes necrotic cell death via suppression of autophagy. Autophagy 5, 824–834. Xu, P., Das, M., Reilly, J. & Davis, R. J. (2011). JNK regulates FoxO-dependent autophagy in neurons. Genes and Development 25, 310–322. Yang, L., Wang, H., Kornblau, S. M., Graber, D. A., Zhang, N., Matthews, J. A., Wang, M., Weber, D. M., Thomas, S. K., Shah, J. J., Zhang, L., Lu, G., Zhao, M., Muddasani, R., Yoo, S. Y., Baggerly, K. A. & Orlowski, R. Z. (2011a). Evidence of a role for the novel zinc-finger transcription factor ZKSCAN3 in modulating Cyclin D2 expression in multiple myeloma. Oncogene 30, 1329–1340. Yang, Z. J., Chee, C. E., Huang, S. & Sinicrope, F. A. (2011b). The role of autophagy in cancer: therapeutic implications. Molecular Cancer Therapeutics 10, 1533–1541. Yang, Y., Fiskus, W., Yong, B., Atadja, P., Takahashi, Y., Pandita, T. K., Wang, H. G. & Bhalla, K. N. (2013). Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy.



24 Proceedings of the National Academy of Sciences of the United States of America 110, 6841–6846. Yang, L., Hamilton, S. R., Sood, A., Kuwai, T., Ellis, L., Sanguino, A., Lopez-Berestein, G. & Boyd, D. D. (2008a). The previously undescribed ZKSCAN3 (ZNF306) is a novel “driver” of colorectal cancer progression. Cancer Research 68, 4321–4330. Yang, L., Zhang, L., Wu, Q. & Boyd, D. D. (2008b). Unbiased screening for transcriptional targets of ZKSCAN3 identifies integrin beta 4 and vascular endothelial growth factor as downstream targets. The Journal of Biological Chemistry 283, 35295–35304. Yorimitsu, T., Nair, U., Yang, Z. & Klionsky, D. J. (2006). Endoplasmic reticulum stress triggers autophagy. The Journal of Biological Chemistry 281, 30299–30304. Young, A. R., Narita, M., Ferreira, M., Kirschner, K., Sadaie, M., Darot, J. F., Tavare, S., Arakawa, S., Shimizu, S. & Watt, F. M. (2009). Autophagy mediates the mitotic senescence transition. Genes and Development 23, 798–803. Yousefi, S., Perozzo, R., Schmid, I., Ziemiecki, A., Schaffner, T., Scapozza, L., Brunner, T. & Simon, H. U. (2006). Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nature Cell Biology 8, 1124–1132. Youssef, J. & Badr, M. (2011). Peroxisome proliferator-activated receptors and cancer: challenges and opportunities. British Journal of Pharmacology 164, 68–82. Yuk, J. M., Shin, D. M., Lee, H. M., Yang, C. S., Jin, H. S., Kim, K. K., Lee, Z. W., Lee, S. H., Kim, J. M. & Jo, E. K. (2009). Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host and Microbe 6, 231–243. Zamoyska, R., Basson, A., Filby, A., Legname, G., Lovatt, M. & Seddon, B. (2003). The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation. Immunological Reviews 191, 107–118. Zhang, H., Bosch-Marce, M., Shimoda, L. A., Tan, Y. S., Baek, J. H., Wesley, J. B., Gonzalez, F. J. & Semenza, G. L. (2008). Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia. The Journal of Biological Chemistry 283, 10892–10903.

Zhang, X., Jing, Y., Qin, Y., Hunsucker, S., Meng, H., Sui, J., Jiang, Y., Gao, L., An, G., Yang, N., Orlowski, R. Z. & Yang, L. (2012). The zinc finger transcription factor ZKSCAN3 promotes prostate cancer cell migration. The International Journal of Biochemistry and Cell Biology 44, 1166–1173. Zhang, L., Yu, J., Pan, H., Hu, P., Hao, Y., Cai, W., Zhu, H., Yu, A. D., Xie, X., Ma, D. & Yuan, J. (2007). Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proceedings of the National Academy of Sciences of the United States of America 104, 19023–19028. Zhou, X., Babu, J. R., da Silva, S., Shu, Q., Graef, I. A., Oliver, T., Tomoda, T., Tani, T., Wooten, M. W. & Wang, F. (2007). Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons. Proceedings of the National Academy of Sciences of the United States of America 104, 5842–5847. Zhao, J., Brault, J. J., Schild, A., Cao, P., Sandri, M., Schiaffino, S., Lecker, S. H. & Goldberg, A. L. (2007). FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metabolism 6, 472–483. Zhao, J., Brault, J. J., Schild, A. & Goldberg, A. L. (2008). Coordinate activation of autophagy and the proteasome pathway by FoxO transcription factor. Autophagy 4, 378–380. Zhao, Y., Li, X., Ma, K., Yang, J., Zhou, J., Fu, W., Wei, F., Wang, L. & Zhu, W. G. (2013). The axis of MAPK1/3-XBP1u-FOXO1 controls autophagic dynamics in cancer cells. Autophagy 9, 794–796. Zhao, Y., Yang, J., Liao, W., Liu, X., Zhang, H., Wang, S., Wang, D., Feng, J., Yu, L. & Zhu, W. G. (2010). Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity. Nature Cell Biology 12, 665–675. Zhou, J., Liao, W., Yang, J., Ma, K., Li, X., Wang, Y., Wang, D., Wang, L., Zhang, Y., Yin, Y., Zhao, Y. & Zhu, W. G. (2012). FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway. Autophagy 8, 1712–1723. Zhou, J., Zhang, W., Liang, B., Casimiro, M. C., Whitaker-Menezes, D., Wang, M., Lisanti, M. P., Lanza-Jacoby, S., Pestell, R. G. & Wang, C. (2009). PPARgamma activation induces autophagy in breast cancer cells. The International Journal of Biochemistry and Cell Biology 41, 2334–2342.

(Received 8 May 2014; revised 4 January 2015; accepted 11 January 2015 )


AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy.

Postnatal signalling with homeoprotein transcription factors.

How do cilia organize signalling cascades?

JNK signalling: a potential autophagy regulation pathway.

Transcription factors NRF2 and HSF1 have opposing functions in autophagy.

Beyond transcription factors: how oncogenic signalling reshapes the epigenetic landscape.

AKT Signalling Cascades inParkinson's Disease.

Regulation of transcription factors by sumoylation.

Regulation of the protein stability of EMT transcription factors.

Plastid sigma factors: Their individual functions and regulation in transcription.

WRKY Transcription Factors: Molecular Regulation and Stress Responses in Plants.

Regulation of the human ABCB10 gene by E2F transcription factors.

Chromatin Kinases Act on Transcription Factors and Histone Tails in Regulation of Inducible Transcription.

Regulation of Cartilage Development and Diseases by Transcription Factors.

Hydrogen peroxide sensing, signaling and regulation of transcription factors.

Dynamic regulation of transcriptional states by chromatin and transcription factors.

Regulation of transcription factors via natural decoys in genomic DNA.

Transcriptome dynamics of developing maize leaves and genomewide prediction of cis elements and their cognate transcription factors.

Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits.

The roles of ethylene and transcription factors in the regulation of onset of leaf senescence.

Transcription factors: specific DNA binding and specific gene regulation.

Robert Feulgen Lecture 1991. Control and role of major signalling cascades of the thyrocyte.

FOXO transcription factors: their clinical significance and regulation.

Regulation of Hippo signalling by p38 signalling.