Commentary 2359 and growth – the yin and yang of nutrient responses

Thomas P. Neufeld Department of Genetics, and Development, University of Minnesota, Minneapolis, MN 55455, USA [email protected]

Journal of Cell Science 125, 2359–2368 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.103333

Summary As a response to nutrient deprivation and other cell stresses, autophagy is often induced in the context of reduced or arrested cell growth. A plethora of signaling molecules and pathways have been shown to have opposing effects on cell growth and autophagy, and results of recent functional screens on a genomic scale support the idea that these processes might represent mutually exclusive cell fates. Understanding the ways in which autophagy and cell growth relate to one another is becoming increasingly important, as new roles for autophagy in tumorigenesis and other growth-related phenomena are uncovered. This Commentary highlights recent findings that link autophagy and cell growth, and explores the mechanisms underlying these connections and their implications for cell physiology and survival. Autophagy and cell growth can inhibit one another through a variety of direct and indirect mechanisms, and can be independently regulated by common signaling pathways. The central role of the mammalian target of rapamycin (mTOR) pathway in regulating both autophagy and cell growth exemplifies one such mechanism. In addition, mTOR-independent signaling and other more direct connections between autophagy and cell growth will also be discussed. This article is part of a Minifocus on Autophagy. For further reading, please see related articles: ‘Ubiquitin-like proteins and autophagy at a glance’ by Tomer Shpilka et al. (J. Cell Sci. 125, 2343-2348) and ‘Autophagy and cancer – issues we need to digest’ by Emma Liu and Kevin Ryan (J. Cell Sci. 125, 2349-2358).

Key words: Autophagy, Cell growth, Mammalian target of rapamycin (mTOR), Protein synthesis, , Cell cycle

Introduction On a fundamental level, autophagy and cell growth are mirror Cells have an intrinsic drive to increase their mass, which is images of one another. If cell growth is defined as the process of illustrated by the ravenous growth of embryos and juveniles mass accumulation through the net uptake and conversion of Journal of Cell Science during development, and the logarithmic proliferation of cells in nutrients into macromolecules, autophagy can be considered to culture. Even in adult organisms, whose size has reached a steady act in opposition to these biosynthetic processes through the state, continued growth and proliferation of differentiated and catabolic breakdown of biomolecules. Thus, under conditions stem cells allows replacement of damaged and senescent cells, conducive to cell growth, the coupling of high rates of and many cells and organs are capable of considerable growth biosynthesis with low rates of autophagy allows cellular through hypertrophy. Cell growth can thus be considered a resources to be channeled towards growth with maximal default state for many cell types, requiring only the presence of efficiency. This synthetic flow is reversed in response to permissive factors such as nutrients and appropriate hormonal nutrient limitation or other stresses that halt cell growth and signals. The process of cell growth, however, has enormous induce autophagy. This inverse correlation between autophagy energetic requirements and is rapidly abandoned when conditions and growth is widely observed under a variety of environmental become unfavorable. In response to nutrient limitation, DNA conditions. For example, growth inhibition and autophagy damage, excessive oxidation, viral infection and other cell induction are coordinated responses to nutrient starvation, stresses, the ATP-consuming processes that underlie cell growth factor withdrawal, cellular stresses such as oxidative growth are rapidly turned off, allowing the cell to conserve or damage, protein misfolding, viral infection, and substrate marshal its resources to deal with the stressor. detachment or contact inhibition of cultured cells (Fig. 1) In addition to switching off energetically demanding growth (reviewed by Neufeld, 2004). processes, cells induce a variety of responses to stress that enable Although the coupling of autophagy and growth might appear them to survive in suboptimal conditions. Central among these is intuitive and logical, the potential cause-and-effect relationships macroautophagy (herein referred to as autophagy), whereby between these two processes and the underlying mechanisms that portions of are sequestered into vesicles known as link them are just beginning to be unraveled. In this Commentary, autophagosomes and degraded by hydrolytic following I highlight key regulatory steps and signaling pathways involved fusion of autophagosomes with the lysosome. This process in autophagy and growth control, and discuss the regulatory supports cell survival by eliminating damaged and potentially relationships by which these processes are coordinated in the cell. harmful cellular structures, and by releasing the breakdown Understanding the physiological benefits of this coordination and products as nutrients that can be re-used by the cell or exported the consequences of its disruption should provide insight into for use by other cells. potential autophagy-based therapeutic approaches. 2360 Journal of Cell Science 125 (10)

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Fig. 1. Correlation between cell size and autophagy. (A) Bax2/2, Bak2/2 mouse bone marrow-derived cells that were cultured in the presence (left) or absence (right) of the growth factor interleukin 3 for six weeks. Of note are the reduced cytoplasmic volume and abundant autophagosomes (arrows). Image reprinted from Lum et al., 2005 with permission from Elsevier (Lum et al., 2005). (B) Camera lucida drawings of sectioned larval fat body cells from fed (left) or three-day starved (right) Drosophila larvae. Dark circular structures represent lipid droplets. Image reprinted from Butterworth et al., 1965 with permission from John Wiley and Sons (Butterworth et al., 1965). C (C) Histogram indicating cell size (forward light scatter) of Atg52/2 mouse embryonic fibroblasts carrying a doxycycline- repressible Atg5 transgene. Cells were starved (ST+, right) in amino-acid-free DMEM or incubated in normal cell culture media (ST-, left), in the presence or absence of doxycycline (DOX). Repression of Atg5 leads to increased cell size under starvation conditions. Image reprinted from Hosokawa et al., 2006 with permission from Elsevier (Hosokawa et al., 2006). Journal of Cell Science Autophagy and the cell growth machinery appropriate for specific environmental conditions (reviewed by Formation of autophagosomes and their fusion with Mizushima et al., 2011). are the defining events of autophagy. Whereas autophagosome– The biosynthetic pathways that promote the accumulation of lysosome fusion largely exploits general factors of the endocytic mass to drive cell growth are also highly coordinated. The best pathway, autophagosome formation is driven by the coordinated understood of these processes is the control of protein synthesis, activity of distinct sets of components that are dedicated to this which is generally rate limiting for cell growth and sufficient to process. Many of these components were first identified through drive cell transformation (reviewed by Proud, 2007). The activity pioneering genetic screens in yeast, and have clear homologs in of translation initiation and elongation factors [e.g. eukaryotic metazoan cells. These include: (1) two ubiquitin-like molecules, translation initiation factors (eIF) 2, 3 and 4 and eukaryotic Atg12 and Atg8 [-associated protein 1 light chain 3 translation elongation factor 2 (eEF2)] are tightly regulated by alpha (LC3) and additional family members in mammals], and protein such as mTOR and (for general control non- the E1-, E2- and E3-like processing machinery that regulates repressed 2, also known as EIF2AK4) in response to nutrient and their conjugation to control autophagosome formation and size; growth factor levels. The production of is similarly (2) a multi-component protein complex containing the class III sensitive to growth conditions, and this regulation encompasses all phosphatidylinositol 3- Vps34 (PIK3C3 in mammals), three RNA polymerases, as well as post-transcriptional controls, to which promotes autophagosome nucleation from cytosolic ensure a balanced production of ribosomal proteins and . membranes; and (3) a complex containing the serine/threonine More recently, we have begun to define the signals that link protein kinase Atg1 (ULK1 and ULK2 in mammals), which cell growth to lipid synthesis and biogenesis, and integrates signals from multiple upstream regulators to control these appear to involve a complex interplay of transcriptional, the localization or activity of Vps34 and other autophagy translational and post-translational control (reviewed by Zoncu components. One such regulator, the protein kinase target of et al., 2011b). It is crucial that the synthesis rates of proteins, lipids rapamycin [Tor1 and Tor2 in yeast; mammalian TOR (mTOR) in and other biomolecules are regulated in parallel, and that these, in mammals], has a central role in controlling Atg1 and ULK1 turn, are linked to the rates of nutrient uptake and, finally, to the activity in response to nutrients and growth conditions. Together, overall growth and division rates of the cell and its . these three systems act in concert, along with a number of Together, these regulatory steps represent nodes of control at accessory proteins, to form autophagosomes at a rate that is which interactions between cell growth and autophagy can occur. Cell growth and autophagy 2361

Coordination of these processes can thus be achieved either regulation, such as EIF4EBP1 or S6K. Instead, mTOR directly through common signaling pathways that intersect at multiple interacts with and phosphorylates components of the Atg1 protein independent control points, or through autophagy- or growth- kinase complex, including Atg1 (ULK1), Atg13 and FIP200 (also dependent effects and signals (Fig. 2). Here, each of these models known as RB1CC1) (reviewed by Mizushima, 2010). In yeast, is considered in turn. Atg13 is phosphorylated on multiple serine and threonine residues under growth conditions, and this disrupts its Coordinated regulation of growth and autophagy association with Atg1. Ohsumi and co-workers recently found by common signals that Atg13 is phosphorylated directly by TOR in vitro, and An efficient means of achieving coordination between autophagy showed that Atg13 lacking these phosphorylation sites stably and cell growth rates is through the joint regulation of these associates with, and activates, Atg1 and is sufficient to promote processes by shared upstream signaling pathways. Although the autophagy under growth conditions, independent of TOR activity molecules that act directly on autophagy and biosynthesis are (Kamada et al., 2010). This ability of non-phosphorylatable distinct, a number of regulators have been shown to control both Atg13 to bypass TOR signaling highlights the Atg1 complex as processes in parallel (Fig. 3). the crucial downstream target mediating the effects of TOR on autophagy. mTOR-dependent regulation of autophagy and growth How TOR-dependent phosphorylation of Atg13 promotes As alluded to above, the kinase mTOR has a central role in Atg1 activation and induction of autophagy remains poorly controlling both cell growth and autophagy. mTOR controls the understood. Interestingly, Herman and colleagues have translation initiation step through direct phosphorylation of two demonstrated that Atg1 forms dimers or oligomers under key targets, EIF4E-binding protein 1 (EIF4EBP1) and S6 kinase conditions that favor autophagy, and that this self-association is (S6K, also known as RPS6KB1), which, in turn, regulate eIF3- promoted by Atg13 (Yeh et al., 2011). Forced dimerization and eIF4-dependent interactions between the mRNA 59 cap, the of Atg1 increases its kinase activity and the level of poly(A)-tail and the 40S and 60S ribosomal subunits (Zoncu et al., autophosphorylation, which is a prerequisite for inducing 2011b). mTOR also has a major impact on protein synthesis and autophagy (Kijanska et al., 2010; Yeh et al., 2010). However, cell growth by promoting biogenesis. In yeast, this this is not sufficient to induce autophagy, suggesting that Atg13 involves inhibitory phosphorylation of three transcription factors, has additional functions. Regulation of metazoan Atg1 (ULK1) Stb3, Dot6 and Tod6, which act as repressors of ribosome appears to differ from that in yeast, as mTOR inhibits ULK1 biogenesis (RiBi) and ribosomal protein (RP) expression without affecting its interaction with Atg13. A functional role (Huber et al., 2011). This phosphorylation is mediated by the for Atg13 phosphorylation has not yet been demonstrated protein kinase Sch9, a yeast homolog of S6K and AKT, and a in mammals, and mTOR-mediated phosphorylation of other direct TOR substrate. mTOR also contributes to ribosome components of the ULK1 complex might have important roles. biogenesis by promoting expression of ribosomal RNA. Recent Recently, Ser757 of ULK1 was identified as an mTOR- studies indicate that there is a nucleolar role for mTOR dependent phosphorylation site that influences the association complexes directly at the promoters of rRNA and tRNA , of ULK1 with the AMP-activated protein kinase (AMPK), which Journal of Cell Science and have identified the RNA polymerase I and III factors TIF1A is an important activator of autophagy in response to cellular (also known as TRIM24), TFIIIC (also known as GTF3) and energy levels (Kim et al., 2011a; Shang et al., 2011) (see below). MAF1 as downstream targets (Kantidakis et al., 2010; Mayer The potential role of mTOR to regulate later steps of et al., 2004; Vazquez-Martin et al., 2011). autophagosome maturation, movement and lysosomal fusion is Independent of these effects on protein synthesis, mTOR less well characterized. At least in some cell types, mTOR regulates the autophagic machinery and suppresses activation can promote autophagosome–lysosome fusion, in part autophagosome formation under conditions that are favorable by facilitating the interaction of Rab7 with its lysosomal effector for growth. Autophagy inhibition by mTOR largely occurs Rab-interacting lysosomal protein (RILP) (Bains et al., 2009; independently of the targets known to be involved in growth Bains et al., 2011; Yamamoto et al., 2006). Furthermore, reactivation of mTOR during sustained nutrient starvation is required to allow re-formation and recycling of lysosomes A B C Signal Signal Signal following the massive delivery of autophagic membrane and cargo. This recovery of mTOR activity and lysosomal integrity is dependent on autophagic degradation and efflux, suggesting that Integrator Autophagy Cell growth nutrients derived from the autolysosome are able to promote mTOR activation (Rong et al., 2011; Shin and Huh, 2011; Yu et al., 2010). Nutrient efflux from autolysosomes also appears Autophagy Cell growth Cell growth Autophagy to promote TOR signaling required for secretory activity in senescent cells (Narita et al., 2011). Fig. 2. Potential signaling relationships between autophagy and cell Recent investigations into the nutrient-dependent regulation growth. The inverse correlation between rates of cell growth and autophagy and localization of mTOR are consistent with this model. In can be accomplished through several distinct mechanisms. Common upstream signaling pathways (A) can provide a coordinated effect on autophagy and response to an increase in nutrient levels, mTOR is recruited to cell growth. Such pathways bifurcate at integration points that independently the surface of lysosomes by members of the Rag family of control the autophagy and growth machinery downstream. Inhibitory effects heterodimeric GTPases (Sancak et al., 2010). The lysosomal of autophagy on cell growth (B) and of cell growth on autophagy (C) can translocation of mTOR requires nutrient-dependent GTP loading provide a direct mechanism for linking these processes. Please refer to the text of the Rag complex, and this recruitment is sufficient to bypass for additional details. the nutrient input to mTOR activation. Interestingly, nucleotide 2362 Journal of Cell Science 125 (10)

Amino acids GlucoseGl

Δψ, Π, pH

tRNAs Energy

AMPK

TOR CKIs Lysosome

CDKs Gcn2

Atg1 Vps34

Actively translating ribosome

Endoplasmic reticulum Gcn4 pRb E2F

Autophagy gene expression

Fig. 3. Effects of cell growth on autophagy. Growth-dependent import of nutrients, such as amino acids and glucose, generates multiple signals that influence the induction of autophagy through their effects on pH, osmotic pressure (P) and the electrochemical gradient (DY), as well as through several key signaling molecules such as Gcn2 (EIF2AK4 in mammals), TOR (or mTOR) and AMPK. The eIF2a kinase Gcn2 senses reduced amino acid levels through the accumulation of uncharged tRNAs, and activates autophagy-related gene expression through the transcription factor Gcn4 (ATG4 in mammals). Amino acids activate the mTOR pathway, in part, by recruiting mTOR complexes to the lysosomal surface through factors such as the Rag GTPases and p62, and through effects on intracellular lysosomal positioning. The AMP-activated protein kinase (AMPK) responds to glucose uptake, and regulates multiple downstream targets that have effects on autophagy, including mTOR, Atg1 (ULK1 in mammals) and CDKs (through cyclin kinase inhibitors; CKIs). The induction of autophagy can

Journal of Cell Science also be suppressed by the presence of actively translating ribosomes on the surface of the , which might limit the availability of sites for autophagosome nucleation. Additional components of TOR, Atg1 and Vps34 complexes are indicated. Please refer to the text for details.

loading of Rag GTPases is sensitive to the amino acid mTOR-independent control of autophagy and growth concentrations within the lysosome, and subunits of the Despite the multiple inputs to mTOR signaling and its pervasive vacuolar ATPase protein pump are required for this signaling effects on autophagy and cell growth, a surprisingly large number (Zoncu et al., 2011a). The microtubule-dependent positioning of of signaling pathways and regulators have been shown to affect lysosomes within the cell has also been shown to influence these processes independently of changes in mTOR activity. mTOR activity, and this appears to be mediated by nutrient- Small-molecule screens have identified several compounds that dependent changes in intracellular pH (Korolchuk et al., 2011). induce autophagy without affecting mTOR. These chemicals Importantly, lysosome-mediated regulation of mTOR influences inhibit a variety of intracellular targets and processes, including multiple, if not all, aspects of mTOR function, including its effects ion channels, Ca2+ homeostasis and G-protein signaling on targets involved in cell growth and autophagy. This implies that (Williams et al., 2008; Zhang et al., 2007). A strong correlation mTOR might retain its activation state for some time after its has been observed between the ability of these compounds to departure from the lysosomal surface and diffusion or transport to promote long-lived protein degradation and to increase cellular other cellular compartments. Indeed, activation of mTOR might be levels of phosphatidylinositol 3-phosphate [PtdIns(3)P], the required for its release from the lysosome (Ohsaki et al., 2010). product of Vps34, thus implicating this kinase as an ultimate Compartmentalization of mTOR from its targets, such as ULK1, autophagic effector. Despite some reports linking Vps34 to has been suggested to promote autophagy during senescence, but mTOR activation (Byfield et al., 2005; Nobukuni et al., 2005), no the extent to which similar mechanisms contribute more generally effect on the phosphorylation of mTOR substrates has been found to mTOR signaling is unclear (Narita et al., 2011). Live imaging for these compounds (Williams et al., 2008; Zhang et al., 2007). and fluorescence recovery after photobleaching (FRAP)-based Similarly, a genomic siRNA screen for autophagy regulatory analysis of mTOR complexes might help to clarify this issue. This genes has found little correlation between autophagy induction centralized control of mTOR activation status probably makes a and mTOR activity, whereas nearly half of the 236 identified hits substantial contribution to coordinating its diverse functions in had a substantial effect on PtdIns(3)P levels (Lipinski et al., autophagy and cell growth. 2010). analysis of these hits has revealed a Cell growth and autophagy 2363

substantial enrichment in signaling molecules and transcription also been described for p53, which induces autophagy in factors, including multiple genes involved in growth factor and response to DNA damage through the activation of target genes cytokine signaling that have well-characterized effects on cell such as those encoding the lysosomal protein DNA-damage growth. Taken together, these results indicate that the ability of regulated autophagy modulator 1 (DRAM1) and ULK1, as well signaling pathways to regulate autophagy and cell growth in as factors, such as sestrin 2, phosphatase and tensin homolog parallel is not confined to the mTOR pathway and might be (PTEN) and tuberous sclerosis 2 (TSC2), that inhibit mTOR widespread. signaling (Crighton et al., 2006; Feng et al., 2005; Gao et al., The oncogenic Ras–RAF–MAPK cascade stimulates cell 2011; Maiuri et al., 2009). These inductive effects on autophagy growth through multiple transcription factors including MYC, through p53-mediated transcription are opposed by a cytoplasmic JUN (also known as transcription factor AP-1) and ETS1, which function of p53, which increases mTOR activity and suppresses target genes that regulate cell cycle progression, ribosome autophagy under basal conditions (Tasdemir et al., 2008). biogenesis and cytokine signaling (McCubrey et al., 2007). Other factors also regulate cell growth and autophagy Ballabio and colleagues recently identified the mammalian through a combination of mTOR-dependent and -independent transcription factor EB (TFEB) as a master regulator of a large mechanisms. For example, in addition to its transcriptional number of autophagy and lysosomal genes that facilitate effects noted above, PKA also inhibits autophagy in yeast by the coordination of autophagosome formation, fusion and directly phosphorylating Atg1 and Atg13, which prevents their degradation (Settembre et al., 2011). Furthermore, this group localization to the sites of autophagosome assembly (Stephan showed that TFEB can be negatively regulated through direct et al., 2009). At the same time, PKA activation increases the phosphorylation by extracellular-signal-regulated kinase 2 sensitivity of cells to rapamycin and exacerbates the growth (ERK2), which is independent of mTOR activity. Similarly in defects of tor mutants, which indicates that PKA has antagonistic yeast, Ras and its downstream target cAMP-dependent protein effects on TOR (Ramachandran and Herman, 2011). Taken kinase (PKA) inhibit the starvation-induced expression of together, these results suggest that PKA sends both positive and autophagy genes, such as ATG8, independently of TOR, and negative signals to the autophagy network, and that the inhibitory PKA promotes expression of growth gene networks such as the effect is dominant under most conditions. Similarly, the energy- RiBi regulon through inactivation of the repressor Tod6 (Graef sensing AMPK regulates autophagy both upstream and and Nunnari, 2011; Lippman and Broach, 2009). Thus both downstream of mTOR. By inhibiting mTOR activity under mammalian and yeast Ras signaling pathways use a coordinated conditions of low energy, AMPK indirectly promotes activation transcriptional mechanism to link growth stimulation and of the ULK1 complex. In addition, AMPK can directly autophagy inhibition in response to nutrients. phosphorylate ULK1, in this case leading to further activation This transcriptional response is probably shared by many other and induction of autophagy (Egan et al., 2011; Kim et al., 2011b; factors that regulate growth in response to multiple stimuli. Shang et al., 2011). Botstein and colleagues found that 25% of all yeast genes display a similar transcriptional response to changes in nutrient Regulation of autophagy by cell growth conditions regardless of carbon source, a pattern they termed a As discussed above, the inverse relationship between cell growth Journal of Cell Science ‘universal’ growth rate response (GRR) (Slavov and Botstein, 2011). Growth-related genes involved in ribosome biogenesis and and autophagy can result in part from their co-regulation. translation display a positive universal GRR (i.e. increased However, this close correlation is also consistent with a cause- transcription in rich medium), whereas autophagy- and vacuolar- and-effect connection between these processes, and suggests associated genes have a negative GRR. Although such that autophagy and cell growth might inhibit each other widespread responses certainly reflect the activity of multiple independently of shared upstream signaling components. transcriptional regulators, individual transcription factors can We can examine this issue from the viewpoint of protein also generate coordinated effects on growth and autophagy. For synthesis and cell cycle progression, two processes that are example, the yeast transcriptional regulator Gcn4 responds to central to cell growth and proliferation. The first obligate step in amino acid starvation by promoting expression of autophagy increasing cellular protein mass is the import of amino acids. The genes and repressing genes encoding ribosome proteins and regulation of this process in metazoan cells is poorly defined, but translation factors (Natarajan et al., 2001). The synthesis of Gcn4 it is clear that growth factor signaling promotes the expression of protein itself increases under conditions of general translation a variety of nutrient transporters on the cell surface, in part by inhibition, providing an additional layer of connection between blocking their endocytic degradation (Edinger, 2007; Edinger and autophagy and cell growth (Hinnebusch, 1997). Thompson, 2002; Hennig et al., 2006). Amino acid uptake in In metazoans, the FOXO family of transcription factors growing cells might directly lead to mTOR activation and hence suppresses cell growth through the expression of inhibitors of suppression of autophagy. However, rapid incorporation of translation and proliferation (Ju¨nger et al., 2003; Stahl et al., amino acids into growing peptide chains might limit their free 2002). Concurrently to this, they stimulate autophagy by intracellular concentration and thus mTOR activation. Transport activating the expression of a large number of autophagy- of amino acids into the cell can also have secondary associated genes (Mammucari et al., 2007; Zhao et al., 2007). consequences, such as changes in osmolarity and cell Interestingly, FOXO1 also has a transcription-independent membrane polarization, which have been shown to influence capacity to promote autophagy. This process involves autophagic signaling (Ha¨ussinger et al., 1990). In addition, there deacetylation of cytoplasmic FOXO1 in response to starvation is evidence that some amino acid transporters might act as or oxidative stress, which promotes its association with ATG7 ‘transceptors’ with dual transport and signaling functions, and and leads to autophagy through an, as yet, unclear mechanism mTOR and PKA have been implicated as targets of such (Zhao et al., 2010). Dual nuclear and cytoplasmic effects have molecules (Taylor, 2009). 2364 Journal of Cell Science 125 (10)

Subsequent steps in protein synthesis can also have substantial upstream pathways. Liang and co-workers have shown that regulatory effects on autophagy. Following their transport into autophagy is inhibited in mouse embryonic fibroblasts that lack the cell, amino acids are ligated to their cognate tRNAs by p27, and that overexpression of p27 or depletion of CDK2 or aminoacyl-tRNA synthetases. Disruption of this process causes CDK4 induces autophagy in breast adenocarcinoma cells (Liang accumulation of uncharged tRNAs, which directly activates the et al., 2007). Both p27 and p16 are linked to autophagy through eIF2a kinase Gcn2 (also known as EIF2AK4 in mammals), the tumor suppressor retinoblastoma 1 (RB1, also known as pRb), leading to translation arrest, upregulation of GCN4 and increased which is a central downstream target of CDK4 in cell cycle expression of autophagy genes (Hinnebusch, 1994). In addition, regulation. RB1 is required for the induction of autophagy by p27 export of tRNA from the nucleus is inhibited by starvation, and and p16, and can trigger autophagy when overexpressed (Jiang depletion of the tRNA nuclear exporter XPOT can activate et al., 2010). RB1 controls cell cycle progression, cell growth and autophagy in the presence of abundant nutrients (Huynh et al., survival by binding to the transcription factor E2F and inhibiting 2010). In this case, autophagy correlates with reduced mTOR expression of E2F target genes. Similarly, induction of autophagy activity, which indicates that tRNAs can influence multiple by RB1 requires binding of RB1 to E2F, and this process can be signaling pathways. antagonized by overexpression of E2F. The effects of RB1 and In rapidly growing cells, the elongation of polypeptide chains E2F are complex, however, and might be influenced by cell type might also inhibit autophagosome formation. Recent studies have and stress conditions. For example, E2F is required for autophagy implied that PtdIns(3)P-rich domains of the rough endoplasmic induction by DNA-damaging agents in U2OS cells, and reticulum (ER) are central staging areas of autophagosome overexpression or activation of E2F can promote autophagy initiation (Axe et al., 2008). The tight juxtaposition of the and increase expression of Atg1 (ULK1), ATG5, LC3 and growing autophagic membrane with the ER surface (Hayashi- DRAM1 in these cells (Polager et al., 2008). In addition, RB1– Nishino et al., 2009; Yla¨-Anttila et al., 2009) suggests that E2F complexes have a negative effect on autophagy in response engagement of active ribosomes with the ER translocon might be to hypoxia, in this case by inhibiting expression of BNIP3 (for incompatible with this initiation process. In rapidly growing BCL2/adenovirus E1B 19kDa interacting protein 3), an activator cells, high occupancy of the surface of the ER with translating of VPS34–beclin-1 complexes and inhibitor of mTOR (Tracy ribosomes might therefore limit its availability as a platform for et al., 2007). autophagosome formation (Blommaart et al., 1995). Even a single CDK can have both positive and negative effects The cell division cycle of proliferating cells provides another on autophagy. Klionsky and co-workers have found that the yeast stage for the interaction between autophagy and growth control. CDK Pho85 has both positive and negative effects on autophagy It has been known for some time that cells in mitosis are resistant induction, depending on the cyclin partner it is associated with to a variety of autophagic stimuli, including starvation and (Yang et al., 2010). The relative expression levels of these cyclins mTOR inhibition (Eskelinen et al., 2002), and this might vary with cell cycle phase and in response to environmental provide a crucial barrier to autophagic degradation of spindle signals, and each phase probably directs Pho85 to different components, genetic material and other exposed cellular cellular compartments or downstream targets, thereby allowing structures. A study using multiple cell cycle markers and a Pho85 to provide an integrated autophagic response to multiple

Journal of Cell Science variety of autophagy inducers found that each stimulus had growth and cell cycle cues. maximal effects in the G1 and S phases of the cell cycle, with In addition to these molecular links, processes that are intrinsic little activity in the G2 and M phases (Tasdemir et al., 2007). to cell growth and proliferation might themselves be incompatible This autophagy timecourse corresponds inversely with activity of with autophagy. For example, periodic reorganization of the mitotic protein cyclin-dependent kinase 1 (CDK1). Yuan and into a mitotic spindle in dividing cells would be colleagues recently identified Vps34 as a substrate of CDK1– expected to disrupt transport of autophagic vesicles or of cyclin-B during mitosis in human cells (Furuya et al., 2010). That components required for their formation. Similarly, dedicating study found that CDK1-mediated phosphorylation of Vps34 on vesicular trafficking pathways towards active synthesis and Thr159 disrupts its association with beclin 1, an essential core secretion might preclude their use in autophagy. Growth and component of Vps34 complexes, thereby reducing the lipid kinase autophagy might also compete for raw materials, such as activity of Vps34. Thr159 phosphorylation of Vps34 increases membrane lipids, or for regulators that function in both during mitosis, correlating with a reduction in autophagy. These processes. For example, Vps34 can assemble into at least three findings suggest that suppression of autophagy during mitosis can complexes with distinct subunits and functions, but only one them result from CDK1-dependent inhibition of Vps34 activity. By (which is defined by the presence of Atg14) is dedicated to contrast, yeast Cdk1 appears to have a positive function in autophagosome formation (Simonsen and Tooze, 2009). Such autophagy because loss of Cdk1 activity leads to G1 arrest and competition-based mechanisms could prevent autophagy from autophagy inhibition (Yang et al., 2010). The region of Vps34 causing damage during vulnerable cell cycle phases. that surrounds Thr159 is not well conserved in yeast, which indicates that other Cdk1 substrates probably mediate its effects Regulation of cell growth by autophagy on autophagy. Bone marrow cells derived from mice that are deficient for the Regulation of autophagy by CDKs might be a general proapoptotic BCL2 family proteins BAX and BAK are able to phenomenon. The CDKN1B (also known as p27 and KIP1) and survive in culture for several weeks in the absence of growth CDKN2A (also known as p16 and INK4) families of G1 CDK factors. This survival is autophagy-dependent and coincides with inhibitors are important regulators of cell cycle and growth in a dramatic reduction in cell mass (Lum et al., 2005). In vivo, response to stress, hormonal and developmental signals. Recent starvation of Drosophila can result in a 90% decrease in the size studies have revealed that these factors have a similarly crucial of larval fat body cells, which is accompanied by a massive role in regulating autophagy by integrating signals from multiple induction of autophagy (Butterworth et al., 1965). Genetic Cell growth and autophagy 2365

disruption of autophagy in this system suppresses the observed where p62 has been depleted or knocked out, mTOR fails to be reduction in growth, and overexpression of Atg1, which induces activated in response to amino acids, which leads to a decrease in autophagy, is sufficient to reduce cell size in the fat body (Scott cell growth. Remarkably, p62 has been shown to stimulate et al., 2007; Scott et al., 2004). In starved mice, the intracellular mTOR signaling by supporting the interaction between Rag protein degradation rate of hepatocytes approaches 40% per day in GTPases, the mTOR partner Raptor and the lysosomal surface, vivo, and starvation-induced size reduction of mouse embryonic which promotes amino-acid-dependent recruitment of mTOR to fibroblasts can be substantially inhibited by disrupting Atg5 the lysosome and its subsequent activation (Duran et al., 2011). (Conde and Scornik, 1976; Hosokawa et al., 2006). Together, These results imply that autophagy can impair mTOR-dependent these examples illustrate the intrinsic ability of autophagy to cell growth through selective degradation of p62. In addition, p62 directly reduce cellular mass through the degradation of bulk contains a C-terminal ubiquitin-associated (UBA) domain and cytoplasm (Fig. 4A). can promote selective autophagy of ubiquitylated soluble or In addition to these presumably non-selective effects in aggregated proteins (Bjørkøy et al., 2005; Kim et al., 2008), growth-arrested cells, autophagy might further reduce the which potentially include other factors involved in cell growth growth rate of actively growing cells through targeted regulation. In a proteomic study tracing the progressive elimination of growth-promoting molecules, complexes and autophagic degradation of proteins in breast cancer cells, it has organelles. The protein p62 (also known as sequestosome 1) is been found that proteins involved in translation, including an intriguing candidate for such a factor. p62 is a multifunctional mTOR, ribosomal protein S6 and tRNA synthetases, are among adapter protein that binds LC3 and other Atg8 family members the most rapidly depleted factors (Kristensen et al., 2008). through its LC3-interacting region (LIR), and thereby becomes Whether these and other growth-promoting factors are selectively incorporated into autophagosomes (Bjørkøy et al., 2005). This targeted for autophagy in a p62-dependent manner remains to be results in efficient autophagy-dependent degradation of p62, and, shown. indeed, p62 protein levels are widely used as indicators of Autophagy can also be employed by cells to selectively autophagic flux (Klionsky et al., 2008). Recently, an important degrade larger complexes and organelles that are required for cell role for p62 in mTOR activation has been described. In cells growth, such as ribosomes and mitochondria. In yeast, starvation results in rapid sequestration and digestion of ribosomes in the A . This process is essential for survival under starvation conditions, is highly selective and involves the ubiquitin protease Ubp3, its activator Bre5, and ubiquitylation of specific ribosomal proteins (Kraft et al., 2008). Interestingly, the mammalian orthologs of Ubp3 and Bre5 interact with ULK1, which is required for the clearance of ribosomes from the cytoplasm Ribophagy of reticulocytes (Behrends et al., 2010; Kundu et al., 2008). Ribosomal degradation represents a potent mechanism to Mitophagy directly reduce cellular growth capacity. In addition, large Journal of Cell Science macromolecular complexes such as P granules in C. elegans and midbody rings of dividing cell populations can indirectly -Ub- promote growth by acting as stem cell factors, and these -Ub- complexes have also been shown to be selective autophagy substrates (Kuo et al., 2011; Pohl and Jentsch, 2009; Zhang et al., Bulk p62-mediated 2009). autophagy selective autophagy Mitochondria can be selectively degraded by autophagy in a process referred to as mitophagy recently (reviewed in Youle and B Narendra, 2011). Both starvation and mitochondrial damage can trigger mitophagy, and this requires LIR-motif-containing ULK1 mTOR WIPI1 proteins [Atg32 in yeast, Nix (also known as BNIP3L) and P- possibly p62 in mammalian cells], which are thought to guide expansion of autophagosomal membranes over the mitochondrial Key surface through interaction with Atg8 family members. In yeast, this interaction is regulated by phosphorylation of Atg32 (Aoki et al., 2011). It was recently demonstrated that mitochondria can Fig. 4. Negative effects of autophagy on cell growth. (A) Multiple forms of be protected from starvation-induced mitophagy by a PKA- autophagy can contribute to growth suppression. From left to right: bulk dependent process of fusion and elongation, which somehow autophagy of cytoplasmic contents including and organelles; spares mitochondria from engulfment into autophagosomes mitophagy, which utilizes adapter molecules such as Atg32, Nix and p62; (Gomes et al., 2011; Rambold et al., 2011). Similarly, in yeast ribophagy, which requires the ubiquitin protease Ubp3 and its activator Bre5; that are grown under conditions in which mitochondria are and p62-dependent degradation of ubiquitylated (Ub) signaling molecules and required for metabolism, mitophagy is not induced, not of p62 itself, which can promote mTOR signaling. (B) Activation of autophagy factors such as ULK1 and WIPI1 might lead to downregulation of even by severe starvation (Kanki and Klionsky, 2008). In mTOR through multiple mechanisms, possibly including ULK1-mediated starved mammalian cells, degradation of mitochondria occurs phosphorylation (P) of Raptor (indicated by the blue oval) and WIPI1- subsequent to degradation of cytosolic proteins and ribosomes dependent effects on maturation of endocytic vesicles. (Kristensen et al., 2008). Thus, mitochondria appear to be the last 2366 Journal of Cell Science 125 (10)

resort for nutrients, which probably reflects their essential role in As new roles of autophagy continue to be revealed, the view both growing and stationary cells. that emerges is that this ancient process is deeply entwined with Several components of the autophagic machinery have the basic cellular functions of growth, proliferation and survival. been shown to have important autophagy-independent roles in Disentangling these connections will be important to our regulating apoptotic cell death, and it is interesting to speculate understanding of the molecular regulation of each of these that similar dual functions might be at work in controlling cell processes. Furthermore, as autophagy-based therapies against a growth. One possible example of this is a feedback signaling loop number of diseases are pursued, it will be important to consider from ULK1 to mTOR, which involves mTOR inhibition by its how potential treatments might impact other processes such as downstream kinase (Fig. 4B). In Drosophila and mammalian cell growth that are linked to autophagy. This will be aided by a cells, Tor (or mTOR)-dependent phosphorylation of its substrates better understanding of growth-dependent and -independent S6K and EIF4EBP1 is increased in Atg1 (or ULK1)-depleted mechanisms that regulate autophagy. cells, and decreased in response to the overexpression of Atg1 (Lee et al., 2007; Scott et al., 2007). These effects correlate with Funding changes in the cytoplasmic localization and trafficking of TOR, The work of our laboratory is supported by the National Institutes of Health [grant number GM62509]. 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