Báo cáo khoa học: Death-associated protein kinase (DAPK) and signal transduction: additional roles beyond cell death ppt

MINIREVIEW Death-associated protein kinase (DAPK) and signal transduction: additional roles beyond cell death Yao Lin, Ted R. Hupp and Craig Stevens CRUK p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh, UK Introduction Death-associated protein kinase-1 (DAPK-1) is the prototypic member of a family of death-related kinases that includes DAPK-1-related protein 1 (also named DAPK-2), Zipper interacting kinase (ZIPK, also named DAPK-3), DAP kinase related apoptosis indu- cing protein kinase 1 (DRAK1) and DRAK2 [1]. These kinases share a high degree of homology in their catalytic domains. However, the extracatalytic domains and biological function of these five proteins differ markedly [1]. DAPK, a calcium ⁄ calmodulin (CaM)- regulated Ser ⁄ Thr protein kinase, was originally identi- fied as a factor that regulates apoptosis in response to the death-inducing cytokine signal interferon-c (INF-c) [2]. In addition to its role in apoptosis, recent advances have established an important role for DAPK in a diverse range of signal transduction pathways, includ- ing growth factor signalling and autophagy. In this review we will integrate these new findings with our existing knowledge of DAPK function and attempt to highlight the areas that remain unresolved and require further investigation. The DAPK interactome A major goal in biological research is to define the system within which a signalling protein operates and Keywords autophagy; DAPK; growth factor; immune response; interactome; kinase; mTOR; peptide Correspondence C. Stevens, CRUK p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh EH4 2XR, UK E-mail: craig.stevens@ed.ac.uk (Received 11 March 2009, revised 12 August 2009, accepted 8 September 2009) doi:10.1111/j.1742-4658.2009.07411.x Death-associated protein kinase (DAPK) is a stress-regulated protein kinase that mediates a range of processes, including signal-induced cell death and autophagy. Although the kinase domain of DAPK has a range of substrates that mediate its signalling, the additional protein interaction domains of DAPK are relatively ill defined. This review will summarize our current knowledge of the DAPK interactome, the use of peptide apta- mers to define novel protein–protein interaction motifs, and how these new protein–protein interactions give insight into DAPK functions in diverse cellular processes, including growth factor signalling, the regulation of autophagy, and its emerging role in the regulation of immune responses. Abbreviations ATM, ataxia telangiectasia mutated; BH3, Bcl-2-homology-3; CaM, calcium ⁄ calmodulin; DAPK, death-associated protein kinase; DIP1, DAPK interacting protein-1; EGF, epidermal growth factor; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; HSP, heat shock protein; INF-c, interferon-c; LAR, leukocyte common antigen related; MAP1B, microtubule-associated protein 1B; MCM3, mini-chromosome maintenance complex component 3; mTOR, mammalian target of rapamycin; NF-jB, nuclear factor kappa-b; PMA, phorbol-12-myristate-13- acetate; RSK, ribosomal S6 kinase; S6K1, ribosomal protein S6 kinase-1; TGF-b, transforming growth factor-b; TNF, tumour necrosis factor; TSC, tuberous sclerosis; ZIPK, Zipper interacting kinase. 48 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS to use this information to understand developmental or disease processes. Classically, genetic screens in trac- table organisms, such as yeast, worms and flies, have been used for defining the landscape of a protein ⁄ path- way. However, many cancer- and immunity-related genes are confined to vertebrates and a full under- standing of how these proteins operate without the use of classic genetics has been relatively difficult. Instead, technologies that define protein–protein interactions have been used to build a protein interaction map (i.e. like a genetic interaction pathway) for a target protein. Such technologies include the yeast two hybrid, mono- clonal antibody co-immunoprecipitation methods coupled to protein sequencing, and tap-tagging molecular biology approaches for trapping a multiprotein com- plex. The yeast two hybrid, for example, has been used to discover a novel interaction between extracellular signal-regulated kinase (ERK) and DAPK, with impli- cations for pro-apoptotic pathways [3]. Furthermore, recent ideas in systems biology hold that many pro- teins have unstructured motifs or linear domains and that dynamic regulation of protein–protein interactions is mediated by the diversity in such small signalling motifs. This property has been exploited using peptide combinatorial libraries to discover novel complexes between DAPK and microtubule-associated protein 1B (MAP1B) [4] and DAPK and tuberous sclerosis 2 (TSC2) [5], with implications for autophagy and mam- malian target of rapamycin (mTOR) signalling. Together, using such distinct approaches, the DAPK interactome is being built up in a range of back- grounds. DAPK is a large 160 kDa protein composed of several functional domains, including a kinase domain, a CaM regulatory domain, eight consecutive ankyrin repeats, two putative nucleotide binding domains (P- loops), a cytoskeletal binding domain and a death domain (Fig. 1). Proteins that interact with DAPK, the domain on DAPK that mediates the interaction and the methods used to discover the interactions are summarized in Table 1. Given that many regions of DAPK can form protein–protein interfaces it is unsur- prising that only a few of the DAPK binding proteins highlighted in Table 1 are substrates of DAPK, sug- gesting that in some circumstances protein interaction alone is sufficient for DAPK to exert its biological effects. Because of the paucity of DAPK substrates, a screen aimed at identifying a consensus DAPK phos- phorylation motif was carried out based on positional scanning peptide substrate library synthesis and activ- ity [6]. The preferred consensus motif for DAPK phosphorylation and substrates for which phospho- acceptor site(s) have been identified are described in Table 2. Of note, mini-chromosome maintenance com- plex component 3 (MCM3), which is a DNA replica- tion licensing factor, was identified using biochemical fractionation and MS analysis to purify and identify potential substrates from Hela cell lysate [7]. This kind of proteomic approach should expedite the identifica- tion of novel, physiologically relevant in vivo substrates of DAPK. Moreover, it could be tailored to reflect DAPK substrate specificity in response to specific signalling events, such as growth factor or cytokine signalling. It is apparent from Table 2 that not all of the DAPK substrates identified are a good match to the identified consensus motif. Chemical genetics, a bio- chemical approach to develop small peptide-mimetic ligands to alter how an enzyme functions, was utilized Ca 2+ /CaM Ankyrin P-loopsrepeats 1 1431 Kinase Death Cytoskeletal Fig. 1. Schematic representation of DAPK. DAPK is a large 160 kDa Ser ⁄ Thr Ca2 + ⁄ CaM-regulated kinase that consists of several functional domains, including a kinase domain, a CaM regulatory domain, eight consecutive ankyrin repeats, two P-loops, a cytoskeletal binding domain and a death domain, which enable it to participate in a wide range of signalling pathways. Table 1. DAPK binding proteins, the region of DAPK important for mediating the protein-protein interaction, and the method used to define the interaction. Binding protein Binding region on DAPK Binding assay used 14-3-3 [65] Not defined Immunoprecipitation Actin [66] Cytoskeletal domain Immunostaining Beclin-1 [36] Not defined Immunoprecipitation CaM [67] Ca 2 + ⁄ CaM regulatory domain Overlay binding assay Cathepsin B [61] C-terminal domain Immunoprecipitation DIP1 [13] Ankyrin repeats Yeast two hybrid a ERK [3] Death domain Yeast two hybrid a FADD [65] Not defined Immunoprecipitation Hsp90 [62] Kinase domain Immunoprecipitation LAR [23] Ankyrin repeats Yeast two hybrid a MAP1B [4] Kinase domain Peptide libraries a PKD [68] Not defined Immunoprecipitation RSK [25] Not defined Immunoprecipitation Src [23] Not defined Yeast two hybrid a TNFR-1 [65] Not defined Immunoprecipitation TSC2 [5] Death domain Peptide libraries a UNC5H2 [69] Death domain Yeast two hybrid a ZIPK [70] Kinase domain Immunoprecipitation a Protein interactions have been confirmed by more physiological methods. Y. Lin et al. DAPK and signal transduction FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 49 recently to develop selective peptide ligands that mod- ulate DAPK activity. For example, DAPK binding to a peptide derived from the amino acid sequence of the cyclin-dependent kinase inhibitor p21 induces a confor- mational change in DAPK that enhances its kinase activity, suggesting that DAPK may require docking in order to phosphorylate a subset of its substrates [8]. It is also possible that the interaction of DAPK with many of its substrates is of too low affinity to detect in cells. In support of this notion, the ataxia telangiecta- sia mutated (ATM) protein kinase, a large > 300 kDa enzyme, does not have an abundance of stable protein–protein partners that would be expected of a protein of its large size. However, a recent MS-linked proteomics screen identifying phospho-Ser-Gln pep- tides that are phosphorylated by ATM identified over 700 substrates [9]. Therefore, it seems that the previ- ously available protein interaction methodologies were not able to faithfully reflect the ATM kinase inter- actome. A future challenge will be the identification of lower affinity or transient DAPK interactions that might otherwise be overlooked in the more traditional assays to further elucidate the functional role of DAPK in diverse signalling pathways. Signalling to DAPK DAPK plays an important role in a wide range of sig- nal transduction pathways with diverse outcomes, such as apoptosis, autophagy and immune responses. The functional outcome of DAPK activity depends largely on the input signal (Fig. 2). For example, DAPK gene expression and apoptotic activity is increased in response to transforming growth factor-b (TGF-b) [10] and to stimuli that activate p53 [11], such as DNA- damaging agents. Other death signals, such as the transforming oncogenes E2F1 and Myc [12], also induce DAPK expression. In addition to its well-docu- mented role in the regulation of apoptosis, DAPK may also play a role in survival pathways, reflected in its activation by growth factor signalling pathways [5], and its ability to counter tumour necrosis factor (TNF)-mediated apoptosis [13]. Table 2. DAPK substrates and the amino acid sequence surround- ing the phosphorylation site. The substrate phosphorylation pattern preferred by DAPK is highlighted in bold; the basic residues also preferred by DAPK are underlined. Substrate Phosphorylation site Beclin-1[36] RLKVT 119 GDL CaMKK [71] GSRREERSLS 511 APG DAPK [72] A R KKW KQS 308 VRLI MCM3 [7] TKKTIERRYS 160 DLTTL MLC [73] TTKKRPQRATS 19 NVF p21 [8] RKRRQT 145 SMTDFYHSK p53 [8] PPLSQET 18 FS 20 DLWKLL S6 [27] QIAKRRRLS 235 SLRAS Syntaxin-1A [38] IIMDSSIS 188 KQALSEIE Tropomyosin-1 [74] HALNDMTS 283 I ZIPK [70] KT 299 TRLKEYTIKS 30 9HS 311 S 312 LPPNNS 318 YADFERFS 326 Consensus KRxxxxxKRRxxS ⁄ T Mitogens EGF Short treatment Long treatment TNF-α TNF-α IFN-γ TGF-β DNA damage oncogenes Growth mTORC1 Gene expression Kinase activity Kinase activity ? Degradation DAPK over-expression AutophagyApoptosisApoptosis Autophagy Inflammation Immune response Blebbing Autophagy AB C DE Beclin-1 phosphorylation MAP1B binding ApoptosisApoptosis Inflammation ?mTORC1? Fig. 2. Signalling to DAPK. DAPK plays an important role in a diverse range of signal transduction pathways. The biological outcome of DAPK activity depends on the input signal and includes cell growth, immune responses, apoptosis and autophagy. (A) Growth factor signal- ling to DAPK is probably the best defined with respect to the proteins that are involved and includes the activities of Src, LAR, ERK and RSK (see text and Fig. 3). (B) The functional outcome of increased DAPK activity in response to short-term treatment with TNF-a is currently unclear, but may contribute to mTORC1 activation and inhibition of inflammatory responses. Longer-term treatment with TNF-a leads to DAPK degradation coincident with apoptosis, suggesting that DAPK may be a resistance factor to TNF-a-induced cell death in some circum- stances. (C) DAPK mediates many cellular responses in response to INF-c, but the molecular mechanisms have not yet been defined. (D) DAPK gene expression and apoptotic activity are increased in response to TGF-b and to stimuli that activate p53, such as DNA-damaging agents. Other death signals, such as the transforming oncogenes E2F1 and Myc, also induce DAPK expression. (E) Overexpression of DAPK can promote autophagy and membrane blebbing via binding to MAP1B, or autophagy via the direct phosphorylation of Beclin-1. The signals that regulate DAPK autophagic activity have yet to be defined. DAPK and signal transduction Y. Lin et al. 50 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS Growth factor signalling/mTOR Serum-induced activation of DAPK catalytic activity has been demonstrated recently [3,5,14] and it is becoming increasingly clear that DAPK is intimately linked to growth factor signalling pathways (Fig. 3). For example, serum-induced phosphorylation of DAPK by ERK enhances its kinase activity and death-promoting effects [3], whereas serum activation of DAPK has also been linked to cell death by suppressing integrin functions and integrin-mediated survival signals [14]. However, in addition to apoptotic signalling, we have recently demonstrated a stimula- tory role for serum-activated DAPK in mTOR signal- ling [5]. mTOR is a member of the phosphoinositide- 3-kinase-related kinase family, which is centrally involved in growth regulation, proliferation control and cell metabolism [15]. In mammalian cells, two structurally and functionally distinct mTOR-containing complexes have been identified, mTORC1 and mTORC2 [15]. mTORC1 directly regulates cell growth by controlling the phosphorylation of a number of components of the translational machinery. In particu- lar, phosphorylation and activation of eukaryotic initi- ation factor 4E binding protein-1 and ribosomal protein S6 kinase-1 (S6K1) are stimulated by serum, insulin and growth factors in an mTORC1-dependent manner [16]. The TSC complex, formed by two proteins, TSC1 and TSC2, is a major regulator of the mTORC1 signalling pathway [17]. TSC2 contains a GTPase-acti- vating protein domain that converts the small GTPase Ras homolog enriched in brain to its inactive GDP-bound form [18]. mTORC1 activity is stimulated by the active GTP-bound form of Ras homolog enriched in brain, thus the TSC complex acts to inhibit mTORC1 function [18]. Growth factor-induced, inacti- vating TSC2 phosphorylation results in mTORC1 acti- vation and is thought to occur primarily through activation of the RAS–extracellular signal-regulated kinase kinase (MEK)–ERK and phosphoinositide-3- kinase–Akt pathways [19,20]. In a protein interaction screen in our laboratory, we identified TSC2 as a novel DAPK death domain interacting protein, and in analy- sing the biological consequences of the DAPK–TSC2 interaction, we were led to the discovery that DAPK can phosphorylate and inactivate TSC2 and functions as a positive cofactor in mTORC1 signalling in response to serum and epidermal growth factor (EGF) stimulation [5]. ERK can directly interact with and phosphorylate DAPK at Ser735, which leads to enhanced kinase activity and pro-apoptotic activity of DAPK [3]. This Ser735 phosphorylation can be stimulated by serum or phorbol-12-myristate-13-acetate (PMA) [3], which acti- vates the RAS–MEK–ERK pathway [21,22]. Interest- Ras Raf MEK ERK RSK DAPK TSC2 TSC1 Apoptosis ?ApoptosisApoptosis DAPK Rheb S6K S6 -T389 -S235/236 P P Cell growth Protein synthesis mTORC1 DAPK P -S289 P -S735 Src LAR P Y491/Y492 - EGF Fig. 3. Growth factor regulation of DAPK. Growth factor signalling to DAPK is complex and regulates a diverse range of biological outcomes. For example, phosphorylation by ERK enhances the apoptotic activity of DAPK, but Src-mediated phosphorylation of DAPK suppresses its apoptotic, antimigra- tion and antiadhesion functions. Under nor- mal growth conditions, DAPK apoptotic activity may also be suppressed until such times as required due to phosphorylation by RSK. DAPK may also act in concert with ERK and RSK to inhibit the TSC complex, resulting in mTORC1 activation. In addition, DAPK and RSK may co-operate to promote protein translation via direct phosphorylation of ribosomal protein S6. Y. Lin et al. DAPK and signal transduction FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 51 ingly, the inactivation of DAPK activity by EGF has been recently described. Wang et al. [23] demonstrated that DAPK is a substrate for leukocyte common antigen related (LAR) tyrosine phosphatase and that dephosphorylation of Y491 ⁄ Y492, located in the ankyrin repeat domain, resulted in activation of the pro-apoptotic activities of DAPK. Reciprocally, Src kinase phosphorylation of Y491⁄ Y492 inhibited DAPK activity [23]. Src kinase was activated in response to EGF stimulation and LAR was downregu- lated, resulting in DAPK inactivation. The ability of EGF signalling to inactivate DAPK is inconsistent with previous findings that DAPK activity can be up- regulated by serum stimulation and ERK, a down- stream effector of the EGF pathway [3], and this is further inconsistent with data showing that in response to PMA, the DAPK–ERK complex induces apoptosis [3]. It is important to note, however, that the apopto- sis-promoting effect of DAPK induced by the ERK activator PMA was only observed in suspension cells [3], whereas in adherent cells the co-expression of a constitutively active mutant of MEK is required for DAPK to induce apoptosis [24]. Therefore, the apop- tosis function of the ERK–DAPK complex may only exist under aberrant conditions, such as when cells are detached, or when the signal to grow is excessive. Other signalling pathways can in turn modify these core activities of DAPK. For example, RAS activation of the ERK–ribosomal S6 kinase (RSK) pathway can attenuate the pro-apoptotic function of DAPK. RSK interacts with DAPK in vitro and in vivo and catalyses the phosphorylation of DAPK on Ser289 in response to PMA [25]. The effect of this phosphorylation on the kinase activity of DAPK was not tested. However, mutation of Ser289 to a nonphosphorylatable Ala results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity [25]. The observation that the Ser289Ala mutant of DAPK is more apoptotic suggests that phosphorylation inhibits the catalytic activity of DAPK [25]. Thus, kinase assays using the Ser289 mutants are required to clearly deter- mine the function of DAPK Ser289 phosphorylation. Interestingly, RSK has also been shown to interact with TSC2, and phosphorylation by RSK inactivates TSC2, resulting in mTORC1 activation [26]. DAPK has also been directly linked to the control of protein translation by phosphorylating ribosomal protein S6 on Ser235 ⁄ 236 [27]. In agreement with this study, we have shown that DAPK can robustly stimu- late the phosphorylation of S6 in cells, even in the presence of the lipophilic macrolide antibiotic rapa- mycin, a potent inhibitor of mTORC1 activity, indicat- ing that DAPK can mediate phosphorylation of S6 in an mTORC1–S6K-dependent and -independent man- ner. Schumacher et al. [27] demonstrated that DAPK phosphorylates S6 directly on Ser235 ⁄ 236 and con- cluded that this is an inhibitory phosphorylation reducing S6 activity and protein translation in vitro.In contrast, Roux et al. [28] demonstrated that RSK kinase phosphorylates the same sites on S6, but they concluded that this was an activating phosphorylation that stimulates S6 activity and promotes assembly of the translation preinitiation complex in cells. Our results are in agreement with the latter study and point towards a role for DAPK in activating S6 and protein translation. Further studies are required to clarify the role of DAPK in the regulation of S6 activity and pro- tein translation in vivo, in particular the interplay between DAPK and RSK signalling to S6 needs to be addressed, and the ability of DAPK to promote cell growth needs to be clearly demonstrated. Taken together, these studies reveal a complex regu- lation of DAPK activity by growth factor signalling pathways mediated by Src, LAR, ERK and RSK. A better understanding of the interplay between signalling to DAPK and TSC2 may explain how the specific activ- ity of DAPK can be modulated to control the balance between pro-apoptotic and pro-survival pathways. DAPK and autophagy DAPK was originally identified as a factor that regu- lates apoptosis in response to various death-inducing signals, including INF-c [2]. DAPK also has auto- phagy signalling activity, which can be either pro-sur- vival or lead to or participate in cell death. Autophagy is a membrane system involved in pro- tein and organelle degradation that probably repre- sents an innate adaptation to starvation. In times of nutrient deficiency, the cell can self-digest and recycle some nonessential components to sustain its minimal growth requirements until a food source becomes available. Over recent years, autophagy has been impli- cated in an increasing number of clinical scenarios, notably infectious diseases, cancer, neurodegenerative diseases and autoimmunity. In some cell types, the overexpression of DAPK can lead to the appearance of autophagic vesicles [29]. However, there is still little known about how DAPK exerts its effects on auto- phagy, and as DAPK is not present in yeast, there have been no classic genetic screens to analyse how DAPK interacts with the core autophagy pathway. Recently, peptide combinatorial libraries identified MAP1B as a DAPK interacting protein that functions as a positive cofactor in DAPK-mediated autophagic DAPK and signal transduction Y. Lin et al. 52 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS vesicle formation and membrane blebbing [4]. MAP1B has been most widely studied as a major component of the neuronal cytoskeleton [30] and relatively little is known about its role outside of these neuronal systems. The cotransfection of both genes stimulated the disruption of microtubules during the induction of membrane blebbing, suggesting that MAP1B–DAPK- induced blebbing involves changes in the dynamics of mictrotubules, as well as changes in the dynamics of contractile cortical actin [4]. This is even more intrigu- ing in light of the recently identified interaction between the essential autophagy protein Atg8 (LC3) and MAP1B [31], and the observation that micro- tubules play an important role in autophagy by support- ing the production and transport of autophagosomes [32]. Future studies will determine whether MAP1B is a key factor that switches DAPK activity towards autophagy induced by certain stresses such as INF-c. Beclin-1, the first identified mammalian autophagy gene [33], interacts with several cofactors to activate the lipid kinase Vps34, thereby inducing autophagy [34]. Beclin-1 is a Bcl-2-homology-3 (BH3) domain-only protein that binds to the BH3 domain of the antiapoptotic proteins Bcl-2 ⁄ Bcl-X L [35]. Under normal conditions, beclin-1 is bound to and inhibited by Bcl-2 or the Bcl-2 homolog Bcl-X L and the dissociation of beclin-1 from Bcl-2 is essential for its autophagic activ- ity [34]. Nutrient deprivation stimulates the dissociation either by activating BH3-only proteins (such as Bad), which can competitively disrupt the interaction, or by post-translational modification [34]. A recent report demonstrated that a constitutively activated form of DAPK triggers autophagy in a beclin-1-dependent manner [36]. DAPK phosphorylates beclin-1 on Thr119 located at a crucial position within its BH3 domain, and thus promotes the dissociation of beclin-1 from Bcl-X L and the induction of autophagy [36]. This study revealed a new substrate for DAPK that acts as one of the core proteins of the autophagic machinery, and provides a new phosphorylation-based mechanism for how DAPK activates autophagy by reducing the inter- action of beclin-1 with its inhibitor Bcl-X L . DAPK has also been directly linked to the regu- lation of endocytosis [37], and can phosphorylate syntaxin-1A, a key component of the soluble N-ethyl- maleimide-sensitive factor (NSF) attachment protein receptors complex essential for synaptic vesicle docking and fusion [38]. Therefore, DAPK may also regulate autophagy via syntaxin-1A. Although most evidence suggests that autophagy acts as a survival response to provide an energy source maintaining cell survival, it has been proposed that autophagy can contribute to cell death in a process termed autophagic (type II) cell death. Disturbance to endoplasmic reticulum (ER) homeostasis that leads to irreparable damage activates ER-specific cell death mechanisms [39]. DAPK was recently identified as an important component in ER stress-induced cell death [40]. DAPK ) ⁄ ) mice are protected from kidney dam- age caused by injection of the ER stress inducer tunicamycin and the cell death response to tunicamy- cin is reduced in DAPK ) ⁄ ) mouse embryonic fibro- blasts [40]. Interestingly, both caspase activation and autophagy induction are attenuated in DAPK) ⁄ ) mouse embryonic fibroblasts, and depletion of ATG5 or beclin-1, essential autophagic proteins, are protected from ER-induced death when combined with caspase-3 depletion [40]. These results suggest that under certain conditions, DAPK-induced autophagy contributes to cell death, possibly through the induction of apoptosis. In the model organism Caenorhabditis elegans,it was recently demonstrated that starvation-induced autophagy is regulated in part through a DAPK sig- nalling pathway and that autophagy levels are critical to drive such cell fate decisions, leading to survival or death of the organism [41] (see the accompanying review by Kang and Avery [42]). In C. elegans, mus- caranic acetylcholine receptor signalling is important in modulating the level of autophagy during starvation [43]. In a simplified model, starvation activates MAPK (MPK-1), the C. elegans ortholog of mammalian ERK, and activated MPK-1 positively regulates auto- phagy, at least in part through DAPK and RGS-2 [43]. It will be interesting to determine whether ERK and DAPK can co-operate to regulate autophagy in higher organisms. The pathway that regulates autophagy also acts through mTORC1 [44]. Rapamycin binds to and inac- tivates mTORC1, leading to an upregulation of auto- phagy [45]. The finding that DAPK is a positive regulator of mTORC1 signalling and a positive regula- tor of autophagy at first seems counterintuitive. There- fore, we would predict that DAPK activity should be activated by starvation, and that its activity would be inversely correlated with that of mTORC1. However, in mammalian cells, although DAPK is reported to be necessary for INF-c-induced autophagy, it seems not to be a crucial element in starvation or rapamycin- induced autophagy [46]. The accompanying review by Kang and Avery [42] proposes an interesting explana- tion for the seemingly contradictory functions of DAPK to promote mTORC1 activity and autophagy. They propose that DAPK may promote mTORC1 activity specifically to mediate S6K activity during starvation, as S6K activity has been shown to promote rather than suppress autophagy in Drosophila [47]. Y. Lin et al. DAPK and signal transduction FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 53 Clearly, further characterization of the interacting proteins and direct substrates of DAPK, as well as differences between simple organisms and complex mammalian systems, are required to clarify how the kinase is linked to the autophagic pathways. DAPK immune responses DAPK has been shown to participate in cell death in response to various cytokine signals, including IFN-c- induced cell death [2], TNF-a and FAS-induced cell death [48], and TGF-b-induced cell death [10]. There are two distinct outcomes of TNF-a signalling, an inflammatory immune response mediated by the nuclear factor kappa-b (NF-jB) signalling pathway, and apoptosis [49]. By comparing the response to TNF-a treatment in DAPK-deficient and wild-type cells, several groups have demonstrated that DAPK is neutral against TNF-a-induced apoptosis [2,10]. More recent studies have indicated that DAPK is in fact a negative regulator of TNF-a-induced apoptosis. For example, antisense depletion of DAPK in Hela cells protects cells from IFN-c-induced apoptosis, but pro- motes TNF-a-induced apoptosis [50], and the expres- sion of DAPK interacting protein-1 (DIP1), a ubiquitin E3 ligase that degrades DAPK, promotes TNF-a-induced apoptosis [13]. Therefore, although it functions as a death-promoting kinase, DAPK can also act as a survival factor and block apoptosis in response to certain cytokine signals. Interestingly, DAPK has recently been shown to function as a neg- ative regulator of T cell activation via NF-jB. How- ever, DAPK had no effect on NF-jB activation by TNF-a, only by T cell receptor activation [51]. In addition, DAPK can act as a negative regulator of inflammatory gene expression in monocytes [52]. In C. elegans, wounding of epidermal layers triggers mul- tiple co-ordinated responses to damage. It was recently shown that the C. elegans ortholog of DAPK acts as a negative regulator of barrier repair and innate immune responses to wounding [53]. Taken together, these studies suggest an intriguing role for DAPK, not only as a modulator of cytokine-induced apoptosis, but as a regulator of various immune responses. Future work It is becoming increasingly clear that DAPK family members have additional roles beyond their functions in cell death. The recent findings that DAPK nega- tively regulates inflammatory gene expression [51,52], responds to mitogenic signals to regulate mTORC1 activity [5] and negatively regulates epidermal damage responses in C. elegans in an apoptosis- and auto- phagy-independent manner [53], highlight the pleo- trophic role of this kinase. What are the crucial questions for the future? Of considerable importance will be to gain a clear under- standing of the role of DAPK in the RAS–MEK– ERK growth factor signalling pathway, in particular the interplay between ERK, RSK and DAPK and the balance between apoptosis and growth needs to be addressed. Gaining a better understanding of DAPK’s role in cancer is particularly important. DAPK hypermethylation is strongly associated with various cancers (see the accompanying review by Michie et al. [54]), but it is not yet clear how reduced levels of DAPK contribute to carcinogenesis. Possible mechanisms include DAPK’s ability to suppress extra- cellular matrix survival signals to regulate anoikis [14] and its ability to inhibit cell polarization and motility [55]. DAPK can suppress transformation by oncoge- nes by activating a pro-apoptotic p53-dependent checkpoint [12], and it can activate autophagy, which has been shown to be antitumorigenic [56–58]. Recent studies indicate that inflammation is an important contributor to tumorigenesis [59]. Therefore, the anti- inflammatory function of DAPK may also contribute to its tumour suppressive activity [52]. Of interest in this regard are recent studies showing that the TSC– mTORC1 pathway regulates inflammatory responses in monocytes, macrophages and primary dendritic cells [60]. The finding that DAPK regulates mTORC1 activity [5], together with the observation that both mTORC1 and DAPK can block NF-jB activation [51,60], raise the intriguing possibility that DAPK may regulate inflammatory immune responses via an mTORC1-dependent mechanism. Further studies are required to determine whether these pathways are related in this context. Mechanisms regulating protein stabilization and turnover are also critical for modulating DAPK activi- ties. Several studies have shown DAPK degradation to be dependent on the ubiquitin–proteasome pathway [13,61–64]. To date, two E3 ubiquitin ligases have been identified that can promote the ubiquitination of DAPK; DIP-1, a ring finger containing E3 that inter- acts directly with the ankyrin repeat region of DAPK [13], and carboxyl terminus of HSC70-interacting pro- tein, a U-box containing E3 ubiquitin ligase that can bind to the heat shock protein (HSP) chaperone pro- teins HSP70 and HSP90, interacts with DAPK indi- rectly via Hsp90 [62]. The identification of additional ubiquitin ligases, and deciphering the degradation pathways that modulate DAPK stability, will shed DAPK and signal transduction Y. Lin et al. 54 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS further light on the role played by DAPK in the regu- lation of cell growth control. There is no doubt that future research into the role of DAPK will yield new and important insights into the mechanisms that integrate the apoptotic, auto- phagic and cell growth regulatory pathways. References 1 Bialik S & Kimchi A (2006) The death-associated protein kinases: structure, function, and beyond. Annu Rev Biochem 75, 189–210. 2 Deiss LP, Feinstein E, Berissi H, Cohen O & Kimchi A (1995) Identification of a novel serine ⁄ threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev 9, 15–30. 3 Chen CH, Wang WJ, Kuo JC, Tsai HC, Lin JR, Chang ZF & Chen RH (2005) Bidirectional signals transduced by DAPK-ERK interaction promote the apoptotic effect of DAPK. EMBO J 24, 294–304. 4 Harrison B, Kraus M, Burch L, Stevens C, Craig A, Gordon-Weeks P & Hupp TR (2008) DAPK-1 binding to a linear peptide motif in MAP1B stimulates auto- phagy and membrane blebbing. J Biol Chem 283, 9999–10014. 5 Stevens C, Lin Y, Harrison B, Burch L, Ridgway RA, Sansom O & Hupp T (2009) Peptide combinatorial libraries identify TSC2 as a death-associated protein kinase (DAPK) death domain-binding protein and reveal a stimulatory role for DAPK in mTORC1 signal- ing. J Biol Chem 284, 334–344. 6 Velentza AV, Schumacher AM, Weiss C, Egli M & Watterson DM (2001) A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates. J Biol Chem 276, 38956–38965. 7 Bialik S, Berissi H & Kimchi A (2008) A high through- put proteomics screen identifies novel substrates of death-associated protein kinase. Mol Cell Proteomics 7, 1089–1098. 8 Fraser JA & Hupp TR (2007) Chemical genetics approach to identify peptide ligands that selectively stimulate DAPK-1 kinase activity. Biochemistry 46, 2655–2673. 9 Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER 3rd, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y et al. (2007) ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160–1166. 10 Jang CW, Chen CH, Chen CC, Chen JY, Su YH & Chen RH (2002) TGF-beta induces apoptosis through Smad-mediated expression of DAP-kinase. Nat Cell Biol 4, 51–58. 11 Martoriati A, Doumont G, Alcalay M, Bellefroid E, Pelicci PG & Marine JC (2005) dapk1, encoding an activator of a p19ARF-p53-mediated apoptotic check- point, is a transcription target of p53. Oncogene 24, 1461–1466. 12 Raveh T, Droguett G, Horwitz MS, DePinho RA & Kimchi A (2001) DAP kinase activates a p19ARF ⁄ p53- mediated apoptotic checkpoint to suppress oncogenic transformation. Nat Cell Biol 3, 1–7. 13 Jin Y, Blue EK, Dixon S, Shao Z & Gallagher PJ (2002) A death-associated protein kinase (DAPK)-inter- acting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factor-induced apoptosis and regulates the cellular levels of DAPK. J Biol Chem 277, 46980–46986. 14 Wang WJ, Kuo JC, Yao CC & Chen RH (2002) DAP- kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals. J Cell Biol 159, 169–179. 15 Rosner M, Hanneder M, Siegel N, Valli A, Fuchs C & Hengstschlager M (2008) The mTOR pathway and its role in human genetic diseases. Mutat Res 659, 284–292. 16 Proud CG (2007) Signalling to translation: how signal transduction pathways control the protein synthetic machinery. Biochem J 403, 217–234. 17 Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC & Blenis J (2002) Tuberous sclerosis com- plex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling. Proc Natl Acad Sci USA 99, 13571–13576. 18 Tee AR, Manning BD, Roux PP, Cantley LC & Blenis J (2003) Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by act- ing as a GTPase-activating protein complex toward Rheb. Curr Biol 13, 1259–1268. 19 Inoki K, Li Y, Zhu T, Wu J & Guan KL (2002) TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol 4, 648–657. 20 Ma L, Chen Z, Erdjument-Bromage H, Tempst P & Pandolfi PP (2005) Phosphorylation and functional inactivation of TSC2 by Erk implications for tuberous sclerosis and cancer pathogenesis. Cell 121, 179–193. 21 Thomas SM, DeMarco M, D’Arcangelo G, Halegoua S & Brugge JS (1992) Ras is essential for nerve growth factor- and phorbol ester-induced tyrosine phosphoryla- tion of MAP kinases. Cell 68, 1031–1040. 22 Wood KW, Sarnecki C, Roberts TM & Blenis J (1992) ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell 68, 1041–1050. 23 Wang WJ, Kuo JC, Ku W, Lee YR, Lin FC, Chang YL, Lin YM, Chen CH, Huang YP, Chiang MJ et al. (2007) The tumor suppressor DAPK is reciprocally Y. Lin et al. DAPK and signal transduction FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 55 regulated by tyrosine kinase Src and phosphatase LAR. Mol Cell 27, 701–716. 24 Stevens C, Lin Y, Sanchez M, Amin E, Copson E, White H, Durston V, Eccles DM & Hupp T (2007) A germ line mutation in the death domain of DAPK-1 inactivates ERK-induced apoptosis. J Biol Chem 282, 13791–13803. 25 Anjum R, Roux PP, Ballif BA, Gygi SP & Blenis J (2005) The tumor suppressor DAP kinase is a target of RSK-mediated survival signaling. Curr Biol 15, 1762– 1767. 26 Roux PP, Ballif BA, Anjum R, Gygi SP & Blenis J (2004) Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase. Proc Natl Acad Sci USA 101, 13489–13494. 27 Schumacher AM, Velentza AV, Watterson DM & Dres- ios J (2006) Death-associated protein kinase phosphory- lates mammalian ribosomal protein S6 and reduces protein synthesis. Biochemistry 45 , 13614–13621. 28 Roux PP, Shahbazian D, Vu H, Holz MK, Cohen MS, Taunton J, Sonenberg N & Blenis J (2007) RAS ⁄ ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J Biol Chem 282, 14056–14064. 29 Inbal B, Bialik S, Sabanay I, Shani G & Kimchi A (2002) DAP kinase and DRP-1 mediate membrane blebbing and the formation of autophagic vesicles dur- ing programmed cell death. J Cell Biol 157, 455–468. 30 Gordon-Weeks PR (1993) Organization of microtubules in axonal growth cones: a role for microtubule-associ- ated protein MAP 1B. J Neurocytol 22, 717–725. 31 Wang QJ, Ding Y, Kohtz DS, Mizushima N, Cristea IM, Rout MP, Chait BT, Zhong Y, Heintz N & Yue Z (2006) Induction of autophagy in axonal dystrophy and degeneration. J Neurosci 26, 8057–8068. 32 Kochl R, Hu XW, Chan EY & Tooze SA (2006) Microtubules facilitate autophagosome formation and fusion of autophagosomes with endosomes. Traffic 7, 129–145. 33 Liang XH, Kleeman LK, Jiang HH, Gordon G, Gold- man JE, Berry G, Herman B & Levine B (1998) Protec- tion against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein. J Virol 72, 8586–8596. 34 Levine B, Sinha S & Kroemer G (2008) Bcl-2 family members: dual regulators of apoptosis and autophagy. Autophagy 4, 600–606. 35 Pattingre S, Tassa A, Qu X, Garuti R, Liang XH, Mizushima N, Packer M, Schneider MD & Levine B (2005) Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell 122, 927–939. 36 Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, Sabanay H, Pinkas-Kramarski R & Kimchi A (2009) DAP-kinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-X(L) and induction of autophagy. EMBO Rep 10, 285–292. 37 Pelkmans L, Fava E, Grabner H, Hannus M, Haber- mann B, Krausz E & Zerial M (2005) Genome-wide analysis of human kinases in clathrin- and caveo- lae ⁄ raft-mediated endocytosis. Nature 436, 78–86. 38 Tian JH, Das S & Sheng ZH (2003) Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18. J Biol Chem 278, 26265–26274. 39 Wu J & Kaufman RJ (2006) From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ 13 , 374–384. 40 Gozuacik D, Bialik S, Raveh T, Mitou G, Shohat G, Sabanay H, Mizushima N, Yoshimori T & Kimchi A (2008) DAP-kinase is a mediator of endoplasmic reticu- lum stress-induced caspase activation and autophagic cell death. Cell Death Differ 15, 1875–1886. 41 Kang C, You YJ & Avery L (2007) Dual roles of auto- phagy in the survival of Caenorhabditis elegans during starvation. Genes Dev 21 , 2161–2171. 42 Kang C & Avery L (2009) Death-associated protein kinase (DAPK) and signal transduction: fine-tuning of autophagy in Caenorhabditis elegans homeostasis. FEBS J. 43 Kang C & Avery L (2008) To be or not to be, the level of autophagy is the question: dual roles of autophagy in the survival response to starvation. Autophagy 4, 82–84. 44 Klionsky DJ (2007) Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 8, 931–937. 45 Rubinsztein DC, Gestwicki JE, Murphy LO & Klionsky DJ (2007) Potential therapeutic applications of auto- phagy. Nat Rev Drug Discov 6, 304–312. 46 Gozuacik D & Kimchi A (2006) DAPk protein family and cancer. Autophagy 2, 74–79. 47 Scott RC, Schuldiner O & Neufeld TP (2004) Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev Cell 7, 167–178. 48 Cohen O, Inbal B, Kissil JL, Raveh T, Berissi H, Spivak-Kroizaman T, Feinstein E & Kimchi A (1999) DAP-kinase participates in TNF-alpha- and Fas- induced apoptosis and its function requires the death domain. J Cell Biol 146, 141–148. 49 Bradley JR (2008) TNF-mediated inflammatory disease. J Pathol 214, 149–160. 50 Jin Y & Gallagher PJ (2003) Antisense depletion of death-associated protein kinase promotes apoptosis. J Biol Chem 278 , 51587–51593. 51 Chuang YT, Fang LW, Lin-Feng MH, Chen RH & Lai MZ (2008) The tumor suppressor death-associated protein kinase targets to TCR-stimulated NF-kappa B activation. J Immunol 180, 3238–3249. 52 Mukhopadhyay R, Ray PS, Arif A, Brady AK, Kinter M & Fox PL (2008) DAPK-ZIPK-L13a axis constitutes DAPK and signal transduction Y. Lin et al. 56 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS a negative-feedback module regulating inflammatory gene expression. Mol Cell 32, 371–382. 53 Tong A, Lynn G, Ngo V, Wong D, Moseley SL, Ewbank JJ, Goncharov A, Wu YC, Pujol N & Chisholm AD (2009) Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase. Proc Natl Acad Sci USA 106, 1457–1461. 54 Michie AM, McCaig AM, Nakagawa R & Vukovic M (2009) Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer. FEBS J. 55 Kuo JC, Wang WJ, Yao CC, Wu PR & Chen RH (2006) The tumor suppressor DAPK inhibits cell motil- ity by blocking the integrin-mediated polarity pathway. J Cell Biol 172, 619–631. 56 Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C et al. (2009) Autophagy suppresses tumorigenesis through elimination of p62. Cell 137, 1062–1075. 57 Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S & White E (2007) Autophagy miti- gates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 21, 1621–1635. 58 Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K, Chen G, Jin S & White E (2007) Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev 21, 1367–1381. 59 Coussens LM & Werb Z (2002) Inflammation and cancer. Nature 420, 860–867. 60 Weichhart T, Costantino G, Poglitsch M, Rosner M, Zeyda M, Stuhlmeier KM, Kolbe T, Stulnig TM, Horl WH, Hengstschlager M et al. (2008) The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity 29, 565–577. 61 Lin Y, Stevens C & Hupp T (2007) Identification of a dominant negative functional domain on DAPK-1 that degrades DAPK-1 protein and stimulates TNFR-1-mediated apoptosis. J Biol Chem 282, 16792– 16802. 62 Zhang L, Nephew KP & Gallagher PJ (2007) Regula- tion of death-associated protein kinase. Stabilization by HSP90 heterocomplexes. J Biol Chem 282, 11795– 11804. 63 Jin Y, Blue EK & Gallagher PJ (2006) Control of death-associated protein kinase (DAPK) activity by phosphorylation and proteasomal degradation. J Biol Chem 281, 39033–39040. 64 Citri A, Harari D, Shohat G, Ramakrishnan P, Gan J, Lavi S, Eisenstein M, Kimchi A, Wallach D, Pietrokov- ski S et al. (2006) Hsp90 recognizes a common surface on client kinases. J Biol Chem 281, 14361–14369. 65 Henshall DC, Araki T, Schindler CK, Shinoda S, Lan JQ & Simon RP (2003) Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures. J Neurochem 86, 1260–1270. 66 Bialik S, Bresnick AR & Kimchi A (2004) DAP-kinase- mediated morphological changes are localization depen- dent and involve myosin-II phosphorylation. Cell Death Differ 11, 631–644. 67 Cohen O, Feinstein E & Kimchi A (1997) DAP-kinase is a Ca2+ ⁄ calmodulin-dependent, cytoskeletal-asso- ciated protein kinase, with cell death-inducing functions that depend on its catalytic activity. Embo J 16, 998– 1008. 68 Eisenberg-Lerner A & Kimchi A (2007) DAP kinase regulates JNK signaling by binding and activating protein kinase D under oxidative stress. Cell Death Differ 14, 1908–1915. 69 Llambi F, Lourenco FC, Gozuacik D, Guix C, Pays L, Del Rio G, Kimchi A & Mehlen P (2005) The depen- dence receptor UNC5H2 mediates apoptosis through DAP-kinase. Embo J 24, 1192–1201. 70 Shani G, Marash L, Gozuacik D, Bialik S, Teitelbaum L, Shohat G & Kimchi A (2004) Death-associated pro- tein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death func- tions. Mol Cell Biol 24, 8611–8626. 71 Schumacher AM, Schavocky JP, Velentza AV, Mirzoeva S & Watterson DM (2004) A calmodulin- regulated protein kinase linked to neuron survival is a substrate for the calmodulin-regulated death-associated protein kinase. Biochemistry 43, 8116–8124. 72 Shohat G, Spivak-Kroizman T, Cohen O, Bialik S, Shani G, Berrisi H, Eisenstein M & Kimchi A (2001) The pro-apoptotic function of death-associated protein kinase is controlled by a unique inhibitory autopho- sphorylation-based mechanism. J Biol Chem 276, 47460–47467. 73 Kuo JC, Lin JR, Staddon JM, Hosoya H & Chen RH (2003) Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase. J Cell Sci 116, 4777– 4790. 74 Houle F, Poirier A, Dumaresq J & Huot J (2007) DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress. J Cell Sci 120, 3666–3677. Y. Lin et al. DAPK and signal transduction FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 57 . MINIREVIEW Death- associated protein kinase (DAPK) and signal transduction: additional roles beyond cell death Yao Lin, Ted R. Hupp and Craig Stevens CRUK p53 Signal Transduction Laboratories,. September 2009) doi:10.1111/j.1742-4658.2009.07411.x Death- associated protein kinase (DAPK) is a stress-regulated protein kinase that mediates a range of processes, including signal- induced cell death and autophagy. Although the kinase domain. responses by death- associated protein kinase. Proc Natl Acad Sci USA 106, 1457–1461. 54 Michie AM, McCaig AM, Nakagawa R & Vukovic M (2009) Death- associated protein kinase (DAPK) and signal transduction: