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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 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 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 aptamers 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. (Received 11 March 2009, revised 12 August 2009, accepted 8 September 2009) doi:10.1111/j.1742-4658.2009.07411.x 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 inducing 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 identified 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, including 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 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-13acetate; 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 Y. Lin et al. DAPK and signal transduction to use this information to understand developmental or disease processes. Classically, genetic screens in tractable organisms, such as yeast, worms and flies, have been used for defining the landscape of a protein ⁄ pathway. However, many cancer- and immunity-related genes are confined to vertebrates and a full understanding 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, monoclonal antibody co-immunoprecipitation methods coupled to protein sequencing, and tap-tagging molecular biology approaches for trapping a multiprotein complex. The yeast two hybrid, for example, has been used to discover a novel interaction between extracellular signal-regulated kinase (ERK) and DAPK, with implications for pro-apoptotic pathways [3]. Furthermore, recent ideas in systems biology hold that many proteins 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 mammalian target of rapamycin (mTOR) signalling. Together, using such distinct approaches, the DAPK interactome is being built up in a range of backgrounds. 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 (Ploops), 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 unsurAnkyrin 2+ Ca /CaM repeats P-loops 1 1431 Kinase Cytoskeletal Death 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] Actin [66] Beclin-1 [36] CaM [67] Not defined Cytoskeletal domain Not defined Ca2+ ⁄ CaM regulatory domain C-terminal domain Ankyrin repeats Death domain Not defined Kinase domain Ankyrin repeats Kinase domain Not defined Not defined Not defined Not defined Death domain Death domain Kinase domain Immunoprecipitation Immunostaining Immunoprecipitation Overlay binding assay Cathepsin B [61] DIP1 [13] ERK [3] FADD [65] Hsp90 [62] LAR [23] MAP1B [4] PKD [68] RSK [25] Src [23] TNFR-1 [65] TSC2 [5] UNC5H2 [69] ZIPK [70] Immunoprecipitation Yeast two hybrida Yeast two hybrida Immunoprecipitation Immunoprecipitation Yeast two hybrida Peptide librariesa Immunoprecipitation Immunoprecipitation Yeast two hybrida Immunoprecipitation Peptide librariesa Yeast two hybrida Immunoprecipitation a Protein interactions have been confirmed by more physiological methods. prising that only a few of the DAPK binding proteins highlighted in Table 1 are substrates of DAPK, suggesting 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 phosphorylation motif was carried out based on positional scanning peptide substrate library synthesis and activity [6]. The preferred consensus motif for DAPK phosphorylation and substrates for which phosphoacceptor site(s) have been identified are described in Table 2. Of note, mini-chromosome maintenance complex component 3 (MCM3), which is a DNA replication 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 identification 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 biochemical approach to develop small peptide-mimetic ligands to alter how an enzyme functions, was utilized FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 49 DAPK and signal transduction Y. Lin et al. 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 peptides that are phosphorylated by ATM identified over 700 substrates [9]. Therefore, it seems that the previously available protein interaction methodologies were not able to faithfully reflect the ATM kinase interactome. 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. Table 2. DAPK substrates and the amino acid sequence surrounding 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] CaMKK [71] DAPK [72] MCM3 [7] MLC [73] p21 [8] p53 [8] S6 [27] Syntaxin-1A [38] Tropomyosin-1 [74] ZIPK [70] RLKVT119GDL GSRREERSLS511APG A R KKW KQS308V R LI TKKTIERRYS160DLTTL TTKKRPQRATS19NVF RKRRQT145SMTDFYHSK PPLSQET18FS20DLWKLL QIAKRRRLS235SLRAS IIMDSSIS188KQALSEIE HALNDMTS283I KT299TRLKEYTIKS309HS311 S312LPPNNS318YADFERFS326 KRxxxxxKRRxxS ⁄ T Consensus Signalling to DAPK DAPK plays an important role in a wide range of signal 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 DNAdamaging agents. Other death signals, such as the transforming oncogenes E2F1 and Myc [12], also induce DAPK expression. In addition to its well-documented 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]. recently to develop selective peptide ligands that modulate DAPK activity. For example, DAPK binding to a peptide derived from the amino acid sequence of the cyclin-dependent kinase inhibitor p21 induces a conformational 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 telangiectasia mutated (ATM) protein kinase, a large > 300 kDa enzyme, does not have an abundance of stable A B C Mitogens EGF Short treatment TNF-α Long treatment TNF-α Kinase activity Kinase activity Degradation Apoptosis Growth mTORC1? Inflammation ? mTORC1 Apoptosis D IFN-γ TGF-β DNA damage oncogenes ? Gene expression Apoptosis Inflammation Autophagy Immune response Apoptosis E DAPK over-expression MAP1B binding Blebbing Autophagy Beclin-1 phosphorylation Autophagy 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 signalling 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 circumstances. (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. 50 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Lin et al. DAPK and signal transduction 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 stimulatory role for serum-activated DAPK in mTOR signalling [5]. mTOR is a member of the phosphoinositide3-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 particular, phosphorylation and activation of eukaryotic initiation 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-activating 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, inactivating TSC2 phosphorylation results in mTORC1 activation and is thought to occur primarily through activation of the RAS–extracellular signal-regulated kinase kinase (MEK)–ERK and phosphoinositide-3kinase–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 analysing 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 activates the RAS–MEK–ERK pathway [21,22]. Interest- EGF Src Ras Raf MEK LAR 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, antimigration and antiadhesion functions. Under normal 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. ERK TSC1 Y491/Y492 - P DAPK P -S735 RSK DAPK TSC2 P -S289 DAPK Rheb mTORC1 Apoptosis Apoptosis FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS Apoptosis ? P -T389 S6K P -S235/236 S6 Cell growth Protein synthesis 51 DAPK and signal transduction Y. Lin et al. 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 downregulated, resulting in DAPK inactivation. The ability of EGF signalling to inactivate DAPK is inconsistent with previous findings that DAPK activity can be upregulated by serum stimulation and ERK, a downstream 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 apoptosis-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 apoptosis 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 determine 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 stimulate the phosphorylation of S6 in cells, even in the presence of the lipophilic macrolide antibiotic rapamycin, a potent inhibitor of mTORC1 activity, indicat52 ing that DAPK can mediate phosphorylation of S6 in an mTORC1–S6K-dependent and -independent manner. Schumacher et al. [27] demonstrated that DAPK phosphorylates S6 directly on Ser235 ⁄ 236 and concluded 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 protein 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 regulation 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 activity 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 regulates apoptosis in response to various death-inducing signals, including INF-c [2]. DAPK also has autophagy signalling activity, which can be either pro-survival or lead to or participate in cell death. Autophagy is a membrane system involved in protein and organelle degradation that probably represents 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 implicated 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 autophagy, 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 FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Lin et al. 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–DAPKinduced blebbing involves changes in the dynamics of mictrotubules, as well as changes in the dynamics of contractile cortical actin [4]. This is even more intriguing in light of the recently identified interaction between the essential autophagy protein Atg8 (LC3) and MAP1B [31], and the observation that microtubules play an important role in autophagy by supporting 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-XL [35]. Under normal conditions, beclin-1 is bound to and inhibited by Bcl-2 or the Bcl-2 homolog Bcl-XL and the dissociation of beclin-1 from Bcl-2 is essential for its autophagic activity [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-XL 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 interaction of beclin-1 with its inhibitor Bcl-XL. DAPK has also been directly linked to the regulation of endocytosis [37], and can phosphorylate syntaxin-1A, a key component of the soluble N-ethylmaleimide-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 DAPK and signal transduction 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 damage caused by injection of the ER stress inducer tunicamycin and the cell death response to tunicamycin is reduced in DAPK ) ⁄ ) mouse embryonic fibroblasts [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 signalling 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, muscaranic 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 autophagy, 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 inactivates mTORC1, leading to an upregulation of autophagy [45]. The finding that DAPK is a positive regulator of mTORC1 signalling and a positive regulator of autophagy at first seems counterintuitive. Therefore, 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 rapamycininduced autophagy [46]. The accompanying review by Kang and Avery [42] proposes an interesting explanation 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]. FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS 53 DAPK and signal transduction Y. Lin et al. 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-cinduced 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 promotes TNF-a-induced apoptosis [50], and the expression 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 negative regulator of T cell activation via NF-jB. However, 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 multiple 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 negatively regulates inflammatory gene expression [51,52], responds to mitogenic signals to regulate mTORC1 54 activity [5] and negatively regulates epidermal damage responses in C. elegans in an apoptosis- and autophagy-independent manner [53], highlight the pleotrophic role of this kinase. What are the crucial questions for the future? Of considerable importance will be to gain a clear understanding 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 extracellular matrix survival signals to regulate anoikis [14] and its ability to inhibit cell polarization and motility [55]. DAPK can suppress transformation by oncogenes 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 antiinflammatory 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 activities. 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 interacts directly with the ankyrin repeat region of DAPK [13], and carboxyl terminus of HSC70-interacting protein, a U-box containing E3 ubiquitin ligase that can bind to the heat shock protein (HSP) chaperone proteins HSP70 and HSP90, interacts with DAPK indirectly via Hsp90 [62]. The identification of additional ubiquitin ligases, and deciphering the degradation pathways that modulate DAPK stability, will shed FEBS Journal 277 (2010) 48–57 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Lin et al. further light on the role played by DAPK in the regulation 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, autophagic 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. 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