Apoptosis DOI 10.1007/s10495-013-0926-3

THE UNIVERSE OF DAPK

The DAP-kinase interactome Shani Bialik • Adi Kimchi

Ó Springer Science+Business Media New York 2013

Abstract DAP-kinase (DAPK) is a Ca2?/calmodulin regulated Ser/Thr kinase that activates a diverse range of cellular activities. It is subject to multiple layers of regulation involving both intramolecular signaling, and interactions with additional proteins, including other kinases and phosphatases. Its protein stability is modulated by at least three distinct ubiquitin-dependent systems. Like many kinases, DAPK participates in several signaling cascades, by phosphorylating additional kinases such as ZIP-kinase and protein kinase D (PKD), or Pin1, a phospho-directed peptidyl-prolyl isomerase that regulates the function of many phosphorylated proteins. Other substrate targets have more direct cellular effects; for example, phosphorylation of the myosin II regulatory chain and tropomyosin mediate some of DAPK’s cytoskeletal functions, including membrane blebbing during cell death and cell motility. DAPK induces distinct death pathways of apoptosis, autophagy and programmed necrosis. Among the substrates implicated in these processes, phosphorylation of PKD, Beclin 1, and the NMDA receptor has been reported. Interestingly, not all cellular effects are mediated by DAPK’s catalytic activity. For example, by virtue of protein–protein interactions alone, DAPK activates pyruvate kinase isoform M2, the microtubule affinity regulating kinases and inflammasome protein NLRP3, to promote glycolysis, influence microtubule dynamics, and enhance interleukin-1b production, respectively. In addition, a number of other substrates and interacting proteins have been identified, the physiological significance of which has not yet been established. All of these substrates, effectors and regulators together comprise S. Bialik  A. Kimchi (&) Department of Molecular Genetics, Weizmann Institute of Science, 76100 Rehovot, Israel e-mail: [email protected]

the DAPK interactome. By presenting the components of the interactome network, this review will clarify both the mechanisms by which DAPK function is regulated, and by which it mediates its various cellular effects. Keywords DAP kinase  Phosphorylation  Programmed cell death  Substrates  Interacting proteins

Introduction DAP-kinase (DAPK or DAPK1) has been linked to the regulation of various cellular processes, including both caspase-dependent (i.e. apoptosis) and independent cell death, anoikis, autophagy, inflammation, cell adhesion, cell motility and more. Understanding how DAPK is involved in this wide range of activities has obviously been a high priority among researchers in the field, especially since it has been shown to be a tumor suppressor with important clinical implications [1]. Initial research into the function of DAPK was aided by the identification of a classic kinase domain and Ca2?-activated calmodulin (CaM) autoregulatory domain at its N-terminus, placing it within the CaM-regulated kinase superfamily. This naturally led to a search for phosphorylation substrates that mediate its many functional arms. Approximately one dozen relevant substrates have been identified to date. These, however, do not account for all of DAPK’s known cellular effects, so there are likely to be many more missing substrates. A second direction lay in the identification of proteins that interact with DAPK. DAPK’s many extra-catalytic domains, including typical protein–protein interaction domains, such as ankyrin repeats and a death domain, have been shown to interact with numerous proteins, the total of which we refer to as the

123

Apoptosis

DAPK interactome. The DAPK interactome includes proteins that function upstream of DAPK, to regulate its catalytic activity, stability and degradation, and/or localization, and proteins that link DAPK to its downstream cellular functions, including substrates and non-substrate effectors. Identification of the DAPK interactome has thus led to a clearer elucidation of mechanisms of regulation and function of this complex protein. This review will present the DAPK interactome, and how each component was identified, subdivided into its regulators and effectors. Note that some of the topics covered by other reviews in this issue will overlap with the description of interacting proteins. Therefore, this review will present these interactors only briefly and the reader is referred to these reviews for further detail.

A Look upstream: regulators of DAPK Direct regulators of catalytic activity DAPK catalytic activity is regulated both intra-molecularly and also by factors that bind and or modify the protein

(Fig. 1). Most prominent is CaM, which binds the calmodulin autoregulatory domain immediately downstream of the catalytic domain [2, 3]. Typically, the CaM autoregulatory domain is positioned inside the catalytic cleft, thereby serving as an auto-inhibitory pseudosubstrate. Interaction of CaM with the domain leads to conformation changes that result in its removal from the catalytic cleft, and access for substrate. In this manner, the CaM-mediated mechanism of regulation links DAPK activity to signals that involve increased intracellular Ca2?. Control of access to the catalytic cleft is aided by an additional mechanism of regulation, involving auto-phosphorylation of Ser308 within the calmodulin autoregulatory domain [4]. This phosphorylation has an inhibitory effect, since it further stabilizes the autoregulatory domain’s docking within the substrate-binding site and also reduces its affinity to CaM. As a result, two inter-related steps are required for full activation of kinase activity: binding of CaM and dephosphorylation of Ser308. Dephosphorylation of Ser308 is a common event following multiple stimuli, including ceramide [4–6], ER stress [7], TNFa [6], ischemia [8], and treatment with several IFN

Ca2+

KLHL20

Cul3

TSC2

CHIP Hsp90

CaM

Kinase domain

CaM AutoReg.

Mib

GTP

Ankyrin Repeats

S289 S308

ROC S735

Y491/2 LAR

Netrin 1

Unc5H

LRRK1/2

COR B (B α ) PP2A (C)

DD

A (PR65β)

Ser rich tail

ERK

Src RSK

Regulation key EGF Inhibition of catalytic activity Activation of catalytic activity

Ceramide ER stress Ischemia TNFα

PMA

Ub-mediated degradation

Unknown Phosphorylation Dephosphorylation Signaling Stimulus

Fig. 1 The DAPK interactome upstream. Depiction of the various proteins that interact with and regulate DAPK, and the stimuli that activate the mode of regulation, when known (dashed arrows). Some interactors modify DAPK by phosphorylation/dephosphorylation (curved arrows), thereby enhancing (blue) or attenuating (red) DAPK catalytic activity. Phosphorylated residues are indicated. Other

123

interactors mediate ubiquitination (Ub) of DAPK (green), thereby affecting protein stability and degradation. Proteins that have been shown to directly bind DAPK through a specific region are positioned above or below that domain of DAPK. CaM Auto-Reg. calmodulin autoregulatory domain, DD death domain, ROC Ras of complex proteins domain, COR C-terminal of ROC domain

Apoptosis

anti-cancer drugs [9–11]. Thus the phosphatase responsible for removing the phospho group from Ser308 is often a direct link between DAPK and the stimuli that lead to its activation. As such, identification of the Ser308 phosphatase was of prime interest in the field. Treatment of cells with various phosphatase inhibitors implicated a PP2A-like phosphatase in dephosphorylating Ser308 following ER stress. Furthermore, incubation of DAPK with purified PP2A in vitro led to reduction in phospho-Ser308 levels [7]. PP2A, one of the major classes of cellular Ser/Thr protein phosphatases, consists of three subunits—the catalytic C subunit, of which there are two isoforms, the structural scaffold A subunit (either PR65a or b), and a regulatory B subunit, which directs substrate binding and sub-cellular localization. There are 15 distinct B subunit genes, which together with their numerous splice variants, are divided into four separate families. Altogether, more than 200 varieties of PP2A holoenzyme can be assembled, providing the specificity of function and substrate recognition. Two additional studies confirmed the identity of PP2A as the DAPK Ser308 phosphatase by studying different subunits. First, the Ba subunit was identified as a DAPK interacting protein via Mass Spec analysis of a TAP-tagged DAPK immune-complex [12]. Co-immunoprecipitation experiments confirmed that DAPK interacted with both Ba and its highly related splice isoform, Bd, as well as the PP2A C and A subunits, at the exogenous and endogenous levels. PP2A is recruited to DAPK’s cytoskeletal domain, a region that is necessary for its association with the actin cytoskeleton, and which overlaps with its ROC–COR domains. Also in this study, purified PP2A holoenzyme containing either Ba or Bd dephosphorylated DAPK in vitro, leading to enhanced catalytic activity and CaM binding. During ceramideinduced anoikis of HeLa cells, knock-down of Ba partially attenuated Ser308 dephosphorylation, indicating that this PP2A holoenzyme plays some role in DAPK activation in response to a physiological death stimulus. In a totally independent manner, Mehlen and coworkers [5] identified the A subunit PR65b. This came about indirectly while addressing the mechanism of action by which the dependence receptor UNC5H induces apoptosis in the absence of its ligand, netrin-1. Previously, this same group showed that DAPK is necessary for apoptosis upon removal of netrin-1 [13]. Unliganded UNC5H binds DAPK via their respective death domains, and this leads to reduced Ser308 phosphorylation and enhanced DAPK catalytic activity. In a further siRNA screen for factors necessary for apoptosis induced by a netrin-1 antagonist in a breast cancer cell line, PR65b emerged [5]. In the absence of netrin-1, UNC5H recruits both DAPK and PP2A to lipid rafts within the cell membrane, leading to Ser308 dephosphorylation, DAPK activation and DAPK-

dependent apoptosis [5]. It is interesting that in this system, the DAPK–PP2A interaction is facilitated by a third factor that directly binds DAPK, UNC5H. Thus UNC5H is an essential component of the phosphoSer308 regulatory mechanism in the specialized setting of dependencereceptor induced apoptosis. The identity of the B subunit, which serves to recruit the specific PP2A subunit to the substrate, was not addressed in the Mehlen study, and it is not known whether Ba is utilized. Conceivably, the third party scaffold could enable interaction with additional PP2A holoenzymes that would otherwise not bind DAPK. This would broaden the scope of the regulatory mechanism, an important factor, especially considering the fact that different subunits have specific and limited patterns of tissue expression. It remains to be determined whether different PP2A holoenzymes regulate DAPK in response to different stimuli or in different cell types, and whether other scaffold proteins serve to recruit PP2A in response to other signals. Yet a third layer of intra-molecular regulation of the kinase domain involves the ROC (Ras of Complex proteins)–COR (C-terminal of ROC) domains of DAPK. These two domains, always found in tandem, define the ROCO family of proteins, of which the most famous mammalian member is the Parkinson Disease associated LRRK2 [14, 15]. In fact, DAPK can form heterodimers with LRRK2 and the closely related LRRK1 [15], the significance of which is not known. Our group has shown that as in other ROCO proteins, DAPK’s ROC domain binds and hydrolyzes GTP [16]. Mutational analysis that abolishes GTP binding to the P-loop within the ROC domain resulted in reduced Ser308 autophosphorylation in a PP2A independent manner, leading to enhanced catalytic and cellular activity. Thus, the loss of GTP binding to the ROC–COR domains is likely to directly impair the ability of DAPK to autophosphorylate Ser308. Based on these results, a model was proposed, which still awaits full validation, in which GTP binding to the ROC domain restrains the kinase domain, resulting in a ‘‘kinase-off’’ state. This is consistent with the greater affinity for GTP than GDP that was observed in vitro [16]; in the basal, unstressed state when DAPK is normally inactive, it is predicted to be bound to GTP. GTP hydrolysis would then serve as a regulated event that leads to activation of the kinase by an intra-molecular mode of signaling involving a conformational change that reduces Ser308’s accessibility to the catalytic cleft. Additional regulators Several additional proteins interact with DAPK and regulate its activity, mainly through phosphorylation events (Fig. 1). These kinases and phosphatases lie downstream to

123

Apoptosis

growth factor signaling pathways, thus connecting DAPK and its potential anti-tumor properties to the signals that promote cell growth and tumorigenesis. Activation of the Ras–Raf–MAPK–ERK pathway can lead to either DAPK activation or inactivation, depending on the context. ERK binds a canonical docking sequence within DAPK’s death domain, and phosphorylates DAPK on Ser735, within the ROC domain [17]. In fact, serum activation or co-transfection with the ERK activator MEK1, led to phosphorylation of Ser735 in vivo in an ERK-dependent manner. This modification enhances DAPK’s catalytic activity towards its substrate, myosin regulatory light chain (MLC), both in vitro and in vivo. This was reflected by lower Km values, while Kcat and Vmax were unaffected, suggesting that Ser735 modification may somehow affect substrate binding. The mechanism by which this occurs is not known; however, considering that it is now known that Ser735 lies within the ROC domain, the phosphorylation may be related to the G-protein cycle. The physiological significance of the regulation of DAPK by ERK was shown in several cell models including anoikis in 3T3 fibroblasts, and PMA-induced apoptosis in detached erythroblasts [17]. ERK can also negatively regulate DAPK. In serum starved HEK 293 cells, treatment with PMA blocked apoptosis induced by DAPK overexpression in an ERKdependent manner [18]. Activation of the Ras–MAPk– ERK pathway by PMA led to an enhanced interaction between DAPK and ERK’s downstream effector, p90 ribosomal S6 kinase (RSK). Furthermore, RSK phosphorylated DAPK both in vivo and in vitro. This phosphorylation was mapped by Mass Spectrometry to Ser289 within the calmodulin autoregulatory domain and shown to be inhibitory; the phosphomimetic Ser289Glu mutant had decreased apoptotic activity, while mutation to Ala led to enhanced activity in vivo. Although this work did not explore the mechanism by which the phosphorylation event inhibits kinase activity, one can speculate that it negatively affects CaM binding, or enhances the effect of the nearby Ser308 phosphorylation. DAPK was also shown to interact with phosphorylated (active) p38 MAP kinase upon TNFa treatment of HCT116 colorectal tumor cells [19]. p38 may also be an upstream regulator of DAPK, as its knock-down led to reduced DAPK in vitro catalytic activity when isolated from TNFa treated cells. Knock-down and/or inhibition of either kinase blocked TNFa-induced apoptosis in these cells. The leukocyte common antigen-related (LAR) Tyr phosphatase was identified as another DAPK interacting protein by a yeast-two hybrid using DAPK’s ankyrin repeats as bait [20]. DAPK interacts with LAR in vivo, and serves as its substrate. The dephosphorylation sites were

123

identified as Tyr491/492, which are targets of the kinase Src. Dephosphorylation by LAR enhanced DAPK’s ability to phosphorylate its substrate MLC in vitro and in vivo, and likewise enhanced its pro-apoptotic, anti-adhesion, and anti-migratory effects. In contrast, LAR knock-down, or phosphorylation by Src, had the opposite effect on DAPK’s biological activities. From these studies we see that different kinases and phosphatases, through their interactions with different domains of DAPK, can regulate DAPK catalytic and cellular activities. Viewed in another way, different domains of DAPK, from the nearby calmodulin autoregulatory domain, to the ROC domain, the ankyrin repeats and the death domain, can fine-tune the activity of the very N-terminal kinase domain by virtue of their interactions with and/or modifications by specific signaling proteins. Thus the modular nature of the DAPK structure and its specific interactome enables it to respond to a diverse range of stimuli. Regulators of protein stability A third paradigm exists for regulation of DAPK by its interactors: factors that indirectly regulate DAPK by affecting protein stability. DAPK is a client protein of the chaperone Heat Shock Protein 90 (Hsp90), which facilitates DAPK maturation, stability and activity [21]. When Hsp90 is inhibited, the activated Ser308 dephosphorylated DAPK is degraded in a proteasome-dependent manner either by the U-box ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP), which forms a ternary complex with DAPK and Hsp90; or Mindbomb 1 (MIB1)/DAPK interacting protein-1 (DIP-1), an E3 ligase identified by yeast two-hybrid using DAPK’s ankyrin repeats as bait [22]. This leads to accumulation of the remaining, inactive Ser308 phosphorylated DAPK [23]. Interestingly, MIB1/DIP-1 has also been proposed to regulate DAPK’s apical localization in differentiated gastric zymogenic cells (ZCs) [24]. This is more consistent with its degradation-independent role as a regulator of the Notch pathway, in which it mono-ubiquitinates Notch ligands Delta and Jagged, promoting their endocytosis [25]. Yet a third ubiquitin E3 ligase system involves KLHL20, a BTB/Kelch protein that acts as an adaptor for Cullin3-based E3 ligases, which was found to bind DAPK by a yeast two-hybrid using DAPK’s death domain [26]. The interaction with KLHL20 is inhibited by interferon (IFN) a and c, so that IFN treatment results in reduced degradation, and thus an increase in DAPK steady state levels. Further details of the regulatory mechanisms controlling DAPK stability can be found in an accompanying review [27].

Apoptosis

cytoskeletal dynamics are the subjects of two additional reviews in this issue, we will focus here on the remaining substrates.

Substrates and effectors DAPK substrates Identification of DAPK interacting proteins that function downstream has been critical to understanding the molecular mechanisms of DAPK function. The vast majority of DAPK functions are mediated by the phosphorylation of various target proteins, so that a significant portion of the DAPK interactome is comprised of its substrates (Fig. 2). DAPK, which localizes to the actin cytoskeleton, has several cytoskeletal associated substrates, including the first recognized substrate, MLC [2, 28], and tropomyosin [29]. These contribute to its cytoskeletal-related effects, including membrane blebbing, by increased acto-myosin contractility, and cell motility, by stress fiber formation. The DAPK substrates Beclin 1 [30] and Protein Kinase D (PKD) [31], mediate DAPK’s effects on autophagy. Both of these proteins interact with and are phosphorylated by DAPK, leading to their activation. Interestingly, phosphorylation of both of these substrates affects the class III phosphatidyl inositol-3 kinase Vps34 complex, of which Beclin 1 is an activating component. Phosphorylation of Beclin 1 by DAPK blocks its interaction with the inhibitor Bcl-2, enabling its association with Vps34 [30]. PKD directly phosphorylates and activates Vps34 [31]. As DAPK’s roles in both autophagy and

Cell death associated substrates One of the first functions of DAPK to be recognized was its role in mediating apoptosis, both p53 dependent and independent [32]. The apoptosis related substrates and interactors have been elusive, however. There are several paradigms for DAPK-regulated apoptosis. DAPK can mediate anoikis (apoptosis induced by loss of matrix attachment) by interfering with integrin function [33], but the direct substrate in this pathway has not been identified. DAPK is required for apoptosis induced by unliganded Unc5H [13]; while the interaction between the two proteins is essential, it is not known what lies downstream of DAPK to activate apoptosis. In addition, DAPK can activate p53 in a p19ARF-dependent manner in response to transforming oncogenes, leading to p53-dependent apoptosis in MEFs [34]. p53 itself is a direct substrate of DAPK; DAPK can phosphorylate tetrameric p53 in vitro on Ser20 within the transactivation domain that binds p300 [35]. In vivo, over-expressed DAPK co-immunoprecipitated with endogenous p53, enhanced Ser20 phosphorylation, and increased p53 transactivation activity. On the other hand, depletion of DAPK by siRNA led to reductions in p53

DAP-kinase ?

NF-κ B

? mdm2

ZIPk

P

NLRP3

P

Tropomyosin

P

MLC

Mcm3

MARK1/2

Acto-myosin contractility

P P

PKD

Stress fiber formation

P

Pin1

Membrane blebbing

Calcium influx

Vps34

P

Tau

Cell polarity motility

Cell adhesion

NR2B NMDA receptor

Beclin 1 P

IL-1β

Inflammatory Genes

P

CaMKK

Syntaxin-1A P

p53

P

P

?

L13 Integrin Inflammasome assembly Talin T cell GAIT assembly activation

P

p19ARF

P

GLUT4 Pyruvate Kinase M2

JNK

Ischemic injury Glucose uptake Programmed necrosis

MT instability Oncogene transactivation Glycolysis

Apoptosis

Autophagy

Cell transformation

Fig. 2 The DAPK interactome downstream. Schematic of the effectors and substrates of DAPK (polygons), and the signaling pathways mediated by them (black lines). Kinase/substrate interactions are indicated by red lines and P, kinase-independent interactions by a red line only. Dashed red lines lead to indirect effectors (ovals) and pathways in which the direct interactor/substrate is not yet known. Arrowheads and a signify that the interaction/phosphorylation activates or suppresses the target, respectively. Black dashed

lines are known functions mediated by DAPK effectors that have not been confirmed to be DAPk-dependent. DAPK family members are in red. Effectors are grouped and color-coded by related function: immune response (shades of purple), cytoskeleton functions, also leading to taupathies and metastasis (shades of blue), oncogenesis (shades of green), autophagy and cell death (shades of pink). Additional colors (yellow, orange) depict ‘‘orphan’’ effectors with no known function

123

Apoptosis

Ser20 phosphorylation, and reduced p53 protein levels. This may explain how DAPK activates p53-dependent apoptosis in response to oncogene expression in MEFs, although it does not account for the requirement for p19ARF [34]. Thus there are still missing pieces to the apoptosis puzzle. Related to its cell death function, DAPK also participates in cerebral ischemia-induced neuronal damage through its interaction with the N-methyl-D-aspartate (NMDA) glutamate receptor [36]. Glutamate accumulation at the synapse following ischemic injury leads to over-stimulation of the glutamate receptors, which results in increased Ca2? influx through receptor gated ion channels, leading ultimately to neuronal cell death. DAPK associates with the NMDA receptor upon induction of focal cerebral ischemia in mice, specifically binding the NR2B regulatory subunit. This leads to enhanced receptor conductance. Furthermore, DAPK phosphorylates NR2B on Ser1303. Significantly, blocking the DAPK–NMDA receptor interaction suppressed stroke-induced brain damage. Likewise, DAPK knock-out blocked neuronal death following ischemia. DAPK is activated by ischemic injury, as assessed by decreased Ser308 phosphorylation, which may explain why its deletion had no affect on basal NMDA receptor activity. Signaling cascades Like many kinases, DAPK can phosphorylate other kinases, initiating kinase cascades with various downstream effects. As mentioned above, one kinase substrate is PKD. Oxidative stress enhances its interaction with DAPK in 293 cells [37] and in cardiomyocytes upon contraction-induced ROS formation [38]. In H2O2-treated 293 cells, in addition to regulating autophagy through Vps34 [31], phosphorylation of PKD by DAPK led to activation of JNK, and consequently, programmed necrosis, thereby linking DAPK to an additional death pathway [37]. In the cardiomyocyte system, activated PKD induced translocation of GLUT4 and enhanced glucose uptake [38]. Thus the DAPK–PKD cascade has several functional outcomes, depending on the stimulus and cell setting (Fig. 3a). A second cascade involves CaM dependent protein kinase kinase2 (CaMKK2), which was identified as an interacting protein of DAPK in a yeast two-hybrid screen of human brain cDNA library [39]. The interaction was confirmed for the endogenous proteins by co-immunoprecipitation in rat brain extracts. DAPK phosphorylates CaMKK2 in vitro and in vivo on Ser511, which is located near the CaM binding domain. The authors showed that Ser511 phosphorylation inhibits CaM-activated autophosphorylation, implying that DAPK inhibits CaMKK2 activity, although this was not directly assessed nor addressed in cells. The physiologic consequence of CaMKK2 as a DAPk substrate is not known.

123

A third kinase phosphorylated by DAPK is the closely related ZIP-Kinase (ZIPK), also known as DAPK3 (see review on family). Members of the DAPK family share a common basic loop within the catalytic domain, which mediates homodimerization and heterodimerization among the family members [40, 41]. In addition to forming a complex, DAPK phosphorylates ZIPK on several sites, including Thr299 [40]. While ZIPK can be found in both the nucleus and cytoplasm, the phosphorylated protein is predominantly diffusely cytoplasmic [40, 42]. Although ZIPK has several nuclear substrates and thus a distinct function in the nucleus (ZIPK review [43], this issue), the cytoplasmic ZIPK is more potent in promoting membrane blebbing and autophagy, similar to DAPK [40]. Thus the DAPK–ZIPK cascade serves to amplify DAPK’s cellular effects. In addition, the DAPK–ZIPK cascade is involved in the inflammatory response to IFN-c through activation of the IFN-c-activated inhibitor of translation (GAIT) complex, which serves to down-regulate the expression of certain inflammatory genes, thereby limiting or terminating the inflammatory response [44]. ZIPK phosphorylates GAIT component ribosomal protein L13A on Ser77. This results in its disassociation from the ribosome and recruitment to the GAIT complex, thereby activating the translation inhibitor complex. DAPK was necessary for L13A phosphorylation by ZIPK in vitro and in vivo, and for translation inhibition, even though DAPK did not directly phosphorylate L13A. Furthermore, DAPK catalytic activity was induced earlier than ZIPK activity, confirming the presence of a DAPK–ZIPK cascade that is activated in response to IFN-c. DAPK has also been linked to translational control by the 40S ribosomal protein S6. DAPK and S6 co-immunoprecipitated in rat brain, and DAPK phosphorylated S6 on Ser235 in vitro and in vivo [45]. Addition of DAPK to reticulocyte lysates inhibited in vitro translation, implying that the phosphorylation of S6 suppressed its function. Furthermore, activation of DAPK in vivo attenuated protein synthesis. While this activity was accompanied by Ser235 phosphorylation, the authors did not rigorously prove that S6 phosphorylation by DAPK was necessary for its effects on translation. This is especially critical, as Ser235 is also the target of other kinases, such as the S6K family, RSK and MAPK/ERK, and DAPK can functionally affect both ERK [17], which activates RSK, and mTOR [46], which phosphorylates and activates p70S6K. Although initially described to inhibit ribosomal function, the role that S6 phosphorylation has in regulating ribosomal mediated translation is actually controversial and unresolved [47]. In addition, S6 phosphorylation has been implicated in cell growth and cell size determination, cell proliferation, and insulin secretion [47], and has more recently been linked to the development of pancreatic cancer [48], functions that are

Apoptosis

a

DAP-kinase

P

PKD

P

P

P

Beclin 1 GLUT4

SSH

Ask1

Bcl2P

PAK4 P

?

P

LIMK

MKK4/7

Vps34

P

P

JNK

cofilin

PI3P

Glucose uptake

Autophagy Endocytosis

Programmed necrosis

Apoptosis

Actin severing remodeling

DAP-kinase b ? p19ARF

?

P

P

NF-κB

Nucleus

p53

Pin1

Pyruvate Kinase M2

mdm2

MARK1/2 P

p53

Tau

Warburg Effect

pSer/Thr-Pro cis/trans Glycolysis MT instability

Transcription (NF-κB, β -catenin, cyclin D)

cyclin D1 stability

Chromosomal instability Centrosome amplification Mitotic spindle deformities Cell transformation

not necessarily related to protein synthesis regulation. Extraribosomal functions may also exist [47], as has been shown for L13A. Thus the relevance of the DAPK phosphorylation

of S6 remains to be shown. Curiously, a proteomics-based substrate searched identified ribosomal protein L5 as an unvalidated candidate DAPK substrate as well [49].

123

Apoptosis b Fig. 3 DAPK-regulated signaling hubs. a DAPK activates PKD, a

master regulator of many signaling pathways, some of which correspond to DAPK functions (red arrows). Solid yellow lines indicate activation of PKD-dependent pathways that have been shown to require DAPK-mediated activation of PKD. Dashed yellow lines represent phosphorylation of substrates by PKD whose dependence on DAPK is hypothetical. DAPK has been shown to interact with LIMK and cofilin and promote the latter’s phosphorylation by an unknown mechanism. b DAPK activates the Pin1 phospho-directed prolyl isomerase, a master regulator of many signaling pathways, some of which correspond to DAPK functions (red arrows). Solid lines indicate functions that have been shown to be regulated by DAPK. Dashed green lines indicate regulation of Pin1 targets that are related to known DAPK pathways, but have not been experimentally proven. Note that the pathways presented are not meant to be complete representations of PKD or Pin1 signaling pathways

Another key component of the downstream DAPK interactome is the phospho-Ser/Thr directed peptidyl-prolyl isomerase Pin1, a master signal transduction regulator (Fig. 3b). Pin1 controls the activity, stability, and/or localization of many phospho-proteins through cis/trans isomerization of Pro residues that follow phosphorylated Ser or Thr. DAPK interacts with Pin1 via a region overlapping DAPK’s ROC–COR domains and the latter’s isomerase domain [50]. It phosphorylates Pin1 on Ser71 in vitro and in vivo, and Ser71 phosphorylation of endogenous Pin1 was reduced in cells depleted of DAPK by siRNA, and in cancer cells lacking DAPK expression. Phosphorylation of Ser71 inactivated Pin1’s catalytic activity, blocked its nuclear localization, and suppressed its cellular functions, such as stabilization of cyclin D1 protein and enhanced transcription of the cyclin D1, b-catenin and NF-jB promoters. Importantly, Pin1 may be a critical DAPK target that mediates its tumor suppressive function. DAPK-mediated Ser71 phosphorylation blocked celltransformation activities of Pin1 in NIH3T3 cells, such as centrosome amplification, abnormal spindle formation and matrix-independent cell growth. Furthermore, Pin1 knockdown suppressed the increased cell migration observed upon knock-down of DAPK in breast cancer cells, implying that phosphorylation/inactivation of Pin1 is critical for DAPK’s anti-metastatic capabilities. Additional substrates Additional members of the DAPK interactome can be classified as ‘‘orphan substrates’’ whose functional significance has yet to be elucidated. Syntaxin-1A, a component of the SNARE [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein receptors] complex that mediates synaptic vesicle docking and fusion, is one such target. DAPK was identified as a syntaxin-1A binding protein in a yeast two-hybrid screen, and was shown to phosphorylate syntaxin-1A on Ser188 [51]. The phosphorylation

123

interfered with syntaxin-1A’s binding to MNK-18-1, an inhibitor of SNARE complex assembly. The physiological significance of this, and moreover, DAPK’s contribution to synaptic vesicle exocytosis, is not known, however. DAPK has been functionally linked to other membrane fusion events, such as endocytosis [52] and autophagy (see DAPK and Autophagy review [53], this issue), so the DAPK– syntaxin-1A connection is intriguing. More work is needed in this direction to clarify the significance of this part of the DAPK interactome. A proteomics screen for DAPK substrates led to the identification of the DNA replication licensing factor Mcm3 as an in vitro and in vivo DAPK substrate [49]. The phosphorylation site was mapped to Ser160, but the significance of this modification is not known. Mcm3 may link DAPK to yet a new function involving DNA replication, or to one of its ‘‘moonlighting’’ activities, such as STAT1 mediated, IFN-c induced gene expression [54] or transcriptional repression of the Ink4/ARF locus [55], both of which may be relevant to DAPK function. Catalytic-independent effectors Although the focus of research into DAPK effectors has been on its substrates, interestingly, some DAPK functions occur independently of its catalytic activity; DAPK can regulate several effector proteins by virtue of interaction alone (Figs. 2, 3b). For example, co-immunoprecipitation experiments revealed an interaction between DAPK and the microtubule affinity regulating kinases (MARK) 1/2, via the former’s death domain [56]. The MARKs phosphorylate microtubule (MT) associated proteins (MAPs), which leads to their dissociation from MTs, thereby affecting MT dynamics, MT-based motor transport and cell polarity [57]. The DAPK interaction disrupts an inhibitory intra-molecular interaction within the MARK proteins. Thus DAPK activates MARK in a manner independent of its catalytic activity. As a result, DAPK expression inhibited MT assembly in MCF7 cells, axon formation in neurons, and led to taupathy and loss of photoreceptor neurons in the Drosophila eye. In contrast, depletion of DAPK by siRNA in hippocampal neurons or knock-out in mouse brain led to reduced tau phosphorylation by MARK [56]. DAPK can also interact with MAP1B, which was discovered through a peptide combinatorial library screen for motifs that bind DAPK’s kinase domain [58]. The interaction was confirmed for the full-length proteins in vivo, and was shown to be enhanced by starvation. Functionally, MAP1B expression synergized with DAPK expression to induce membrane blebbing and loss of clonogenicity. Conversely, MAP1B knock-down attenuated DAPK’s ability to promote membrane blebbing or autophagy. These results indicate that MAP1B is an effector of DAPK,

Apoptosis

particularly functioning in cytoskeletal events, such as acto-myosin contraction or potentially, MT-based trafficking events that are required for autophagosome formation and/or maturation. Interestingly, Capoccia et al. [24] noted a correlation between DAPK apical localization and phosphorylation of MAP1B in gastric ZCs, which in turn regulated microtubule-based trafficking events in the differentiated cells. Based on the earlier MAP1B/DAPK interaction paper, they propose that DAPK is directly responsible for MAP1B phosphorylation. However, there is no evidence that DAPK can phosphorylate MAP1B directly; it is not known how the interaction with MAP1B relates to the functional connection between the two genes. A second protein that is activated by DAPK by virtue of its interaction is pyruvate kinase M2 (PKM2). PKM2 was identified as a DAPK interactor by yet another yeast twohybrid screen with DAPK’s death domain [59]. Pyruvate kinase is a key glycolytic enzyme that mediates the conversion of phosphoenolpyruvate to pyruvate, generating ATP in the process. The M2 isoform, normally restricted to embryonic development, is re-expressed in tumor cells, and is believed to be essential for the Warburg effect, in which cancer cells upregulate glycolysis and lactate production at the expense of oxidative respiration [60]. DAPK activates PKM2, leading to enhanced catalytic rate in vitro and increased glycolysis in cells, as indicated by elevated lactate secretion [59]. The interaction between DAPK and PKM does not require the kinase domain, and DAPK failed to phosphorylate PKM2, indicating that its effects on PKM2 activity occur through the interaction between the two proteins (Figs. 2, 3b). The relationship between DAPK and PKM2 is counterintuitive: why would a tumor suppressor activate an oncogene? This can be explained by a paradox of PKM2 biology [60]. PKM2, which is catalytically functional as an allosterically activated tetramer, is less efficient than the normal adult isoform, PKM1, even though cancer cells have a higher ATP requirement than normal cells. The slower glycolytic rate is believed to allow for rerouting of glycolytic intermediates to biosynthetic pathways for the generation of lipid and nucleic acid precursors necessary for the highly proliferative cancer cell. Thus the switch from the M1 to the M2 isoform promotes tumor growth. Furthermore, in its dimeric form, PKM2 can act as a protein kinase, and can localize to the nucleus, where it functions as a transcriptional co-activator in promoting expression of metabolic and proliferative genes. It has been proposed that PKM2’s nuclear functions mediate the Warburg effect [61]. Interestingly, the PKM2 and DAPK interaction is linear with respect to concentration even at a ratio of 4:1, indicating that DAPK can interact with the tetramer [59]. It is not known whether DAPK can affect dimeric PKM2’s nuclear functions. Hypothetically, if

DAPK promotes the glycolytic function of the tetramer at the expense of its nuclear and dimeric functions, it can subvert PKM2 away from its pro-proliferative and progrowth functions; by activating PKM2’s enzymatic activity it may actually inhibit its oncogenic potential [60]. Significantly, Mor et al. [59] showed that expression of the kinase domain-deleted DAPK had a mild suppressive effect on cell proliferation, indicating that its kinase-independent functions, possibly involving PKM2, can contribute to its tumor suppressive activity. Additional kinase-independent interactions may also contribute to the latter observation. For example, DAPK can sequester ERK in the cytoplasm, thereby suppressing the latter’s nuclear pro-growth functions [17]. In fact, an increase in the DAPK–ERK cytoplasmic interaction, and a decrease in levels of nuclear ERK, were observed in CNE1 nasopharyngeal carcinoma cells upon treatment with grifolin, a mushroom metabolite with anti-cancer properties, which may contribute to the latter’s ability to induce G1arrest in these cells [9]. Yet another functional arm of DAPK is involved in immune responses and inflammation, a topic that will be discussed in full detail in the accompanying review [27]. At least one of these functions was shown to depend on an interacting partner of DAPK, independently of kinase activity. DAPK interacts with NACHT domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3), a component of the inflammasome complex that serves to activate caspase-1 in response to microbial infections and danger signals. DAPK is required for NLRP3 inflammasome assembly and function, affecting caspase-1 activation and interleukin (IL)-1b production [62]. This has implications for inflammatory disease and for caspase-1 mediated cell death, a process called pyroptosis. DAPK was also shown to negatively regulate STAT3 and as a result, IL-6 production, in response to TNFa treatment in intestinal epithelial cells. This may involve complex formation between DAPK and STAT3, and may be important for the development of ulcerative colitis and its associated carcinomas [63].

The greater interactome: perspective and future directions As can be seen from the data presented here, the DAPK interactome is large and multi-functional. Yet, as depicted it cannot be complete, as there are still many unexplained functions that have not yet been attributed to a specific interactor or substrate, for example, its inhibitory effects on integrin function that reduce cell adhesion, on the association of talin and integrin, which blocks cell polarization and migration [33, 64], and its role in immune responses, such as inhibition of NF-jB and T cell activation [65].

123

Apoptosis

There have been several attempts at identifying the full repertoire of DAPK substrates, through proteomics approaches [39, 49, 66]. The results of these studies imply that there are additional substrates that have not yet been validated or functionally assessed. A database search of a proposed DAPK optimal substrate motif, generated by means of a positional scanning peptide library [67], indicates nearly 200 proteins that contain this ‘‘consensus’’ and thus are potential substrates. Most of these candidates have not been tested. While some true substrates such as Beclin1 [30], PKD [37], CaMKK [39], RPS6 [45] and MLC [67], contain an exact or near match to the consensus sequence, many of the known substrates sites are only loosely related (e.g. p53 [35]) or not at all (syntaxin-1A [51]). Thus additional substrates may exist that do not conform to the proposed consensus and cannot be thus predicted. As is the case for other multi-potent signaling kinases, such as PKA, MAPK, etc., DAPK’s functions must be individually regulated so that its activation in a particular cell setting will turn on a limited number of downstream pathways at one time, as appropriate. A main unresolved issue is what then determines the choice of effector once DAPK is activated? Some of DAPK’s effectors and substrates are expressed in a tissue/cell specific manner, and therefore those pathways will be functional only within that cell type, such as immune related substrates or neuronal proteins like the NMDA receptor, MARKs or CaMKK. Within a cell type, DAPK’s intracellular localization can also determine which substrates/effectors are regulated. For example, DAPK’s localization at actin filaments [2, 28] will bring DAPK into proximity of substrates such as MLC and tropomyosin. It can also bind tubulin [58], and presumably that interaction enables its recognition of microtubule-associated proteins, MARK and MAP1B. Unc5H interacts with DAPK specifically at membrane lipid rafts [68], where a different set of substrates may be found that lead to apoptosis upon activation of this signaling pathway. Thus, for these effectors, the critical factor will be the (as of yet unknown) mechanisms that control DAPK localization. Effector selectivity may also stem from the different mechanisms that regulate DAPK function and stability as described in the first section of this review. For example, IFN activates the DAPK–ZIK–L13A pathway [69], and oxidative stress activates the DAPK–PKD signaling cascade [37]. These are probably not the only functional arms activated by these triggers, and there are likely to be additional stimuli that activate these same pathways. As of yet there is no comprehensive data indicating which triggers activate which pathways. Even with this information, there would still remain the question of ‘‘how’’ a specific trigger activates a specific pathway at the exclusion of other pathways.

123

The issue of which effector pathway is activated in a particular setting becomes even more complex when one considers that many DAPK interacting proteins/substrates are themselves critical signaling hubs and master regulators. For example, PKD has numerous substrates that mediate a wide range of functions [70], and even the substrate shown relevant to DAPK-induced autophagy, Vps34, has additional membrane trafficking functions [71] (Fig. 3a). Pin1 also influences diverse cellular functions through the isomerization of numerous substrates [72] (Fig. 3b). And of course, p53 has both transcriptional-dependent and independent activities, which regulate apoptosis, cell cycle arrest, senescence and autophagy. Are all downstream pathways activated/inhibited by DAPK, or only a subset? If the latter, what determines which pathways are DAPK-dependent? Interestingly, some of the pathways that these master signaling proteins activate are related to known DAPK functions, such as p53-induced autophagy, and it is tempting to speculate that they are also dependent on DAPK for activation. Also, several functions of Pin1, which is inhibited by DAPK-mediated phosphorylation, are inversely related with other DAPk functions. Pin1 promotes tau dephosphorylation [72], and binds and isomerizes ERKphosphorylated PKM2, thereby promoting the latter’s nuclear translocation and transactivation activity, and the Warburg effect [61]. Inhibition of Pin1 by DAPK to block these functions may be essential to enable the dominance of its own signaling pathways that promote tau phosphorylation (via MARK1/2), and the glycolytic (cytoplasmic) function of PKM2 (via direct interaction) (Fig. 3b). Expanding the interactome to include downstream pathways of the major signaling proteins that are DAPK substrates can sometimes shed light on missing pieces in known DAPK pathways. A case-in-point is DAPK’s effects on the F-actin severing protein cofilin, which promotes actin filament turn-over and remodeling, for example, during cell migration. Cofilin function is regulated by the balance of phosphorylation by LIM-kinases (LIMK), which blocks its interaction with actin, and dephosphorylation by the slingshot (SSH) and chronophin phosphatases, which activates it [73]. DAPK was recently shown to enhance cofilin phosphorylation by an unknown mechanism during TNFa-induced apoptosis [74]. Although DAPK inhibition or depletion led to reduced LIMK and cofilin phosphorylation, it was not determined whether either was a direct DAPK substrate. Rather, the authors propose that DAPK acts as a scaffold that binds both LIMK and cofilin, enhancing the interaction between them. Intriguingly, PKD can promote cofilin phosphorylation (i.e. inactivation) by phosphorylating and inactivating SSH [73], and by phosphorylating and activating PAK4, which in turn phosphorylates LIMK [75]. It is very compelling to suggest that PKD connects DAPK to LIMK/cofilin.

Apoptosis

On the other hand, close examination of the expanded interactome indicates that several pathways intersect with opposing effects. What then is the net result of DAPK signaling? For example, several PKD functions counter DAPK activities, such as its effects on cell survival and proliferation [70]. Also, Pin1 stabilizes p53 by blocking its interaction with Mdm2 [72]. If the DAPK a Pin1 ? p53 pathway is a valid cellular pathway, then it would counter the known DAPK ? p19ARF ? p53 pathway (Fig. 3b). Obviously, in order to generate a certain cellular effect, there must be mechanisms in place to selectively activate only a subset of the pathways that are activated by DAPK substrates. From these examples, it is clear that the DAPK interactome is complex and has many missing pieces. Much work is still required to fully understand the DAPK interactome and its functional ramifications. Acknowledgments This work was supported by Grants from the Flight Attendants Medical Research Institute (FAMRI) and the European Research Council (ERC) FP7. AK is the incumbent of the Helena Rubinstein Chair of Cancer Research.

References 1. Bialik S, Kimchi A (2004) DAP-kinase as a target for drug design in cancer and diseases associated with accelerated cell death. Semin Cancer Biol 14(4):283–294. doi:10.1016/j.semcancer. 2004.04.008 2. Cohen O, Feinstein E, Kimchi A (1997) DAP-kinase is a Ca2?/ calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J 16(5):998–1008. doi:10.1093/emboj/16.5.998 3. de Diego I, Kuper J, Bakalova N, Kursula P, Wilmanns M (2010) Molecular basis of the death-associated protein kinase-calcium/ calmodulin regulator complex. Sci Signal 3(106):ra6. doi:10. 1126/scisignal.2000552 4. 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 autophosphorylation-based mechanism. J Biol Chem 276(50):47460–47467. doi:10.1074/jbc.M105133200 5. Guenebeaud C, Goldschneider D, Castets M, Guix C, Chazot G, Delloye-Bourgeois C, Eisenberg-Lerner A, Shohat G, Zhang M, Laudet V, Kimchi A, Bernet A, Mehlen P (2010) The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase. Mol Cell 40(6):863–876. doi:10.1016/j.molcel.2010.11.021 6. Jin Y, Blue EK, Gallagher PJ (2006) Control of death-associated protein kinase (DAPK) activity by phosphorylation and proteasomal degradation. J Biol Chem 281(51):39033–39040. doi:10. 1074/jbc.M605097200 7. 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 reticulum stress-induced caspase activation and autophagic cell death. Cell Death Differ 15(12):1875–1886. doi:10.1038/cdd.2008.121 8. Shamloo M, Soriano L, Wieloch T, Nikolich K, Urfer R, Oksenberg D (2005) Death-associated protein kinase is activated by

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

dephosphorylation in response to cerebral ischemia. J Biol Chem 280:42290–42299. doi:10.1074/jbc.M505804200 Luo XJ, Li W, Yang LF, Yu XF, Xiao LB, Tang M, Dong X, Deng QP, Bode AM, Liu JK, Cao Y (2011) DAPK1 mediates the G1 phase arrest in human nasopharyngeal carcinoma cells induced by grifolin, a potential antitumor natural product. Eur J Pharmacol 670(2–3):427–434. doi:10.1016/j.ejphar.2011.08.026 Ling YH, Aracil M, Zou Y, Yuan Z, Lu B, Jimeno J, Cuervo AM, Perez-Soler R (2011) PM02734 (elisidepsin) induces caspaseindependent cell death associated with features of autophagy, inhibition of the Akt/mTOR signaling pathway, and activation of death-associated protein kinase. Clin Cancer Res 17(16): 5353–5366. doi:10.1158/1078-0432.CCR-10-1948 Jiang Q, Li F, Shi K, Yang Y, Xu C (2012) Sodium seleniteinduced activation of DAPK promotes autophagy in human leukemia HL60 cells. BMB Rep 45(3):194–199. doi:10.5483/ BMBRep.2012.45.3.194 Widau RC, Jin Y, Dixon SA, Wadzinski BE, Gallagher PJ (2010) Protein phosphatase 2A (PP2A) holoenzymes regulate deathassociated protein kinase (DAPK) in ceramide-induced anoikis. J Biol Chem 285(18):13827–13838. doi:10.1074/jbc.M109. 085076 Llambi F, Lourenco FC, Gozuacik D, Guix C, Pays L, Del Rio G, Kimchi A, Mehlen P (2005) The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase. EMBO J 24(6):1192–1201. doi:10.1038/sj.emboj.7600584 Marin I, van Egmond WN, van Haastert PJ (2008) The Roco protein family: a functional perspective. FASEB J 22(9): 3103–3110. doi:10.1096/fj.08-111310 Klein CL, Rovelli G, Springer W, Schall C, Gasser T, Kahle PJ (2009) Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment. J Neurochem 111(3):703–715. doi:10.1111/j.1471-4159.2009.06358.x Carlessi R, Levin-Salomon V, Ciprut S, Bialik S, Berissi H, Albeck S, Peleg Y, Kimchi A (2011) GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling. EMBO Rep 12(9):917–923. doi:10.1038/embor. 2011.126 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(2):294–304. doi:10.1038/sj.emboj.7600510 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(19):1762–1767. doi:10.1016/j.cub. 2005.08.050 Bajbouj K, Poehlmann A, Kuester D, Drewes T, Haase K, Hartig R, Teller A, Kliche S, Walluscheck D, Ivanovska J, Chakilam S, Ulitzsch A, Bommhardt U, Leverkus M, Roessner A, SchneiderStock R (2009) Identification of phosphorylated p38 as a novel DAPK-interacting partner during TNFalpha-induced apoptosis in colorectal tumor cells. Am J Pathol 175(2):557–570. doi:10.2353/ ajpath.2009.080853 Wang WJ, Kuo JC, Ku W, Lee YR, Lin FC, Chang YL, Lin YM, Chen CH, Huang YP, Chiang MJ, Yeh SW, Wu PR, Shen CH, Wu CT, Chen RH (2007) The tumor suppressor DAPK is reciprocally regulated by tyrosine kinase Src and phosphatase LAR. Mol Cell 27(5):701–716. doi:10.1016/j.molcel.2007.06.037 Citri A, Harari D, Shohat G, Ramakrishnan P, Gan J, Lavi S, Eisenstein M, Kimchi A, Wallach D, Pietrokovski S, Yarden Y (2006) Hsp90 recognizes a common surface on client kinases. J Biol Chem 281:14361–14369. doi:10.1074/jbc.M512613200 Jin Y, Blue EK, Dixon S, Shao Z, Gallagher PJ (2002) A deathassociated protein kinase (DAPK)-interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factorinduced apoptosis and regulates the cellular levels of DAPK.

123

Apoptosis

23.

24.

25.

26.

27. 28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

J Biol Chem 277(49):46980–46986. doi:10.1074/jbc. M208585200 Zhang L, Nephew KP, Gallagher PJ (2007) Regulation of deathassociated protein kinase. Stabilization by HSP90 heterocomplexes. J Biol Chem 282(16):11795–11804. doi:10.1074/jbc. M610430200 Capoccia BJ, Jin RU, Kong YY, Peek RM Jr, Fassan M, Rugge M, Mills JC (2013) The ubiquitin ligase Mindbomb 1 coordinates gastrointestinal secretory cell maturation. J Clin Invest 123(4):1475–1491. doi:10.1172/JCI65703 Koo BK, Lim HS, Song R, Yoon MJ, Yoon KJ, Moon JS, Kim YW, Kwon MC, Yoo KW, Kong MP, Lee J, Chitnis AB, Kim CH, Kong YY (2005) Mind bomb 1 is essential for generating functional notch ligands to activate notch. Development 132(15):3459–3470. doi:10.1242/dev.01922 Lee YR, Yuan WC, Ho HC, Chen CH, Shih HM, Chen RH (2010) The Cullin 3 substrate adaptor KLHL20 mediates DAPK ubiquitination to control interferon responses. EMBO J 29(10):1748–1761. doi:10.1038/emboj.2010.62 Lai M-Z, Chen R-H (2013) Regulation of inflammation by DAPK. Apoptosis. doi:10.1007/s10495-013-0933-4 Bialik S, Bresnick AR, Kimchi A (2004) DAP-kinase-mediated morphological changes are localization dependent and involve myosin-II phosphorylation. Cell Death Differ 11(6):631–644. doi:10.1038/sj.cdd.4401386 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(20):3666–3677. doi:10.1242/jcs.003251 Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, Sabanay H, Pinkas-Kramarski R, Kimchi A (2009) DAPkinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-XL and induction of autophagy. EMBO Rep 10(3):285–292. doi:10.1038/embor.2008. 246 Eisenberg-Lerner A, Kimchi A (2011) PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of DAPk. Cell Death Differ 19(5):788–797. doi:10.1038/cdd.2011.149 Bialik S, Kimchi A (2006) The death-associated protein kinases: structure, function and beyond. Annu Rev Biochem 75:189–210. doi:10.1146/annurev.biochem.75.103004.142615 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(1):169–179. doi:10.1083/ jcb.200204050 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):1–7. doi:10.1038/35050500 Craig AL, Chrystal JA, Fraser JA, Sphyris N, Lin Y, Harrison BJ, Scott MT, Dornreiter I, Hupp TR (2007) The MDM2 ubiquitination signal in the DNA-binding domain of p53 forms a docking site for calcium calmodulin kinase superfamily members. Mol Cell Biol 27(9):3542–3555. doi:10.1128/MCB.01595-06 Tu W, Xu X, Peng L, Zhong X, Zhang W, Soundarapandian MM, Belal C, Wang M, Jia N, Zhang W, Lew F, Chan SL, Chen Y, Lu Y (2010) DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke. Cell 140(2):222–234. doi:10.1016/j.cell.2009.12.055 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. doi:10.1038/sj. cdd.4402212 Dirkx E, Schwenk RW, Coumans WA, Hoebers N, Angin Y, Viollet B, Bonen A, van Eys GJ, Glatz JF, Luiken JJ (2012)

123

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes. J Biol Chem 287(8):5871–5881. doi:10.1074/jbc.M111. 281881 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(25):8116–8124. doi:10.1021/bi049589v Shani G, Marash L, Gozuacik D, Bialik S, Teitelbaum L, Shohat G, Kimchi A (2004) Death-associated protein kinase phosphorylates ZIP kinase, forming a unique kinase hierarchy to activate its cell death functions. Mol Cell Biol 24(19):8611–8626. doi:10. 1128/MCB.24.19.8611-8626.2004 Zimmermann M, Atmanene CD, Xu Q, Fouillen L, Van Dorsselaer A, Bonnet D, Marsol C, Hibert M, Sanglier-Cianferani S, Pigault C, McNamara LK, Watterson DM, Haiech J, Kilhoffer M-C (2010) Homodimerization of the death-associated protein kinase catalytic domain: development of a new small molecule fluorescent reporter. PLoS One 5(11):e14120. doi:10.1371/ journal.pone.0014120 Graves PR, Winkfield KM, Haystead TA (2005) Regulation of zipper-interacting protein kinase activity in vitro and in vivo by multisite phosphorylation. J Biol Chem 280(10):9363–9374. doi:10.1074/jbc.M412538200 Usui T, Okada M, Yamawaki H (2013) Zipper interacting protein kinase (ZIPK): function and signaling. Apoptosis. doi:10.1007/ s10495-013-0934-3 Mukhopadhyay R, Jia J, Arif A, Ray PS, Fox PL (2009) The GAIT system: a gatekeeper of inflammatory gene expression. Trends Biochem Sci 34(7):324–331. doi:10.1016/j.tibs.2009.03. 004 Schumacher A, Velentza A, Watterson D, Dresios J (2006) Death-associated protein kinase phosphorylates mammalian ribosomal protein s6 and reduces protein synthesis. Biochemistry 45(45):13614–13621. doi:10.1021/bi060413y 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 signaling. J Biol Chem 284(1):334–344. doi:10.1074/jbc. M805165200 Meyuhas O (2008) Physiological roles of ribosomal protein S6: one of its kind. Int Rev Cell Mol Biol 268:1–37. doi:10.1016/ S1937-6448(08)00801-0 Khalaileh A, Dreazen A, Khatib A, Apel R, Swisa A, KidessBassir N, Maitra A, Meyuhas O, Dor Y, Zamir G (2013) Phosphorylation of ribosomal protein S6 attenuates DNA damage and tumor suppression during development of pancreatic cancer. Cancer Res 73(6):1811–1820. doi:10.1158/0008-5472.CAN-122014 Bialik S, Berissi H, Kimchi A (2008) A high throughput proteomics screen identifies novel substrates of death-associated protein kinase. Mol Cell Proteomics 7(6):1089–1098. doi:10. 1074/mcp.M700579-MCP200 Lee TH, Chen C-H, Suizu F, Huang P, Schiene-Fischer C, Daum S, Zhang YJ, Goate A, Chen R-H, Zhou XZ, Lu KP (2011) Death-associated protein kinase 1 phosphorylates Pin1 and inhibits its prolyl isomerase activity and cellular function. Mol Cell 42(2):147–159. doi:10.1016/j.molcel.2011.03.005 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(28):26265–26274. doi:10.1074/jbc.M300492200 Pelkmans L, Fava E, Grabner H, Hannus M, Habermann B, Krausz E, Zerial M (2005) Genome-wide analysis of human

Apoptosis

53. 54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436(7047):78–86. doi:10.1038/nature03571 Levin-Salomon V, Bialik S, Kimchi A (2013) DAP-kinase and autophagy. Apoptosis. doi:10.1007/s10495-013-0918-3 Snyder M, He W, Zhang JJ (2005) The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation. Proc Natl Acad Sci USA 102(41):14539–14544. doi:10.1073/ pnas.0507479102 Gonzalez S, Klatt P, Delgado S, Conde E, Lopez-Rios F, Sanchez-Cespedes M, Mendez J, Antequera F, Serrano M (2006) Oncogenic activity of Cdc6 through repression of the INK4/ARF locus. Nature 440(7084):702–706. doi:10.1038/nature04585 Wu PR, Tsai PI, Chen GC, Chou HJ, Huang YP, Chen YH, Lin MY, Kimchi A, Chien CT, Chen RH (2011) DAPK activates MARK1/2 to regulate microtubule assembly, neuronal differentiation, and tau toxicity. Cell Death Differ 18(9):1507–1520. doi:10.1038/cdd.2011.2 Matenia D, Mandelkow E-M (2009) The tau of MARK: a polarized view of the cytoskeleton. Trends Biochem Sci 34(7):332–342. doi:10.1016/j.tibs.2009.03.008 Harrison B, Kraus M, Burch L, Stevens C, Craig A, GordonWeeks P, Hupp T (2008) DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing. J Biol Chem 283:9999–10014. doi:10.1074/jbc.M706040200 Mor I, Carlessi R, Ast T, Feinstein E, Kimchi A (2011) Deathassociated protein kinase increases glycolytic rate through binding and activation of pyruvate kinase. Oncogene 31(6):683–693. doi:10.1038/onc.2011.264 Luo W, Semenza GL (2012) Emerging roles of PKM2 in cell metabolism and cancer progression. Trends Endocrinol Metab 23(11):560–566. doi:10.1016/j.tem.2012.06.010 Yang W, Zheng Y, Xia Y, Ji H, Chen X, Guo F, Lyssiotis CA, Aldape K, Cantley LC, Lu Z (2012) ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol 14 (12):1295–1304. http://www. nature.com/ncb/journal/v14/n12/abs/ncb2629.html#supplementaryinformation Chuang YT, Lin YC, Lin KH, Chou TF, Kuo WC, Yang KT, Wu PR, Chen RH, Kimchi A, Lai MZ (2010) Tumor suppressor death-associated protein kinase is required for full IL-1{beta} production. Blood 17(3):960–970. doi:10.1182/blood-2010-08303115 Chakilam S, Gandesiri M, Rau TT, Agaimy A, Vijayalakshmi M, Ivanovska J, Wirtz RM, Schulze-Luehrmann J, Benderska N, Wittkopf N, Chellappan A, Ruemmele P, Vieth M, Rave-Frank M, Christiansen H, Hartmann A, Neufert C, Atreya R, Becker C, Steinberg P, Schneider-Stock R (2013) Death-associated protein kinase controls STAT3 activity in intestinal epithelial cells. Am J Pathol 182(3):1005–1020. doi:10.1016/j.ajpath.2012.11.026

64. Kuo J, Wang W, Yao C, Wu P, Chen R (2006) The tumor suppressor DAPK inhibits cell motility by blocking the integrinmediated polarity pathway. J Cell Biol 172(4):619–631. doi:10. 1083/jcb.200505138 65. 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(5):3238–3249 66. Douglass J, Gunaratne R, Bradford D, Saeed F, Hoffert JD, Steinbach PJ, Knepper MA, Pisitkun T (2012) Identifying protein kinase target preferences using mass spectrometry. Am J Physiol Cell Physiol 303(7):C715–C727. doi:10.1152/ajpcell.00166.2012 67. 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(42):38956–38965. doi:10.1074/jbc. M104273200 68. Maisse C, Rossin A, Cahuzac N, Paradisi A, Klein C, Haillot ML, Herincs Z, Mehlen P, Hueber AO (2008) Lipid raft localization and palmitoylation: identification of two requirements for cell death induction by the tumor suppressors UNC5H. Exp Cell Res 314(14):2544–2552. doi:10.1016/j.yexcr.2008.06.001 69. Mukhopadhyay R, Ray PS, Arif A, Brady AK, Kinter M, Fox PL (2008) DAPK-ZIPK-L13a axis constitutes a negative-feedback module regulating inflammatory gene expression. Mol Cell 32(3):371–382. doi:10.1016/j.molcel.2008.09.019 70. Rozengurt E (2011) Protein kinase D signaling: multiple biological functions in health and disease. Physiology (Bethesda) 26(1):23–33. doi:10.1152/physiol.00037.2010 71. Backer JM (2008) The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem J 410(1):1–17. doi:10.1042/ BJ20071427 72. Lu KP, Zhou XZ (2007) The prolyl isomerase PIN1: a pivotal new twist in phosphorylation signalling and disease. Nat Rev Mol Cell Biol 8(11):904–916. doi:10.1038/nrm2261 73. Mizuno K (2013) Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation. Cell Signal 25(2):457–469. doi:10.1016/j.cellsig.2012.11.001 74. Ivanovska J, Tregubova A, Mahadevan V, Chakilam S, Gandesiri M, Benderska N, Ettle B, Hartmann A, Soder S, Ziesche E, Fischer T, Lautscham L, Fabry B, Segerer G, Gohla A, Schneider-Stock R (2013) Identification of DAPK as a scaffold protein for the LIMK/cofilin complex in TNF-induced apoptosis. Int J Biochem Cell Biol 45(8):1720–1729. doi:10.1016/j.biocel.2013. 05.013 75. Spratley SJ, Bastea LI, Doppler H, Mizuno K, Storz P (2011) Protein kinase D regulates cofilin activity through p21-activated kinase 4. J Biol Chem 286(39):34254–34261. doi:10.1074/jbc. M111.259424

123