THE UNIVERSE OF DAPK

The DAP-kinase interactome

Shani Bialik • Adi Kimchi

� 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 regu-

lation involving both intramolecular signaling, and inter-

actions 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 mem-

brane 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 microtu-

bule 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

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 pri-

ority 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-regu-

lated 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

S. Bialik � A. Kimchi (&)

Department of Molecular Genetics, Weizmann Institute

of Science, 76100 Rehovot, Israel

e-mail: adi.kimchi@weizmann.ac.il

123

Apoptosis

DOI 10.1007/s10495-013-0926-3

DAPK interactome. The DAPK interactome includes pro-

teins that function upstream of DAPK, to regulate its cata-

lytic 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 func-

tion of this complex protein. This review will present the

DAPK interactome, and how each component was identi-

fied, 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. There-

fore, 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 cal-

modulin autoregulatory domain immediately downstream

of the catalytic domain [2, 3]. Typically, the CaM aut-

oregulatory 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-phos-

phorylation of Ser308 within the calmodulin autoregula-

tory 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

S308

Src

ERK

Unc5H

PP2A (C)

RSK

Mib

S735

CaM LRRK1/2

TSC2

S289 Y491/2

Hsp90

KLHL20

CHIP

GTP Kinase domain Ankyrin Repeats ROC COR DDCaM Auto- Reg.

Cul3

LAR B (B

A (PR65 )

Ser rich tail

Inhibition of catalytic activity

Activation of catalytic activity

Ub-mediated degradation

Unknown

Phosphorylation

Dephosphorylation

Signaling Stimulus

Regulation key EGF

PMA

Netrin 1

Ceramide ER stress Ischemia

TNF

Ca2+ IFN

α) β

α

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

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

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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 phospha-

tase 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 sub-

strate 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 Bddephosphorylated DAPK in vitro, leading to enhanced

catalytic activity and CaM binding. During ceramide-

induced 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 dependence-

receptor 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 mecha-

nism, 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 pro-

teins)–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 val-

idation, 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 conforma-

tional change that reduces Ser308’s accessibility to the

catalytic cleft.

Additional regulators

Several additional proteins interact with DAPK and regu-

late its activity, mainly through phosphorylation events

(Fig. 1). These kinases and phosphatases lie downstream to

Apoptosis

123

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 ERK-

dependent 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 phosphor-

ylated DAPK both in vivo and in vitro. This phosphory-

lation 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 TNFatreated 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

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 cel-

lular 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 facili-

tates 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 ter-

nary 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 gas-

tric 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 con-

trolling DAPK stability can be found in an accompanying

review [27].

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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 cyto-

skeletal associated substrates, including the first recognized

substrate, MLC [2, 28], and tropomyosin [29]. These con-

tribute to its cytoskeletal-related effects, including mem-

brane 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 compo-

nent. 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

cytoskeletal dynamics are the subjects of two additional

reviews in this issue, we will focus here on the remaining

substrates.

Cell death associated substrates

One of the first functions of DAPK to be recognized was its

role in mediating apoptosis, both p53 dependent and inde-

pendent [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 interfer-

ing 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-immunopre-

cipitated with endogenous p53, enhanced Ser20 phosphory-

lation, and increased p53 transactivation activity. On the other

hand, depletion of DAPK by siRNA led to reductions in p53

DAP-kinase

p53

Apoptosis

MARK1/2

Tau

MT instability

PKD

Programmed necrosis

MLC

Membrane blebbing

Tropomyosin

Acto-myosin contractility

Stress fiber formation

Integrin

Cell adhesion

Ischemic injury Pin1

Cell transformation

NLRP3

Inflammasome assembly

Vps34

Talin

Cell polarity motility

Glycolysis Autophagy

NMDA receptor

?

?

T cell activation

?

Calcium influx

NR2B

P

P

P

P

P

P

P P

mdm2

Oncogene transactivation

CaMKK

Mcm3

P P

P

ZIPk p19ARF

Syntaxin-1A

P

Pyruvate

Kinase M2

L13

P

GAIT assembly

Inflammatory Genes

Beclin 1 P

GLUT4

Glucose uptake

JNK

NF- B κ

IL-1β

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 interac-

tions 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/phosphoryla-

tion 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

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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) gluta-

mate 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 sup-

pressed 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 kina-

ses, 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], phosphory-

lation of PKD by DAPK led to activation of JNK, and

consequently, programmed necrosis, thereby linking

DAPK to an additional death pathway [37]. In the cardio-

myocyte 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-immunopre-

cipitation 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 autophos-

phorylation, 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.

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 cata-

lytic activity was induced earlier than ZIPK activity, con-

firming 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-immunopre-

cipitated 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 phos-

phorylates 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

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not necessarily related to protein synthesis regulation. Extra-

ribosomal 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].

PKD

Programmed necrosis

Vps34

Beclin 1

Autophagy

PI3P

LIMK

PAK4

SSH

cofilin

Actin severing

remodeling

? P

P

P P

P P

P

GLUT4

Glucose uptake

DAP-kinase

P

JNK

Endocytosis

Bcl-2

P Ask1

MKK4/7

Apoptosis

Pin1

P

Glycolysis

Warburg Effect

Pyruvate

Kinase M2

MARK1/2

Tau

P

MT instability

DAP-kinase

Chromosomal instability Centrosome amplification Mitotic spindle deformities

Cell transformation

Transcription (NF- B, -catenin, cyclin D) cyclin D1 stability

pSer/Thr-Pro cis/trans

NF- B

Nucleus

p53

?

P

p19ARF

p53

mdm2

?

a

b

κ β

κ

Apoptosis

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Another key component of the downstream DAPK in-

teractome 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 over-

lapping 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 endog-

enous 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 cell-

transformation activities of Pin1 in NIH3T3 cells, such as

centrosome amplification, abnormal spindle formation and

matrix-independent cell growth. Furthermore, Pin1 knock-

down suppressed the increased cell migration observed

upon knock-down of DAPK in breast cancer cells, imply-

ing 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 signifi-

cance 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

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 sig-

nificance of this modification is not known. Mcm3 may

link DAPK to yet a new function involving DNA replica-

tion, 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 phos-

phorylate 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 inhib-

ited MT assembly in MCF7 cells, axon formation in neu-

rons, 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 dis-

covered through a peptide combinatorial library screen for

motifs that bind DAPK’s kinase domain [58]. The inter-

action 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,

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

b

Apoptosis

123

particularly functioning in cytoskeletal events, such as

acto-myosin contraction or potentially, MT-based traf-

ficking events that are required for autophagosome for-

mation 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 two-

hybrid screen with DAPK’s death domain [59]. Pyruvate

kinase is a key glycolytic enzyme that mediates the con-

version 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 lac-

tate 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 counter-

intuitive: 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 pro-

tein 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 pro-

growth functions; by activating PKM2’s enzymatic activity

it may actually inhibit its oncogenic potential [60]. Sig-

nificantly, 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 grif-

olin, a mushroom metabolite with anti-cancer properties,

which may contribute to the latter’s ability to induce G1-

arrest 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 compo-

nent 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 nega-

tively 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 associ-

ation 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].

Apoptosis

123

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], indi-

cates 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 Beclin-

1 [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 sub-

strates 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 pre-

sumably that interaction enables its recognition of micro-

tubule-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 cas-

cade [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 trig-

gers 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.

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 isomeriza-

tion 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 tempt-

ing to speculate that they are also dependent on DAPK for

activation. Also, several functions of Pin1, which is inhib-

ited by DAPK-mediated phosphorylation, are inversely

related with other DAPk functions. Pin1 promotes tau

dephosphorylation [72], and binds and isomerizes ERK-

phosphorylated 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 phosphoryla-

tion (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 phosphory-

lation, 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 phos-

phorylating and activating PAK4, which in turn phos-

phorylates LIMK [75]. It is very compelling to suggest that

PKD connects DAPK to LIMK/cofilin.

Apoptosis

123

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 inter-

actome is complex and has many missing pieces. Much

work is still required to fully understand the DAPK inter-

actome 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.

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