THE UNIVERSE OF DAPK

DAP-kinase and autophagy

Vered Levin-Salomon • Shani Bialik •

Adi Kimchi

Published online: 22 November 2013

� Springer Science+Business Media New York 2013

Abstract DAP-kinase (DAPK) is a Ca2?-calmodulin

regulated kinase with various, diverse cellular activities,

including regulation of apoptosis and caspase-independent

death programs, cytoskeletal dynamics, and immune func-

tions. Recently, DAPK has also been shown to be a critical

regulator of autophagy, a catabolic process whereby the cell

consumes cytoplasmic contents and organelles within spe-

cialized vesicles, called autophagosomes. Here we present

the latest findings demonstrating how DAPK modulates

autophagy. DAPK positively contributes to the induction

stage of autophagosome nucleation by modulating the

Vps34 class III phosphatidyl inositol 3-kinase complex by

two independent mechanisms. The first involves a kinase

cascade in which DAPK phosphorylates protein kinase D,

which then phosphorylates and activates Vps34. In the

second mechanism, DAPK directly phosphorylates Beclin 1,

a necessary component of the Vps34 complex, thereby

releasing it from its inhibitor, Bcl-2. In addition to these

established pathways, we will discuss additional connec-

tions between DAPK and autophagy and potential mecha-

nisms that still remain to be fully validated. These include

myosin-dependent trafficking of Atg9-containing vesicles to

the sites of autophagosome formation, membrane fusion

events that contribute to expansion of the autophagosome

membrane and maturation through the endocytic pathway,

and trafficking to the lysosome on microtubules. Finally, we

discuss how DAPK’s participation in the autophagic process

may be related to its function as a tumor suppressor protein,

and its role in neurodegenerative diseases.

Keywords DAP-kinase � Autophagy � Beclin 1 �Protein kinase D � Programmed cell death

Introduction

Death associated protein kinase (DAPK, also DAPK1) is a

Ca2?/calmodulin (CaM) regulated Ser/Thr kinase that was

originally described as a positive regulator of interferon

(IFN)-c-mediated cell death [1]. While its necessity for

apoptosis induced by various triggers has been established

[2], it has since been recognized that in certain cellular

settings, DAPK is capable of inducing a non-apoptotic

caspase-independent programmed cell death [3]. Further-

more, DAPK activity promotes the formation of auto-

phagosomes by increasing the autophagy flux [3–5]. These

two latter observations have led to the conclusion that

DAPK is a mediator of autophagic cell death.

Autophagy is a catabolic process whereby the cell

engulfs cellular components in a double membrane vesicle

that eventually fuses with the lysosome, upon which its

contents are degraded [6]. Autophagy is active at a basal

rate to degrade long-lived proteins and scavenge damaged

organelles and misfolded proteins, serving as a quality

control mechanism in the cytoplasm. It also plays an

important role during development, for example, by

removing maternal protein and maintaining a source of

energy and nutrients in the early embryo, eliminating cel-

lular organelles, such as paternal mitochondria upon fer-

tilization and nuclei in developing erythrocytes, and

facilitating cellular remodeling of adipocytes [7–9]. Under

stress conditions (e.g. starvation, hypoxia, oxidative stress),

autophagic activity is greatly enhanced, to facilitate

removal of damaged proteins and organelles and/or provide

a source of recycled macromolecular building blocks when

V. Levin-Salomon � S. Bialik � A. Kimchi (&)

Department of Molecular Genetics, Weizmann Institute of

Science, 76100 Rehovot, Israel

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

123

Apoptosis (2014) 19:346–356

DOI 10.1007/s10495-013-0918-3

nutrients and energy are lacking. Autophagy also plays an

important immune function as it serves to remove intra-

cellular pathogens. Thus, autophagy is critical for the

maintenance of cellular homeostasis and survival during

periods of cellular stress. Dysregulation of the autophagic

process is known to be involved in the development of

cancer, neurodegenerative disorders and myopathies [10,

11].

Hypothetically, excessive autophagy can actually kill a

cell, by over-consuming cellular contents and organelles,

or by eliminating critical survival factors. Alternatively,

autophagy in itself may not kill the cell, but rather may

activate or facilitate other death pathways. In fact, pro-

longed autophagy has been functionally coupled to the

induction of caspase-dependent apoptosis [12] and pro-

grammed necrosis [13]. Notably, in many of the previous

cases where autophagy and cell death were observed, a

clear causative relationship was not definitively estab-

lished, implying that in these cases, autophagy may actu-

ally represent a failed attempt to mitigate stress-induced

damage [14]. Yet, it is clear that DAPK is implicated in

cell death processes that are associated with increased

autophagy. This review will first introduce the reader to the

autophagic process at the molecular level, and then sum-

marize the main issues known in the literature regarding

DAPK and its relationship with autophagy. It will further

describe the mechanisms by which DAPK regulates the

autophagic process, and how DAPK’s functions in tumor

suppression and neuronal damage may be related to its role

as an autophagy regulator.

The basic autophagy machinery and associated

regulators

The autophagosome forms de novo at specialized regions

of the ER called the omegasome [15]. As the nascent

membrane, or phagophore, grows, it surrounds organelles,

proteins and portions of the cytoplasm. The autophago-

some membrane expands and fuses into a sealed vesicle,

which then matures by fusion with components of the

endocytic pathway to form the amphisome. Finally, the

amphisome fuses with the lysosome to obtain the auto-

lysosome, wherein its contents are degraded by lysosomal

enzymes and recycled to the cytoplasm.

Induction of autophagy is tightly regulated by several

signaling pathways that are sensitive to environmental

conditions, such as growth factors, amino acids, glucose

and ATP levels. A key pathway regulating autophagy is the

mTOR signaling pathway [16] (Fig. 1a). The mTOR

kinase, as part of the mTORC1 complex, is activated by

Rheb, a small GTPase that is active when bound to GTP.

Rheb is in turn inhibited by the TSC1/TSC2 dimer, which

acts as a GTPase-activating protein (GAP) to facilitate

GTP hydrolysis. mTOR signaling is positively regulated by

the class I PI3-kinase/AKT and MAPK signaling pathways,

which respond to insulin and growth factors [16]. Amino

acids activate mTOR through a heterodimer of Rag GTP-

ases, and the Ragulator complex, a guanine nucleotide

exchange factor (GEF) for the Rags. Rags and Ragulator

sense amino acids in an as yet unknown mechanism and

recruit mTOR to the lysosome, where it is activated by

Rheb [17]. The Rag dimer is also regulated by the Gator

complex, which functions as its GAP [18]. Conversely,

AMPK, which is activated by changes in the AMP:ATP

ratio upon energy depletion, negatively regulates mTORC1

[19]. Kinases of the MAPk cascade, Akt and AMPk con-

verge on both TSC2 and on components of the mTORC1

complex, such as Raptor, which recruits mTOR to its

substrates. When active, mTORC1 activates protein syn-

thesis and other metabolic processes necessary for cell

growth, and simultaneously inhibits autophagy. In contrast,

under starvation conditions, mTOR is inactive and

autophagy is thus induced.

The genes that encode the core machinery of autoph-

agy were initially identified through genetic screens per-

formed in yeast [15]. Most of these genes, named ATG,

are also found in human, suggesting that autophagy is a

conserved mechanism throughout evolution. The autoph-

agy process can be divided into several steps that include

autophagy induction and vesicle nucleation (Fig. 1b),

cargo recognition and selection (in the case of selective

autophagy, not discussed here), vesicle elongation

(Fig. 1c), and the final maturation stages that include

trafficking, autophagosome-lysosome fusion and cargo

degradation (Fig. 1d).

The first step of autophagy is the formation of the

phagophore, which occurs following recruitment of the

autophagy machinery to the omegasomes. The ER is

believed to be the main source for phagophore membrane,

although the Golgi, endosomes, mitochondria and the

plasma membrane have also been shown to contribute [20,

21]. The fusion and tethering machinery of the endocytic

pathway, such as SNARE [soluble N-ethylmaleimide-sen-

sitive fusion (NSF) attachment protein receptors] proteins

and the small GTPase Rabs, are important for the homo-

typic fusion events that expand the phagophore [20, 22].

The precise roles that these endocytic proteins play in

autophagy, and their contribution to the initial formation of

the phagophore, and later, to fusion with the lysosome, is a

topic of current, active research and beyond the scope of

this review. Another critical regulator of autophagosome

nucleation and expansion is Atg9, a membrane spanning

protein that redistributes from the TGN to the site of

autophagosome formation upon autophagy initiation [23].

By shuttling from sites of pre-existing membrane, Atg9-

Apoptosis (2014) 19:346–356 347

123

containing vesicles are believed to deliver a source of

membrane to the expanding phagophore (Fig. 1b).

The nucleation of the phagophore membrane is medi-

ated by the class III phosphatidyl inositol (PtdIns) 3-kinase

(PI3K) complex (Fig. 1b). Key players in this complex are

the catalytic unit Vps34, the myristoylated membrane tar-

geting unit Vps15/p150 and the positive modulator Beclin

1 [15]. Additional interactions with other proteins (e.g.

Atg14, Ambra1, UVRAG, Bif1, Rubicon and Bcl-2) fur-

ther modulate the activity of the entire complex. The

function of Vps34 is to generate PtdIns 3-phosphate (PI3P)

from PtdIns, serving multiple roles within the cell. Another

component of the Vps34 complex, Atg14, recruits this

activity specifically for autophagosome formation [24].

c

ATG12

ATG7

ATG10

ATG3

ATG5

PE

E1

E2

E2

ATG4

-GlyLC3

ATG16 ATG16ATG16ATG16

ATG16ATG5

ATG12

LC3 LC3

ATG5/12/16complex

ATG5

ATG12

ATG5

ATG12

ATG5ATG12

ATG5ATG12

Membrane elongationVesicle

closure

Autophagosome

Phagophore

Endosome/MVB

Amphisome Autolysosome

Lysosome

Autophagosome

Lysosomal proteasesMicrotubulesSNAREsMT motor

d

Atg101 FIP200ATG13

Ulk1/2

P

P

P

a

PI(3)K

PTEN AKT PDK1

MEK1/2

ERK1/2

RSK1

AMPK

AMPATP

growth factor/receptor

Ras

Raf1

PIP2 PIP3P P

Atg101 FIP200ATG13

Ulk1/2

Inactive P

Active

mTORRaptor mLST8

RhebGTP

ActivemTORC1

PRAS40

FKBP38mTOR

Raptor mLST8

RhebGDP

PmTORC1

Inactive

TSC1

LKB1

TSC2

p53

Amino acids

RagA/BRagulato

rGTP

b

actin

Active Class III PI(3)K

Vps34ATG14L

Vps15

Beclin 1

PI PI(3)PP

P

PMyosin/MLC

P

dyneinAmbra1P

Sqa(MLCK?)

Vps34Beclin 1

Ambra1

microtubules

ATG9

RagC/DGDP

ATG9

Atg101 FIP200

Ulk1/2

PP ATG13

LC3

Fig. 1 Regulation of autophagy by mTOR and Atg genes. a mTOR

negatively regulates autophagy. mTOR senses energy, nutrients and

growth factors through the MAPk and Akt signaling pathways, which

lead to phosphorylation and inactivation of TSC2, the GAP for Rheb.

GTP-bound Rheb activates mTOR within the mTORC1 complex. In

response to amino acids, Rag recruits mTOR to the lysosome, where

it is activated by Rheb. When active, mTOR phosphorylates

components of the Uk1 complex, leading to its inactivation. In the

absence of growth factors/nutrients, mTOR is inactive. Changes in the

AMP:ATP ratio as a result of energy depletion also inactivate mTOR,

through AMPK, which phosphorylates and activates TSC2 and

phosphorylates and inactivates Raptor. As a result, the Ulk1 complex

is dephosphorylated and becomes active to initiate autophagy.

b Autophagy induction and nucleation of the phagophore membrane.

Ulk1 initiates phagophore formation by three mechanisms. Ulk1

phosphorylates Beclin 1, which activates the Vps34 complex. It also

phosphorylates the Beclin 1 binding protein Ambra1, which disrupts

its interaction with dynein. This releases Beclin–Vps34 from the

microtubules, enabling its delivery to the site of phagophore

formation. Finally, Ulk1 activates myosin through the phosphoryla-

tion of MLC. In Drosophila, the Ulk1 orthologue Atg1

phosphorylates Sqa, which phosphorylates the fly MLC. In mammals,

the precise identity of the MLC kinase has yet to be fully established,

but may include DAPK family members. Myosin drives the

redistribution of Atg9-containing vesicles along actin to the phago-

phore, where it contributes membrane to the nascent vesicle.

c Membrane elongation by ubiquitin-like conjugation schemes. In

the first scheme, Atg12 is conjugated to Atg5 via the E1 and E2

ligases Atg7 and Atg10. Atg5–Atg12 then associates with Atg16 to

form a large complex that acts as an E3 ligase for the second scheme,

in which LC3 (Atg8) is conjugated to PE via Atg7 and Atg3.

Conjugated LC3 binds the phagophore membrane, and is critical for

its elongation. The phagophore membrane elongates and closes to

form the double-membrane autophagosome. d Maturation of the

autophagosome. The autophagosome trafficks along microtubules and

fuses with early/late endosomes and MVBs to form the amphisome.

The docking and fusion machinery of the endocytic pathway mediates

these events. For simplicity, only SNAREs are shown. The outer

membrane of the amphisome is then competent to fuse with the

lysosome to form the autolysosome. In the final step, the inner

autophagosomal membrane breaks down and its contents are

degraded by lysosomal enzymes

348 Apoptosis (2014) 19:346–356

123

PI3P serves as a signaling molecule controlling autophagic

vesicle formation by mediating the binding of PI3P effector

proteins such as DFCP1 and WIPI-1, and -2, whose exact

functions are not yet understood [15].

Both Vps34 complex activity and Atg9 trafficking are

regulated by the Ulk1 complex, which is composed of the

Ulk1 or Ulk2 kinases (mammalian equivalent of Atg1),

Atg13, FIP200 and Atg101 [25]. mTORC1 phosphorylates

several components of this complex, thereby inactivating

them (Fig. 1a). Upon mTOR suppression, such as during

starvation, Ulk1 is activated and autophosphorylates itself

and additional members of the complex, thereby initiating

autophagy. The contribution of Ulk1 to phagophore for-

mation is still not fully understood, yet several mechanisms

have been proposed (Fig. 1b). First, Ulk1 can phosphory-

late Ambra1 [26], which releases it from an interaction

with dynein light chain. When bound to dynein, Ambra1

brings the Beclin 1–Vps34 complex to the microtubules,

removed from the autophagosome machinery. Ulk1 phos-

phorylation of Ambra1 thus allows Vps34 to relocalize to

the ER membrane, from where it can initiate autophagy

[26]. Ulk1 can also phosphorylate Beclin 1 directly, only

within the context of the Atg14 containing Vps34 complex,

thereby enhancing PI3K activity to induce autophagy. Thus

starvation, which suppresses Vps34 activity in general,

activates the autophagy-specific Beclin–Vps34 complex

[24]. Ulk1 also is necessary for the redistribution of Atg9

[27]. The mechanism responsible for this was described in

Drosophila [28]. The Drosophila orthologue of Ulk1,

Atg1, phosphorylates the myosin light chain kinase Sqa,

which in turn phosphorylates Sqh, the fly myosin light

chain. This activates myosin II contraction on actin fila-

ments to drive Atg9 trafficking [28]. While the equivalent

pathway has not been fully validated in mammalian cells, it

is likely that a similar mechanism is involved, as both Ulk1

and myosin II are required for Atg9 redistribution (see

more details below on the possible involvement of the

DAPK family in this process).

Expansion of the phagophore membrane to a complete

autophagosome requires the activity of two ubiquitin-like

conjugating systems, resulting in the covalent conjugation

of Atg12 to Atg5 and LC3 (Atg8) to the lipid phosphati-

dylethanolamine (PE) [15] (Fig. 1c). Conjugation of the

latter is often used as a marker for autophagosome for-

mation, as the lipidated form of LC3 (LC3-II) migrates

faster on western blots compared to the unlipidated form

(LC3-I). Lipidated LC3 is recruited to autophagosome

membranes, where it interacts with various cargo receptors,

facilitating engulfment of specific autophagy cargos, such

as protein aggregates or damaged mitochondria [15]. LC3-

II appears in cells as punctate structures, which can be

visualized by light microscopy upon fusion with GFP

(GFP-LC3).

The fully expanded autophagosome membrane under-

goes fusion to form a closed, double membrane vesicle. It

then passes through several maturation steps in which it

fuses with components of the endocytic pathway, such as

early and late endosomes and multivesicular bodies

(MVBs) to form the amphisome [29] (Fig. 1d). Various

proteins involved in the normal trafficking and fusion of

these vesicles are necessary at this stage, including small

GTPase proteins Rabs, SNAREs, and endosomal sorting

complex required for transport (ESCRT) proteins [22, 30].

This is believed to provide the vesicle with the necessary

fusion machinery to eventually enable delivery to and

fusion with the lysosome. Trafficking of the autophago-

some along this pathway also requires an intact microtu-

bule cytoskeleton, which brings the autophagosome to the

lysosome [31]. Within the autolysosome, the inner auto-

phagosomal membrane breaks down, the engulfed contents

are degraded by lysosomal enzymes, and then recycled to

the cytoplasm.

Functional connection between DAPK and autophagy

Expression of DAPK in various cell types leads to the

enhanced formation of autophagosomes. This was

observed by electron microscopy as an increase in the

appearance of double membrane vesicles enclosing cyto-

plasmic contents, indicative of autophagosomes in varying

states of maturation, including autolysosomes [3]. In

addition, increased accumulation of the autophagy marker

GFP-LC3 in puncta representing the autophagosome

membrane was observed upon DAPK expression [3, 5, 32].

Significantly, DAPK is activated by various stimuli that

induce autophagy. Activation of DAPK has been shown to

involve several inter-related mechanisms that include

binding of Ca2?-activated CaM to the CaM regulatory

domain [33], dephosphorylation of Ser308 within the CaM

regulatory domain by the PP2A phosphatase [34–37], and

potentially, hydrolysis of GTP to GDP by the Ras of

complex proteins (ROC)–C-terminal of ROC (COR)

domains [38]. Since these different mechanisms are

reflected by the phosphorylation status of Ser308 [39, 40],

the latter is often used as a marker of the activation of

DAPK in vivo, by means of western blotting with phospho-

specific antibodies [34]. In this manner, it was found that

Ser308 undergoes dephosphorylation during autophagy

resulting from ER stress induced by the N-linked glyco-

sylation inhibitor tunicamycin [35]. Agents that lead to

increased intracellular Ca2?, such as the Ca2? channel

blocker thapsigargin, and the Ca2? ionophore ionomycin,

also activated DAPK via Ser308 dephosphorylation and led

to autophagy [35] (G. Oberkovitz and A. Kimchi, unpub-

lished data, see also Fig. 2, inset a). Reductions in Ser308

Apoptosis (2014) 19:346–356 349

123

phosphorylation were also observed during autophagy

induced by several anti-neoplastic drugs, such as the phase

II clinical drug PM02734 (Elisidepsin) in non-small cell

lung cancer (NSCLC) cells [41] and the histone deacetyl-

ase inhibitor LBH589 (Panobinostat) in colon cancer [42].

Similar effects on DAPK’s phosphorylation/activation state

were observed in hepatocellular carcinoma cells treated

with the p38 MAPK inhibitor SB203580. Interestingly, the

anti-proliferative and autophagy effects of this drug are

independent of p38, and may instead involve activation of

AMPK and inhibition of Akt/PKB [43].

Moreover, DAPK was shown to be necessary for

autophagy induced by these stimuli. For example, in the

first study connecting DAPK to autophagy, expression of

DAPK antisense inhibited autophagy and cell death induced

by IFNc [3]. Subsequently, DAPK knock-out MEFs were

resistant to tunicamycin-induced autophagosome induction,

as compared to their wild-type counterparts [35]. In this

system, cell death was attributed to both apoptotic and

autophagic processes, and cell viability was rescued only by

blocking both pathways. DAPK deletion, however, was

sufficient in itself to attenuate cell death in tunicamycin

treated MEFs and reduced tunicamycin toxicity to the

mouse kidney in vivo [35]. In addition, DAPK depletion by

siRNA blocked autophagy induced by oxidative stress

that resulted from H2O2 treatment [4]. This stimulus also

triggered cell death, at least part of which was attributed

to DAPK-dependent programmed necrosis. Similarly,

SB203580-induced autophagy in hepatocellular carcinoma

cells and PM02734-induced autophagy in NSCLC were

partially suppressed by DAPK knock-down [41, 43]. Nei-

ther of these drugs induced apoptotic cell death, and, in

Vps34

ATG14

Vps15

Vps34ATG14

Vps15

P

P

PI PI(3)P

P

DAPK

JNK

ASK1

TRAF6

Ub

P

PKD

Beclin 1BH3 HMGB1

ER/Omegasome

ROCK1

NT Ion

Beclin pThr119 -*

Ion: NT 2h 6h

DAPK pSer308 -

DAPK -

*

GFP-DFCP1 GFP-DFCP1

a

b

c d

Fig. 2 Activation of autophagy by DAPK by two different mecha-

nisms targeting the Beclin 1/Vps34 complex. Bcl-2 binds and inhibits

Beclin 1, preventing its association with the Vps34 complex. In

response to ionomycin, DAPK disrupts the Beclin 1/Bcl-2 interaction

through phosphorylation of Beclin 1 within the BH3 domain (inset b,

western blot using anti-phospho-Thr119 antibodies). ROCK1 can also

phosphorylate this residue in response to starvation. Under oxidative

stress, DAPK phosphorylates PKD, which in turn phosphorylates and

activates Vps34 directly. Activation of Vps34 leads to PI3P forma-

tion, which recruits PI3P effectors such as DFCP1, which can be seen

as a shift from a mainly diffuse localization of GFP-DFCP1 (inset c),

to its enhanced accumulation in puncta (inset d). The interaction of

Beclin 1 with Bcl-2 can also be disrupted by JNK-mediated

phosphorylation of Bcl-2, ubiquitination of Beclin 1 by TRAF6, or

binding of HMGB1 to Beclin 1. During oxidative stress, PKD can

also lead to JNK phosphorylation through the MAP3K ASK1; it is not

known if and when DAPK can modulate Bcl-2 through a PKD-JNK

pathway. Inset a shows activation of DAPK by Ser308 dephospho-

rylation in response to ionomycin (Ion) treatment by western blotting

with anti-phospho-Ser308. NT non-treated control. Asterisks indicate

non-specific bands

350 Apoptosis (2014) 19:346–356

123

particular in the case of PM02734, disruption of the

autophagy program, or DAPK depletion, rescued cell via-

bility [41].

Interestingly, the various scenarios in which DAPK was

shown to be necessary for autophagy have another factor in

common: they all involved cell death. While in some

scenarios (e.g. oxidative stress, ER stress) other death

programs were also evident, in others, a strong case for

autophagic cell death was presented, i.e. death was blocked

by inhibition of autophagy and occurred in the absence of

another death program. This correlation between autoph-

agy-associated cell death and DAPK is even more striking

considering that we have observed that DAPK is not nec-

essary for starvation induced autophagy, which has a pro-

survival role (unpublished observations, G. Oberkovitz,

Gozuacik, D. and Kimchi, A.). This suggests that DAPK is

specifically linked to autophagy activated in pathologic and

cell death settings. It may thus act as a factor that converts

the pro-survival mechanism to a death mechanism. Below

we will discuss how DAPK activates autophagy, and

speculate as to how this may lead to cell death.

Mechanistic connection between DAPK and autophagy

DAPK and the Vps34 PI3K complex

DAPK has been linked to the regulation of Vps34 by two

independent mechanisms. The first mechanism involves

Beclin 1 [5]. While it is presently unclear how Beclin 1

activates Vps34, it has been shown to be a key regulatory

component of the complex (Fig. 2). Association of the anti-

apoptotic protein Bcl-2 or Bcl-XL with Beclin 1 via the

latter’s BH3 domain interferes with the interaction of Beclin

1 with Vps34, thus inhibiting PI3K complex activity [44].

Interestingly, the Beclin 1/Bcl-2 interaction serves as a

converging point for diverse signaling pathways that control

Vps34 activation. For example, HMGB1 interacts with Be-

clin 1, leading to dissociation of Bcl-2, thus supporting

autophagic activity [45]. In macrophages, under amino acid

starvation and in response to LPS, Beclin 1 undergoes

ubiquitination on Lys117, mediated by the E3 ligase TRAF6.

This non-proteolytic Lys63-linked modification disrupts the

interaction between Beclin 1 and Bcl-2, thereby providing a

mechanism by which pathogen invasion can lead to the

induction of autophagy [46]. The Beclin 1/Bcl-2 interaction

can also be modulated through the modification of Bcl-2. In

response to starvation or ceramide, JNK1 phosphorylates

Bcl-2 at Thr69, Ser70 and Ser87, blocking the interaction

with Beclin 1 and increasing autophagy [47, 48].

DAPK provides yet another Beclin 1-dependent mech-

anism to regulate autophagy (Fig. 2). Zalckver, et al. [5]

identified Beclin 1 as a novel interacting partner and

substrate of DAPK. DAPK phosphorylates Beclin 1 at

Thr119, which is located within the BH3 domain, thereby

causing to a strong inhibition of the interaction with both

Bcl-2 and Bcl-XL [5, 49]. Moreover, the authors showed

that a phospho-mimicking (T119E) mutant of Beclin 1

interacted less efficiently with Bcl-XL compared to the

phospho-silencing mutant (T119A), which resulted in

increased formation of autophagosomes [5]. This site was

recently reported to also be the target of the Rho-associated

kinase ROCK1, which can activate Beclin 1 by the same

mechanism during nutrient-starvation induced autophagy

[50]. Furthermore, ROCK1 was shown to be necessary for

autophagy following nutrient withdrawal in cells, and in

the heart of starved mice. The fact that DAPK and ROCK1

both recognize the Thr119 phosphorylation site within

Beclin 1 is not surprising considering that the two kinases

have similar consensus motifs and that they share two

additional substrates, the myosin regulatory light chain

(MLC) and ZIP-kinase (ZIPK) [2, 32, 51]. They may each

activate Beclin 1 in response to different signals. For

example, in contrast to ROCK1, DAPK does not activate

Beclin 1 during starvation-induced autophagy. However,

unpublished data from our lab (Oberkovitz, G. and Kimchi,

A.) indicate that stimuli that activate DAPK through

increased intracellular Ca2?, such as the Ca2? ionophore

ionomycin, lead to phosphorylation of Beclin 1 on Thr119

in cells (Fig. 2, inset b).

The second alternative mechanism linking DAPK to

autophagy activation is through phosphorylation of protein

kinase D (PKD). It has been previously shown in our

laboratory that in response to oxidative stress, DAPK

interacts with, phosphorylates and activates PKD [52].

PKD in turn mediates DAPK’s autophagy function under

these conditions by a mechanism linked to Vps34 that is

independent of the Bcl-2/Beclin 1 interaction [4]. Signifi-

cantly, PKD interacts with Vps34 in cells, and moreover,

can phosphorylate Vps34 in vitro. Expression of PKD, but

not a kinase inactive variant, led to an accumulation of

GFP-DFCP1 in punctate structures representing PI3P-

enriched phagophore membranes (Fig. 2 inset d), which

reflect activation of Vps34 in cells. Thus during oxidative

stress, DAPK regulates a signaling pathway that directly

activates the key initiator of autophagosome formation,

Vps34 (Fig. 2). It should be noted that during oxidative

stress, phosphorylation of PKD by DAPK can also lead to

JNK activation [52]. As mentioned above, activated JNK

was previously reported to phosphorylate Bcl-2, and by

that means, disrupt the Beclin 1/Bcl-2 interaction to pro-

mote Vps34 activity and autophagy. However, it is not

known if and when DAPK can also modulate Bcl-2 through

a PKD-JNK pathway. As for the two established DAPK-

mediated mechanisms, it will be interesting to determine

whether DAPK-mediated phosphorylation of Beclin 1 and

Apoptosis (2014) 19:346–356 351

123

PKD-mediated phosphorylation of Vps34 are mutually

exclusive events that occur in response to different stimuli

(e.g., oxidative stress and elevated intracellular Ca2?,

respectively), or if they represent a coordinated response to

DAPK activity.

DAPK’s cytoskeletal function and autophagy

One of DAPK’s most prominent functional outcomes is its

effects on the cytoskeleton. It can associate with both actin

filaments [33, 53] and microtubules [54]. DAPK activity

leads to membrane blebbing and stress fiber formation as a

consequence of MLC phosphorylation and activation of

myosin II [53, 55]. Furthermore, it suppresses integrin

function and disrupts its interaction with talin, thereby

interfering with cell adhesion, cell polarization and direc-

ted migration [56, 57]. It is therefore not surprising that its

cytoskeletal functions can be recruited to facilitate the

autophagy process.

The first potential example of this involves Ulk1 and

Atg9. As stated above, Atg1/Ulk1 directs Atg9 trafficking

through the activation of myosin II, as a result of MLC

phosphorylation [28]. In the fly, Atg1 directly phosphory-

lated the MLC kinase Sqa. Sqa most closely resembles the

mammalian MLCK proteins, which belong to the CaM

kinase superfamily. The DAPK family (including DAPK,

DRP-1 (DAPK2) and ZIPK (DAPK3)) also belong to this

superfamily, although they form a branch distinct from the

MLCK proteins [58]. DAPK’s ability to phosphorylate

MLC and induce autophagosome formation is shared by its

close relatives DRP-1 and ZIPK [2]. Interestingly, there is

no Drosophila DAPK orthologue, although other insects

possess orthologues of both DAPK and Sqa [58]. However,

during autophagy, Sqa may substitute for DAPK and act as

a functional homologue of the DAPK family in flies. In

support of this, ZIPK was shown to possibly connect Ulk1

to MLC phosphorylation in human cells [28]. Starvation-

induced autophagy was associated with enhanced MLC

phosphorylation, which required both Ulk1 and ZIPK.

Furthermore, depletion of ZIPk partially suppressed star-

vation-induced autophagy. Significantly, Ulk1 and ZIPk

were shown to interact, but whether a kinase-substrate

relationship exists between them was not assessed [28].

However, the Thr in the fly Sqa that is targeted by Atg1 is

conserved within ZIPk, and is a known phosphorylation

site that promotes ZIPk activity [59]. Of note, these data do

not exclude a role for DAPK, or for that matter, any other

MLC kinase, in this pathway. Significantly, DAPK has

been shown to activate ZIPk for membrane blebbing and

autophagy by trans-phosphorylation [32]. It will be inter-

esting to determine if DAPK or any of its family members

are in fact substrates of Ulk1 and involved in MLC phos-

phorylation during autophagy.

Microtubule-based trafficking is required for the late

stages of delivery of the autophagosome to the lysosome to

form the autolysosome [31]. DAPK has been shown to

regulate microtubule dynamics through activation of

microtubule affinity regulating kinases (MARKS), the

kinases that phosphorylate microtubule associated proteins

(MAPs) [60]. Binding of MAPs (MAP1A, 1B, tau) to

microtubules stabilizes them, and competes with the

binding of microtubule-based motor proteins. The extent of

bound MAPs is thus critical for both microtubule assembly/

disassembly and transport, and is dynamically regulated by

phosphorylation of the MAPs, which promotes their dis-

sociation from the microtubules [61]. Thus, by activating

MARK to phosphorylate MAPs, DAPK has been shown to

lead to microtubule disassembly, affecting axonal out-

growth in neurons [60]. One could speculate that this might

also promote microtubule-based trafficking of vesicles such

as autophagosomes.

Interestingly, another link between DAPK, MAPs and

autophagy was raised in a paper by Harrison et al. [62].

They identified MAP1B as an interacting protein of DAPK

that serves as a positive regulator of DAPK’s functions.

MAP1B is a microtubule binding protein that may be

involved in autophagosome trafficking in neurons [63].

DAPK-mediated membrane blebbing and DAPK-mediated

autophagosome accumulation were both stimulated by co-

expression of MAP1B [62]. Overexpression of these pro-

teins in A375 cells resulted in the disruption of microtu-

bules in conjunction with extensive membrane blebbing.

Moreover, DAPK and MAP1B co-localized both to

microtubules and actin microfilaments. The results descri-

bed in the work are interesting, considering the fact that

phospho-MAP1B is associated with Atg8/LC3 on the

autophagosome, and may mediate autophagosome traf-

ficking in neurons [64]. This raises the possibility that

DAPK not only interacts with MAP1B on microtubules,

but also phosphorylates MAP1B, thereby enhancing its

interaction with Atg8/LC3. In further support of this,

DAPK localization correlated with increased MAP1B

phosphorylation in differentiated gastric zymogenic cells

[65]. These results suggest that by interacting with both

LC3-II and DAPK, MAP1B may bring DAPK into prox-

imity of the autophagy-inducing machinery, including the

latter’s potential substrates.

Additional hypothetical integration points

between DAPK and autophagy

As stated above, autophagy is dependent on the endocytic

pathway, both during early stages of phagophore forma-

tion, and later trafficking to the lysosome. DAPK too has

been linked to endocytosis. It was isolated in a large-scale

siRNA-based kinase screen as necessary for clathrin-

352 Apoptosis (2014) 19:346–356

123

mediated endocytosis; upon DAPK knock-down, uptake of

Vesicular stomatitis virus (VSV), a clathrin-dependent

endocytic process, was inhibited, and early/late endosomes

accumulated [66]. The mechanism by which DAPK regu-

lates endocytosis was not demonstrated. Interestingly,

additional components of clathrin-mediated endocytosis

have been shown to be necessary for the formation of pre-

autophagosomal structures, including clathrin heavy chain,

the GTPase dynamin, and the SNAREs syntaxin-7 and -8

and VAMP7 [20, 22]. SNARES are a large family of

proteins that mediate fusion between two opposing mem-

branes through the formation of complexes of different

SNARES on either membrane. Intriguingly, an additional

paper has linked DAPK function to SNARES, specifically

to the synaptic vesicle SNARE syntaxin-1A [67]. Using a

yeast two-hybrid screen, the authors identified an interac-

tion between syntaxin-1A and DAPK. Furthermore, DAPK

phosphorylated syntaxin-1A on Ser188, resulting in

decreased binding between syntaxin-1A and its regulator

Munc18-1. One of the known functions of Munc18-1 is to

sequester syntaxin-1A from binding to the SNARE com-

ponent SNAP25. Surprisingly, however, the reduction in

interaction between phospho-syntaxin-1A and Munc18-1

had no influence on the assembly of the SNARE complex.

Thus the physiological significance of syntaxin-1A phos-

phorylation by DAPK is still unknown. However, the link

between DAPK and components of the endocytic pathway

is highly suggestive, especially considering that the DAPK

phosphorylation site within syntaxin-1A is conserved in

other syntaxin proteins. The precise connection between

DAPK, endocytosis and autophagy remains an exciting

avenue of future research.

Interestingly, DAPK has also been linked to the regu-

latory stage of DAPK induction through its interaction with

TSC2, the GAP for the mTOR regulator Rheb. DAPK

binding to TSC2 competes with the TSC1/TSC2 interac-

tion, thereby inhibiting the latter’s activity [54]. Thus, in

the presence of DAPK, Rheb, and therefore mTOR, is

constitutively active. Since mTOR suppresses autophagy

through Ulk1, this would imply that autophagy is inhibited

by DAPK, contrary to the role that DAPK normally plays

in activating autophagy. The authors of that paper did not

examine the effect of the DAPK–TSC2 interaction on

autophagy, although they did show enhanced ribosomal

protein S6 and S6 kinase phosphorylation, as an indication

of increased mTOR activity. Thus, it is not known whether

DAPK does in fact regulate autophagy through TSC2.

Perspective and conclusions

Both cancer and neurodegenerative disease have been

associated with misregulated autophagy, and have been

linked to loss- or gain-of-function of DAPK, respectively.

It will thus be interesting to determine if DAPK’s auto-

phagic functions contribute to its role in these pathologic

conditions. The relationship between autophagy and cancer

is a complex and often contradictory one [68]. In early

stages, autophagy has a tumor suppressive function

resulting, in part, from its ability to suppress accumulation

of p62 and the generation of radical oxygen species, and

limit genomic instability in response to oxidative and

metabolic stress. It also serves to prevent tumor necrosis

and inflammation [69–72, and reviewed in 73]. In contrast,

in later stages, autophagy is necessary for tumor progres-

sion, for example, by providing energy and nutrients to the

rapidly dividing and poorly vascularized tumor. Further-

more, it contributes to the resistance of tumor cells to

chemotherapy and radiation treatment by mitigating cel-

lular stress and blocking apoptosis [68]. Thus, many tumors

become ‘‘addicted’’ to autophagy. Significantly, several

autophagy genes, such as Beclin 1, have been shown to

function as tumor suppressors [74, 75]. Similarly, DAPKs

ability to regulate autophagy at different stages and its

frequent epigenetic loss in tumors [76] may be connected.

Since these genes also have non-autophagic roles, however,

it is still not clear if the tumor suppressive function stems

from their roles as modulators of autophagy. Beclin 1, for

example, has been shown to inhibit the functions of de-

ubiquitinating enzymes that in turn affect p53 stability, a

master tumor suppressor in its own right [77]. Similarly,

DAPK has many different functions that contribute to its

ability to suppress tumor growth and metastasis. These

include its ability to upregulate p53 upon oncogene

expression [78], to induce apoptosis, especially in response

to loss of matrix attachment and cytotoxic cytokines such

as TNFa [56, 79], to activate pyruvate kinase M’s glyco-

lytic function [80], and to block cell motility and metastasis

[57]. Thus, it is uncertain whether or not DAPK’s function

as an autophagy inducer leads to tumor suppression.

DAPK has also been implicated in the pathogenesis of

neuronal disorders such as Alzheimer’s disease [81], epi-

lepsy [82] and ischemic brain injury [83]. This can be

related to its ability to induce neuronal cell death in

response to ceramide, ischemia and glutamate toxicity [84–

86]. In addition, DAPK can affect microtubule dynamics

through its activation of the MARK kinases, which phos-

phorylate MAPs such as tau, leading to tauopathies [60].

However, the connection between DAPK’s role in

autophagy and neuropathologies is still unclear and

remains an important area for future research.

There are many regulators of autophagy. DAPK is

unique in its capability to impact several different stages

within the autophagy flux pathway, including autophago-

some induction, trafficking, and fusion. Some of these

regulatory events are more well-grounded and proven than

Apoptosis (2014) 19:346–356 353

123

others. Furthermore, it is not known what triggers these

regulatory mechanisms, and whether one or more are

activated concurrently. Nevertheless, by enhancing multi-

ple steps in the process, DAPK has the potential for being

an autophagy ‘‘super-activator’’. This may explain its

connection specifically to autophagy associated with cell

death. Additional research into the mechanisms by which

DAPK regulates autophagy will shed light not only on the

function of DAPK, but also on the connection between

autophagy and cell death.

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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