The DAPK interactome A major goal in biological research is to define the system within which a signalling protein operates and Keywords autophagy; DAPK; growth factor; immune response; i
Trang 1Death-associated protein kinase (DAPK) and signal
transduction: additional roles beyond cell death
Yao Lin, Ted R Hupp and Craig Stevens
CRUK p53 Signal Transduction Laboratories, Institute of Genetics and Molecular Medicine, University of Edinburgh, UK
Introduction
Death-associated protein kinase-1 (DAPK-1) is the
prototypic member of a family of death-related kinases
that includes DAPK-1-related protein 1 (also named
DAPK-2), Zipper interacting kinase (ZIPK, also
named DAPK-3), DAP kinase related apoptosis
indu-cing protein kinase 1 (DRAK1) and DRAK2 [1]
These kinases share a high degree of homology in their
catalytic domains However, the extracatalytic domains
and biological function of these five proteins differ
markedly [1] DAPK, a calcium⁄ calmodulin
(CaM)-regulated Ser⁄ Thr protein kinase, was originally
identi-fied as a factor that regulates apoptosis in response to
the death-inducing cytokine signal interferon-c (INF-c)
[2] In addition to its role in apoptosis, recent advances have established an important role for DAPK in a diverse range of signal transduction pathways, includ-ing growth factor signallinclud-ing and autophagy In this review we will integrate these new findings with our existing knowledge of DAPK function and attempt to highlight the areas that remain unresolved and require further investigation
The DAPK interactome
A major goal in biological research is to define the system within which a signalling protein operates and
Keywords
autophagy; DAPK; growth factor;
immune response; interactome; kinase;
mTOR; peptide
Correspondence
C Stevens, CRUK p53 Signal Transduction
Laboratories, Institute of Genetics and
Molecular Medicine, University of
Edinburgh, Edinburgh EH4 2XR, UK
E-mail: craig.stevens@ed.ac.uk
(Received 11 March 2009, revised
12 August 2009, accepted 8 September
2009)
doi:10.1111/j.1742-4658.2009.07411.x
Death-associated protein kinase (DAPK) is a stress-regulated protein kinase that mediates a range of processes, including signal-induced cell death and autophagy Although the kinase domain of DAPK has a range
of substrates that mediate its signalling, the additional protein interaction domains of DAPK are relatively ill defined This review will summarize our current knowledge of the DAPK interactome, the use of peptide apta-mers to define novel protein–protein interaction motifs, and how these new protein–protein interactions give insight into DAPK functions in diverse cellular processes, including growth factor signalling, the regulation of autophagy, and its emerging role in the regulation of immune responses
Abbreviations
ATM, ataxia telangiectasia mutated; BH3, Bcl-2-homology-3; CaM, calcium ⁄ calmodulin; DAPK, death-associated protein kinase; DIP1, DAPK interacting protein-1; EGF, epidermal growth factor; ER, endoplasmic reticulum; ERK, extracellular signal-regulated kinase; HSP, heat shock protein; INF-c, interferon-c; LAR, leukocyte common antigen related; MAP1B, microtubule-associated protein 1B; MCM3, mini-chromosome maintenance complex component 3; mTOR, mammalian target of rapamycin; NF-jB, nuclear factor kappa-b; PMA, phorbol-12-myristate-13-acetate; RSK, ribosomal S6 kinase; S6K1, ribosomal protein S6 kinase-1; TGF-b, transforming growth factor-b; TNF, tumour necrosis factor; TSC, tuberous sclerosis; ZIPK, Zipper interacting kinase.
Trang 2to use this information to understand developmental
or disease processes Classically, genetic screens in
trac-table organisms, such as yeast, worms and flies, have
been used for defining the landscape of a protein⁄
path-way However, many cancer- and immunity-related
genes are confined to vertebrates and a full
under-standing of how these proteins operate without the use
of classic genetics has been relatively difficult Instead,
technologies that define protein–protein interactions
have been used to build a protein interaction map (i.e
like a genetic interaction pathway) for a target protein
Such technologies include the yeast two hybrid,
mono-clonal antibody co-immunoprecipitation methods
coupled to protein sequencing, and tap-tagging molecular
biology approaches for trapping a multiprotein
com-plex The yeast two hybrid, for example, has been used
to discover a novel interaction between extracellular
signal-regulated kinase (ERK) and DAPK, with
impli-cations for pro-apoptotic pathways [3] Furthermore,
recent ideas in systems biology hold that many
pro-teins have unstructured motifs or linear domains and
that dynamic regulation of protein–protein interactions
is mediated by the diversity in such small signalling
motifs This property has been exploited using peptide
combinatorial libraries to discover novel complexes
between DAPK and microtubule-associated protein 1B
(MAP1B) [4] and DAPK and tuberous sclerosis 2
(TSC2) [5], with implications for autophagy and
mam-malian target of rapamycin (mTOR) signalling
Together, using such distinct approaches, the DAPK
interactome is being built up in a range of
back-grounds
DAPK is a large 160 kDa protein composed of
several functional domains, including a kinase domain,
a CaM regulatory domain, eight consecutive ankyrin
repeats, two putative nucleotide binding domains
(P-loops), a cytoskeletal binding domain and a death
domain (Fig 1) Proteins that interact with DAPK,
the domain on DAPK that mediates the interaction
and the methods used to discover the interactions are
summarized in Table 1 Given that many regions of
DAPK can form protein–protein interfaces it is
unsur-prising that only a few of the DAPK binding proteins highlighted in Table 1 are substrates of DAPK, sug-gesting that in some circumstances protein interaction alone is sufficient for DAPK to exert its biological effects Because of the paucity of DAPK substrates, a screen aimed at identifying a consensus DAPK phos-phorylation motif was carried out based on positional scanning peptide substrate library synthesis and activ-ity [6] The preferred consensus motif for DAPK phosphorylation and substrates for which phospho-acceptor site(s) have been identified are described in Table 2 Of note, mini-chromosome maintenance com-plex component 3 (MCM3), which is a DNA replica-tion licensing factor, was identified using biochemical fractionation and MS analysis to purify and identify potential substrates from Hela cell lysate [7] This kind
of proteomic approach should expedite the identifica-tion of novel, physiologically relevant in vivo substrates
of DAPK Moreover, it could be tailored to reflect DAPK substrate specificity in response to specific signalling events, such as growth factor or cytokine signalling
It is apparent from Table 2 that not all of the DAPK substrates identified are a good match to the identified consensus motif Chemical genetics, a bio-chemical approach to develop small peptide-mimetic ligands to alter how an enzyme functions, was utilized
Ca2+/CaM
Ankyrin P-loops repeats
Fig 1 Schematic representation of DAPK DAPK is a large
160 kDa Ser ⁄ Thr Ca2 + ⁄ CaM-regulated kinase that consists of
several functional domains, including a kinase domain, a CaM
regulatory domain, eight consecutive ankyrin repeats, two P-loops,
a cytoskeletal binding domain and a death domain, which enable it
to participate in a wide range of signalling pathways.
Table 1 DAPK binding proteins, the region of DAPK important for mediating the protein-protein interaction, and the method used to define the interaction.
Binding protein Binding region on DAPK Binding assay used 14-3-3 [65] Not defined Immunoprecipitation Actin [66] Cytoskeletal domain Immunostaining Beclin-1 [36] Not defined Immunoprecipitation CaM [67] Ca 2 + ⁄ CaM
regulatory domain
Overlay binding assay Cathepsin B [61] C-terminal domain Immunoprecipitation DIP1 [13] Ankyrin repeats Yeast two hybrid a
ERK [3] Death domain Yeast two hybrida FADD [65] Not defined Immunoprecipitation Hsp90 [62] Kinase domain Immunoprecipitation LAR [23] Ankyrin repeats Yeast two hybrida MAP1B [4] Kinase domain Peptide libraries a
Src [23] Not defined Yeast two hybrid a
TNFR-1 [65] Not defined Immunoprecipitation TSC2 [5] Death domain Peptide libraries a
UNC5H2 [69] Death domain Yeast two hybrida ZIPK [70] Kinase domain Immunoprecipitation
a Protein interactions have been confirmed by more physiological methods.
Trang 3recently to develop selective peptide ligands that
mod-ulate DAPK activity For example, DAPK binding to
a peptide derived from the amino acid sequence of the
cyclin-dependent kinase inhibitor p21 induces a
confor-mational change in DAPK that enhances its kinase
activity, suggesting that DAPK may require docking in
order to phosphorylate a subset of its substrates [8] It
is also possible that the interaction of DAPK with
many of its substrates is of too low affinity to detect in
cells In support of this notion, the ataxia
telangiecta-sia mutated (ATM) protein kinase, a large > 300 kDa
enzyme, does not have an abundance of stable
protein–protein partners that would be expected of a protein of its large size However, a recent MS-linked proteomics screen identifying phospho-Ser-Gln pep-tides that are phosphorylated by ATM identified over
700 substrates [9] Therefore, it seems that the previ-ously available protein interaction methodologies were not able to faithfully reflect the ATM kinase inter-actome
A future challenge will be the identification of lower affinity or transient DAPK interactions that might otherwise be overlooked in the more traditional assays
to further elucidate the functional role of DAPK in diverse signalling pathways
Signalling to DAPK
DAPK plays an important role in a wide range of sig-nal transduction pathways with diverse outcomes, such
as apoptosis, autophagy and immune responses The functional outcome of DAPK activity depends largely
on the input signal (Fig 2) For example, DAPK gene expression and apoptotic activity is increased in response to transforming growth factor-b (TGF-b) [10] and to stimuli that activate p53 [11], such as DNA-damaging agents Other death signals, such as the transforming oncogenes E2F1 and Myc [12], also induce DAPK expression In addition to its well-docu-mented role in the regulation of apoptosis, DAPK may also play a role in survival pathways, reflected in its activation by growth factor signalling pathways [5], and its ability to counter tumour necrosis factor (TNF)-mediated apoptosis [13]
Table 2 DAPK substrates and the amino acid sequence
surround-ing the phosphorylation site The substrate phosphorylation pattern
preferred by DAPK is highlighted in bold; the basic residues also
preferred by DAPK are underlined.
Syntaxin-1A [38] IIMDSSIS 188 KQALSEIE
Tropomyosin-1 [74] HALNDMTS283I
S 312 LPPNNS 318 YADFERFS 326
Mitogens
EGF
Short treatment Long treatment TNF- α TNF- α IFN- γ
TGF- β DNA damage oncogenes
Growth
mTORC1
Gene expression Kinase activity Kinase activity Degradation ?
DAPK over-expression
Autophagy Apoptosis
Apoptosis Autophagy
Inflammation Immune response
Blebbing Autophagy
Beclin-1 phosphorylation MAP1B binding
Apoptosis Apoptosis mTORC1? Inflammation ?
Fig 2 Signalling to DAPK DAPK plays an important role in a diverse range of signal transduction pathways The biological outcome of DAPK activity depends on the input signal and includes cell growth, immune responses, apoptosis and autophagy (A) Growth factor signal-ling to DAPK is probably the best defined with respect to the proteins that are involved and includes the activities of Src, LAR, ERK and RSK (see text and Fig 3) (B) The functional outcome of increased DAPK activity in response to short-term treatment with TNF-a is currently unclear, but may contribute to mTORC1 activation and inhibition of inflammatory responses Longer-term treatment with TNF-a leads to DAPK degradation coincident with apoptosis, suggesting that DAPK may be a resistance factor to TNF-a-induced cell death in some circum-stances (C) DAPK mediates many cellular responses in response to INF-c, but the molecular mechanisms have not yet been defined (D) DAPK gene expression and apoptotic activity are increased in response to TGF-b and to stimuli that activate p53, such as DNA-damaging agents Other death signals, such as the transforming oncogenes E2F1 and Myc, also induce DAPK expression (E) Overexpression of DAPK can promote autophagy and membrane blebbing via binding to MAP1B, or autophagy via the direct phosphorylation of Beclin-1 The signals that regulate DAPK autophagic activity have yet to be defined.
Trang 4Growth factor signalling/mTOR
Serum-induced activation of DAPK catalytic activity
has been demonstrated recently [3,5,14] and it is
becoming increasingly clear that DAPK is intimately
linked to growth factor signalling pathways (Fig 3)
For example, serum-induced phosphorylation of
DAPK by ERK enhances its kinase activity and
death-promoting effects [3], whereas serum activation
of DAPK has also been linked to cell death by
suppressing integrin functions and integrin-mediated
survival signals [14] However, in addition to apoptotic
signalling, we have recently demonstrated a
stimula-tory role for serum-activated DAPK in mTOR
signal-ling [5] mTOR is a member of the
phosphoinositide-3-kinase-related kinase family, which is centrally
involved in growth regulation, proliferation control
and cell metabolism [15] In mammalian cells, two
structurally and functionally distinct mTOR-containing
complexes have been identified, mTORC1 and
mTORC2 [15] mTORC1 directly regulates cell growth
by controlling the phosphorylation of a number of
components of the translational machinery In
particu-lar, phosphorylation and activation of eukaryotic
initi-ation factor 4E binding protein-1 and ribosomal
protein S6 kinase-1 (S6K1) are stimulated by serum,
insulin and growth factors in an mTORC1-dependent
manner [16]
The TSC complex, formed by two proteins, TSC1 and TSC2, is a major regulator of the mTORC1 signalling pathway [17] TSC2 contains a GTPase-acti-vating protein domain that converts the small GTPase Ras homolog enriched in brain to its inactive GDP-bound form [18] mTORC1 activity is stimulated
by the active GTP-bound form of Ras homolog enriched in brain, thus the TSC complex acts to inhibit mTORC1 function [18] Growth factor-induced, invating TSC2 phosphorylation results in mTORC1 acti-vation and is thought to occur primarily through activation of the RAS–extracellular signal-regulated kinase kinase (MEK)–ERK and phosphoinositide-3-kinase–Akt pathways [19,20] In a protein interaction screen in our laboratory, we identified TSC2 as a novel DAPK death domain interacting protein, and in analy-sing the biological consequences of the DAPK–TSC2 interaction, we were led to the discovery that DAPK can phosphorylate and inactivate TSC2 and functions
as a positive cofactor in mTORC1 signalling in response to serum and epidermal growth factor (EGF) stimulation [5]
ERK can directly interact with and phosphorylate DAPK at Ser735, which leads to enhanced kinase activity and pro-apoptotic activity of DAPK [3] This Ser735 phosphorylation can be stimulated by serum or phorbol-12-myristate-13-acetate (PMA) [3], which acti-vates the RAS–MEK–ERK pathway [21,22]
Interest-Ras Raf MEK ERK RSK
DAPK
TSC2 TSC1
Apoptosis ? Apoptosis
Apoptosis
DAPK
Rheb
S6K
S6
-T389
-S235/236 P P
Cell growth Protein synthesis
mTORC1
DAPK
P -S289
P -S735
Src
LAR
P Y491/Y492
-EGF
Fig 3 Growth factor regulation of DAPK.
Growth factor signalling to DAPK is complex
and regulates a diverse range of biological
outcomes For example, phosphorylation by
ERK enhances the apoptotic activity of
DAPK, but Src-mediated phosphorylation of
DAPK suppresses its apoptotic,
antimigra-tion and antiadhesion funcantimigra-tions Under
nor-mal growth conditions, DAPK apoptotic
activity may also be suppressed until such
times as required due to phosphorylation by
RSK DAPK may also act in concert with
ERK and RSK to inhibit the TSC complex,
resulting in mTORC1 activation In addition,
DAPK and RSK may co-operate to promote
protein translation via direct phosphorylation
of ribosomal protein S6.
Trang 5ingly, the inactivation of DAPK activity by EGF has
been recently described Wang et al [23] demonstrated
that DAPK is a substrate for leukocyte common
antigen related (LAR) tyrosine phosphatase and that
dephosphorylation of Y491⁄ Y492, located in the
ankyrin repeat domain, resulted in activation of the
pro-apoptotic activities of DAPK Reciprocally, Src
kinase phosphorylation of Y491⁄ Y492 inhibited
DAPK activity [23] Src kinase was activated in
response to EGF stimulation and LAR was
downregu-lated, resulting in DAPK inactivation The ability of
EGF signalling to inactivate DAPK is inconsistent
with previous findings that DAPK activity can be
up-regulated by serum stimulation and ERK, a
down-stream effector of the EGF pathway [3], and this is
further inconsistent with data showing that in response
to PMA, the DAPK–ERK complex induces apoptosis
[3] It is important to note, however, that the
apopto-sis-promoting effect of DAPK induced by the ERK
activator PMA was only observed in suspension cells
[3], whereas in adherent cells the co-expression of a
constitutively active mutant of MEK is required for
DAPK to induce apoptosis [24] Therefore, the
apop-tosis function of the ERK–DAPK complex may only
exist under aberrant conditions, such as when cells are
detached, or when the signal to grow is excessive
Other signalling pathways can in turn modify these
core activities of DAPK For example, RAS activation
of the ERK–ribosomal S6 kinase (RSK) pathway can
attenuate the pro-apoptotic function of DAPK RSK
interacts with DAPK in vitro and in vivo and catalyses
the phosphorylation of DAPK on Ser289 in response
to PMA [25] The effect of this phosphorylation on the
kinase activity of DAPK was not tested However,
mutation of Ser289 to a nonphosphorylatable Ala
results in a DAPK mutant with enhanced apoptotic
activity, whereas the phosphomimetic mutation
(Ser289Glu) attenuates its apoptotic activity [25] The
observation that the Ser289Ala mutant of DAPK is
more apoptotic suggests that phosphorylation inhibits
the catalytic activity of DAPK [25] Thus, kinase assays
using the Ser289 mutants are required to clearly
deter-mine the function of DAPK Ser289 phosphorylation
Interestingly, RSK has also been shown to interact with
TSC2, and phosphorylation by RSK inactivates TSC2,
resulting in mTORC1 activation [26]
DAPK has also been directly linked to the control
of protein translation by phosphorylating ribosomal
protein S6 on Ser235⁄ 236 [27] In agreement with this
study, we have shown that DAPK can robustly
stimu-late the phosphorylation of S6 in cells, even in the
presence of the lipophilic macrolide antibiotic
rapa-mycin, a potent inhibitor of mTORC1 activity,
indicat-ing that DAPK can mediate phosphorylation of S6 in
an mTORC1–S6K-dependent and -independent man-ner Schumacher et al [27] demonstrated that DAPK phosphorylates S6 directly on Ser235⁄ 236 and con-cluded that this is an inhibitory phosphorylation reducing S6 activity and protein translation in vitro In contrast, Roux et al [28] demonstrated that RSK kinase phosphorylates the same sites on S6, but they concluded that this was an activating phosphorylation that stimulates S6 activity and promotes assembly of the translation preinitiation complex in cells Our results are in agreement with the latter study and point towards a role for DAPK in activating S6 and protein translation Further studies are required to clarify the role of DAPK in the regulation of S6 activity and pro-tein translation in vivo, in particular the interplay between DAPK and RSK signalling to S6 needs to be addressed, and the ability of DAPK to promote cell growth needs to be clearly demonstrated
Taken together, these studies reveal a complex regu-lation of DAPK activity by growth factor signalling pathways mediated by Src, LAR, ERK and RSK A better understanding of the interplay between signalling
to DAPK and TSC2 may explain how the specific activ-ity of DAPK can be modulated to control the balance between pro-apoptotic and pro-survival pathways
DAPK and autophagy
DAPK was originally identified as a factor that regu-lates apoptosis in response to various death-inducing signals, including INF-c [2] DAPK also has auto-phagy signalling activity, which can be either pro-sur-vival or lead to or participate in cell death
Autophagy is a membrane system involved in pro-tein and organelle degradation that probably repre-sents an innate adaptation to starvation In times of nutrient deficiency, the cell can self-digest and recycle some nonessential components to sustain its minimal growth requirements until a food source becomes available Over recent years, autophagy has been impli-cated in an increasing number of clinical scenarios, notably infectious diseases, cancer, neurodegenerative diseases and autoimmunity In some cell types, the overexpression of DAPK can lead to the appearance
of autophagic vesicles [29] However, there is still little known about how DAPK exerts its effects on auto-phagy, and as DAPK is not present in yeast, there have been no classic genetic screens to analyse how DAPK interacts with the core autophagy pathway Recently, peptide combinatorial libraries identified MAP1B as a DAPK interacting protein that functions
as a positive cofactor in DAPK-mediated autophagic
Trang 6vesicle formation and membrane blebbing [4] MAP1B
has been most widely studied as a major component of
the neuronal cytoskeleton [30] and relatively little is
known about its role outside of these neuronal
systems The cotransfection of both genes stimulated
the disruption of microtubules during the induction of
membrane blebbing, suggesting that
MAP1B–DAPK-induced blebbing involves changes in the dynamics of
mictrotubules, as well as changes in the dynamics of
contractile cortical actin [4] This is even more
intrigu-ing in light of the recently identified interaction
between the essential autophagy protein Atg8 (LC3)
and MAP1B [31], and the observation that
micro-tubules play an important role in autophagy by
support-ing the production and transport of autophagosomes
[32] Future studies will determine whether MAP1B is
a key factor that switches DAPK activity towards
autophagy induced by certain stresses such as INF-c
Beclin-1, the first identified mammalian autophagy
gene [33], interacts with several cofactors to activate
the lipid kinase Vps34, thereby inducing autophagy
[34] Beclin-1 is a Bcl-2-homology-3 (BH3) domain-only
protein that binds to the BH3 domain of the
antiapoptotic proteins Bcl-2⁄ Bcl-XL[35] Under normal
conditions, beclin-1 is bound to and inhibited by Bcl-2
or the Bcl-2 homolog Bcl-XL and the dissociation of
beclin-1 from Bcl-2 is essential for its autophagic
activ-ity [34] Nutrient deprivation stimulates the dissociation
either by activating BH3-only proteins (such as Bad),
which can competitively disrupt the interaction, or by
post-translational modification [34] A recent report
demonstrated that a constitutively activated form of
DAPK triggers autophagy in a beclin-1-dependent
manner [36] DAPK phosphorylates beclin-1 on Thr119
located at a crucial position within its BH3 domain,
and thus promotes the dissociation of beclin-1 from
Bcl-XLand the induction of autophagy [36] This study
revealed a new substrate for DAPK that acts as one of
the core proteins of the autophagic machinery, and
provides a new phosphorylation-based mechanism for
how DAPK activates autophagy by reducing the
inter-action of beclin-1 with its inhibitor Bcl-XL
DAPK has also been directly linked to the
regu-lation of endocytosis [37], and can phosphorylate
syntaxin-1A, a key component of the soluble
N-ethyl-maleimide-sensitive factor (NSF) attachment protein
receptors complex essential for synaptic vesicle docking
and fusion [38] Therefore, DAPK may also regulate
autophagy via syntaxin-1A
Although most evidence suggests that autophagy
acts as a survival response to provide an energy source
maintaining cell survival, it has been proposed that
autophagy can contribute to cell death in a process
termed autophagic (type II) cell death Disturbance to endoplasmic reticulum (ER) homeostasis that leads to irreparable damage activates ER-specific cell death mechanisms [39] DAPK was recently identified as an important component in ER stress-induced cell death [40] DAPK) ⁄ ) mice are protected from kidney dam-age caused by injection of the ER stress inducer tunicamycin and the cell death response to tunicamy-cin is reduced in DAPK ) ⁄ ) mouse embryonic fibro-blasts [40] Interestingly, both caspase activation and autophagy induction are attenuated in DAPK) ⁄ ) mouse embryonic fibroblasts, and depletion of ATG5
or beclin-1, essential autophagic proteins, are protected from ER-induced death when combined with caspase-3 depletion [40] These results suggest that under certain conditions, DAPK-induced autophagy contributes to cell death, possibly through the induction of apoptosis
In the model organism Caenorhabditis elegans, it was recently demonstrated that starvation-induced autophagy is regulated in part through a DAPK sig-nalling pathway and that autophagy levels are critical
to drive such cell fate decisions, leading to survival or death of the organism [41] (see the accompanying review by Kang and Avery [42]) In C elegans, mus-caranic acetylcholine receptor signalling is important
in modulating the level of autophagy during starvation [43] In a simplified model, starvation activates MAPK (MPK-1), the C elegans ortholog of mammalian ERK, and activated MPK-1 positively regulates auto-phagy, at least in part through DAPK and RGS-2 [43] It will be interesting to determine whether ERK and DAPK can co-operate to regulate autophagy in higher organisms
The pathway that regulates autophagy also acts through mTORC1 [44] Rapamycin binds to and inac-tivates mTORC1, leading to an upregulation of auto-phagy [45] The finding that DAPK is a positive regulator of mTORC1 signalling and a positive regula-tor of autophagy at first seems counterintuitive There-fore, we would predict that DAPK activity should be activated by starvation, and that its activity would be inversely correlated with that of mTORC1 However,
in mammalian cells, although DAPK is reported to be necessary for INF-c-induced autophagy, it seems not
to be a crucial element in starvation or rapamycin-induced autophagy [46] The accompanying review by Kang and Avery [42] proposes an interesting explana-tion for the seemingly contradictory funcexplana-tions of DAPK to promote mTORC1 activity and autophagy They propose that DAPK may promote mTORC1 activity specifically to mediate S6K activity during starvation, as S6K activity has been shown to promote rather than suppress autophagy in Drosophila [47]
Trang 7Clearly, further characterization of the interacting
proteins and direct substrates of DAPK, as well as
differences between simple organisms and complex
mammalian systems, are required to clarify how the
kinase is linked to the autophagic pathways
DAPK immune responses
DAPK has been shown to participate in cell death in
response to various cytokine signals, including
IFN-c-induced cell death [2], TNF-a and FAS-IFN-c-induced cell
death [48], and TGF-b-induced cell death [10] There
are two distinct outcomes of TNF-a signalling, an
inflammatory immune response mediated by the
nuclear factor kappa-b (NF-jB) signalling pathway,
and apoptosis [49] By comparing the response to
TNF-a treatment in DAPK-deficient and wild-type
cells, several groups have demonstrated that DAPK is
neutral against TNF-a-induced apoptosis [2,10] More
recent studies have indicated that DAPK is in fact a
negative regulator of TNF-a-induced apoptosis For
example, antisense depletion of DAPK in Hela cells
protects cells from IFN-c-induced apoptosis, but
pro-motes TNF-a-induced apoptosis [50], and the
expres-sion of DAPK interacting protein-1 (DIP1), a
ubiquitin E3 ligase that degrades DAPK, promotes
TNF-a-induced apoptosis [13] Therefore, although it
functions as a death-promoting kinase, DAPK can
also act as a survival factor and block apoptosis in
response to certain cytokine signals Interestingly,
DAPK has recently been shown to function as a
neg-ative regulator of T cell activation via NF-jB
How-ever, DAPK had no effect on NF-jB activation by
TNF-a, only by T cell receptor activation [51] In
addition, DAPK can act as a negative regulator of
inflammatory gene expression in monocytes [52] In
C elegans, wounding of epidermal layers triggers
mul-tiple co-ordinated responses to damage It was
recently shown that the C elegans ortholog of DAPK
acts as a negative regulator of barrier repair and
innate immune responses to wounding [53] Taken
together, these studies suggest an intriguing role for
DAPK, not only as a modulator of cytokine-induced
apoptosis, but as a regulator of various immune
responses
Future work
It is becoming increasingly clear that DAPK family
members have additional roles beyond their functions
in cell death The recent findings that DAPK
nega-tively regulates inflammatory gene expression [51,52],
responds to mitogenic signals to regulate mTORC1
activity [5] and negatively regulates epidermal damage responses in C elegans in an apoptosis- and auto-phagy-independent manner [53], highlight the pleo-trophic role of this kinase
What are the crucial questions for the future? Of considerable importance will be to gain a clear under-standing of the role of DAPK in the RAS–MEK– ERK growth factor signalling pathway, in particular the interplay between ERK, RSK and DAPK and the balance between apoptosis and growth needs to
be addressed Gaining a better understanding of DAPK’s role in cancer is particularly important DAPK hypermethylation is strongly associated with various cancers (see the accompanying review by Michie et al [54]), but it is not yet clear how reduced levels of DAPK contribute to carcinogenesis Possible mechanisms include DAPK’s ability to suppress extra-cellular matrix survival signals to regulate anoikis [14] and its ability to inhibit cell polarization and motility [55] DAPK can suppress transformation by oncoge-nes by activating a pro-apoptotic p53-dependent checkpoint [12], and it can activate autophagy, which has been shown to be antitumorigenic [56–58] Recent studies indicate that inflammation is an important contributor to tumorigenesis [59] Therefore, the anti-inflammatory function of DAPK may also contribute
to its tumour suppressive activity [52] Of interest in this regard are recent studies showing that the TSC– mTORC1 pathway regulates inflammatory responses
in monocytes, macrophages and primary dendritic cells [60] The finding that DAPK regulates mTORC1 activity [5], together with the observation that both mTORC1 and DAPK can block NF-jB activation [51,60], raise the intriguing possibility that DAPK may regulate inflammatory immune responses via an mTORC1-dependent mechanism Further studies are required to determine whether these pathways are related in this context
Mechanisms regulating protein stabilization and turnover are also critical for modulating DAPK activi-ties Several studies have shown DAPK degradation to
be dependent on the ubiquitin–proteasome pathway [13,61–64] To date, two E3 ubiquitin ligases have been identified that can promote the ubiquitination of DAPK; DIP-1, a ring finger containing E3 that inter-acts directly with the ankyrin repeat region of DAPK [13], and carboxyl terminus of HSC70-interacting pro-tein, a U-box containing E3 ubiquitin ligase that can bind to the heat shock protein (HSP) chaperone pro-teins HSP70 and HSP90, interacts with DAPK indi-rectly via Hsp90 [62] The identification of additional ubiquitin ligases, and deciphering the degradation pathways that modulate DAPK stability, will shed
Trang 8further light on the role played by DAPK in the
regu-lation of cell growth control
There is no doubt that future research into the role
of DAPK will yield new and important insights into
the mechanisms that integrate the apoptotic,
auto-phagic and cell growth regulatory pathways
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