Báo cáo khoa học: Apoptosis and autophagy: Regulation of apoptosis by DNA damage signalling – roles of p53, p73 and HIPK2 ppt

Recent studies strongly suggest an important tumour-suppressive role of the DNA damage response DDR in humans: molecular markers indicative for an active DDR, including site-specifically

Apoptosis and autophagy: Regulation of apoptosis

by DNA damage signalling – roles of p53, p73 and HIPK2 Nadja Bitomsky and Thomas G Hofmann

German Cancer Research Center (DKFZ), Cellular Senescence Group, DKFZ-ZMBH Alliance, Heidelberg, Germany

Introduction

Protection of the genome and maintenance of genomic

integrity following genotoxic stress is a crucial step in

counteracting tumorigenesis Eukaryotic organisms,

from yeasts to humans, have developed efficient

molec-ular mechanisms to sense different types of DNA

damage These different qualities of DNA damage

include DNA double-strand breaks (potently induced

by ionizing radiation), base modifications (e.g induced

by alkyllating agents such as N-methyl-N-nitrosourea), DNA crosslinks (e.g induced by cisplatin) and stalling

of replication forks in the S phase of the cell cycle (e.g elicited by topoisomerase inhibitors such as camptothe-cin and etoposide) [1] Recent studies strongly suggest

an important tumour-suppressive role of the DNA damage response (DDR) in humans: molecular markers indicative for an active DDR, including site-specifically

Keywords

apoptosis; ataxia-telangiectasia mutated

(ATM); ataxia-telangiectasia mutated

and Rad3-related (ATR); DNA damage;

homeodomain-interacting protein

kinase 2 (HIPK2); nuclear bodies; p53;

p73; promyelocytic leukaemia

(PML)

Correspondence

T G Hofmann, German Cancer

Research Center, Cellular Senescence

Group A210, DKFZ-ZMBH Alliance, Im

Neuenheimer Feld 242, 69120 Heidelberg,

Germany

Fax: +49 (0)6221 424902

Tel: +49 (0)6221 424631

E-mail: t.hofmann@dkfz.de

(Received 13 March 2009, revised 14

August 2009, accepted 27 August

2009)

doi:10.1111/j.1742-4658.2009.07331.x

Genomic stability is constantly threatened by DNA damage, caused by numerous environmental and intrinsic sources, including radiation, chemi-cals and oncogene expression Consequently, cells have evolved a sophisti-cated signal transduction network to sense DNA damage and to mount an appropriate DNA damage response Dysregulation of the DNA damage response leads to genomic instability and cancer Dependent on the cellular background and extent of DNA damage, the DNA damage response trig-gers cell cycle arrest and DNA repair, or in the case of irreparable damage, inactivation of the cells by senescence or apoptosis In this minireview, we concentrate on the apoptotic response to DNA damage and signalling pathways linked to the cell nucleus and nuclear bodies, with a particular focus on the molecular players p53 and p73 and on the DNA damage-acti-vated kinase homeodomain-interacting protein kinase 2 (HIPK2)

Abbreviations

ATM, ataxia-telangiectasia mutated; ATR, ATM and Rad3-related; Bak, Bcl-2 homologous antagonist ⁄ killer; CBP, CREB binding protein; Bax, breakpoint cluster-2-associated x protein; Bcl-2, B-cell lymphoma 2; CtBP, C-terminal binding protein; DDR, DNA damage response; HDM2/ MDM2, human double minute/murine double minute 2; HIPK2, homeodomain-interacting protein kinase 2; IR, ionizing radiation; JNK, c-Jun N-terminal kinase; NB, nuclear body; PML, promyelocytic leukaemia; Puma, p53-upregulated modulator of apoptosis; Siah1, seven in absentia homologue 1; TGF-b, transforming growth factor-b; YAP1, Yes-associated protein 1.

phosphorylated ataxia-telangiectasia mutated (ATM),

p53 and histone H2AX, have been found in early

neo-plastic lesions, but not in full-blown cancerous lesions

where the DDR is typically compromised [2]

The first signal transduction wave in response to

DNA damage is governed by rapid activation of

checkpoint kinases belonging to the family of

phospha-tidylinositol-3-OH-kinase-like protein kinases The

currently best studied

phosphatidylinositol-3-OH-kinase-like protein kinase members are ATM, and

ATM and Rad3-related (ATR) [3–5] Following DNA

double-strand breakage, cells primarily activate ATM,

which becomes recruited as an inactive dimer to the

DNA lesion by a sensor complex comprising the

pro-teins Mre11, Rad50 and NBS1 (i.e the MRN

com-plex) The MRN complex and ATM locate at the

damaged DNA foci marked by phosphorylated histone

H2AX (c-H2AX) where ATM becomes fully activated

by autophosphorylation and

phosphorylation-depen-dently regulates numerous downstream mediators to

coordinate the DDR [1] By contrast, the ATR kinase

mainly senses stress during DNA replication in the S

phase Here, single-stranded DNA becomes opsonized

by the replication protein A, which recruits ATR via

the ATR-interacting protein to the DNA lesions and

orchestrates DNA-topoisomerase II beta-binding

pro-tein (TopBP1)-dependent ATR activation [6] Both

ATM and ATR phosphorylate, and thereby activate,

further checkpoint kinases, including Chk1 and Chk2,

to transmit the damage signal to effector molecules

such as the tumour suppressor protein p53

Interest-ingly, recent studies identified coordinated crosstalk

mechanisms between ATM and ATR, indicating that

their downstream signalling routes are actually not

running separately, as supposed initially [7–9]

Depending on the cellular context and the extent of

DNA damage – which determines whether or not

dam-age is reparable – the activated DDR can trigger

differ-ent cellular responses Mild DNA damage is usually

handled through induction of cell cycle arrest through

the upregulation of cyclin-dependent kinase inhibitors,

such as p21, and subsequent repair of the lesions To

achieve faithful repair, cells can engage numerous

sophisticated DNA-repair mechanisms [10] In response

to irreparable DNA damage, the cellular response

switches towards induction of the senescence or

cell-death programme The molecular basis underlying the

decision making is currently subject of intense

investi-gation Although the cellular background appears to

play a major role, as for instance fibroblasts prefer to

undergo senescence whereas thymocytes favour cell

death induction, the molecular switch remains largely

unclear In this minireview we focus on the apoptotic

signalling routes of the DDR regulated mainly from the cell nucleus and the key molecules p53, p73 and homeodomain-interacting protein kinase 2 (HIPK2)

Multiple functions of p53 in DNA damage-induced apoptosis

The tumour suppressor and transcription factor p53 is

a major regulator of the cellular defence against neo-plastic transformation and cancer development Up to 50% of all human tumours show mutations in the p53 gene, which result in the expression of functionally inactive p53 or in complete loss of p53 expression In tumours expressing wild-type p53, its ability to repress cancer development often becomes functionally inacti-vated via the upregulation of critical negative regula-tors of p53, including its ubiquitin ligase HDM2⁄ MDM2 [11,12] Polyubiquitination and subse-quent proteasomal degradation are major efforts to keep the p53 protein levels low in healthy cells [12] In general, p53 activity in response to DNA damage is tightly controlled by its post-translational modification status and that of its E3 ubiquitin ligases, in particular through site-specific phosphorylation, acetylation and ubiquitination [13,14] Consistently, p53 is phosphory-lated by numerous DNA damage-activated protein kinases, including ATM, ATR, Chk1, Chk2 and HIPK2 [14]

Nuclear p53: regulation by HIPK2 and promyelocytic leukemia protein nuclear bodies

Upon DNA damage, as triggered by UV light, ionizing radiation (IR) and chemotherapeutic drug treatment, p53 is stabilized and activated DNA damage-induced p53 stabilization and activation is mediated primarily

by inactivating the negative regulatory effect of the p53 ubiquitin ligase HDM2 (MDM2 in mouse) In this context, ATM- and ATR-mediated phosphorylation of p53 at Ser15, and Chk1⁄ 2-mediated phosphorylation

at Ser20, as well as phosphorylation of MDM2 at Ser395 by ATM, are critical events (Fig 1) [13,14] Depending on the extent of damage, p53 induces transcription of different sets of target genes, leading

to cell cycle arrest, apoptosis or cellular senescence Interestingly, nuclear p53 has been demonstrated to be recruited to promyelocytic leukemia protein (PML) nuclear bodies (NBs) through interaction with the tumour suppressor PML in response to IR-induced apoptosis as well as oncogene-induced senescence after expression of oncogenic variant of the small GTPase Ras [15,16] In this context, the PML isoform PML IV

has been shown to act as a cofactor in p53-dependent

transcription In addition, a further critical p53

regula-tor, the acetyltransferase CREB binding protein (CBP),

is corecruited by PML to PML-NBs, which results in

increased p53 Lys382 acetylation and p53 activation

[15] Consequently, PML-NBs are envisioned as

macromolecular multiprotein complexes with a critical

role in regulating cell death, senescence and

differentia-tion [17–19]

Presumably the most prominent mark in priming the

apoptotic activity of p53 is phosphorylation at Ser46

[20] This phosphorylation mark is clearly associated

with severe DNA damage (elicited by UV light, IR,

adriamycin⁄ doxorubicin or cisplatin) and has been shown to drive the expression of apoptotic p53 target genes, such as p53-upregulated modulator of apoptosis (Puma), p53 regulated apoptosis-inducing protein 1 (p53AIP1) and breakpoint cluster-2-associated x pro-tein (Bax) [11] Subsequently, HIPK2, a conserved Ser⁄ Thr kinase predominantly localizing to NBs, has been identified as the p53 Ser46 kinase [21,22] HIPK2 phosphorylates p53 at Ser46 in response to UV light,

IR and treatment with adriamycin and cisplatin [21–25] Upon severe damage induced by UV light, HIPK2 binds p53 and is recruited to PML-NBs in a PML-dependent manner [21,22,26] Consistently, PML

Fig 1 Regulation of DNA damage-induced cell death by p53 and HIPK2 Genotoxic stress-induced DNA damage facilitates activation of the DNA damage-activated protein kinases ATM and ATR ATR and ATM in turn phosphorylation-dependently activate the downstream check-point kinases Chk1 and Chk2, respectively, and the tumour suppressor p53 Furthermore, ATM and ATR mediate HIPK2 activation by facili-tating its stabilization through phosphorylation of the HIPK2 ubiquitin ligase, Siah1, which facilitates disruption of the HIPK2–Siah1 complex Once stabilized and activated, HIPK2 can bind p53 and is recruited to PML-NBs via interacting with PML HIPK2 phosphorylates p53 at Ser46 and stimulates pro-apoptotic p53 target genes, including caspase-6 (Casp-6) and Pml In an autoregulatory feedback mechanism, cas-pase-6 potentiates HIPK2 activity by removing its C-terminal autoinhibitory domain In addition, HIPK2, and also JNK, can induce cell death in

a p53-independent manner by phosphorylation-dependent degradation of the anti-apoptotic corepressor CtBP Beyond its nuclear function, p53 is shuttled into the cytoplasm in response to DNA damage where it targets the mitochondria by activation of Bax and Bak, resulting in the release of pro-apoptotic factors and apoptotic cell death.

is a critical cofactor for efficient HIPK2-driven p53

Ser46 phosphorylation upon treatment with adriamycin

[26,27] In addition, HIPK2 also interacts with the

CBP acetyltransferase and colocalizes with CBP and

p53 at PML-NBs [21] HIPK2-mediated p53 Ser46

phosphorylation enhances CBP-mediated p53

acetyla-tion at Lys382, which leads to full transcripacetyla-tional

activation of p53, thereby potentiating the expression

of pro-apoptotic target genes [21] The apoptotic signal

can be additionally boosted through the p53-dependent

upregulation of PML expression This

positive-feed-back loop leads to PML accumulation and potentiation

of the apoptotic signal [29]

HIPK2 regulation

Tumour suppressor p53 not only serves as a critical

HIPK2 substrate, but also potentiates HIPK2 activity

by transcriptional upregulation of caspase-6 in response

to adriamycin-induced apoptosis Caspase-6 cuts off the

C-terminal negative-regulatory domain of HIPK2,

which results in a hyperactivated truncated HIPK2

iso-form and increased p53 Ser46 phosphorylation and

apoptosis induction [25] As p53 can also negatively

reg-ulate HIPK2 stability (see below) in response to damage

by sublethal concentrations of adriamycin [30], or

during recovery from reparable UV damage [31], p53 shows an apparent split personality in regard to HIPK2 regulation The switch between these opposing p53 func-tions appears to be regulated by the extent of DNA damage, which in turn determines whether DNA lesions can, or cannot, be repaired As unrepaired DNA dam-age is characterized by constant activity of the DNA damage checkpoint kinase ATM and⁄ or ATR, continu-ous ATM⁄ ATR activity may represent a key regulatory switch in apoptosis induction through facilitating prolonged HIPK2 stabilization and activation [31] Similarly to p53, HIPK2 is an unstable protein in unstressed cells because it is constantly degraded through the ubiquitin–proteasome system HIPK2 pro-tein levels are kept low in unstressed cells by polyubiq-uitination, which is carried out by the E3 ubiquitin ligases seven in absentia homolog-1 (Siah1), seven in absentia homolog-2 (Siah2) and WD-repeat and sup-pressor of cytokine signalling (SOCS) box-containing-1 (WSB1) [31,32] In response to treatment with UV light and adriamycin, HIPK2 degradation by Siah1 and WSB-1 is released, resulting in the accumulation

of HIPK2 In this context, Siah1 becomes phosphory-lated by ATM and ATR at Ser19, which leads to dis-ruption of the HIPK2–Siah1 complex, thus allowing HIPK2 stabilization and activation [31] Remarkably,

Fig 2 Regulation of DNA damage-induced

cell death by the p73 pathway In response

to DNA damage, JNK

phosphorylation-dependently liberates the tyrosine kinase

c-Abl from its cyctoplasmic anchor protein

14-3-3f Subsequently, c-Abl is translocated

into the nucleus where it becomes fully

activated through site-specific

phosphoryla-tion by ATM c-Abl regulates p73-induced

pro-apoptotic target gene expression by

direct phosphorylation of p73 at Tyr99 and

through phosphorylating YAP1, the cofactor

of p73 YAP1, in addition, stabilizes p73 by

protecting it against ubiquitination by the E3

ubiquitin ligase Itch Furthermore, YAP1

interacts physically with PML, which in turn

stabilizes YAP1 through SUMOylation p73,

YAP1, PML and p300 form a potent

plat-form for transcriptional activation of critical

pro-apoptotic target genes As PML is a p73

target gene, p73 activates a positive

feedback loop, further stimulating p73

activity.

during cellular recovery from reparable UV damage,

accumulated HIPK2 is rapidly removed through

Siah1-mediated degradation, which inhibits cell death

[31] Although HIPK2 is stabilized upon repairable

UV damage, p53 Ser46 phosphorylation remains

absent under these conditions [31] The function and

the substrates of HIPK2, in response to repairable

damage, remain elusive; however, it is tempting to

speculate that HIPK2 is also implicated in

nonapop-totic pathways, such as coordination of DNA repair

Furthermore, HIPK2 also appears to be degraded

through involvement of the SCFFbx3 E3 ubiquitin

ligase complex, and the degradation is inhibited by

PML, thereby resulting in increased p53 transcriptional

activity [33] Remarkably, overexpression of the acute

promyelocytic leukemia (APL)-causing chromosomal

translocation-derived fusion protein PML–RARa

dras-tically destabilizes HIPK2 [33] How these pathways

affect HIPK2 activity and p53 Ser46 phosphorylation

in response to DNA damage remains to be elucidated

Another means to keep the proapoptotic activity of

HIPK2 in check is sequestration to the cytoplasm

Overexpression of the high-mobility group protein A1

(HMGA1) oncoprotein relocalizes HIPK2 into the

cytoplasm and inhibits p53 Ser46 phosphorylation

upon UV light-induced damage in HCT116 cells [34]

Collectively, these findings indicate that the apoptotic

function of HIPK2 is vulnerable and can be

dysregu-lated at different levels

Cytoplasmic p53: targeting the

mitochondria

It is well established that p53 acts as a transcription

factor primarily located to the nucleus However, there

is emerging experimental evidence that p53 has

additional functions in apoptosis induction in the

cyto-plasm (see Fig 1) In the mid-1990s it was discovered

that p53 is capable of inducing apoptosis upon

expo-sure to UV light, not only by transcription-dependent

mechanisms but also by transcription-independent

mechanisms [35] Remarkably, it has been

demon-strated that transactivation activity-deficient p53 is still

capable of inducing programmed cell death through

the intrinsic pathway in response to ectopic p53

expression [36], and that recombinant p53 is capable

of triggering mitochondrial membrane

permeabiliza-tion in cell-free systems [37,38] Later on, p53 has been

reported to translocate to the cytoplasm in response to

numerous stress signals, including DNA damage,

hypoxia and oncogene expression, where it drives

mitochondrial outer membrane permeabilization and

caspase activation [39,40]

Transcription-independent cytoplasmic apoptosis-inducing functions of p53 are carried out by regulating the activity of Bcl-2 family members in IR-treated and camptothecin-treated cells p53 interacts with both pro-apoptotic and anti-apoptotic members of the Bcl-2 protein family p53 is able to interact (via its core DNA-binding domain) with the anti-apoptotic mole-cules B-cell lymphoma-extra large (Bcl-xL) and Bcl-2 [40] Remarkably, nuclear p53-dependent upregulation

of Puma results in increased cytoplasmic Puma levels, which facilitate liberation of cytoplasmic p53 from Bcl-xL, thus contributing directly to the mitochondrial cell-death route in response to UV light-induced dam-age [41] Additionally, p53 also interacts with the pro-apoptotic Bcl-2 homologous antagonist⁄ killer (Bak) protein This interaction seems to liberate Bak from its inhibitor protein, myeloid cell leukaemia 1 (Mcl-1), to induce Bak oligomerization, pore formation and subse-quent mitochondrial outer membrane permeabilization after treatment with adriamycin [42] In the case of the pro-apoptotic Bax protein, no physical interaction of p53 and Bax was observed, although p53 can induce Bax oligmerization and cytochrome c release [41,43]

So, how does p53 receive its signal for cytoplasmic and mitochondrial translocation in respect of lacking a classical mitochondrial translocation motif? As p53 post-translational modification is the most prominent event to regulate its function in response to DNA damage, it seemed to be promising to search for altera-tions between cytoplasmic p53 and nuclear p53 How-ever, the first analyses of the phosphorylation and acetylation patterns of active cytoplasmic p53 failed to detect any major differences between nuclear and cyto-plasmic p53 in IR-damaged cells [44] Interestingly, ubiquitin ligase HDM2⁄ MDM2 has been previously demonstrated to regulate p53 also by mono-ubiquitina-tion Mono-ubiquitination is not sufficient to target p53 for proteasomal degradation Consistently, it has been shown that p53 mono-ubiquitination within its C-terminus indeed assists its nucleocytoplasmic trans-location [45–47] A recently discovered novel p53 E3 ubiquitin ligase, called MSL2, which, unlike MDM2, does not regulate p53 turnover, mediates p53 mono-ubiquitination at Lys351 and Lys357, and MDM2-independent nucleo-cytoplasmic translocation upon treatment with etoposide [48] Whether this simply leads to p53 inactivation by removing it from its target gene promotors, or contributes to the apoptotic func-tion of p53 in the cytoplasm, remains to be investi-gated In addition, it is also conceivable that MSL2 contributes to the tumour suppressor function of p53

by inhibition of autophagy (self-eating), a recently uncovered novel function for cytoplasmic (but not

nuclear) p53 [49] Through promoting catabolic

reac-tions, autophagy facilitates the maintenance of high

ATP levels and survival in response to nutrient

deple-tion, hypoxia and the DNA damage-inducing drug

etoposide [50,51] Depletion, inhibition or loss of p53

leads to the induction of autophagy and increases cell

survival in response to stress [49]

p73 function in DNA damage-induced

cell death

Another key molecule critically involved in DNA

dam-age-induced cell death signalling is the p53-related

tumour suppressor and transcription factor p73 (see

Fig 2) Similarly to p53, p73 is an unstable molecule

and is expressed in various isoforms [52] In unstressed

cells, p73 forms a complex with the E3 ubiquitin ligase

Itch, which marks it for degradation by the ubiquitin–

proteasome system Upon DNA damage (by UV

irradiation or the DNA-damaging chemotherapeutics

adriamycin, etoposide and cisplatin), the levels of Itch

become reduced and allow the accumulation of p73

[53] p73 displays functions in apoptosis induction, and

many of its pro-apoptotic target genes indeed overlap

with those of p53, for example Puma, caspase-6 or

CD95 [54] Like p53, p73 is also recruited to

PML-NBs upon DNA damage, like other key players in

DNA damage-induced cell death signalling (see below)

Moreover, p73 also binds to HIPK2, and both factors

colocalize in NBs [55] Although HIPK2 has been

shown to drive p73-dependent transcription of an

arti-ficial reporter construct, the physiological role of the

HIPK2-p73 interaction is currently unclear [55]

Whether there exists a similar activation loop between

p73 and HIPK2, as previously described for HIPK2

and p53, also remains to be clarified

Post-translational modifications of p73 by

acetyla-tion through p300 and by phosphorylaacetyla-tion by the

DNA damage-activated, nonreceptor tyrosine kinase

c-Abl were found to be crucial for transactivating its

pro-apoptotic target genes after treatment with

adria-mycin [56] In undamaged cells c-Abl is sequestered to

the cytoplasm by its interaction with 14-3-3f, which

becomes phosphorylated by c-Jun N-terminal kinase

(JNK) upon damage caused by treatment with

adriamycin, thus triggering the release of 14-3-3f and

translocation of c-Abl to the nucleus [57]

Once translocated to the nucleus, c-Abl is

phosphor-ylated by ATM at Ser465 after IR [58,59]

Phosphory-lation at Ser465 leads to subsequent activation of

c-Abl and facilitates p73 transcriptional activation

through c-Abl-mediated phosphorylation of p73 at

Tyr99 [60] In addition, a key regulator of p73 activity,

Yes-associated protein 1 (YAP1), also becomes phos-phorylated by c-Abl This phosphorylation mark is essential to drive p73-mediated apoptosis by focussing the co-activator function of YAP1 on p73 in cells exposed to IR or cisplatin [61] YAP1 is also critical to protect p73 from proteasomal degradation upon dam-age caused by treatment with cisplatin by competing with its E3 ubiquitin ligase Itch for p73 binding Accordingly, YAP1 downregulation by RNA interfer-ence decreases induction of apoptosis in p53-deficient, p73-proficient H1299 cells following treatment with cisplatin [62]

Recently, it has been demonstrated that PML is also

a direct target gene of the p73–YAP1 complex in response to treatment with cisplatin Moreover, PML also interacts physically with YAP1 and promotes YAP1 stabilization through facilitating modification

of YAP1 with the small ubiquitin-like modifier 1 (SUMO-1) [63] Thus, p73 – similarly to what has been previously reported for p53 [29] – further enhances its pro-apoptotic activity through an autoregulatory feed-back loop

Interestingly, p73 becomes processed by caspases, and the truncated versions of the p73 protein localize

at mitochondria and augment apoptosis induction in response to treatment with the death receptor ligand TRAIL (tumour necrosis factor related apoptosis indu-cing ligand) [64] However, whether p73 exerts a simi-lar function after DNA damage-induced cell death is currently unclear It will be interesting to see whether

or not p73 has cytoplasmic functions similar to those

of p53 In summary, p73 apparently shares numerous – but probably not all – regulatory principles and effector pathways with its famous brother p53

HIPK2 in p53-independent apoptosis routes

In addition to its fundamental role in p53-driven apop-tosis, HIPK2 also facilitates DNA damage-induced cell death in the absence of p53 In UV light-damaged cells, HIPK2 phosphorylation-dependently targets the anti-apoptotic transcriptional corepressor C-terminal binding protein (CtBP) for proteasomal destruction (see Fig 1) [65] CtBP plays a critical role in repressing pro-apoptic target genes, such as Bax [65,66] After treatment with UV light and cisplatin, HIPK2, and also the stress-activated protein kinase JNK1, phos-phorylate CtBP at Ser422 and thereby mark it for degradation [67] In addition, HIPK2 was also shown

to activate the JNK signalling pathway in hepatoma cells after treatment with transforming growth factor-b (TGF-b), making it likely that HIPK2 also contributes

to p53-independent cell death in response to DNA

damage, both directly and via the JNK signalling

path-way [68,69], which is also capable of stimulating cell

death via the mitochondrial pathway [70]

Interest-ingly, a recent report indicates that TGF-b mediates

activation of ATM in 293 cells, which results in p53

Ser15 phosphorylation [71] Therefore, it will be

inter-esting to study whether HIPK2 also plays a role in

such a cell death-inducing setting

Even though PML is a direct pro-apoptotic p53 and

p73 target gene in response to apoptotic stimuli, an

additional regulatory principle of PML regulation was

recently demonstrated: HIPK2 is able to stabilize PML

in a p53-independent manner following treatment with

doxorubicin by phosphorylating Ser8 and Ser38 [72]

PML phosphorylation is accompanied by an increased

SUMOylation and stability of PML, suggesting an

additional role of HIPK2 in regulating DNA

damage-induced cell death

Collectively, these findings indicate that HIPK2 is

involved in DNA damage-induced cell death signalling

by using different downstream signalling routes

involv-ing p53, CtBP, PML and JNK

Concluding remarks

Cell death activation from the nucleus is an important

regulatory principle in regulating the apoptotic

response to DNA damage In the past decade,

numer-ous pathways and molecular players responsible for

controlling DNA damage-induced apoptosis have been

identified, including sensors, mediators and

execution-ers In particular, tumour suppressor PML and its

associated PML-NB turned out to be a critical

signal-ling hub in coordinating the apoptotic arm of the

DDR PML-NBs functionally cooperate with pivotal

apoptotic molecules, including p53, p73 and HIPK2,

by regulating their localization and pro-apoptotic

func-tion However, much needs to be learned about

poten-tial crosstalk between these signalling pathways and

the molecular mechanisms underlying their regulation

An additional pressing question is whether these

sig-nalling pathways operate in parallel in a given cell or

whether they act in a cell type- or tissue-restricted

manner Last, but not least, the tumour-suppressive

activities of these players make it an attractive

approach to systematically mine their pathways for

novel targets in anticancer drug discovery

Acknowledgements

We want to apologize to all the authors who made

important contributions to the field that could not be

cited here because of space restrictions Work in our laboratory is funded by the Landesstiftung foundation

of the State of Baden-Wu¨rttemberg, the German Research Foundation, the German Cancer Aid, the DKFZ-ZMBH Alliance, the Network Ageing Research

in Heidelberg and the Helmholtz Association

References

1 Harper JW & Elledge SJ (2007) The DNA damage response: ten years after Mol Cell 28, 739–745

2 Bartek J, Bartkova J & Lukas J (2007) DNA damage signalling guards against activated oncogenes and tumour progression Oncogene 26, 7773–7779

3 Shiloh Y (2003) ATM and related protein kinases: safe-guarding genome integrity Nat Rev Cancer 3, 155–168

4 Kastan MB & Lim DS (2000) The many substrates and functions of ATM Nat Rev Mol Cell Biol 1, 179–186

5 Abraham RT (2004) PI 3-kinase related kinases: ‘big’ players in stress-induced signaling pathways DNA Repair (Amst) 3, 883–887

6 Kumagai A & Dunphy WG (2006) How cells activate ATR Cell Cycle 5, 1265–1268

7 Cuadrado M, Martinez-Pastor B, Murga M, Toledo LI, Gutierrez-Martinez P, Lopez E & Fernandez-Capetillo

O (2006) ATM regulates ATR chromatin loading in response to DNA double-strand breaks J Exp Med

203, 297–303

8 Jazayeri A, Falck J, Lukas C, Bartek J, Smith GC, Lukas J & Jackson SP (2006) ATM- and cell cycle-dependent regulation of ATR in response to DNA double-strand breaks Nat Cell Biol 8, 37–45

9 Stiff T, Walker SA, Cerosaletti K, Goodarzi AA, Petermann E, Concannon P, O’Driscoll M & Jeggo PA (2006) ATR-dependent phosphorylation and activation

of ATM in response to UV treatment or replication fork stalling EMBO J 25, 5775–5782

10 Rouse J & Jackson SP (2002) Interfaces between the detection, signaling, and repair of DNA damage Science 297, 547–551

11 Vousden KH & Lu X (2002) Live or let die: the cell’s response to p53 Nat Rev Cancer 2, 594–604

12 Brooks CL & Gu W (2006) p53 ubiquitination: Mdm2 and beyond Mol Cell 21, 307–315

13 Maya R, Balass M, Kim ST, Shkedy D, Leal JF, Shifman O, Moas M, Buschmann T, Ronai Z, Shiloh Y

et al.(2001) ATM-dependent phosphorylation of Mdm2

on serine 395: role in p53 activation by DNA damage Genes Dev 15, 1067–1077

14 Bode AM & Dong Z (2004) Post-translational modifica-tion of p53 in tumorigenesis Nat Rev Cancer 4, 793– 805

15 Pearson M, Carbone R, Sebastiani C, Cioce M, Fagioli

M, Saito S, Higashimoto Y, Appella E, Minucci S,

Pandolfi PP et al (2000) PML regulates p53 acetylation

and premature senescence induced by oncogenic Ras

Nature 406, 207–210

16 Guo A, Salomoni P, Luo J, Shih A, Zhong S, Gu W &

Pandolfi PP (2000) The function of PML in

p53-depen-dent apoptosis Nat Cell Biol 2, 730–736

17 Hofmann TG & Will H (2003) Body language: the

function of PML nuclear bodies in apoptosis regulation

Cell Death Differ 10, 1290–1299

18 Bernardi R & Pandolfi PP (2007) Structure, dynamics

and functions of promyelocytic leukaemia nuclear

bodies Nat Rev Mol Cell Biol 8, 1006–1016

19 Krieghoff-Henning E & Hofmann TG (2008) Role of

nuclear bodies in apoptosis signalling Biochim Biophys

Acta 1783, 2185–2194

20 Oda K, Arakawa H, Tanaka T, Matsuda K, Tanikawa

C, Mori T, Nishimori H, Tamai K, Tokino T,

Nakamura Y et al (2000) p53AIP1, a potential

mediator of p53-dependent apoptosis, and its regulation

by Ser-46-phosphorylated p53 Cell 102, 849–862

21 Hofmann TG, Moller A, Sirma H, Zentgraf H, Taya

Y, Droge W, Will H & Schmitz ML (2002) Regulation

of p53 activity by its interaction with

homeodomain-interacting protein kinase-2 Nat Cell Biol 4, 1–10

22 D’Orazi G, Cecchinelli B, Bruno T, Manni I,

Higashim-oto Y, Saito S, Gostissa M, Coen S, Marchetti A, Del

Sal G et al (2002) Homeodomain-interacting protein

kinase-2 phosphorylates p53 at Ser 46 and mediates

apoptosis Nat Cell Biol 4, 11–19

23 Dauth I, Kruger J & Hofmann TG (2007)

Homeodo-main-interacting protein kinase 2 is the ionizing

radia-tion-activated p53 serine 46 kinase and is regulated by

ATM Cancer Res 67, 2274–2279

24 Di Stefano V, Rinaldo C, Sacchi A, Soddu S & D’Orazi

G (2004) Homeodomain-interacting protein kinase-2

activity and p53 phosphorylation are critical events for

cisplatin-mediated apoptosis Exp Cell Res 293, 311–

320

25 Gresko E, Roscic A, Ritterhoff S, Vichalkovski A, Del

Sal G & Schmitz ML (2006) Autoregulatory control of

the p53 response by caspase-mediated processing of

HIPK2 EMBO J 25, 1883–1894

26 Moller A, Sirma H, Hofmann TG, Rueffer S, Klimczak

E, Droge W, Will H & Schmitz ML (2003) PML is

required for homeodomain-interacting protein kinase 2

(HIPK2)-mediated p53 phosphorylation and cell cycle

arrest but is dispensable for the formation of HIPK

domains Cancer Res 63, 4310–4314

27 Sombroek D & Hofmann TG (2009) How cells switch

HIPK2 on and off Cell Death Differ 16, 187–194

28 Reference withdrawn

29 de Stanchina E, Querido E, Narita M, Davuluri RV,

Pandolfi PP, Ferbeyre G & Lowe SW (2004) PML is a

direct p53 target that modulates p53 effector functions

Mol Cell 13, 523–535

30 Rinaldo C, Prodosmo A, Mancini F, Iacovelli S, Sacchi

A, Moretti F & Soddu S (2007) MDM2-regulated degra-dation of HIPK2 prevents p53Ser46 phosphorylation and DNA damage-induced apoptosis Mol Cell 25, 739–750

31 Winter M, Sombroek D, Dauth I, Moehlenbrink J, Scheuermann K, Crone J & Hofmann TG (2008) Control of HIPK2 stability by ubiquitin ligase Siah-1 and checkpoint kinases ATM and ATR Nat Cell Biol

10, 812–824

32 Choi DW, Seo YM, Kim EA, Sung KS, Ahn JW, Park

SJ, Lee SR & Choi CY (2008) Ubiquitination and deg-radation of homeodomain-interacting protein kinase 2

by WD40 repeat⁄ SOCS box protein WSB-1 J Biol Chem 283, 4682–4689

33 Shima Y, Shima T, Chiba T, Irimura T, Pandolfi PP & Kitabayashi I (2008) PML activates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation Mol Cell Biol 28, 7126–7138

34 Pierantoni GM, Rinaldo C, Mottolese M, Di Benedetto

A, Esposito F, Soddu S & Fusco A (2007) High-mobil-ity group A1 inhibits p53 by cytoplasmic relocalization

of its proapoptotic activator HIPK2 J Clin Invest 117, 693–702

35 Caelles C, Helmberg A & Karin M (1994) p53-depen-dent apoptosis in the absence of transcriptional activa-tion of p53-target genes Nature 370, 220–223

36 Haupt Y, Rowan S, Shaulian E, Vousden KH & Oren

M (1995) Induction of apoptosis in HeLa cells by trans-activation-deficient p53 Genes Dev 9, 2170–2183

37 Ding HF, McGill G, Rowan S, Schmaltz C, Shimamura

A & Fisher DE (1998) Oncogene-dependent regulation

of caspase activation by p53 protein in a cell-free system J Biol Chem 273, 28378–28383

38 Schuler M, Bossy-Wetzel E, Goldstein JC, Fitzgerald P

& Green DR (2000) p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

J Biol Chem 275, 7337–7342

39 Marchenko ND, Zaika A & Moll UM (2000) Death signal-induced localization of p53 protein to mitochon-dria A potential role in apoptotic signaling J Biol Chem 275, 16202–16212

40 Mihara M, Erster S, Zaika A, Petrenko O, Chittenden

T, Pancoska P & Moll UM (2003) p53 Has a Direct Apoptogenic Role at the Mitochondria Mol Cell 11, 577–590

41 Chipuk JE, Bouchier-Hayes L, Kuwana T, Newmeyer

DD & Green DR (2005) PUMA couples the nuclear and cytoplasmic proapoptotic function of p53 Science

309, 1732–1735

42 Leu JI, Dumont P, Hafey M, Murphy ME & George

DL (2004) Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex Nat Cell Biol 6, 443–450

43 Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M & Green DR (2004) Direct

activation of Bax by p53 mediates mitochondrial

mem-brane permeabilization and apoptosis Science 303,

1010–1014

44 Nemajerova A, Erster S & Moll UM (2005) The

post-translational phosphorylation and acetylation

modifica-tion profile is not the determining factor in targeting

endogenous stress-induced p53 to mitochondria Cell

Death Differ 12, 197–200

45 Carter S, Bischof O, Dejean A & Vousden KH (2007)

C-terminal modifications regulate MDM2 dissociation

and nuclear export of p53 Nat Cell Biol 9, 428–435

46 Marchenko ND, Wolff S, Erster S, Becker K & Moll

UM (2007) Monoubiquitylation promotes

mitochon-drial p53 translocation EMBO J 26, 923–934

47 Li M, Brooks CL, Wu-Baer F, Chen D, Baer R & Gu

W (2003) Mono- versus polyubiquitination: differential

control of p53 fate by Mdm2 Science 302, 1972–1975

48 Kruse JP & Gu W (2009) MSL2 promotes

Mdm2-inde-pendent cytoplasmic localization of p53 J Biol Chem

284, 3250–3263

49 Tasdemir E, Maiuri MC, Galluzzi L, Vitale I,

Djava-heri-Mergny M, D’Amelio M, Criollo A, Morselli E,

Zhu C, Harper F et al (2008) Regulation of autophagy

by cytoplasmic p53 Nat Cell Biol 10, 676–687

50 Maiuri MC, Zalckvar E, Kimchi A & Kroemer G

(2007) Self-eating and self-killing: crosstalk between

autophagy and apoptosis Nat Rev Mol Cell Biol 8,

741–752

51 Katayama M, Kawaguchi T, Berger MS & Pieper RO

(2007) DNA damaging agent-induced autophagy

pro-duces a cytoprotective adenosine triphosphate surge in

malignant glioma cells Cell Death Differ 14, 548–558

52 Melino G (2003) p73, the ‘‘assistant’’ guardian of the

genome? Ann N Y Acad Sci 1010, 9–15

53 Rossi M, De Laurenzi V, Munarriz E, Green DR,

Liu YC, Vousden KH, Cesareni G & Melino G (2005)

The ubiquitin-protein ligase Itch regulates p73 stability

EMBO J 24, 836–848

54 Dobbelstein M, Strano S, Roth J & Blandino G (2005)

p73-induced apoptosis: a question of compartments and

cooperation Biochem Biophys Res Commun 331, 688–

693

55 Kim EJ, Park JS & Um SJ (2002) Identification and

characterization of HIPK2 interacting with p73 and

modulating functions of the p53 family in vivo J Biol

Chem 29, 29

56 Costanzo A, Merlo P, Pediconi N, Fulco M, Sartorelli

V, Cole PA, Fontemaggi G, Fanciulli M, Schiltz L,

Blandino G et al (2002) DNA damage-dependent

acetylation of p73 dictates the selective activation of

apoptotic target genes Mol Cell 9, 175–186

57 Yoshida K, Yamaguchi T, Natsume T, Kufe D &

Miki Y (2005) JNK phosphorylation of 14-3-3 proteins

regulates nuclear targeting of c-Abl in the apoptotic

response to DNA damage Nat Cell Biol 7, 278–285

58 Shafman T, Khanna KK, Kedar P, Spring K, Kozlov

S, Yen T, Hobson K, Gatei M, Zhang N, Watters D

et al.(1997) Interaction between ATM protein and c-Abl in response to DNA damage Nature 387, 520–523

59 Baskaran R, Wood LD, Whitaker LL, Canman CE, Morgan SE, Xu Y, Barlow C, Baltimore D, Wynshaw-Boris A, Kastan MB et al (1997) Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation Nature 387, 516– 519

60 Agami R, Blandino G, Oren M & Shaul Y (1999) Inter-action of c-Abl and p73alpha and their collaboration to induce apoptosis Nature 399, 809–813

61 Levy D, Adamovich Y, Reuven N & Shaul Y (2008) Yap1 phosphorylation by c-Abl is a critical step in selective activation of proapoptotic genes in response to DNA damage Mol Cell 29, 350–361

62 Levy D, Adamovich Y, Reuven N & Shaul Y (2007) The Yes-associated protein 1 stabilizes p73 by prevent-ing Itch-mediated ubiquitination of p73 Cell Death Differ 14, 743–751

63 Lapi E, Di Agostino S, Donzelli S, Gal H, Domany E, Rechavi G, Pandolfi PP, Givol D, Strano S, Lu X et al (2008) PML, YAP, and p73 are components of a proapoptotic autoregulatory feedback loop Mol Cell

32, 803–814

64 Sayan AE, Sayan BS, Gogvadze V, Dinsdale D, Nyman U, Hansen TM, Zhivotovsky B, Cohen GM, Knight RA & Melino G (2008) P73 and caspase-cleaved p73 fragments localize to mitochondria and augment TRAIL-induced apoptosis Oncogene 27, 4363–4372

65 Zhang Q, Yoshimatsu Y, Hildebrand J, Frisch SM & Goodman RH (2003) Homeodomain Interacting Pro-tein Kinase 2 Promotes Apoptosis by Downregulating the Transcriptional Corepressor CtBP Cell 115, 177– 186

66 Grooteclaes M, Deveraux Q, Hildebrand J, Zhang Q, Goodman RH & Frisch SM (2003) C-terminal-binding protein corepresses epithelial and proapoptotic gene expression programs Proc Natl Acad Sci U S A 100, 4568–4573

67 Wang SY, Iordanov M & Zhang Q (2006) c-Jun NH2-terminal kinase promotes apoptosis by down-regulating the transcriptional co-repressor CtBP J Biol Chem 281, 34810–34815

68 Hofmann TG, Stollberg N, Schmitz ML & Will H (2003) HIPK2 regulates transforming growth factor-beta-induced c-Jun NH(2)-terminal kinase activation and apoptosis in human hepatoma cells Cancer Res 63, 8271–8277

69 Hofmann TG, Jaffray E, Stollberg N, Hay RT & Will H (2005) Regulation of homeodomain-interacting protein kinase 2 (HIPK2) effector function through

dynamic small ubiquitin-related modifier-1 (SUMO-1)

modification J Biol Chem 280, 29224–29232

70 Lei K & Davis RJ (2003) JNK phosphorylation of

Bim-related members of the Bcl2 family induces

Bax-dependent apoptosis Proc Natl Acad Sci U S A

100, 2432–2437

71 Zhang S, Ekman M, Thakur N, Bu S, Davoodpour P,

Grimsby S, Tagami S, Heldin CH & Landstrom M

(2006) TGFbeta1-induced activation of ATM and p53

mediates apoptosis in a Smad7-dependent manner Cell Cycle 5, 2787–2795

72 Gresko E, Ritterhoff S, Sevilla-Perez J, Roscic A, Frobius K, Kotevic I, Vichalkovski A, Hess D, Hemmings BA & Schmitz ML (2009) PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage

Oncogene 28, 698–708