Autophosphorylation and Pin1 binding coordinate DNAdamage-induced HIPK2 activation and cell deathNadja Bitomskya, Elisa Conrada, Christian Moritzb, Tilman Polonio-Vallona, Dirk Sombroeka, Kathrin Schultheissa,Carolina Glasa, Vera Greinera, Christoph Herbela, Fiamma Mantovanic,d, Giannino del Salc,d, Francesca Perib,and Thomas G. Hofmanna,1

aGerman Cancer Research Center, Cellular Senescence Group, Deutsches Krebsforschungszentrum-Zentrum für Molekulare Biologie der UniversitätHeidelberg Alliance (DKFZ-ZMBH), 69120 Heidelberg, Germany; bEuropean Molecular Biology Laboratory (EMBL), 69117 Heidelberg, Germany;cLaboratorio Nazionale del Consorzio Interuniversitario per le Biotecnologie (LNCIB), 34012 Trieste, Italy; and dDipartimento di Scienze della Vita,Università degli Studi di Trieste, 34128 Trieste, Italy

Edited by Carol Prives, Columbia University, New York, NY, and approved September 23, 2013 (received for review May 28, 2013)

Excessive genome damage activates the apoptosis response. Proteinkinase HIPK2 is a key regulator of DNA damage-induced apoptosis.Here, we deciphered the molecular mechanism of HIPK2 activa-tion and show its relevance for DNA damage-induced apoptosisin cellulo and in vivo. HIPK2 autointeracts and site-specificallyautophosphorylates upon DNA damage at Thr880/Ser882. Auto-phosphorylation regulates HIPK2 activity and mutation of thephosphorylation-acceptor sites deregulates p53 Ser46 phosphory-lation and apoptosis in cellulo. Moreover, HIPK2 autophosphory-lation is conserved between human and zebrafish and is importantfor DNA damage-induced apoptosis in vivo. Mechanistically, auto-phosphorylation creates a binding signal for the phospho-specificisomerase Pin1. Pin1 links HIPK2 activation to its stabilization byinhibiting HIPK2 polyubiquitination and modulating Siah-1–HIPK2interaction. Concordantly, Pin1 is required for DNA damage-inducedHIPK2 stabilization and p53 Ser46 phosphorylation and is essentialfor induction of apotosis both in cellulo and in zebrafish. Our resultsidentify an evolutionary conserved mechanism regulating DNAdamage-induced apoptosis.

Activation of the apoptotic response upon severe genomedamage plays a crucial role in suppression of cellular trans-

formation and cancer development. In addition, apoptosis is amajor mechanism of action underlying the efficacy of widely usedDNA-damaging cancer therapies including radiotherapy andchemotherapy. To gain insight into the mechanisms underlyingcancer cell resistance to therapy, detailed knowledge about themolecular players and the regulatory network governing theDNA damage-induced apoptosis response is of fundamentalimportance.Tumor suppressor p53 is a master regulator of the DNA

damage response and drives expression of different sets of targetgenes that regulate cell fate decisions as DNA repair, senes-cence, and cell death (1, 2). p53 target gene selection after DNAdamage is controlled in part at the level of its posttranslationalmodifications, including site-specific phosphorylation and acety-lation. p53 phosphorylation is mediated by the DNA damagecheckpoint kinases ATM and ATR as well as their downstreamkinases Chk1, Chk2, and HIPK2, which control p53 stability,activity, and target gene selection through a complex signalingnetwork (3, 4).The Ser/Thr protein kinase homeodomain interacting protein

kinase 2 (HIPK2) is an evolutionarily conserved regulator of celldeath and cell growth during development and in response tocellular stress (5, 6). There is growing evidence that HIPK2 actsas a tumor suppressor both in mice and men (7–10) and that thekinase is functionally deregulated by cellular and candidate viraloncogenes (11). In addition to its role in cancer, HIPK2 dys-regulation has been linked to pathophysiology including neu-rodegeneration and kidney fibrosis (7, 12, 13).HIPK2 channels the apoptotic response upon DNA damage

induced by UV irradiation, ionizing radiation (IR), and chemo-

therapeutic drug treatment through different signaling pathwaysincluding phosphorylation of p53 at serine 46 and phosphoryla-tion-mediated degradation of antiapoptotic molecules such ascorepressor CtBP and transcription factor ΔNp63α (14–20).Unstressed cells and cells recovering from DNA damage keepHIPK2 activity low through targeting the kinase for protea-some-dependent degradation by the ubiquitin ligases WSB1and Siah-1 (21-23). In response to DNA damage, HIPK2 isstabilized through a mechanism involving the DNA damagecheckpoint kinases ATM and ATR, which facilitate dissociationof the HIPK2–Siah-1 complex, at least in part, by phosphor-ylation of Siah-1 (16, 22). However, the detailed mechanismsunderlying HIPK2 activation upon genotoxic stress remainsstill unclear.In the present study, we investigated the molecular mecha-

nism regulating HIPK2 activation in response to DNA damage.We show that HIPK2 activation is facilitated through a mecha-nism involving HIPK2 oligomerization and site-specific auto-phosphorylation at Thr880/Ser882. HIPK2 autophosphorylationincreases its kinase activity and apoptotic function both in celluloand in vivo. Mechanistically, we show that phosphorylation atThr880/Ser882 serves as a binding signal for the prolyl-peptidylcis/trans isomerase Pin1, which links HIPK2 activation to itsstabilization.

Significance

Activation of the cell death (apoptosis) program is a majorprinciple of DNA-damaging cancer treatments including ion-izing radiation and chemotherapeutic drug treatment. Theprotein kinase HIPK2 plays a key role in radiosensitivity andchemosensitivity. Here, we found that HIPK2 autointeractsand autophosphorylates after DNA damage. HIPK2 auto-phosphorylation is conserved in evolution and regulates itsapoptosis-inducing activity by facilitating binding of theisomerase Pin1. Pin1 couples HIPK2 activation to its stabili-zation and is essential for DNA damage-induced apoptosis incancer cells and in zebrafish. Our findings identify a mecha-nism linking HIPK2 activation to its stabilization and high-light a conserved function of HIPK2 and Pin1 in the DNAdamage-induced apoptosis response.

Author contributions: N.B. and T.G.H. designed research; N.B., E.C., C.M., T.P.-V., D.S., K.S.,C.G., V.G., C.H., and F.M. performed research; G.d.S. and F.P. contributed new reagents/analytic tools; N.B., E.C., C.M., T.P.-V., D.S., K.S., C.G., V.G., C.H., F.M., G.d.S., and T.G.H.analyzed data; and T.G.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: t.hofmann@dkfz-heidelberg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1310001110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1310001110 PNAS | Published online October 21, 2013 | E4203–E4212

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ResultsHIPK2 Autophosphorylates at Thr880/Ser882 in Vitro. To determinewhether HIPK2 autophosphorylates, we expressed kinase-proficient His-HIPK2 and kinase-deficient His-HIPK2K221A inEscherichia coli and purified the proteins by metal-affinitychromatography. In vitro kinase assays using [γ-32P]-ATP as aphosphate donor showed that only wild-type HIPK2 proteinautophosphorylated and phosphorylated the substrate myelinbasic protein (MBP) (Fig. 1A). Immunoblot analysis with apan-phospho-specific Ser/Thr (pSer/pThr) antibody showedstrong reactivity with the wild-type form of HIPK2, but notwith kinase-deficient HIPK2K221A (Fig. 1B). In addition, pre-treatment of HIPK2 with λ-phosphatase abrogated reactivity ofthe pSer/pThr antibody (Fig. 1C).For mapping of the autophosphorylation sites, we focused on the

C-terminal regulatory part of HIPK2, which contains an utoinhi-bitory domain (24). We speculated that phosphorylation in this partmay modulate HIPK2 activity by attenuating the autoinhibitoryfunction of the C terminus. In vitro mapping experiments of theHIPK2 autophosphorylation sites using site-directed mutagenesisidentified Thr880/Ser882 as the main HIPK2 autophosphorylationsite (Fig. 1D). Additional minor sites of autophosphorylation weredetectable that were largely abolished through mutation of addi-tional six Ser/Thr residues (S668, S827, T838, S848, S924, S934),resulting in a HIPK2S/T8A mutant (Fig. 1D).We next raised phosphorylation-specific antibodies against the

phospho-Thr880/Ser882 motif. The pThr880/pSer882 HIPK2 anti-bodies recognized bacterially expressed wild-type HIPK2,but failed to detect the HIPK2T880A/S882A mutant, HIPK2S/T8A,and the autophosphorylation-deficient HIPK2K221A protein (Fig.1D, Middle Lower blots). These results show that HIPK2 auto-

phosphorylates at Thr880/Ser882 in vitro and that our antibodiesspecifically recognize Thr880/Ser882-phosphorylated HIPK2.To gain insight into the mechanism underlying HIPK2 auto-

phosphorylation, we examined whether HIPK2 may autophos-phorylate in trans through an intermolecular mechanism. Thus,we performed in vitro kinase assays by using bacterially ex-pressed His-HIPK2K221A as substrate for Flag-HIPK2 and Flag-HIPK2K221A. To subsequently separate the autophosphorylatedFlag-HIPK2 from its substrate His-HIPK2K221A, we purified theHis-HIPK2K221A protein from the kinase reaction under dena-turing conditions by using metal-affinity purification on the Histag of the substrate protein. Immunoblot analysis of the His-HIPK2K221A showed clear phosphorylation at Thr880/Ser882 inthe presence of wild-type HIPK2, whereas virtually no phos-phorylation was found using HIPK2K221A (Fig. 1E). These resultssuggest that HIPK2 autophosphorylates in trans through anintermolecular mechanism.

HIPK2 Autophosphorylates at Thr880/Ser882 upon DNA Damage. Wenext investigated whether HIPK2 autophosphorylates in cells.The pThr880/pSer882 HIPK2 antibody specifically recognizedectopically expressed Thr880/Ser882-phosphorylated HIPK2 incells (Fig. S1A). To investigate whether HIPK2 autophosphor-ylation is stimulated upon DNA damage, we analyzed Thr880/Ser882 phosphorylation of ectopically expressed HIPK2. DNAdamage was induced by using the chemotherapeutic drugAdriamycin (Adria), a Topoisomerase II inhibitor inducing DNAdouble-strand breaks (25). Of note, DNA damage resulted in anincreased Thr880/Ser882 phosphorylation of HIPK2 (Fig. 2A).In addition, we were able to detect endogenous HIPK2 to bephosphorylated at Thr880/Ser882 in response to DNA damage

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Fig. 1. HIPK2 autophosphorylates at Thr880/Ser882 invitro. (A) HIPK2 autophosphorylates in vitro. Bac-terially expressed 6xHis-HIPK2 and kinase-deficient6xHis-HIPK2K221A proteins were incubated with MBPin the presence of [γ-32P]-ATP. Reactions were an-alyzed by SDS-PAGE and autoradiography. MBPinput was analyzed by Coomassie brilliant bluestaining; His-HIPK2 input by immunoblotting. n = 2.(B) HIPK2 autophosphorylates at Ser/Thr residues invitro. Bacterially expressed 6xHis-HIPK2 proteinswere analyzed by immunoblotting using pan-pSer/pThr- and HIPK2-specific antibodies. n = 2. (C) His-HIPK2 treated with λ-phosphatase or left untreatedwas analyzed by immunoblotting as in B. (D) Iden-tification of Thr880/Ser882 as the main HIPK2 auto-phosphorylation site. Bacterially expressed His-HIPK2proteins were analyzed by immunoblotting using pan-pSer/pThr- specific and HIPK2 pThr880/pSer882-specificantibodies. n = 2. (E) HIPK2 autophosphorylates byan intermolecular mechanism. Bacterially expressedHis-HIPK2K221A was used as substrate for Flag-HIPK2in an in vitro kinase reaction. His-HIPK2K221A was pu-rified from the kinase reaction under denaturing con-ditions by Ni-NTA beads, and the eluted proteintogether with the input was analyzed by immmunoblotting. n = 2. Representative experimentsare shown.

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(Fig. 2B). Endogenous HIPK2 Thr880/Ser882 phosphorylationwas evident at the early phase of HIPK2 activation after DNAdamage and dropped to background levels 20 h after damagewhen HIPK2 was stabilized.We used confocal microscopy to visualize the subcellular

compartment of HIPK2 autophosphorylation. First, we validated

the specificity of the pThr880/Ser882 HIPK2 antibody by usingoverexpression of wild-type HIPK2 and HIPK2T880A/S882A andknockdown of endogenous HIPK2 expression by RNAi (Fig. S2).Interestingly, Thr880/Ser882-phosphorylated HIPK2 localizedto the cell nucleus and to nuclear bodies (Fig. 2C). Stabilizationof HIPK2 and induction of DNA strand breaks was visualizedby staining with antibodies recognizing pan-HIPK2 and H2AXphosphorylated at Ser139 (γ-H2AX), respectively (Fig. 2C).These data suggest that HIPK2 autophosphorylation takesplace in the cell nucleus and nuclear bodies.

HIPK2 Autointeracts and Oligomerizes upon DNA Damage. To gainfurther insight into the mechanism underlying HIPK2 autophos-phorylation, we investigated whether HIPK2 autointeracts inresponse to DNA damage. To this end, we expressed differen-tially tagged HIPK2 proteins in human cells and performedcoimmunoprecipitation analysis in the absence and presence ofDNA damage. Indeed, immunoblotting revealed pronouncedHIPK2 autointeraction upon DNA damage, whereas only littleHIPK2 autointeraction was evident in undamaged cells (Fig.2D), suggesting that DNA damage potentiates complex forma-tion between HIPK2 molecules. Furthermore, in vitro GST-pulldown assays independently confirmed autointeraction prop-erties of HIPK2 (Fig. S1B). To address whether HIPK2 can formdimers or oligomers, we performed in vivo cross-linking experi-ments on ectopically expressed HIPK2 and analyzed the sampleson nonreducing gels. To control our experimental condition,we used p53, which is known to form dimers and tetramers(Fig. S1C). Exogenously expressed HIPK2 formed higher-ordercomplexes, suggesting that HIPK2 oligomerizes (Fig. S1C). Inaddition, endogenous HIPK2 also formed higher-molecularprotein complexes reminiscent of dimers and oligomers, whenstimulated by DNA damage (Fig. 2E). Autophosphorylation-defective HIPK2 mutants showed comparable autointeractionas wild-type HIPK2 (Fig. S2), indicating autophosphorylation isdispensable for HIPK2 autointeraction.

Autophosphorylation Modulates HIPK2 Kinase Activity and ItsApoptotic Function. We next investigated whether autophos-phorylation is critical for the apoptotic function of ectopicallyexpressed HIPK2 (15, 26). To this end, we compared theapoptotic activity of phosphorylation-deficient and mimeticHIPK2 mutants by using FACS analysis. In comparison with thewild-type HIPK2, HIPK2T880A/S882A showed a reduced apoptoticactivity, whereas phospho-mimetic HIPK2T880D/S882D showed in-creased apoptosis-activating function (Fig. 3A). Accordingly,HIPK2T880A/S882A exhibits reduced capacity to phosphory-late p53 at Ser46 (Fig. 3B), whereas HIPK2T880D/S882D showed in-creased phosphorylation of p53 at Ser46 and PARP cleavagecompared with the wild-type kinase (Fig. 3C). Because theautophosphorylation-defective HIPK2 point mutants showcomparable p53 interaction as their wild-type counterpart (Fig.S3), reduced p53 Ser46 phosphorylation is not due to alteredp53 binding.To test whether autophosphorylation of HIPK2 modulates its

kinase activity, we compared the kinase activity of the wild-typeprotein to the autophosphorylation-defective mutants. To thisend, we performed in vitro kinase assays by using [γ-32P]-ATP asa phosphate donor with bacterially expressed His-HIPK2 pro-teins purified by metal-affinity chromatography. HIPK2 kinaseactivity was determined by using the model substrate MBP.Compared with the wild-type kinase, HIPK2T880A/S882A exhibitsa reduced (40 ± 4%) kinase activity (Fig. 3D). In addition, a similarreduction in autophosphorylation was observed in HIPK2T880A/S882A

compared with the wild-type kinase (Fig. 3D). In contrast,phospho-mimetic HIPK2T880D/S882D showed a slightly increasedkinase activity in comparison with wild-type kinase (26 ± 3%)(Fig. S4A). Kinase-dead HIPK2K221A lacked detectably kinase

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Fig. 2. HIPK2 autophosphorylates at Thr880/Ser882 upon DNA damage. (A)DNA damage triggers autophosphorylation of ectopically expressed HIPK2.The 293T cells expressing Flag-HIPK2 were treated as indicated and analyzedby immunoblotting. n = 3. (B) Endogenous HIPK2 is phosphorylated atThr880/Ser882 upon DNA damage. HCT116 cells were treated with Adria-mycin (1 μg/mL) or solvent (control), and cell lysates were analyzed by im-munoblotting. (C) Thr880/Ser882-phosphorylated HIPK2 localizes to nuclearbodies. U2OS cells were treated with Adriamycin or solvent for 6 h andsubsequently analyzed by indirect immunofluorescence using HIPK2 pT880/pS882, HIPK2, and γ-H2AX antibodies. (Scale bars, 20 μm) (D) HIPK2 auto-interaction is potentiated upon DNA damage. HA-tagged and Flag-taggedHIPK2 were expressed in 293T cells as indicated, cells were treated withAdriamycin as shown, and immunoprecipitation and immunoblotting anal-ysis was performed. (E) Endogenous HIPK2 dimerizes/oligomerizes uponDNA damage. HCT116 cells were incubated either in the presence or absenceof Adriamycin (16 h, 1 μg/mL) or solvent. Protein complexes were cross-linkedwith dimethyl 3,3′-dithiobispropionimidate (2.5 mM in PBS) for 15 min.Cross-linking was resolved by treatment with β-mercaptoethanol (β-ME) asindicated. Total cell lysates were analyzed by immunoblotting using HIPK2-specific antibodies.

Bitomsky et al. PNAS | Published online October 21, 2013 | E4205

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activity and autophosphorylation. Similarly, the HIPK2S/T8A

mutant exhibited only faint kinase activity and autophosphor-ylation. Taken together, these results show that HIPK2 activityand apoptotic function is modulated by autophosphorylationat Thr880/Ser882.

Autophosphorylation of HIPK2 Is Conserved in Zebrafish andRegulates IR-Induced Apoptosis in Vivo. The HIPK2 autophos-phorylation site at Thr880/Ser882 is conserved between humanand zebrafish (Danio rerio) (Fig. 3E). Moreover, D. rerio HIPK2expressed in human cells is recognized by the phospho-specificHIPK2 antibody (Fig. 3F), demonstrating that HIPK2 auto-phosphorylation is conserved between human and zebrafish.Because zebrafish embryos have been recently established as

an in vivo model system to analyze DNA damage-induced apo-ptosis in response to IR (27), we addressed the effect of HIPK2autophosphorylation on IR-induced apoptosis in zebrafish

embryos. To this end, we injected embryos with mRNA encodingwild-type human HIPK2, kinase-dead HIPK2K221A, autophos-phorylation-defective HIPK2T880A/S882A, and an autophos-phorylation-mimetic HIPK2T880D/S882D mutant and controlledcomparable expression levels by immunoblotting of whole-bodyzebrafish embryo lysates (Fig. 3G). Injection of the HIPK2mRNA did not result in a changed developmental phenotypeand looked as empty vector-injected animals (Fig. 3G). Thus, 24 hafter fertilization, these embryos were exposed to whole-body IRof 12.5 Gy and analyzed 7.5 h after IR exposure by TUNEL andacridine orange (AO) staining. We quantified apoptosis in thespinal cord of the embryos (n = 60 for each construct forTUNEL and n > 10 for AO) and found that wild-type HIPK2, incontrast to kinase-dead HIPK2K221A, increased IR-inducedapoptosis in the embryos, indicating that the kinase-dependent,apoptosis-potentiating function of HIPK2 is conserved betweenhuman and fish (Fig. 3G). Interestingly, autophosphorylation-

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Fig. 3. HIPK2 autophosphorylation at Thr880/Ser882 regulates its apoptotic function in humancells and zebrafish embryos. (A) HIPK2 autophos-phorylation is critical for its apoptotic function.HCT116 cells were transfected with empty vectorsand HIPK2 expression vectors as indicated. Cellswere analyzed by FACS for apoptosis-associatedannexin V-positivity after annexin V/propidium io-dide staining. The graph shows means and SD. n =3. siGL indicates control siRNA transfected cells(black bars); siPin1 indicates cells depleted of Pin1(gray bars). Flag-HIPK2, Pin1, and p53 levels weredetermined by immunoblotting. A representativeresult is shown. (B and C) HIPK2 autophosphorylationat Thr880/Ser882 regulates p53 Ser46 phosphoryla-tion capacity. H1299 cells were transfected with theexpression vectors indicated, and cell lysates wereanalyzed by immunoblotting. (D) HIPK2 autophos-phorylation correlates with its kinase activity. Bacte-rially expressed 6xHis-HIPK2 proteins were used forradioactive in vitro kinase assay with MBP as sub-strate and analyzed by autoradiography. Input wascontrolled by Coomassie brilliant blue staining andHIPK2 immunoblotting. Input levels of the His-HIPK2proteins are shown in Fig. 1C, which was performedin parallel to this experiment here by using the samebatch and amounts of His-HIPK2 proteins. (E) TheHIPK2 autophosphorylation site is conserved be-tween zebrafish and humans. (F) D. rerio HIPK2 isautophosphorylated. H1299 cells were transfectedwith expression vectors coding for wild-type or ki-nase-deficient zebrafish HIPK2. Total cell extractswere analyzed by immunoblotting using the in-dicated antibodies. (G) HIPK2 autophosphorylation atT880/S882 potentiates apoptosis induction in zebra-fish embryos. Embyos were microinjected withmRNAs coding for HIPK2 proteins as indicated, ex-posed to IR (12.5 Gy), and analyzed and quantifiedfor TUNEL positivity.

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defective HIPK2T880A/S882A exhibited reduced apoptotic activity,whereas a phospho-mimetic HIPK2S880D/T882D mutant showedpotentiation of IR-induced apoptosis in the embryos (Fig. 3G).We also knocked-down endogenous HIPK2 in zebrafish embryosby using a HIPK2 Morpholino oligonucleotide (Fig. S5). Un-expectedly, depletion of HIPK2 resulted in a strong spontaneousapoptosis response (Fig. S5), suggesting HIPK2 is importantfor early zebrafish development. Because of the high apoptosislevels in HIPK2-depleted zebrafish embryos, we were not ableto determine the effect of HIPK2 depletion on IR-induced celldeath. In summary, our results indicate that autophosphorylationmodulates the apoptotic activity of HIPK2 both in cellulo andin vivo.

Pin1 Isomerase Binds Autophosphorylated HIPK2. HIPK2 autophos-phorylation sites consist of Ser/Thr residues followed by Pro. Ifsuch motifs are phosphorylated, they can, in principle, serve asbinding sites for the phosphorylation-specific prolyl-peptidyl cis/trans isomerase Pin1 (28). Pin1 translates specific phosphorylationmarks into changed protein conformation by isomerization of thepSer/pThr-Pro bond and, thus, can alter the function of its client

proteins. This feature prompted us to study whether Pin1 interactswith HIPK2 in an autophosphorylation-regulated manner.To investigate whether DNA damage-induced HIPK2 auto-

phosphorylation leads to complex formation between HIPK2and Pin1, we performed coimmunoprecipitation assays. Indeed,endogenous Pin1 coimmunoprecipitated with endogenousHIPK2 after DNA damage, whereas almost no Pin1–HIPK2complex formation was evident in undamaged cells (Fig. 4A).Pin1–HIPK2 interaction correlated with HIPK2 Thr880/Ser882phosphorylation and occurred early (6 h) after DNA damageinduction (Fig. 4A). Twenty hours after damage, when HIPK2is fully stabilized, Thr880/Ser882 autophosphorylation and Pin1binding were again reduced to background levels. This resultsuggests that Pin1–HIPK2 binding occurs at an early time windowafter DNA damage and that HIPK2 autophosphorylation isreversible.To determine whether HIPK2 autophosphorylation regulates

its complex formation with Pin1, we compared Pin1 bindingof wild-type HIPK2 and autophosphorylation-defective HIPK2using coimmunoprecipitation assays. In contrast to wild-typeHIPK2, Pin1 showed only very little interaction with autophos-phorylation-defective HIPK2 mutants in cells (Fig. 4B), whereas

B

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Fig. 4. Pin1 interacts with HIPK2 phosphorylatedat Thr880/Ser882. (A) Endogenous HIPK2-Pin1 com-plex formation upon DNA damage. EndogenousHIPK2 (Left) was immunoprecipitated from lysatesof HCT116 cells treated as indicated. Precipitatedcomplexes and cell lysate controls were analyzedby immunoblotting. (B–D) Pin1 and HIPK2 interactin a phosphorylation-dependent fashion in cells.Pin1 and HIPK2 wild-type or mutant proteins wereexpressed in H1299 cells transfected as indicated.Immunoprecipitation targeting the Flag epitopewere performed; lysates and precipitated com-plexes were analyzed by immunoblotting. (E andF) Phosphorylation-specific binding of Pin1 andHIPK2 in vitro. (E) Bacterially expressed His-HIPK2was preincubated with λ-phosphatase or in bufferlacking the enzyme. Subsequently, pulldowns wereperformed with bacterially expressed GST-Pin1and GST proteins. Protein complexes were ana-lyzed by autoradiography (E Top and Middle, FTop) and Coomassie brilliant blue staining (E andF, Lower). (F) GST-pulldown assay were performedwith in vitro translated wild-type and autophos-phorylation-deficient HIPK2 as indicated andanalyzed as in E. (G) Pin1 protects HIPK2 fromsubtilisin-dependent proteolysis through its isom-erase activity. In vitro translated 35S-labeled HIPK2was immunoprecipitated and preincubated withGST, GST-Pin1, or GST-Pin1C113S expressed in E. coli.Subsequently, proteolysis assays using subtilisinwere performed and analyzed by SDS-PAGE andautoradiography (Top andMiddle). Loading of GSTand GST-Pin1 proteins used was visualized by Coo-massie brilliant blue staining (Bottom). (H) Thr880/Ser882 is important for Pin1-mediated protection ofHIPK2 from subtilisin-dependent proteolysis. HIPK2and HIPK2T880A/S882A were in vitro translated, immu-noprecipitated, preincubated with GST-Pin1, andsubsequently limited proteolysis was performed withsubtilisin. Samples were analyzed as in G. Represen-tative experiments are shown. n = 3.

Bitomsky et al. PNAS | Published online October 21, 2013 | E4207

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autophosphorylation-mimetic HIPK2T880D/S882D readily inter-acted with Pin1 (Fig. 4C). Kinase-dead HIPK2K221A showed nointeraction with Pin1 (Fig. 4D).Next, we performed in vitro pulldown assays by using bacte-

rially expressed GST-Pin1 and in vitro translated HIPK2. Ofnote, wild-type HIPK2, but not the kinase-deficient form, auto-phosphorylates during in vitro translation (Fig. S6A). In vitropulldown assays demonstrated that Pin1 interacts with wild-typeHIPK2 (Fig. 4E). No interaction occurs when HIPK2 was pre-treated with λ-phosphatase (Fig. 4E), showing that Pin1 bindsHIPK2 in a phosphorylation-dependent manner. Furthermore,although the wild-type form of HIPK2 readily interacted withPin1 in vitro, autophosphorylation-defective HIPK2T880A/S882A

almost entirely failed to bind Pin1 (Fig. 4F), suggesting thatphosphorylation at these residues is critical for Pin1 binding.

Pin1 Alters HIPK2 Conformation. Because Pin1 can alter the con-formation of its substrate proteins (28), we aimed to determinewhether Pin1 alters HIPK2 conformation. Thus, we performedpartial proteolysis assays with subtilisin, a protease that is par-ticularly sensitive to substrate conformation (28). Preincubationof 35S-labeled HIPK2 with recombinant GST-Pin1 (to facilitatePin1-mediated isomerization) revealed a decreased sensitivity tosubtilisin, whereas preincubation with catalytically inactivePin1C113A (which interacts with HIPK2; Fig. S6B) or GST alonedid virtually not change subtilisin sensitivity of HIPK2 (Fig. 4G).These results indicate that the isomerase activity of Pin1 isimportant for the observed effect. To address the relevance ofHIPK2 autophosphorylation, we compared proteolysis sensitivityof wild-type HIPK2 and HIPK2T880A/S882A after preincubation/isomerization with GST-Pin1. HIPK2T880A/S882A was less effec-tively protected from proteolysis compared with wild-type HIPK2(Fig. 4H). Together these results suggest that Pin1 mediatesHIPK2 isomerization and, thereby, alters HIPK2 conformation.

Pin1 Couples HIPK2 Activation to Its Stabilization. Because Pin1–HIPK2 binding takes place early after DNA damage inductionduring the HIPK2 stabilization phase, we hypothesized that Pin1might contribute to HIPK2 stabilization. Consistent with thishypothesis, depletion of endogenous Pin1 by RNAi preventedHIPK2 stabilization upon DNA damage (Fig. 5A). The role ofPin1 for DNA damage-regulated HIPK2 stabilization was fur-ther characterized by using Pin1−/− and Pin1+/+ mouse embry-onic fibroblasts (MEFs). We first confirmed the specificity of theHIPK2 antibody for mouse HIPK2 by using Hipk2+/+ andHipk2−/− MEF lysates (Fig. S7A). DNA damage-induced HIPK2stabilization was reduced in Pin1−/− MEFs (Fig. 5B). In addition,reconstitution of Pin1 expression in Pin1−/− cells by retroviralgene transfer rescued HIPK2 stabilization after DNA damage(Fig. 5C), indicating that Pin1 is essential for DNA damage-induced HIPK2 stabilization. In addition, we compared the pro-teins half-life of wild-type HIPK2 and the phospho-mutants.Although autophosphorylation-defective HIPK2T880A/S882A showeda shorter half-life than the wild-type protein, kinase-mimeticHIPK2T880S/S882D showed an increased one (Fig. S7 B and C),suggesting that autophosphorylation at Thr880/Ser882 regulatesHIPK2 stability.Moreover, we used gain-of-function approaches to analyze the

effect of Pin1 on HIPK2 stability. Ectopic expression of Pin1resulted in increased HIPK2 protein levels in a dose-dependentmanner (Fig. 5D). In contrast to wild-type Pin1, an isomerase-deficient Pin1C113A mutant failed to mediate a substantialHIPK2 stabilization, suggesting that the catalytic activity of Pin1is required for this effect (Fig. 5E). Furthermore, the stabilizingeffect of Pin1 on HIPK2 requires HIPK2 autophosphorylationbecause autophosphorylation-defective HIPK2T880A/S882A was onlymildly affected by Pin1 (Fig. 5F). Taken together, these results

suggest that Pin1 mediates stabilization of activated, autophos-phorylated HIPK2.

Pin1 Inhibits HIPK2 Polyubiquitination and Degradation. To de-termine the mechanism by which Pin1 links HIPK2 activation toits stabilization, we investigated the effect of Pin1 on the regu-lation of HIPK2 by its ubiquitin ligase Siah-1. Whereas Pin1efficiently rescued wild-type HIPK2 from Siah-1–mediated deg-radation, it was less efficient in rescuing HIPK2T880A/S882A (Fig.6A), suggesting that Pin1 can protect HIPK2 from degradation inan autophosphorylation-dependent fashion. Consistent with this

HIPK2

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Fig. 5. Pin1 regulates DNA damage-induced HIPK2 stabilization. (A) Pin1knockdown prevents HIPK2 stabilization upon DNA damage induction.HCT116 cells were transfected with Pin1-specific or control siRNAs, and DNAdamage was applied with Adriamycin (1 μg/mL) for 20 h. Total cell lysateswere analyzed by immunoblotting with HIPK2 and Pin1-specific antibodies.(B) Reduced HIPK2 stabilization in Pin1-deficient cells. Pin1−/− and Pin1+/+

MEFs were treated as indicated, and cell lysates were analyzed by immu-noblotting. (C) Rescue of HIPK2 expression in Pin1−/− MEFs by stable re-constitution of Pin1 expression. Pin1−/− MEFs transduced with a retrovirusharboring a Pin1 expression cassette or with empty vector were treated asindicated. Cell lysates were analyzed by immunoblotting. (D) Ectopicallyexpressed Pin1 stabilizes HIPK2. The 293T cells were transfected with theexpression construct indicated, and total cell lysates were analyzed by im-munoblotting. (E) Isomerase activity-deficient Pin1 fails to stabilize HIPK2.The 293T cells were transfected as indicated, and cell lysates were analyzedby immunoblotting. Of note, the increased molecular weight of Pin1 incomparison with Pin1C113S originates from a few additional amino acidsbetween the HA tag and the start methionine in this Pin1 construct. (F)Effect of Pin1 on HIPK2 autophosphorylation mutants. The 293T cells weretransfected as indicated, and cell lysates were analyzed by immunoblotting.The relative HIPK2/GFP ratio (fold induction) was determined by usingdensitometry. Statistical analysis of three experiments revealed the follow-ing fold inductions (average ± SD): HIPK2, 4.2 ± 0.4; HIPK2T880/S882, 1.9 ± 0.1;HIPK2S/T8A, 1.2 ± 0.1. Representative experiments are shown. n = 3.

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observation, we found that Pin1 interferes with Siah-1–mediatedHIPK2 polyubiquitination both in cellulo and in vitro, which re-quires HIPK2 autophosphorylation (Fig. 6 B and C) and Pin1isomerase activity (Fig. 6C). Interestingly, overexpression of wild-type Pin1 inhibits HIPK2–Siah-1 interaction in a dose-dependentfashion (Fig. 6D). In contrast, overexpression of isomerase-de-ficient Pin1C113A did not result in disruption of the HIPK2–Siah-1complex (Fig. 6E). These results suggest that the isomerase activityof Pin1 is critical for HIPK2–Siah-1 complex disruption.Additionally, we aimed to evaluate the relevance of Pin1 in

DNA damage-induced dissociation of the HIPK2–Siah-1 com-plex. To this end, we used Pin1−/− MEFs stably reconstituted forPin1 expression and vector-transduced Pin1−/− control MEFs.Because of a lack of suited Siah-1 antibodies to analyze theendogenous HIPK2–Siah-1 complex, we expressed Flag-HIPK2and HA-Siah-1 in Pin1-reconstituted and Pin1-deficient cells.HIPK2–Siah-1 complex disruption was found upon DNA dam-age in Pin1-expressing cells, whereas in Pin-deficient cells, stillinteraction of HIPK2 and Siah-1 was evident (Fig. 6F). Theseresults indicate that Pin1 regulates dissociation of the HIPK2–Siah-1 complex upon DNA damage.

Pin1 Regulates HIPK2 Function in Human Cells. To address the roleof Pin1 in HIPK2-mediated apoptosis, we determined the impactof Pin1 depletion on induction of apoptosis by ectopic expression of

wild-type HIPK2 and HIPK2 phospho-mutants. Depletion of Pin1reduced the apoptotic activity both of wild-type and the phos-pho-mimetic HIPK2T880D/S882D mutant (Fig. 3A). These resultsindicate that Pin1 is critical for HIPK2-mediated apoptosis, whichis in line with a recent finding (26).Next, we aimed to determine the role of endogenous Pin1 for

endogenous HIPK2 stabilization and p53 Ser46 phosphorylationin response to DNA damage. Depletion of Pin1 resulted in ablunted HIPK2 stabilization and, consequently, in reduced p53Ser46 phosphorylation (Fig. 7A). In consequence, Pin1 depletionresulted in a substantial reduction in apoptosis-associated DNAfragmentation (by approximately 65%) and, consistently, also indiminished cleavage of the caspase substrate PARP (Fig. 7B).Thus, Pin1 regulates HIPK2-mediated p53 Ser46 phosphoryla-tion and apoptosis commitment.To further define the effect of Pin1 on p53 Ser46 phosphor-

ylation, we used an overexpression-based gain-of-function approachthat revealed that Pin1 expression leads to a dose-dependent in-crease in HIPK2-mediated p53 Ser46 phosphorylation (Fig. 7C).Pin1 failed to efficiently potentiate p53 Ser46 phosphorylation inthe presence of HIPK2T880A/S882A, again suggesting that HIPK2autophosphorylation is important for the functional interplaybetween Pin1 and HIPK2 (Fig. 7D). Together, these results sup-port the conclusion that Pin1 regulates HIPK2-mediated p53 Ser46phosphorylation based on HIPK2 autophosphorylation.

A

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Fig. 6. Pin1 regulates Siah-1–mediated HIPK2ubiquitination and degradation. (A) Pin1 protectsHIPK2 from Siah-1–induced degradation in a fashionthat requires HIPK2 autophosphorylation. H1299cells were transfected with the expression vectorsindicated, and cell lysates were analyzed by immu-noblotting. (B) Pin1 inhibits Siah-1–mediated HIPK2polyubiquitination in vitro. In vitro ubiquitinationreactions were performed. Subsequently, His-HIPK2was purified from the reactions by using Ni-NTAbeads under denaturing conditions and analyzed bySDS/PAGE, and immunoblotting. GST-fusion proteinswere visualized by Coomassie brilliant blue staining.(C ) Pin1 regulates HIPK2 polyubiquitination inintact cells. H1299 cells were transfected with theexpression constructs indicated, and cell lysateswere subjected to immunoprecipitation by usingFlag antibodies. Immunoprecipitated HIPK2 wasanalyzed by immunoblotting using polyubiquitinand Flag antibodies. Aliquots of the cell lysatesused for immunoprecipitation were analyzed byimmunoblotting for HIPK2 and Pin1. (D) Pin1 over-expression triggers disruption of the HIPK2–Siah-1complex. H1299 cells were transfected and treatedas indicated. Flag-HIPK2 was immunoprecipitatedwith Flag antibodies. Cell lysates and immunecomplexes were analyzed by immunoblotting. (E)Overexpression of isomerase-deficient Pin1 fails todisrupt the HIPK2–Siah-1 complex. Experiment wasperformed as described in D except that Pin1C113A

was used instead of wild-type Pin1. (F) Pin1 is criticalfor disruption of the HIPK2–Siah1 complex after DNAdamage. Pin1−/− MEFs virally transduced with emptyvector or HA-Pin1 expression vector were transfectedas indicated. Cells were treated with Adriamycin andMG132 as indicated, and Flag-HIPK2 was immuno-precipitated from cell lysates. Immune complexes andcell lysates were analyzed by immunoblotting. Rep-resentative experiments are shown. n = 3.

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Pin1 Is Required for IR-Induced Cell Death in Vivo in ZebrafishEmbryos. To our knowledge, there is no published data ad-dressing the relevance of Pin1 in DNA damage-induced apoptosisin vivo in an intact organism. To this end, we knocked-down Pin1

in zebrafish embryos using a Pin1 specific morpholino oligonucle-otide. Indeed, immunoblotting of whole-body embryo lysates con-firmed reduction in endogenously expressed Pin1 (Fig. 7E).Although Pin1 depletion did not lead to obvious developmental

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Fig. 7. Pin1 regulates HIPK2-mediated p53 Ser46 phosphorylation in human cells and is required for DNA damage-induced apoptosis in zebrafish. (A) Pin1knockdown inhibits Adriamycin-induced HIPK2 stabilization and p53 Ser46 phosphorylation. HCT116 cells were transfected with control (GL2, luciferase) or Pin1-specific siRNA as indicated and treated with 1 μg/mL Adriamycin. Cells were lysed at the timepoints indicated and analyzed by immunoblotting. (B) Pin1 depletionimpairs apoptosis induction. HCT116 cells were transfected with Pin1-specific or control siRNAs. Cells were treated 24 h with 1 μg/mL Adriamycin. Sub-G1 DNAcontent analysis was performed by propidium-iodide staining and FACS analysis. The graph shows means and SD from three independent experiments.Knockdown efficiency and apoptosis induction was controlled by immunoblotting. (C ) Pin1 potentiates HIPK2-mediated p53 Ser46 phosphorylation.H1299 cells were transfected as indicated. Cell lysates were analyzed by immunoblotting. (D) Pin1–HIPK2 interaction seems to be important for in-duction of p53 Ser46 phosphorylation by Pin1. H1299 cells were transfected with the expression vectors indicated. Amounts of transfected HIPK2expression vectors were adjusted to result in a comparable HIPK2 expression. Cell lysates were analyzed by immunoblotting. (E–H) Pin1 depletion inzebrafish embryos results in decreased apoptosis induction upon IR in vivo. (E ) Zebrafish eggs were injected with Pin1-specific or control morpholinos.Knockdown of endogenous Pin1 was controlled by immunoblotting of whole-body embryo lysates. (F and G) At 24 h after fertilization (hpf) embryoswere exposed to 12.5 Gy IR and analyzed 30 hpf for apoptosis induction by using either TUNEL (F ) or acridine orange (AO) (G) staining. Representativeimages are shown. (H) Quantification of the AO staining in the zebrafish embryos. (A–G) Representative experiments are shown. (I) Proposed mech-anism of HIPK2 activation upon DNA damage. For details, see Discussion.

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abnormalities in 24-h-old zebrafish embryos, we observed a mildincrease in TUNEL positive and AO positive nuclei suggesting anincrease in apoptotic cell death (Fig. 7 F andG). Interestingly, bothTUNEL and AO staining revealed that in Pin1-depleted embryosthere is an efficient protection against induction of apoptosis inresponse to whole-body IR (Fig. 7 F–H). These results demonstratethat Pin1 is essential for DNA damage-induced cell death in vivo.

DiscussionHere we report an evolutionary conserved mechanism couplingactivation of DNA damage-responsive HIPK2 kinase to its sta-bilization. Based on our findings, we propose a model in whichHIPK2 activation upon DNA damage is facilitated downstreamof the ATM checkpoint through a mechanism that involvesHIPK2 oligomerization and site-specific autophosphorylation(Fig. 7I). HIPK2 autophoshorylation presumably takes place inthe cell nucleus and, at least in part, in association with nuclearbodies, which is consistent with the previously reported HIPK2localization (14, 15). Concentration of HIPK2 molecules at thesemacromolecular structures may foster HIPK2 oligomerization.Furthermore, in vitro phosphorylation experiments support themodel that HIPK2 autophosphorylates by an intermolecularmechanism, suggesting that HIPK2 molecules phosphorylateeach other upon DNA damage. HIPK2 colocalizes with itsubiquitin ligase Siah-1 at nuclear bodies, suggesting that thesenuclear domains are the site of HIPK2 ubiquitination (22). Ourfindings that autophosphorylated HIPK2 localizes to nuclearbodies, along with our results showing that Pin1 interferes withSiah-1–mediated HIPK2 ubiquitination, suggests that auto-phosphorylation may interfere with HIPK2 ubiquitination at thesubcellular site of HIPK2–Siah-1 colocalization.Our data indicate that autophosphorylated—and thereby acti-

vated—HIPK2 is recognized by the prolyl-peptidyl cis/trans isom-erase Pin1. Thus, Pin1 may facilitate stabilization of activatedHIPK2. Accordingly, HIPK2 autophosphorylation and the in-terplay with Pin1 is important for HIPK2 stabilization and apo-ptotic function. Because our data also suggest that Pin1 changesHIPK2 conformation, similar to what has been shown for numer-ous other Pin1 client proteins (28), we assume that a conforma-tional change is important to allow efficient dissociation of theHIPK2–Siah-1 complex. In accordance with these findings, ourprevious work showed that, although phosphorylation of Siah-1 atSer19 by ATM substantially weakens HIPK2–Siah-1 binding andcontributes to HIPK2 stabilization, it is not sufficient to triggercomplete dissociation of the HIPK2–Siah-1 complex (22).Using mass spectrometry, two recent reports showed that

HIPK2 autophosphorylates in cis at numerous sites includingTyr354 and Ser357 within its activation loop (29, 30). BecauseTyr354 and Ser357 are critical for HIPK2 activity regulation,these findings suggest a mechanism for HIPK2 activation throughcis autophosphorylation (29, 30). The Thr880/Ser882 autophos-phorylation site identified in our study was not identified in thesestudies. Possible explanations for this discrepancy might be limitedcoverage of the entire HIPK2 amino acid sequence in the massspectrometry experiments and/or limitations in the ionization ofthe HIPK2 phospho-peptides.Our analysis of HIPK2 Thr880/Ser882 autophosphorylation in

response to DNA damage suggests that autophosphorylation atthis site is reversible and appears to be removed by a currentlyunknown phosphatase. Because cis/trans isomerase Pin1 cancatalyse both directions of the isomerization reaction (28), wehypothesize that timed removal of the Pin1 binding signal drivesthe isomerization reaction into a defined direction to lock HIPK2in a particular conformation.Through signaling acting in parallel to the HIPK2 activation

pathway, ATM directly phosphorylates p53 at Ser15 and stim-ulates p53 Ser20 phosphorylation via activation of Chk2 (31).These phosphorylation marks contribute to p53 stabilization and

activation and accompany HIPK2-mediated p53 Ser46 phosphor-ylation (32). Interestingly, one of the phosphorylation markspreviously shown to guide Pin1–p53 interaction is Ser46 phos-phorylation (33–35). Thus, by facilitating stabilization of auto-phosphorylated, activated HIPK2, Pin1 stimulates p53 Ser46phosphorylation and, thereby, may regulate its own complexformation with p53 by virtue of a positive feed-forward loop.Standard cancer treatments such as radiation therapy and

chemotherapy rely on DNA damage-induced activation of apo-ptosis. Because HIPK2 is a mediator of radiosensitivity andchemosensitivity in cancer cells (36), our findings, along withprevious ones demonstrating that Pin1 regulates p53 activity,argue that Pin1 contributes to the outcome of cancer therapy, inparticular in wild-type p53-expressing cancer cells (33, 34, 37).However, because HIPK2 can also mediate cell death also inabsence of p53 (19, 38), Pin1 is presumably also beneficial forresponsiveness of p53-deficient cancer cells. Because Pin1 alsoregulates cell proliferation, transformation, and tumor growth,pharmacological inhibition of Pin1 has been suggested for cancertreatment (28, 37, 39). In the light of our results, it will be im-portant to develop strategies to target selective Pin1–substrateinteractions in cancer cells to maintain efficient induction ofapoptosis after radiotherapy or chemotherapy.

Materials and MethodsCell Lines, Cell Culture, Transduction, and Transfection. H1299, 293T, (bothobtained from American Type Culture Collection), HCT116, and HCT116 p53−/−

(40) (kindly provided by Bert Vogelstein, The Johns Hopkins University,Baltimore) were maintained in DMEM (Gibco) supplemented with 10% (vol/vol) FCS, 1% (wt/vol) penicillin/streptomycin, 2 mM L-Glutamine, 1 mM so-dium Pyruvat, and 20 mM Hepes buffer at 37 °C at 5% (vol/vol) CO2. Primaryhuman fibroblasts (GM08399, population doubling 23) were obtained fromCoriell Cell Repositories. Hipk2+/+ and Hipk2−/− MEFs were generated fromembryonic day 12.5 embryos. Pin1−/− and Pin+/+ MEF cells were maintainedin supplemented DMEM by using 10% (vol/vol) FCS. Pin1 expression wasreconstituted in Pin1−/− cells by retroviral transduction. Cell pools weregenerated by selection with Puromycin (2 μg/mL). Transient transfectionswere carried out by using Lipofectamine 2000 (Invitrogen) or by standardcalcium phosphate precipitation.

Antibodies. The antibodies used were as follows: Pin1 (Ab-1) from Calbio-chem, p53 (DO-1 and FL-393), GFP (FL), Pin1 (H-123), and polyubiquitin (P4D1)from Santa Cruz Biotechnologies; Flag (M2) from Sigma; Flag polyclonalrabbit from Sigma; actin (C4) from MP Biomedicals; HA (clones 12CA5 and3F10) from Roche; HA.11 clone 16B12 from Covance; phospho-Ser/Thr (22A)from BD Biosciences; and p53 phospho-Ser46, phospho-Ser15 (16G8), andγ-H2AX from Cell Signaling Technologies. The affinity-purified rabbit HIPK2antibody has been described (14, 22). Phospho-specific Thr880/Ser882 HIPK2antibodies were generated by immunizing rabbits with the following KLH-coupled peptide: NH2-CVIPD(pT)P(pS)PT-CONH2. Rabbit sera were affinitypurified against the phospho-peptide, and nonphosphorylation-specificantibodies were removed by the nonphospho-peptide.

Expression Constructs. Human HIPK2, Siah-1, and ubiquitin constructs havebeen described (22, 41). HIPK2S/T8A constructs were generated by sub-stitution of the following serines and threonines to alanines: S668, S827,T838, S848, T880, S882, S924, and S934. The HIPK2 Ser/Thr mutants and theD. rerio HIPK2 constructs were generated by using synthetic biology(GeneArt). Pin1 constructs were either obtained from G.d.S. or generated bystandard PCR techniques and molecular cloning, or by shuttling GatewayFull ORF clones (obtained from the Genomics and Proteomics Core Facility,German Cancer Research Center) into the respective destination vector byusing the Gateway (Invitrogen) recombination system. All constructs wereverified by DNA sequencing.

RNA Interference. siRNA duplexes were produced by Dharmacon Research andQiagen. For Pin1 knockdown, either the ON-TARGETplus SMARTpool againsthuman Pin1 (Dharmacon; L-003291-00) was used or a siRNA targeting5′-GGCCGAGTGTACTACTTCAdTdT (Qiagen). The Smart pool and the singlesiRNA for Pin1 revealed comparable knockdown efficiencies for endogenousPin1. For functional experiments, both knockdown strategies were used andshowed comparable results. For control experiments, the nonspecific control

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duplex IX siRNA (Dharmacon; D-001206-09-20) and a GL2 luciferase duplexwas used. siRNA duplexes (final concentration 75–100 nM) were transfectedby using Dharmafect 4 (Dharmacon) or HiPerFect (Qiagen) as specified bythe manufacturer.

Induction of DNA Damage. Cells were continuously incubated with culturemedium supplemented with Adriamycin (solved in water) at the specifiedconcentrations. The ATM inhibitor (Ku55933) was solved in DMSO. In general,also if not indicated separately, all control cells were treated with the sameamounts of the solvent(s), respectively. After incubation, cells were sub-sequently harvested and processed as indicated.

Apoptosis Measurement. Cells were transfected with siRNA as indicated, andapoptosis was determined by FACS analysis (Becton Dickinson) of 30,000 cellsusing either the Annexin V-FITC Apoptosis detection kit (Sigma-Aldrich)according to the manufacturers’ instructions or by analysis of DNA content(sub-G1 analysis) as reported previously (22).

Immunofluorescence Staining and Confocal Microscopy. HCT116 cells wereseeded onto coverslips and treated with 0.75 μg/mL Adriamycin and in-cubated for 6 h or left untreated. Preextraction was performed by in-cubating the cells with CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mMMgCl2 and 10 mM Pipes at pH 6.8) for 5 min, CSK buffer with 0.5% TritonX-100 for 2 min, and washing with CSK buffer before fixation with 4% PFAin PBS for 40 min at room temperature (RT). After washing once with PBS,the cells were blocked in 10% goat serum in PBS for 1 h at RT. Cells wereincubated with the rabbit anti-phospho-Thr880/Ser882 HIPK2 antibodies for

1 h at RT (22 °C). After washing with PBS, cells were incubated with sec-ondary antibodies Alexa Fluor 488 donkey anti-rabbit (Invitrogen) in PBSwith Hoechst 1:1,000 wt/vol (Sigma) and mounted on glass slides withMowiol (Sigma). Images were taken by using a confocal laser scanning mi-croscope (FluoView1000; Olympus) with a 60× oil objective using the se-quential scanning mode. All images were collected and processed by usingthe FluoView Software (Olympus) and ImageJ.

In Vitro Kinase Assays Analysis. Bacterially expressed and purified 6×His-HIPK2proteins (0.2–0.5 μg) were incubated in 30 μL of kinase buffer containing40 μM cold ATP and 5 μCi [γ-32P] ATP and 1 μg of Myelin Basic Protein (MBP;Sigma) as described (14). After incubation for 30 min at 30 °C, the reactionwas stopped by adding 5× SDS loading buffer. After separation by SDS-PAGE, gels were fixed, dried, and exposed to X-ray films. For immunoblot-based autophosphorylation analysis, either bacterially expressed 6×His-HIPK2 proteins or Flag-tagged HIPK2 proteins immunopurified from 293Tcell lysates were used.

Additional laboratory techniques are described in SI Materialsand Methods.

ACKNOWLEDGMENTS. We thank Karin Scheuermann for technical assis-tance and A. Ucar and to J. S. Malter for providing MEFs. This work wasfunded by Deutsche Forschungsgemeinschaft Grant HO 2438/3-2, DeutscheKrebshilfe Grant 108528 (to T.G.H.), the Sonderforschungsbereich (SFB)1036(T.G.H.), the Associazione Italiana per la Ricerca sul Cancro (to G.d.S.), theMinistero dell’Istruzione dell’Università e della Ricerca (G.d.S.), the Geron-toSys Network AGENET funded by Bundesministerium für Bildung und For-schung Grant FKZ 0315898A (to T.G.H.).

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