Autophosphorylation and Pin1 binding coordinate DNA PNAS PLUS damage-induced HIPK2 activation and cell death Nadja 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 Universität Heidelberg 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. Protein therapeutic drug treatment through different signaling pathways kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. including phosphorylation of p53 at serine 46 and phosphoryla- Here, we deciphered the molecular mechanism of HIPK2 activa- tion-mediated degradation of antiapoptotic molecules such as tion and show its relevance for DNA damage-induced apoptosis corepressor CtBP and transcription factor ΔNp63α (14–20). in cellulo fi and in vivo. HIPK2 autointeracts and site-speci cally Unstressed cells and cells recovering from DNA damage keep autophosphorylates upon DNA damage at Thr880/Ser882. Auto- HIPK2 activity low through targeting the kinase for protea- phosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphory- some-dependent degradation by the ubiquitin ligases WSB1 lation and apoptosis in cellulo. Moreover, HIPK2 autophosphory- and Siah-1 (21-23). In response to DNA damage, HIPK2 is lation is conserved between human and zebrafish and is important stabilized through a mechanism involving the DNA damage for DNA damage-induced apoptosis in vivo. Mechanistically, auto- checkpoint kinases ATM and ATR, which facilitate dissociation phosphorylation creates a binding signal for the phospho-specific of the HIPK2–Siah-1 complex, at least in part, by phosphor- isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by ylation of Siah-1 (16, 22). However, the detailed mechanisms CELL BIOLOGY inhibiting HIPK2 polyubiquitination and modulating Siah-1–HIPK2 underlying HIPK2 activation upon genotoxic stress remains interaction. Concordantly, Pin1 is required for DNA damage-induced still unclear. HIPK2 stabilization and p53 Ser46 phosphorylation and is essential In the present study, we investigated the molecular mecha- in cellulo fi for induction of apotosis both and in zebra sh. Our results nism regulating HIPK2 activation in response to DNA damage. identify an evolutionary conserved mechanism regulating DNA We show that HIPK2 activation is facilitated through a mecha- damage-induced apoptosis. nism involving HIPK2 oligomerization and site-specific auto- phosphorylation at Thr880/Ser882. HIPK2 autophosphorylation ctivation of the apoptotic response upon severe genome increases its kinase activity and apoptotic function both in cellulo damage plays a crucial role in suppression of cellular trans- A and in vivo. Mechanistically, we show that phosphorylation at formation and cancer development. In addition, apoptosis is a major mechanism of action underlying the efficacy of widely used Thr880/Ser882 serves as a binding signal for the prolyl-peptidyl DNA-damaging cancer therapies including radiotherapy and cis/trans isomerase Pin1, which links HIPK2 activation to its chemotherapy. To gain insight into the mechanisms underlying stabilization. cancer cell resistance to therapy, detailed knowledge about the molecular players and the regulatory network governing the Significance DNA damage-induced apoptosis response is of fundamental importance. Activation of the cell death (apoptosis) program is a major Tumor suppressor p53 is a master regulator of the DNA principle of DNA-damaging cancer treatments including ion- damage response and drives expression of different sets of target izing radiation and chemotherapeutic drug treatment. The genes that regulate cell fate decisions as DNA repair, senes- protein kinase HIPK2 plays a key role in radiosensitivity and cence, and cell death (1, 2). p53 target gene selection after DNA chemosensitivity. Here, we found that HIPK2 autointeracts damage is controlled in part at the level of its posttranslational and autophosphorylates after DNA damage. HIPK2 auto- modifications, including site-specific phosphorylation and acety- phosphorylation is conserved in evolution and regulates its lation. p53 phosphorylation is mediated by the DNA damage apoptosis-inducing activity by facilitating binding of the checkpoint kinases ATM and ATR as well as their downstream isomerase Pin1. Pin1 couples HIPK2 activation to its stabili- kinases Chk1, Chk2, and HIPK2, which control p53 stability, zation and is essential for DNA damage-induced apoptosis in activity, and target gene selection through a complex signaling cancer cells and in zebrafish. Our findings identify a mecha- network (3, 4). nism linking HIPK2 activation to its stabilization and high- The Ser/Thr protein kinase homeodomain interacting protein light a conserved function of HIPK2 and Pin1 in the DNA kinase 2 (HIPK2) is an evolutionarily conserved regulator of cell damage-induced apoptosis response. death and cell growth during development and in response to Author contributions: N.B. and T.G.H. designed research; N.B., E.C., C.M., T.P.-V., D.S., K.S., cellular stress (5, 6). There is growing evidence that HIPK2 acts C.G., V.G., C.H., and F.M. performed research; G.d.S. and F.P. contributed new reagents/ as a tumor suppressor both in mice and men (7–10) and that the 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. kinase is functionally deregulated by cellular and candidate viral analyzed data; and T.G.H. wrote the paper. oncogenes (11). In addition to its role in cancer, HIPK2 dys- The authors declare no conflict of interest. regulation has been linked to pathophysiology including neu- This article is a PNAS Direct Submission. rodegeneration and kidney fibrosis (7, 12, 13). 1To whom correspondence should be addressed. E-mail: [email protected]. HIPK2 channels the apoptotic response upon DNA damage This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. induced by UV irradiation, ionizing radiation (IR), and chemo- 1073/pnas.1310001110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1310001110 PNAS Early Edition | 1of10 Downloaded by guest on September 29, 2021 Results phosphorylates at Thr880/Ser882 in vitro and that our antibodies HIPK2 Autophosphorylates at Thr880/Ser882 in Vitro. To determine specifically recognize Thr880/Ser882-phosphorylated HIPK2. whether HIPK2 autophosphorylates, we expressed kinase- To gain insight into the mechanism underlying HIPK2 auto- proficient His-HIPK2 and kinase-deficient His-HIPK2K221A in phosphorylation, we examined whether HIPK2 may autophos- Escherichia coli and purified the proteins by metal-affinity phorylate in trans through an intermolecular mechanism. Thus, chromatography. In vitro kinase assays using [γ-32P]-ATP as a we performed in vitro kinase assays by using bacterially ex- K221A phosphate donor showed that only wild-type HIPK2 protein pressed His-HIPK2 as substrate for Flag-HIPK2 and Flag- K221A autophosphorylated and phosphorylated the substrate myelin HIPK2 . To subsequently separate the autophosphorylated basic protein (MBP) (Fig. 1A). Immunoblot analysis with a Flag-HIPK2 from its substrate His-HIPK2K221A, we purified the pan-phospho-specific Ser/Thr (pSer/pThr) antibody showed His-HIPK2K221A protein from the kinase reaction under dena- strong reactivity with the wild-type form of HIPK2, but not turing conditions by using metal-affinity purification on the His with kinase-deficient HIPK2K221A (Fig. 1B). In addition, pre- tag of the substrate protein. Immunoblot analysis of the His- treatment of HIPK2 with λ-phosphatase abrogated reactivity of HIPK2K221A showed clear phosphorylation at Thr880/Ser882 in the pSer/pThr antibody (Fig. 1C). the presence of wild-type HIPK2, whereas virtually no phos- For mapping of the autophosphorylation sites, we focused on the phorylation was found using HIPK2K221A (Fig. 1E). These results C-terminal regulatory part of HIPK2, which contains an utoinhi- suggest that HIPK2 autophosphorylates in trans through an bitory domain (24). We speculated that phosphorylation in this part intermolecular mechanism. may modulate HIPK2 activity by attenuating the autoinhibitory function of the C terminus. In vitro mapping experiments of the HIPK2 Autophosphorylates at Thr880/Ser882 upon DNA Damage. We HIPK2 autophosphorylation sites using site-directed mutagenesis next investigated whether HIPK2 autophosphorylates in cells. identified Thr880/Ser882 as the main HIPK2 autophosphorylation The pThr880/pSer882 HIPK2 antibody specifically recognized site (Fig. 1D). Additional minor sites of autophosphorylation were ectopically expressed Thr880/Ser882-phosphorylated HIPK2 in detectable that were largely abolished through mutation of addi- cells (Fig. S1A). To investigate whether HIPK2 autophosphor- tional six Ser/Thr residues (S668,