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Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63

Nature Structural & Molecular Biology volume 25, pages 261–269 (2018)Cite this article

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Abstract

The survival rate of cancer patients is steadily increasing, owing to more efficient therapies. Understanding the molecular mechanisms of chemotherapy-induced premature ovarian insufficiency (POI) could identify targets for prevention of POI. Loss of the primordial follicle reserve is the most important cause of POI, with the p53 family member p63 being responsible for DNA-damage-induced apoptosis of resting oocytes. Here, we provide the first detailed mechanistic insight into the activation of p63, a process that requires phosphorylation by both the priming kinase CHK2 and the executioner kinase CK1 in mouse primordial follicles. We further describe the structural changes induced by phosphorylation that enable p63 to adopt its active tetrameric conformation and demonstrate that previously discussed phosphorylation by c-Abl is not involved in this process. Inhibition of CK1 rescues primary oocytes from doxorubicin and cisplatin-induced apoptosis, thus uncovering a new target for the development of fertoprotective therapies.

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Fig. 1: Activation of TAp63α requires consecutive phosphorylation by CHK2 and CK1 N terminally to the TI domain.
Fig. 2: Efficient tetramerization requires accumulation of multiple phosphorylation events directly adjacent to the TI domain.
Fig. 3: Charge repulsion of spatially proximal aspartate residues within the TA domain.
Fig. 4: Phosphorylation of TAp63α by CK1 is essential for tetramerization, whereas tyrosine phosphorylation by c-Abl is dispensable.
Fig. 5: Inhibition of CK1 kinase activity preserves primary oocytes in cultured mouse ovaries treated with doxorubicin or cisplatin.
Fig. 6: Proposed activation mechanism of TAp63α in primary oocytes.

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Acknowledgements

The authors thank M. B. Gill, T. J. Wu and J. S. Carroll for their review of and input into this manuscript. Further, we thank F. Pampaloni (Goethe University) for providing the FEP foil holders. The research was funded by the DFG (DO 545/8-1), the Centre for Biomolecular Magnetic Resonance (BMRZ) and the Cluster of Excellence Frankfurt (Macromolecular Complexes). M.T. was supported by a Fellowship from the Funds of the Chemical Industry and Boehringer Ingelheim travel grant. V.R. was funded by Covert Italia S.p.A. The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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Author notes

  1. These authors contributed equally: Marcel Tuppi, Sebastian Kehrloesser and Daniel W. Coutandin.

Authors and Affiliations

  1. Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance and Cluster of Excellence Macromolecular Complexes (CEF), Goethe University, Frankfurt, Germany

    Marcel Tuppi, Sebastian Kehrloesser, Daniel W. Coutandin, Laura M. Luh, Alexander Strubel, Birgit Schäfer & Volker Dötsch

  2. Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy

    Valerio Rossi, Massimo De Felici & Francesca Gioia Klinger

  3. Physical Biology/Physikalische Biologie (IZN, FB 15), Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Frankfurt, Germany

    Katharina Hötte & Ernst H. K. Stelzer

  4. Institute of Biochemistry, Brandenburg Medical School (MHB) Theodor Fontane, Neuruppin and Brandenburg an der Havel, Germany

    Meike Hoffmeister

  5. Georg-Speyer-Haus, Institute for Biomedical Research, Frankfurt, Germany

    Tiago De Oliveira & Florian Greten

  6. German Cancer Network (DKTK), Frankfurt, Germany

    Florian Greten & Stefan Knapp

  7. Nuffield Department of Medicine, Structural Genomics Consortium, Oxford University, Oxford, UK

    Stefan Knapp

  8. Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt, Germany

    Stefan Knapp

  9. Munich Cluster for Systems Neurology, Ludwig-Maximilians-University, Munich, Germany

    Christian Behrends

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Contributions

M.T., S. Kehrloesser, D.W.C., S. Knapp and V.D. contributed to study design; M.T., V.R., L.M.L., A.S., K.H., M.H. and B.S. performed experiments. M.T., S. Kehrloesser and V.R. analyzed data and generated figures. L.M.L., A.S., K.H., M.H., B.S., T.D.O., F.G., E.H.K.S, S. Knapp, M.D.F., C.B. and F.G.K. contributed to data interpretation. M.T., S. Keherloesser and V.D. wrote the manuscript with input from S. Knapp, M.D.F., C.B. and F.G.K.

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Correspondence to Volker Dötsch.

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Supplementary Figure 1 In vitro phosphorylation of TAp63α wt and TAp63α S582A S583A using preactivated MK2 and MS analysis of DNA-damage- induced phosphorlyation of TAp63α in H1299 cells

a, Purified TAp63α wt and the S582A/S583A mutant were incubated with pre-activated MK2 for indicated periods. Oligomeric state was monitored by BN-PAGE. b, The same BN-PAGE from a was also analyzed using the α-pS582 antibody. c, SDS PAGE analysis of the in vitro reaction from a and b demonstrating that the α-pS582 antibody is specific for phosphorylation at S582 in TAp63α. d, Separate, extended time course of experiment shown in a. e, Summary of combined sequence coverage (bold characters) and identified phospho-sites (yellow characters) obtained from two sets of samples (Trypsin, AspN and Trypsin + AspN) and (Trypsin-low, Trypsin-high and Trypsin + LysC). f, Annotated MS/MS spectrum of the TI peptide (AA580-595) harboring phosphor-groups at the CHK2 site S582 and along the CK1 pattern (S585, T586 and S588).

Supplementary Figure 2 Most of the DNA-damage-induced phosphorylation events identified by MS are dispensable for TAp63α tetramerization, while inhibition of CK1δ abrogates TAp63α tetramerization, and in vitro phosphorylation of TAp63α pS582 by CK1 is sufficient to induce tetramerization

TAp63α wt and various Ser or Thr to Ala and Tyr to Phe mutants as well as deletion constructs covering the TAD (a), the linker between TD and SAM (b), phosphorylated residues directly preceding the CHK2 site (AA572-580) (c), T610 and S612 in the TID (d) as well as several individual sites identified throughout this study (T178, S233, S356, S381 and Y387) (e) were transiently expressed in U2OS cells and tetramerization was induced by Dox treatment (10 µM, 6 h). Oligomeric state and phosphorylation induced electrophoretic mobility shift were monitored by BN-PAGE and SDS-PAGE, respectively. f, TAp63α stable expressing H1299 cells and cultured mouse ovaries (g) were treated with increasing concentrations (5, 25 and 50 µM) of the CK1 inhibitors PF5006739 (IC50(CK1δ) = 3.9 nM, IC50(CK1ε) = 17 nM) and PF4800567 (IC50(CK1δ) = 711 nM, IC50(CK1ε) = 32 nM) 1 h prior to induction of tetramerization with Dox (10 µM, 6 h). Oligomeric state and phosphorylation induced electrophoretic mobility shift were monitored by BN-PAGE and SDS-PAGE, respectively. h, U2OS cells were co-transfected with TAp63α and constitutive active or dominant negative mutants of CK1 AA1-319 and tetramerization was induced with 10 µM Dox for 6 h. Oligomeric state was monitored by BN-PAGE. i, Bacterially expressed TAp63α was successively in vitro phosphorylated by pre-activated MK2 and in a second step by constitutively active CK1 (black line) and analyzed using size exclusion chromatography (Superose 6 10/300GL). Purified dimeric, MK2 phosphorylated TAp63α (red line) served as a retention time reference.

Supplementary Figure 3 Flowchart of the CHK2- and CK1-mediated disruption of the autoinhibitory structure of TAp63α

a, Schematic representation of the structural model of the autoinhibitory state formed by TAp63α. Within a monomer of the dimer the TA2A and TA2B domains together with the TID form an anti-parallel three-stranded β-sheet. Together with the second monomer of the dimer the anti-parallel six-stranded β-sheet blocks the tetramerization interface of the TD (Cyan). This closed conformation is further locked by the α-helical TA1 domain occupying the binding site of the second helix (H2) of the TD that only forms upon tetramerization. In close proximity of the CK1 phosphorylation sites, which are N-terminal to the TID, is a stretch of three negative charged aspartates (D61, D63, D66) C-terminal to the TA2B within each monomer. Color code of the core structure of the autoinhibitory conformation of TAp63α according to schematic domain representation in Figure 1c. b, Upon DNA damage active CHK2 phosphorylates TAp63α on S582. c, Phosphorylation on S582 turns TAp63α into a substrate for CK1, which subsequently phosphorylates S585. d, Phosphorylation of S585 generates again a consensus motif for CK1 to phosphorylate S588. e, As in c and d phosphorylation of S588 triggers the phosphorylation of S591 by CK1. f, Finally CK1 phosphorylates T594 without generating a new consensus motif. g, Thus the DNA damage induced phosphorylation events lead to an accumulation of negative charges in close proximity to the intrinsically negatively charged sequence harboring D61, D63 and D66. h and i, The generated forces due to charge repulsion overcome the energy barrier of the kinetically trapped dimer and break up the anti-parallel six-stranded β-sheet. j, Two open dimers will subsequently form an open tetramer. The tetrameric TAp63α is then capable to bind DNA and induce target gene expression that ultimately leads to death of the primordial follicle.

Supplementary Figure 4 c-Abl is not required for TAp63α tetramerization, and activation of TAp63α follows a slower kinetics after cisplatin compared to doxorubicin treatment and LH treatment does not abrogate Cs induced tetramerization in oocytes

a, TAp63α stable expressing H1299 cells were treated with increasing concentrations (5, 25 and 50 µM) of the c-Abl inhibitor imatinib 1 h prior to induction of tetramerization with Dox (10 µM, 6 h). Oligomeric state and phosphorylation induced electrophoretic mobility shift were monitored by BN-PAGE and SDS-PAGE, respectively. b, CD-1 P8 ovaries were cultured with or without 10 µM Dox or Cs for the indicated periods. Oligomeric state was monitored by BN-PAGE and phosphorylation induced electrophoretic mobility shift were SDS-PAGE, respectively. ATM total protein abundance as well as pATM levels were assessed by Western blotting. Ovaries were further subjected to 10 µM Cs in the presence of 200 mIU/ml LH.

Supplementary Figure 5 Inhibition of CHK2 and CK1 markedly reduce doxorubicin induced oocyte death

a, Ovarian fragments isolated from P4 ovaries of p-18 c-Kit/GFP mice cultured for 4 days (0 h) before beginning of indicated treatment for further 24 h. Oocytes were imaged at 0 h, 14 h and 24 h. 0 h and 24 h time points of the untreated, Dox and Dox + 25 µM inhibitor treated are shown in Figure 4c. b, Oocytes were imaged at 0 h, 14 h and 24 h. 0 h and 24 h time points of the untreated, Cs and Cs + 25 µM inhibitor treated are shown in Figure 4d. Scale bar, 75 μm.

Supplementary Figure 6 Imatinib reduces only cisplatin induced oocyte death and shows no effect against doxorubicin

I Ovarian fragments isolated from P4 ovaries of p-18 c-Kit/GFP mice cultured for 4 days (0 h) before beginning of indicated treatment for further 24 h. Oocytes were imaged at 0 h, 14 h and 24 h. Scale bar, 75 μm. 0 h and 24 h time points of the untreated, Dox, Dox + 25 µM Imatinib, Cs, and Cs + 25 µM inhibitor, treated are shown in Figure 4b & d.

Supplementary Figure 7 Inhibition of CK1 markedly reduce cisplatin- and doxorubicin-induced oocyte death

a, Ovarian fragments isolated from P4 ovaries of p-18 c-Kit/GFP mice cultured for 4 days (0 h) before treatment for further 24 h (Ctrl – untreated, Dox – 0.4 µM, Cs – 10 µM, PF5006739 5, 25 and 50 µM. Scale bar, 75 μm. b, Percentage of healthy POs scored after 24 h culture with or without Cs or Dox and increasing concentrations (5, 25 and 50 µM) of PF5006739. Data presented as mean ± SEM (3 fragments each). n.s., not significant. **P < 0.01, ***P < 0.001, ****P < 0.001 two-tailed t-test.

Supplementary information

Videos

Supplementary Video 1

3D staining of whole ovaries after doxorubicin treatment. Upper left, untreated ovary and stained using the oocyte marker DDX-4. Upper right, ovary treated 48 h with 0.4 µM Dox. Lower right, ovary treated 48 h with 0.4 µM Dox and 25 µM CK1 inhibitor. Lower left, ovary treated 48 h with 0.4 µM Dox and 25 µM CHK2 inhibitor. 360° rotation, increments set to 1°.

Supplementary Video 2

3D staining of whole ovaries after cisplatin treatment Upper left, untreated ovary and stainedusing the oocyte marker DDX-4. Upper right, ovary treated 48 h with 10 µM Cs. Lower right, ovary treated 48 h with 10 µM Dox and 25 µM CK1 inhibitor. Lower left, ovary treated 48 h with 10 µM Dox and 25 µM CHK2 inhibitor. 360° rotation, increments set to 1°.

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Tuppi, M., Kehrloesser, S., Coutandin, D.W. et al. Oocyte DNA damage quality control requires consecutive interplay of CHK2 and CK1 to activate p63. Nat Struct Mol Biol 25, 261–269 (2018). https://doi.org/10.1038/s41594-018-0035-7

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