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p63 uses a switch-like mechanism to set the threshold for induction of apoptosis

Nature Chemical Biology volume 16, pages 1078–1086 (2020)Cite this article

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Abstract

The p53 homolog TAp63α is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63α phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one.

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Fig. 1: TAp63α-mediated PF death follows an overall sigmoidal kinetics.
Fig. 2: The third CK1 phosphorylation is the slowest step in the phosphorylation of a p63-derived peptide (PAD).
Fig. 3: The third CK1 phosphorylation is the slowest step in the TAp63α phosphorylation and constitutes the ‘point of no return’.
Fig. 4: Crystal structures of CK1δ in complexes with different PAD peptides.
Fig. 5: MD simulations indicate that E593 and V589 of the p63 peptide are important for interaction with CK1.
Fig. 6: The interaction affinity between CK1 and PAD peptides does not depend on the phospho-state, but KM values differ by more than an order of magnitude.

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Data availability

All data are fully available upon request. PDB accession codes for the three crystal structures are 6RU6, 6RU7 and 6RU8.

References

  1. Johnston, R. J. & Wallace, W. H. Normal ovarian function and assessment of ovarian reserve in the survivor of childhood cancer. Pediatr. Blood Cancer 53, 296–302 (2009).

    Article  PubMed  Google Scholar 

  2. Maltaris, T., Beckmann, M. W. & Dittrich, R. Review. Fertility preservation for young female cancer patients. Vivo 23, 123–130 (2009).

    Google Scholar 

  3. Suh, E. K. et al. p63 protects the female germ line during meiotic arrest. Nature 444, 624–628 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Wallace, W. H., Thomson, A. B. & Kelsey, T. W. The radiosensitivity of the human oocyte. Hum. Reprod. 18, 117–121 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Quast, U. Whole body radiotherapy: a TBI-guideline. J. Med. Phys. 31, 5–12 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Woodard, T. L. & Bolcun-Filas, E. Prolonging reproductive life after cancer: the need for fertoprotective therapies. Trends Cancer 2, 222–233 (2016).

    Article  PubMed  Google Scholar 

  7. Livera, G. et al. p63 null mutation protects mouse oocytes from radio-induced apoptosis. Reproduction 135, 3–12 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Deutsch, G. B. et al. DNA damage in oocytes induces a switch of the quality control factor TAp63α from dimer to tetramer. Cell 144, 566–576 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kerr, J. B. et al. DNA damage-induced primordial follicle oocyte apoptosis and loss of fertility require TAp63-mediated induction of Puma and Noxa. Mol. Cell 48, 343–352 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Kim, S. Y. et al. Transient inhibition of p53 homologs protects ovarian function from two distinct apoptotic pathways triggered by anticancer therapies. Cell Death Differ. 26, 502–515 (2019).

    Article  CAS  PubMed  Google Scholar 

  11. Bolcun-Filas, E., Rinaldi, V. D., White, M. E. & Schimenti, J. C. Reversal of female infertility by Chk2 ablation reveals the oocyte DNA damage checkpoint pathway. Science 343, 533–536 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Tuppi, M. 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).

    Article  CAS  PubMed  Google Scholar 

  13. Cesaro, L. & Pinna, L. A. The generation of phosphoserine stretches in phosphoproteins: mechanism and significance. Mol. Biosyst. 11, 2666–2679 (2015).

    Article  CAS  PubMed  Google Scholar 

  14. Knippschild, U. et al. The CK1 family: contribution to cellular stress response and its role in carcinogenesis. Front Oncol. 4, 96 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  15. Schittek, B. & Sinnberg, T. Biological functions of casein kinase 1 isoforms and putative roles in tumorigenesis. Mol. Cancer 13, 231 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Coutandin, D. et al. Quality control in oocytes by p63 is based on a spring-loaded activation mechanism on the molecular and cellular level. eLife 5, e13909 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Prehoda, K. E., Scott, J. A., Mullins, R. D. & Lim, W. A. Integration of multiple signals through cooperative regulation of the N-WASP-Arp2/3 complex. Science 290, 801–806 (2000).

    Article  CAS  PubMed  Google Scholar 

  18. Chen, L., Glover, J. N., Hogan, P. G., Rao, A. & Harrison, S. C. Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature 392, 42–48 (1998).

    Article  CAS  PubMed  Google Scholar 

  19. Rinaldi, V. D., Hsieh, K., Munroe, R., Bolcun-Filas, E. M. & Schimenti, J. C. Pharmacological inhibition of the DNA damage checkpoint prevents radiation-induced oocyte death. Genetics 206, 1823–1828 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Carmell, M. A. et al. A widely employed germ cell marker is an ancient disordered protein with reproductive functions in diverse eukaryotes. eLife 5, e19993 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Keller, P. J., Schmidt, A. D., Wittbrodt, J. & Stelzer, E. H. Digital scanned laser light-sheet fluorescence microscopy (DSLM) of zebrafish and Drosophila embryonic development. Cold Spring Harb. Protoc. 2011, 1235–1243 (2011).

    Article  PubMed  Google Scholar 

  22. Susaki, E. A. et al. Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis. Cell 157, 726–739 (2014).

    Article  CAS  PubMed  Google Scholar 

  23. Hotte, K. et al. Ultra-thin fluorocarbon foils optimise multiscale imaging of three-dimensional native and optically cleared specimens. Sci. Rep. 9, 17292 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Ferrell, J. E. Jr & Ha, S. H. Ultrasensitivity part III: cascades, bistable switches and oscillators. Trends Biochem. Sci. 39, 612–618 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ferrell, J. E. Jr & Ha, S. H. Ultrasensitivity part II: multisite phosphorylation, stoichiometric inhibitors and positive feedback. Trends Biochem. Sci. 39, 556–569 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ferrell, J. E. Jr & Ha, S. H. Ultrasensitivity part I: Michaelian responses and zero-order ultrasensitivity. Trends Biochem. Sci. 39, 496–503 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ferrell, J. E. Jr & Bhatt, R. R. Mechanistic studies of the dual phosphorylation of mitogen-activated protein kinase. J. Biol. Chem. 272, 19008–19016 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Serber, Z. et al. A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism. Mol. Cell Biol. 22, 8601–8611 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Straub, W. E. et al. The C-terminus of p63 contains multiple regulatory elements with different functions. Cell Death Dis. 1, e5 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Selenko, P. et al. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Nat. Struct. Mol. Biol. 15, 321–329 (2008).

    Article  CAS  PubMed  Google Scholar 

  31. Cordier, F. et al. Ordered phosphorylation events in two independent cascades of the PTEN C-tail revealed by NMR. J. Am. Chem. Soc. 134, 20533–20543 (2012).

    Article  CAS  PubMed  Google Scholar 

  32. Narasimamurthy, R. et al. CK1ẟε protein kinase primes the PER2 circadian phosphoswitch. Proc. Natl Acad. Sci. USA 115, 5986–5991 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Mylona, A. et al. Opposing effects of Elk-1 multisite phosphorylation shape its response to ERK activation. Science 354, 233–237 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Leroy, A. et al. Spectroscopic studies of GSK3β phosphorylation of the neuronal Tau protein and its interaction with the N-terminal domain of apolipoprotein E. J. Biol. Chem. 285, 33435–33444 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Philpott, J. M. Casein kinase 1 dynamics underlie substrate selectivity and the PER2 circadian phosphoswitch. eLife 9, e52343 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Theillet, F. X. et al. Sensitivity-enhanced 13C-NMR for monitoring multisite phosphorylation at physiological temperature and pH. Angew. Chem. Int. Ed. 59, 10411–10415 (2020).

    Article  CAS  Google Scholar 

  37. Favier, A. & Brutscher, B. Recovering lost magnetization: polarization enhancement in biomolecular NMR. J. Biomol. NMR 49, 9–15 (2011).

    Article  CAS  PubMed  Google Scholar 

  38. Zhao, B., Li, L., Tumaneng, K., Wang, C. Y. & Guan, K. L. A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCF(β-TRCP). Genes Dev. 24, 72–85 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Shinohara, Y. et al. Temperature-sensitive substrate and product binding underlie temperature-compensated phosphorylation in the clock. Mol. Cell 67, 783–798 (2017).

    Article  CAS  PubMed  Google Scholar 

  40. Rossi, V. et al. LH prevents cisplatin-induced apoptosis in oocytes and preserves female fertility in mouse. Cell Death Differ. 24, 72–82 (2017).

    Article  CAS  PubMed  Google Scholar 

  41. Pampaloni, F., Stelzer, E. H. K. & Mattheyer, C. Capillary cell, arrangement and method for accommodating, positioning and examining a microscopic specimen. US patent 20150211981A1 (2015).

  42. Lohr, F., Gebel, J., Henrich, E., Hein, C. & Dotsch, V. Towards complete polypeptide backbone NH assignment via combinatorial labeling. J. Magn. Reson. 302, 50–63 (2019).

    Article  PubMed  CAS  Google Scholar 

  43. Gil, S. et al. NMR spectroscopic studies of intrinsically disordered proteins at near-physiological conditions. Angew. Chem. Int. Ed. 52, 11808–11812 (2013).

    Article  CAS  Google Scholar 

  44. Bermel, W. et al. Complete assignment of heteronuclear protein resonances by protonless NMR spectroscopy. Angew. Chem. Int. Ed. 44, 3089–3092 (2005).

    Article  CAS  Google Scholar 

  45. McIntosh, L. P. et al. Detection and assignment of phosphoserine and phosphothreonine residues by 13C-31P spin-echo difference NMR spectroscopy. J. Biomol. NMR 43, 31–37 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. McCoy, A. J. Acknowledging errors: advanced molecular replacement with phaser. Methods Mol. Biol. 1607, 421–453 (2017).

    Article  CAS  PubMed  Google Scholar 

  49. Long, A. M., Zhao, H. & Huang, X. Structural basis for the potent and selective inhibition of casein kinase 1 epsilon. J. Med. Chem. 55, 10307–10311 (2012).

    Article  CAS  PubMed  Google Scholar 

  50. Emsley, P. Tools for ligand validation in COOT. Acta Crystallogr. D Struct. Biol. 73, 203–210 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Skubak, P., Murshudov, G. N. & Pannu, N. S. Direct incorporation of experimental phase information in model refinement. Acta Crystallogr. D Biol. Crystallogr. 60, 2196–2201 (2004).

    Article  PubMed  CAS  Google Scholar 

  52. Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  CAS  PubMed  Google Scholar 

  53. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 27–28 (1996).

    Article  Google Scholar 

  54. Bekker, H. et al. GROMACS—a parallel computer for molecular-dynamics simulations. Phys. Comput. 92, 252–256 (1993).

    Google Scholar 

  55. Best, R. B. & Hummer, G. Optimized molecular dynamics force fields applied to the helix-coil transition of polypeptides. J. Phys. Chem. B 113, 9004–9015 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Lindorff-Larsen, K. et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 78, 1950–1958 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Mamatkulov, S. & Schwierz, N. Force fields for monovalent and divalent metal cations in TIP3P water based on thermodynamic and kinetic properties. J. Chem. Phys. 148, 074504 (2018).

    Article  PubMed  CAS  Google Scholar 

  58. Meagher, K. L., Redman, L. T. & Carlson, H. A. Development of polyphosphate parameters for use with the AMBER force field. J. Comput. Chem. 24, 1016–1025 (2003).

    Article  CAS  PubMed  Google Scholar 

  59. Homeyer, N., Horn, A. H. C., Lanig, H. & Sticht, H. AMBER force-field parameters for phosphorylated amino acids in different protonation states: phosphoserine, phosphothreonine, phosphotyrosine and phosphohistidine. J. Mol. Model. 12, 281–289 (2006).

    Article  CAS  PubMed  Google Scholar 

  60. Greis, K. D. et al. MALDI-TOF MS as a label-free approach to rapid inhibitor screening. J. Am. Soc. Mass Spectrom. 17, 815–822 (2006).

    Article  CAS  PubMed  Google Scholar 

  61. Heap, R. E. et al. Identifying inhibitors of inflammation: a novel high-throughput MALDI-TOF screening assay for salt-inducible kinases (SIKs). SLAS Discov. 22, 1193–1202 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank I. Theofel, S. Young and E. Chih-Chao Liang for their review of and input into this manuscript. The research was funded by the DFG (DO 545/18-1), the Centre for Biomolecular Magnetic Resonance (BMRZ) and the Cluster of Excellence Frankfurt (Macromolecular Complexes). M.T. was supported by a Fellowship from the fund of the German Chemical Industry. L.S. and G.H. were supported by the Max Planck Society. F.P., K.H. and E.H.K.S. thank the EU Horizon2020 project LSFM4LIFE (grant no. 668350-2) and the ZonMw-BMBF joint sponsored project ‘The Onconoid Hub’ (grant no. 114027003) for funding. The Structural Genomics Consortium is a registered charity (no. 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 Institute, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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  1. Marcel Tuppi

    Present address: The Francis Crick Institute, London, UK

  2. These authors contributed equally: Jakob Gebel, Marcel Tuppi, Apirat Chaikuad.

Authors and Affiliations

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

    Jakob Gebel, Marcel Tuppi, Frank Löhr, Niklas Gutfreund, Franziska Finke, Erik Henrich, Julija Mezhyrova & Volker Dötsch

  2. Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main, Germany

    Apirat Chaikuad, Martin Schröder & Stefan Knapp

  3. Physikalische Biologie, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University, Frankfurt am Main, Germany

    Katharina Hötte, Francesco Pampaloni & Ernst H. K. Stelzer

  4. Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Frankfurt am Main, Germany

    Laura Schulz & Gerhard Hummer

  5. Mathezentrum, Goethe University, Frankfurt am Main, Germany

    Ralf Lehnert

  6. Institute of Biophysics, Goethe University, Frankfurt am Main, Germany

    Gerhard Hummer

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Contributions

J.G. and M.T. performed NMR and kinetic assays in vitro and in ovaries. A.C. crystallized and solved the structures of the kinase–peptide complexes. K.H. and F.P. performed microscopy experiments and quantitative semi-automated segmentation. L.S. carried out MD simulations. F.L. performed NMR experiments. N.G. measured tetramerization kinetics. F.F., E.H. and J.M. expressed and purified proteins. M.S. measured phosphorylation kinetics. R.L. helped to analyze the data. M.T., G.H., E.H.K.S., S.K. and V.D. designed experiments and analyzed data. J.G., M.T., A.C. and V.D. wrote the manuscript.

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Correspondence to Marcel Tuppi or Volker Dötsch.

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Extended data

Extended Data Fig. 1 Phosphorylation kinetics of the PAD peptide.

Phosphorylation kinetics of the PAD peptide. a, Overlay of HSQC spectra of unphosphorylated (cyan) and MK2 phosphorylated PAD peptide (red). A large chemical shift change in S582 can be observed as a result of phosphorylation. b, Phosphorylation kinetics of S582 pre-phosphorylated PAD peptide (250 µM) and 12.5 nM CK1 kinase. The phosphorylation of S585 (red) is faster compared to the S588 phosphorylation (yellow) to the extent that the pS582/pS585 intermediate state (cyan) is populated to >1000x of the kinase concentration. c, Phosphorylation kinetics of S582, S585, and S588 pre-phosphorylated PAD peptide (250 µM) with 2.5 µM CK1 kinase. The phosphorylation of S591 (red) is faster than T594 (yellow). The difference is smaller, but the concentration of the intermediate state is at least 4x larger than the kinase concentration. d, Overlay of [15 N, 1H]-HSQC spectra of a pS582/S585A PAD peptide (red) and the same peptide after 500 min of exposure to CK1 (blue, 1:2000 kinase:substrate ratio, 25 °C). Only partial phosphorylation of a single residue in the vicinity of S582 is visible, indicated by the splitting of the signal, accounting for 45% of the population of S582. e, f, Overlay of [15 N, 1H]-HSQC spectra of a pS582/S588A PAD peptide (red) and the same peptide after 500 min of exposure to CK1 and phosphorylation kinetics, demonstrating that S585 and T586 are the only residues phosphorylated by CK1 when the second phosphorylation site for CK1 is eliminated. g, Mutation of T586 to alanine does not change the biphasic behavior of the CK1 phosphorylation of the PAD peptide. All experiments were repeated twice and reproducible. Only one replicate is shown in each case.

Extended Data Fig. 2 Phosphorylation kinetics of PAD peptide mutants.

Phosphorylation kinetics of PAD peptide mutants. a, Phosphorylation kinetics of selected PAD mutants showing a reduction in the kinetic difference between S588 and S591. b, Measured kinetic constants for the phosphorylation of S588 (k2) and S591 (k3) in wild type and different mutant PAD peptides as well as the constants for the second and third phosphorylation event in the YAP1 peptide. Measurements were repeated twice under identical conditions. Individual data points are shown. The height of the bar represents the mean value and single measurements are indicated. The phosphorylation kinetics of YAP was measured once.

Extended Data Fig. 3 Crystallographic details.

Crystallographic details. a–c, Binding of the PAD peptides within CK1δ in the crystal structures. |Fo | -|Fc| omitted electron density map contoured at three-sigma for the bound PAD peptides. d–f, Detailed interactions at the N termini of the PAD peptides within the kinase.

Extended Data Fig. 4 MD simulation and kinetics with CK1 mutants.

MD simulation and kinetics with CK1 mutants. a, MD simulation of CK1 in complex with a shorter PAD-3P peptide (ACE-TPpSSApSTVpSVGSSETRG-NME) with N-terminal acetyl and C-terminal methylamino capping groups showing similar results as the longer peptide (Fig. 5a, b). b, Snapshot at 1 µs, zooming in on the C-terminal region of the shorter p63 peptide. CK1 is shown as a transparent electrostatic surface (blue/red for positive/negative charge) and the p63 peptide is represented as a cyan cartoon. The residues E593, Arg127 and Lys154 are highlighted. The minimum distances between E593 and the basic residues are indicated. c, Phosphorylation kinetics of wild type and selected CK1δ mutants showing a reduction in the kinetic difference between S588 and S591 compared to the wild type kinase for Lys171Glu and Lys154Glu mutants. For Arg127Glu the kinetics is, however, slower. CK1 amino acids are labeled in three-letter code and PAD residues are labeled in one-letter code. All kinetic experiments involving kinase mutants were measured once.

Extended Data Fig. 5 Measurements of binding affinity and enzyme kinetics.

Measurements of binding affinity and enzyme kinetics. a, Three independent KD measurements for each phosphorylation state of the peptide are shown (see also Fig. 6a and Supplementary Table 3). Values given in Supplementary Table 3 represent mean + /-SD. b, KM/vmax measurements for different phosphorylation reactions were performed in biological triplicates for S585 as well as S588 and duplicates for S591. In all subpanels single experiments are shown individually (see also Fig. 6b and Supplementary Tables 4 and 5). Values given in Supplementary Table 5 represent mean + /-SD. c–e, Linearization curves to account for nonlinear ionization behavior of PAD peptides with different phosphorylation states. Linearization curves were determined once.

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Gebel, J., Tuppi, M., Chaikuad, A. et al. p63 uses a switch-like mechanism to set the threshold for induction of apoptosis. Nat Chem Biol 16, 1078–1086 (2020). https://doi.org/10.1038/s41589-020-0600-3

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