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A novel WEE1 pathway for replication stress responses

Nature Plants volume 7, pages 209–218 (2021)Cite this article

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Abstract

DNA replication stress poses a severe threat to genome stability and is a hallmark of cancer as well as a target for cancer therapy. It is well known that the evolutionarily conserved protein kinase WEE1 regulates replication stress responses by directly phosphorylating and inhibiting the major cell cycle driver CDKs in many organisms. Here, we report a novel WEE1 pathway. We found that Arabidopsis WEE1 directly interacts with and phosphorylates the E3 ubiquitin ligase FBL17 that promotes the degradation of CDK inhibitors. The phosphorylated FBL17 is further polyubiquitinated and degraded, thereby leading to the accumulation of CDK inhibitors and the inhibition of CDKs. In strong support for this model, either loss of function of FBL17 or overexpression of CDK inhibitors suppresses the hypersensitivity of the wee1 mutant to replication stress. Intriguingly, human WEE1 also phosphorylates and destabilizes the FBL17 equivalent protein SKP2, indicating that this is a conserved mechanism. This study reveals that the WEE1-FBL17/SKP2-CKIs-CDKs axis is a molecular framework for replication stress responses, which may have clinical implications because the WEE1 inhibitor AZD1775 is currently in phase II clinical trial as an anticancer drug.

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Fig. 1: Loss of function of FBL17 suppresses the hypersensitivity of atr and wee1 to replication stress.
Fig. 2: FBL17 interacts with WEE1.
Fig. 3: WEE1 phosphorylates FBL17.
Fig. 4: WEE1-mediated FBL17 phosphorylation promotes FBL17 degradation.
Fig. 5: KRP7 functions downstream of FBL17 in the WEE1 pathway.
Fig. 6: Human WEE1A phosphorylates and negatively regulates SKP2.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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References

  1. Zeman, M. K. & Cimprich, K. A. Causes and consequences of replication stress. Nat. Cell Biol. 16, 2–9 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Macheret, M. & Halazonetis, T. D. DNA replication stress as a hallmark of cancer. Annu. Rev. Pathol. Mech. Dis. 10, 425–448 (2015).

    Article  CAS  Google Scholar 

  3. Gaillard, H., García-Muse, T. & Aguilera, A. Replication stress and cancer. Nat. Rev. Cancer 15, 276–280 (2015).

    Article  CAS  PubMed  Google Scholar 

  4. Blackford, A. N. & Jackson, S. P. ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response. Mol. Cell 66, 801–817 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Maréchal, A. et al. PRP19 transforms into a sensor of RPA–ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry. Mol. Cell 53, 235–246 (2014).

    Article  PubMed  CAS  Google Scholar 

  6. Lanz, M. C., Dibitetto, D. & Smolka, M. B. DNA damage kinase signaling: checkpoint and repair at 30 years. EMBO J. 38, e101801 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Saldivar, J. C., Cortez, D. & Cimprich, K. A. The essential kinase ATR: ensuring faithful duplication of a challenging genome. Nat. Rev. Mol. Cell Biol. 18, 622–636 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cortez, D. ATR and ATRIP: partners in checkpoint signaling. Science 294, 1713–1716 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Kumagai, A., Lee, J., Yoo, H. Y. & Dunphy, W. G. TopBP1 activates the ATR–ATRIP complex. Cell 124, 943–955 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Zou, L. Sensing DNA damage through ATRIP recognition of RPA–ssDNA complexes. Science 300, 1542–1548 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Bass, T. E. et al. ETAA1 acts at stalled replication forks to maintain genome integrity. Nat. Cell Biol. 18, 1185–1195 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Haahr, P. et al. Activation of the ATR kinase by the RPA-binding protein ETAA1. Nat. Cell Biol. 18, 1196–1207 (2016).

    Article  CAS  PubMed  Google Scholar 

  13. Lee, Y.-C., Zhou, Q., Chen, J. & Yuan, J. RPA-binding protein ETAA1 is an ATR activator involved in DNA replication stress response. Curr. Biol. 26, 3257–3268 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bermudez, V. P. et al. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc. Natl Acad. Sci. USA 100, 1633–1638 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Delacroix, S., Wagner, J. M., Kobayashi, M., Yamamoto, K.-i & Karnitz, L. M. The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1. Genes Dev. 21, 1472–1477 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sancar, A., Lindsey-Boltz, L. A., Ünsal-Kaçmaz, K. & Linn, S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu. Rev. Biochem. 73, 39–85 (2004).

    Article  CAS  PubMed  Google Scholar 

  17. Hu, Z., Cools, T. & De Veylder, L. Mechanisms used by plants to cope with DNA damage. Annu. Rev. Plant Biol. 67, 439–462 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Harashima, H., Dissmeyer, N. & Schnittger, A. Cell cycle control across the eukaryotic kingdom. Trends Cell Biol. 23, 345–356 (2013).

    Article  CAS  PubMed  Google Scholar 

  19. Brown, E. J. & Baltimore, D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 14, 397–402 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Tominaga, Y., Li, C., Wang, R. H. & Deng, C. X. Murine Wee1 plays a critical role in cell cycle regulation and pre-implantation stages of embryonic development. Int. J. Biol. Sci. 2, 161–170 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Culligan, K., Tissier, A. & Britt, A. ATR regulates a G2-phase cell-cycle checkpoint in Arabidopsis thaliana. Plant Cell 16, 1091–1104 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. De Schutter, K. et al. Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint. Plant Cell 19, 211–225 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Dissmeyer, N. et al. Control of cell proliferation, organ growth, and DNA damage response operate independently of dephosphorylation of the Arabidopsis Cdk1 homolog CDKA;1. Plant Cell 21, 3641–3654 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Wachsman, G., Modliszewski, J. L., Valdes, M. & Benfey, P. N. A SIMPLE pipeline for mapping point mutations. Plant Physiol. 174, 1307–1313 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhao, X. et al. A general G1/S-phase cell-cycle control module in the flowering plant Arabidopsis thaliana. PLoS Genet. 8, e1002847 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Noir, S. et al. The control of Arabidopsis thaliana growth by cell proliferation and endoreplication requires the F-box protein FBL17. Plant Cell 27, 1461–1476 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gusti, A. et al. The Arabidopsis thaliana F-box protein FBL17 is essential for progression through the second mitosis during pollen development. PLoS ONE 4, e4780 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Kim, H. J. et al. Control of plant germline proliferation by SCF(FBL17) degradation of cell cycle inhibitors. Nature 455, 1134–1137 (2008).

    Article  CAS  PubMed  Google Scholar 

  29. Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Walter, M. et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40, 428–438 (2004).

    Article  CAS  PubMed  Google Scholar 

  31. Chen, H. et al. Firefly luciferase complementation imaging assay for protein–protein interactions in plants. Plant Physiol. 146, 368–376 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gao, J., Thelen, J. J., Dunker, A. K. & Xu, D. Musite, a tool for global prediction of general and kinase-specific phosphorylation sites. Mol. Cell. Proteom. 9, 2586–2600 (2010).

    Article  CAS  Google Scholar 

  33. Yi, D. et al. The Arabidopsis SIAMESE-RELATED cyclin-dependent kinase inhibitors SMR5 and SMR7 regulate the DNA damage checkpoint in response to reactive oxygen species. Plant Cell 26, 296–309 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yu, Z. K., Gervais, J. L. & Zhang, H. Human CUL-1 associates with the SKP1/SKP2 complex and regulates p21(CIP1/WAF1) and cyclin D proteins. Proc. Natl Acad. Sci. USA 95, 11324–11329 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Sutterlüty, H. et al. p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells. Nat. Cell Biol. 1, 207–214 (1999).

    Article  PubMed  Google Scholar 

  36. Wei, W. et al. Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex. Nature 428, 194–198 (2004).

    Article  CAS  PubMed  Google Scholar 

  37. Watanabe, N. et al. M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFbeta-TrCP. Proc. Natl Acad. Sci. USA 101, 4419–4424 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Heijink, A. M. et al. A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity. Proc. Natl Acad. Sci. USA 112, 15160–15165 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ogita, N. et al. Identifying the target genes of SUPPRESSOR OF GAMMA RESPONSE 1, a master transcription factor controlling DNA damage response in Arabidopsis. Plant J. 94, 439–453 (2018).

    Article  CAS  PubMed  Google Scholar 

  40. Lim, S. & Kaldis, P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development 140, 3079–3093 (2013).

    Article  CAS  PubMed  Google Scholar 

  41. Topacio, B. R. et al. Cyclin D-Cdk4,6 drives cell-cycle progression via the retinoblastoma protein’s C-terminal helix. Mol. Cell 74, 758–770 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Hydbring, P., Malumbres, M. & Sicinski, P. Non-canonical functions of cell cycle cyclins and cyclin-dependent kinases. Nat. Rev. Mol. Cell Biol. 17, 280–292 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Guardavaccaro, D. & Pagano, M. Stabilizers and destabilizers controlling cell cycle oscillators. Mol. Cell 22, 1–4 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. Bukhari, A. B. et al. Inhibiting Wee1 and ATR kinases produces tumor-selective synthetic lethality and suppresses metastasis. J. Clin. Invest. 129, 1329–1344 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Hirai, H. et al. Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol. Cancer Ther. 8, 2992–3000 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Do, K., Doroshow, J. H. & Kummar, S. Wee1 kinase as a target for cancer therapy. Cell Cycle 12, 3348–3353 (2013).

    Article  CAS  Google Scholar 

  47. Schwob, E. The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell 79, 233–244 (1994).

    Article  CAS  PubMed  Google Scholar 

  48. Verma, R. Phosphorylation of Sic1p by G1 Cdk required for its degradation and entry into S phase. Science 278, 455–460 (1997).

    Article  CAS  PubMed  Google Scholar 

  49. Dissmeyer, N., Weimer, A. K., de Veylder, L., Novak, B. & Schnittger, A. The regulatory network of cell cycle progression is fundamentally different in plants versus yeast or metazoans. Plant Signal. Behav. 5, 1613–1618 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Kalhorzadeh, P. et al. Arabidopsis thaliana RNase H2 deficiency counteracts the needs for the WEE1 checkpoint kinase but triggers genome instability. Plant Cell 26, 3680–3692 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Gentric, N. et al. The F-box-like protein FBL17 is a regulator of DNA-damage response and colocalizes with RETINOBLASTOMA RELATED1 at DNA lesion sites. Plant Physiol. 183, 1295–1305 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Yoo, S.-D., Cho, Y.-H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  PubMed  Google Scholar 

  53. Wang, Y. et al. Strigolactone/MAX2-induced degradation of brassinosteroid transcriptional effector BES1 regulates shoot branching. Dev. Cell 27, 681–688 (2013).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We are grateful to H. G. Nam for providing the pFBL17:FBL17-GFP vector and L. De Veylder for critically revising the manuscript. This work was supported by the National Natural Science Foundation of China (grant nos. 31571253, 31771355 and 31970311); Fundamental Research Funds for the Central Universities (grant no. 2662019PY029); Thousand Talents Plan of China-Young Professionals Grant; and Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (grant no. 2014RC004).

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Authors and Affiliations

  1. College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China

    Ting Pan, Qi Qin, Chubing Nong, Shan Gao, Lili Wang, Bingcheng Cai, Ming Zhang, Chong Wu, Hanchen Chen, Tong Li & Shunping Yan

  2. College of Informatics, Huazhong Agricultural University, Wuhan, Hubei, China

    Dan Xiong & Guoliang Li

  3. College of Life Sciences, Shanghai Normal University, Shanghai, China

    Shui Wang

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Contributions

T.P. and S.Y. designed the experiments. T.P., Q.Q., C.N., S.G., L.W., B.C., M.Z., C.W., H.C., T.L., D.X., G.L. and S.W. carried out the experiments. T.P. and S.Y. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Shunping Yan.

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Peer review information Nature Plants thanks Pascal Genschik, Jingsong Yuan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Pan, T., Qin, Q., Nong, C. et al. A novel WEE1 pathway for replication stress responses. Nat. Plants 7, 209–218 (2021). https://doi.org/10.1038/s41477-021-00855-8

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