Degradation of Cdc25A by β-TrCP during S phase and in response to DNA damage

Abstract

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A1,2,3. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A1,4,5 and contribute to delaying cell-cycle progression, thereby preventing genomic instability1,2,3,4,5,6,7. Here we report that β-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of β-TrCP1 and β-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that β-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of β-TrCP expression results in radioresistant DNA synthesis in response to DNA damage—a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that β-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Cdc25A interacts with β-TrCP1 and β-TrCP2 in vivo.
Figure 2: Interaction with β-TrCP protein through a phosphorylated DSG motif is required for Cdc25A degradation and polyubiquitination.
Figure 3: β-TrCP controls Cdc25A abundance during progression of S phase.
Figure 4: β-TrCP is required for degradation of Cdc25A induced by ionizing radiation in the intra-S-phase checkpoint.

References

  1. 1

    Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J. & Lukas, J. The ATM–Chk2–Cdc25A checkpoint pathway guards against radioresistant DNA synthesis. Nature 410, 842–847 (2001)

    CAS  Article  Google Scholar 

  2. 2

    Zhao, H., Watkins, J. L. & Piwnica-Worms, H. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints. Proc. Natl Acad. Sci. USA 99, 14795–14800 (2002)

    CAS  Article  Google Scholar 

  3. 3

    Sorensen, C. S. et al. Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell 3, 247–258 (2003)

    CAS  Article  Google Scholar 

  4. 4

    Mailand, N. et al. Rapid destruction of human Cdc25A in response to DNA damage. Science 288, 1425–1429 (2000)

    CAS  Article  Google Scholar 

  5. 5

    Molinari, M., Mercurio, C., Dominguez, J., Goubin, F. & Draetta, G. F. Human Cdc25 A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep. 1, 71–79 (2000)

    CAS  Article  Google Scholar 

  6. 6

    Bartek, J. & Lukas, J. Mammalian G1- and S-phase checkpoints in response to DNA damage. Curr. Opin. Cell Biol. 13, 738–747 (2001)

    CAS  Article  Google Scholar 

  7. 7

    Falck, J., Petrini, J. H., Williams, B. R., Lukas, J. & Bartek, J. The DNA damage-dependent intra-S phase checkpoint is regulated by parallel pathways. Nature Genet. 30, 290–294 (2002)

    Article  Google Scholar 

  8. 8

    Donzelli, M. et al. Dual mode of degradation of Cdc25A phosphatase. EMBO J. 21, 4875–4884 (2002)

    CAS  Article  Google Scholar 

  9. 9

    Jackson, P. K. & Eldridge, A. G. The SCF ubiquitin ligase: an extended look. Mol. Cell 9, 923–925 (2002)

    CAS  Article  Google Scholar 

  10. 10

    Patton, E. E., Willems, A. R. & Tyers, M. Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. Trends Genet. 14, 236–243 (1998)

    CAS  Article  Google Scholar 

  11. 11

    Spruck, C. H. & Strohmaier, H. M. Seek and destroy: SCF ubiquitin ligases in mammalian cell cycle control. Cell Cycle 1, 250–254 (2002)

    CAS  Article  Google Scholar 

  12. 12

    Cenciarelli, C. et al. Identification of a family of human F-box proteins. Curr. Biol. 9, 1177–1179 (1999)

    CAS  Article  Google Scholar 

  13. 13

    Kipreos, E. T. & Pagano, M. The F-box protein family. Genome Biol. 1, Reviews3002.1–3002.7 [online] 〈http://genomebiology.com/2000/1/5/reviews/3002〉 (2000)

  14. 14

    Yaron, A. et al. Identification of the receptor component of the IκBα-ubiquitin ligase. Nature 396, 590–594 (1998)

    CAS  Article  Google Scholar 

  15. 15

    Winston, J. T. et al. The SCF β-TRCP–ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro. Genes Dev. 13, 270–283 (1999)

    CAS  Article  Google Scholar 

  16. 16

    Lassot, I. et al. ATF4 degradation relies on a phosphorylation-dependent interaction with the SCFβTrCP ubiquitin ligase. Mol. Cell. Biol. 21, 2192–2202 (2001)

    CAS  Article  Google Scholar 

  17. 17

    Lang, V. et al. βTrCP-mediated proteolysis of NF-κB1 p105 requires phosphorylation of p105 serines 927 and 932. Mol. Cell. Biol. 23, 402–413 (2003)

    CAS  Article  Google Scholar 

  18. 18

    Liu, C. et al. Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108, 837–847 (2002)

    CAS  Article  Google Scholar 

  19. 19

    Guardavaccaro, D. et al. Control of meiotic and mitotic progression by the F box protein β-Trcp1 in vivo. Dev. Cell 4, 799–812 (2003)

    CAS  Article  Google Scholar 

  20. 20

    Margottin-Goguet, F. et al. Prophase destruction of Emi1 by the SCFβTrCP/Slimb ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase. Dev. Cell 4, 813–826 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Hsu, J. Y., Reimann, J. D., Sorensen, C. S., Lukas, J. & Jackson, P. K. E2F-dependent accumulation of hEmi1 regulates S phase entry by inhibiting APCCdh1. Nature Cell Biol. 4, 358–366 (2002)

    CAS  Article  Google Scholar 

  22. 22

    Painter, R. B. & Young, B. R. Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc. Natl Acad. Sci. USA 77, 7315–7317 (1980)

    CAS  Article  Google Scholar 

  23. 23

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

    CAS  Article  Google Scholar 

  24. 24

    Nash, P. et al. Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521 (2001)

    CAS  Article  Google Scholar 

  25. 25

    Wu, G. et al. Structure of a β-TrCP1–Skp1–β-catenin complex: destruction motif binding and lysine specificity of the SCFβ-TrCP1 ubiquitin ligase. Mol. Cell 11, 1445–1456 (2003)

    CAS  Article  Google Scholar 

  26. 26

    Clucas, C., Cabello, J., Bussing, I., Schnabel, R. & Johnstone, I. L. Oncogenic potential of a C. elegans cdc25 gene is demonstrated by a gain-of-function allele. EMBO J. 21, 665–674 (2002)

    CAS  Article  Google Scholar 

  27. 27

    Bell, D. W. et al. Heterozygous germ line hCHK2 mutations in Li–Fraumeni syndrome. Science 286, 2528–2531 (1999)

    CAS  Article  Google Scholar 

  28. 28

    Carrano, A. C., Eytan, E., Hershko, A. & Pagano, M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nature Cell Biol. 1, 193–199 (1999)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank T. K. Ko for his contribution; Z. G. Pan and S. Reed for reagents; G. Ossolengo for producing and purifying the anti-phosphoS82/S88 antibody; and M. Squatrito and E. De Billy for discussion. This work was supported by an Italian American Cancer Foundation fellowship and a Susan Komen Breast Cancer Foundation fellowship to D.G.; a Irma Hirschl Scholarship and grants from the NIH to M.P.; and grants from the Italian Association for Cancer Research (AIRC), the Italian Foundation for Cancer Research (FIRC) and Telethon to G.F.D.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Maddalena Donzelli.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Busino, L., Donzelli, M., Chiesa, M. et al. Degradation of Cdc25A by β-TrCP during S phase and in response to DNA damage. Nature 426, 87–91 (2003). https://doi.org/10.1038/nature02082

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing