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Acetylation by GCN5 regulates CDC6 phosphorylation in the S phase of the cell cycle

Nature Structural & Molecular Biology volume 16, pages 412–420 (2009)Cite this article

Abstract

In eukaryotic cells, the cell-division cycle (CDC)-6 protein is essential to promote the assembly of pre-replicative complexes in the early G1 phase of the cell cycle, a process requiring tight regulation to ensure that proper origin licensing occurs once per cell cycle. Here we show that, in late G1 and early S phase, CDC6 is found in a complex also containing Cyclin A, cyclin-dependent kinase (CDK)-2 and the acetyltransferase general control nonderepressible 5 (GCN5). GCN5 specifically acetylates CDC6 at three lysine residues flanking its cyclin-docking motif, and this modification is crucial for the subsequent phosphorylation of the protein by Cyclin A–CDKs at a specific residue close to the acetylation site. GCN5-mediated acetylation and site-specific phosphorylation of CDC6 are both necessary for the relocalization of the protein to the cell cytoplasm in the S phase, as well as to regulate its stability. This two-step, intramolecular regulatory program by sequential modification of CDC6 seems to be essential for proper S-phase progression.

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Figure 1: CDC6 is acetylated by GCN5 on lysines 92, 105 and 109 both in vitro and in vivo.
Figure 2: Specific CDC6 phosphorylation on Ser106 depends on GCN5-mediated CDC6 acetylation.
Figure 3: CDC6 acetylation is cell cycle dependent.
Figure 4: GCN5-dependent CDC6 acetylation regulates its subcellular localization.
Figure 5: Characterization of the CDC6 K3R and S106A mutants.
Figure 6: Model showing the regulation of CDC6 by sequential modification by acetylation and phosphorylation in early S phase.

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References

  1. Lei, M. & Tye, B.K. Initiating DNA synthesis: from recruiting to activating the MCM complex. J. Cell Sci. 114, 1447–1454 (2001).

    CAS  PubMed  Google Scholar 

  2. Bell, S.P. The origin recognition complex: from simple origins to complex functions. Genes Dev. 16, 659–672 (2002).

    Article  CAS  Google Scholar 

  3. Blow, J.J. & Dutta, A. Preventing re-replication of chromosomal DNA. Nat. Rev. Mol. Cell Biol. 6, 476–486 (2005).

    Article  CAS  Google Scholar 

  4. Nishitani, H. & Lygerou, Z. Control of DNA replication licensing in a cell cycle. Genes Cells 7, 523–534 (2002).

    Article  CAS  Google Scholar 

  5. Nasmyth, K. Viewpoint: putting the cell cycle in order. Science 274, 1643–1645 (1996).

    Article  CAS  Google Scholar 

  6. Diffley, J.F. Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786 (2004).

    Article  CAS  Google Scholar 

  7. Dutta, A. & Bell, S.P. Initiation of DNA replication in eukaryotic cells. Annu. Rev. Cell Dev. Biol. 13, 293–332 (1997).

    Article  CAS  Google Scholar 

  8. Stillman, B. Initiation of eukaryotic DNA replication in vitro. Annu. Rev. Cell. Biol. 1989, 197–245 (1989).

    Article  Google Scholar 

  9. Leatherwood, J. Emerging mechanisms of eukaryotic DNA replication initiation. Curr. Opin. Cell Biol. 10, 742–748 (1998).

    Article  CAS  Google Scholar 

  10. Coleman, T.R., Carpenter, P.B. & Dunphy, W.G. The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts. Cell 87, 53–63 (1996).

    Article  CAS  Google Scholar 

  11. Tanaka, T., Knapp, D. & Nasmyth, K. Loading of an Mcm protein onto DNA replication origins is regulted by Cdc6p and CKDs. Cell 90, 649–660 (1997).

    Article  CAS  Google Scholar 

  12. Donovan, S., Harwood, J., Drury, L.S. & Diffley, J.F. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proc. Natl. Acad. Sci. USA 94, 5611–5616 (1997).

    Article  CAS  Google Scholar 

  13. Jiang, W., Wells, N.J. & Hunter, T. Multistep regulation of DNA replication by Cdk phosphorylation of HsCdc6. Proc. Natl. Acad. Sci. USA 96, 6193–6198 (1999).

    Article  CAS  Google Scholar 

  14. Petersen, B.O., Lukas, J., Sorensen, C.S., Bartek, J. & Helin, K. Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization. EMBO J. 18, 396–410 (1999).

    Article  CAS  Google Scholar 

  15. Petersen, B.O. et al. Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1. Genes Dev. 14, 2330–2343 (2000).

    Article  CAS  Google Scholar 

  16. Saha, P . et al. Human CDC6/Cdc18 associates with Orc1 and Cyclin-CDK and is selectively eliminated from the nucleus at the onset of S-phase. Mol. Cell. Biol. 18, 2758–2767 (1998).

    Article  CAS  Google Scholar 

  17. Williams, R.S., Shohet, R.V. & Stillman, B. A human protein related to yeast Cdc6p. Proc. Natl. Acad. Sci. USA 94, 142–147 (1997).

    Article  CAS  Google Scholar 

  18. Mailand, N. & Diffley, J.F. CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122, 915–926 (2005).

    Article  CAS  Google Scholar 

  19. Mendez, J. & Stillman, B. Chromatin association of human origin recognition complex, CDC6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol. Cell. Biol. 20, 8602–8612 (2000).

    Article  CAS  Google Scholar 

  20. Delmolino, L.M., Saha, P. & Dutta, A. Multiple mechanisms regulate subcellular localization of human CDC6. J. Biol. Chem. 276, 26947–26954 (2001).

    Article  CAS  Google Scholar 

  21. Herbig, U., Griffith, J.W. & Fanning, E. Mutation of Cyclin/CDK phosphorylation sites in HsCdc6 disrupts a late step in initiation of DNA replication in human cells. Mol. Biol. Cell 11, 4117–4130 (2000).

    Article  CAS  Google Scholar 

  22. Alexandrow, M.G. & Hamlin, J.L. Cdc6 chromatin affinity is unaffected by serine-54 phosphorylation, S-phase progression, and overexpression of cyclin A. Mol. Cell. Biol. 24, 1614–1627 (2004).

    Article  CAS  Google Scholar 

  23. Timmers, H.T. & Tora, L. SAGA unveiled. Trends Biochem. Sci. 30, 7–10 (2005).

    Article  CAS  Google Scholar 

  24. Burgess, S.M., Ajimura, M. & Kleckner, N. GCN5-dependent histone H3 acetylation and RPD3-dependent histone H4 deacetylation have distinct, opposing effects on IME2 transcription, during meiosis and during vegetative growth, in budding yeast. Proc. Natl. Acad. Sci. USA 96, 6835–6840 (1999).

    Article  CAS  Google Scholar 

  25. Howe, L. et al. Histone H3 specific acetyltransferases are essential for cell cycle progression. Genes Dev. 15, 3144–3154 (2001).

    Article  CAS  Google Scholar 

  26. Krebs, J.E., Kuo, M.H., Allis, C.D. & Peterson, C.L. Cell cycle-regulated histone acetylation required for expression of the yeast HO gene. Genes Dev. 13, 1412–1421 (1999).

    Article  CAS  Google Scholar 

  27. Zhang, W., Bone, J.R., Edmondson, D.G., Turner, B.M. & Roth, S.Y. Essential and redundant functions of histone acetylation revealed by mutation of target lysines and loss of the Gcn5p acetyltransferase. EMBO J. 17, 3155–3167 (1998).

    Article  CAS  Google Scholar 

  28. Paulson, M., Press, C., Smith, E., Tanese, N. & Levy, D.E. IFN-stimulated transcription through a TBP-free acetyltransferase complex escapes viral shutoff. Nat. Cell Biol. 4, 140–147 (2002).

    Article  CAS  Google Scholar 

  29. Palhan, V.B. et al. Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration. Proc. Natl. Acad. Sci. USA 102, 8472–8477 (2005).

    Article  CAS  Google Scholar 

  30. Takei, Y. et al. MCM3AP, a novel acetyltransferase that acetylates replication protein MCM3. EMBO Rep. 2, 119–123 (2001).

    Article  CAS  Google Scholar 

  31. Zarkowska, T. & Mittnacht, S. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J. Biol. Chem. 272, 12738–12746 (1997).

    Article  CAS  Google Scholar 

  32. Galbiati, L., Mendoza-Maldonado, R., Gutierrez, M.I. & Giacca, M. Regulation of E2F–1 after DNA damage by p300-mediated acetylation and ubiquitination. Cell Cycle 4, 930–939 (2005).

    Article  CAS  Google Scholar 

  33. Takahashi, T., Ohara, E., Nishitani, H. & Masukata, H. Multiple ORC-binding sites are required for efficient MCM loading and origin firing in fission yeast. EMBO J. 22, 964–974 (2003).

    Article  CAS  Google Scholar 

  34. Muratoglu, S. et al. Two different Drosophila ADA2 homologues are present in distinct GCN5 histone acetyltransferase-containing complexes. Mol. Cell. Biol. 23, 306–321 (2003).

    Article  CAS  Google Scholar 

  35. Guelman, S. et al. Host cell factor and an uncharacterized SANT domain protein are stable components of ATAC, a novel dAda2A/dGcn5-containing histone acetyltransferase complex in Drosophila. Mol. Cell. Biol. 26, 871–882 (2006).

    Article  CAS  Google Scholar 

  36. Vaziri, C. et al. A p53-dependent checkpoint pathway prevents rereplication. Mol. Cell 11, 997–1008 (2003).

    Article  CAS  Google Scholar 

  37. Nishitani, H., Lygerou, Z. & Nishimoto, T. Proteolysis of DNA replication licensing factor Cdt1 in S-phase is performed independently of geminin through its N-terminal region. J. Biol. Chem. 279, 30807–30816 (2004).

    Article  CAS  Google Scholar 

  38. Drury, L.S., Perkins, G. & Diffley, J.F. The Cdc4/34/53 pathway targets Cdc6p for proteolysis in budding yeast. EMBO J. 16, 5966–5976 (1997).

    Article  CAS  Google Scholar 

  39. Duursma, A. & Agami, R. p53-dependent regulation of Cdc6 protein stability controls cellular proliferation. Mol. Cell. Biol. 25, 6937–6947 (2005).

    Article  CAS  Google Scholar 

  40. Coverley, D., Laman, H. & Laskey, R.A. Distinct roles for cyclins E and A during DNA replication complex assembly and activation. Nat. Cell Biol. 4, 523–528 (2002).

    Article  CAS  Google Scholar 

  41. Pelizon, C., Madine, M.A., Romanowski, P. & Laskey, R.A. Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle. Genes Dev. 14, 2526–2533 (2000).

    Article  CAS  Google Scholar 

  42. Oehlmann, M., Score, A.J. & Blow, J.J. The role of Cdc6 in ensuring complete genome licensing and S phase checkpoint activation. J. Cell Biol. 165, 181–190 (2004).

    Article  CAS  Google Scholar 

  43. Clay-Farrace, L., Pelizon, C., Santamaria, D., Pines, J. & Laskey, R.A. Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1. EMBO J. 22, 704–712 (2003).

    Article  CAS  Google Scholar 

  44. Kikuchi, H., Takami, Y. & Nakayama, T. GCN5: a supervisor in all-inclusive control of vertebrate cell cycle progression through transcription regulation of various cell cycle-related genes. Gene 347, 83–97 (2005).

    Article  CAS  Google Scholar 

  45. Iizuka, M., Matsui, T., Takisawa, H. & Smith, M.M. Regulation of replication licensing by acetyltransferase Hbo1. Mol. Cell. Biol. 26, 1098–1108 (2006).

    Article  CAS  Google Scholar 

  46. Burke, T.W., Cook, J.G., Asano, M. & Nevins, J.R. Replication factors MCM2 and ORC1 interact with the histone acetyltransferase HBO1. J. Biol. Chem. 276, 15397–15408 (2001).

    Article  CAS  Google Scholar 

  47. Iizuka, M. & Stillman, B. Histone acetyltransferase HBO1 interacts with the ORC1 subunit of the human initiator protein. J. Biol. Chem. 274, 23027–23034 (1999).

    Article  CAS  Google Scholar 

  48. Yang, X.J. Multisite protein modification and intramolecular signaling. Oncogene 24, 1653–1662 (2005).

    Article  CAS  Google Scholar 

  49. Bode, A.M. & Dong, Z. Post-translational modification of p53 in tumorigenesis. Nat. Rev. Cancer 4, 793–805 (2004).

    Article  CAS  Google Scholar 

  50. Matsuzaki, H. et al. Acetylation of FoxO1 alters its DNA-binding ability and sensitivity to phosphorylation. Proc. Natl. Acad. Sci. USA 102, 11278–11283 (2005).

    Article  CAS  Google Scholar 

  51. Ozaki, T. et al. Functional implication of p73 protein stability in neuronal cell survival and death. Cancer Lett. 228, 29–35 (2005).

    Article  CAS  Google Scholar 

  52. Vervoorts, J., Luscher-Firzlaff, J.M. & Luscher, B. The ins and outs of MYC regulation by posttranslational mechanisms. J. Biol. Chem. 281, 34725–34729 (2006).

    Article  CAS  Google Scholar 

  53. Chan, H.M., Krstic-Demonacos, M., Smith, L., Demonacos, C. & La Thangue, N.B. Acetylation control of the retinoblastoma tumour-suppressor protein. Nat. Cell Biol. 3, 667–674 (2001).

    Article  CAS  Google Scholar 

  54. Marzio, G. et al. E2F family members are differentially regulated by reversible acetylation. J. Biol. Chem. 275, 10887–10892 (2000).

    Article  CAS  Google Scholar 

  55. Marcello, A., Massimi, P., Banks, L. & Giacca, M. Adeno-associated virus type 2 rep protein inhibits human papillomavirus type 16 E2 recruitment of the transcriptional coactivator p300. J. Virol. 74, 9090–9098 (2000).

    Article  CAS  Google Scholar 

  56. Marcello, A. et al. Recruitment of human cyclin T1 to nuclear bodies through direct interaction with the PML protein. EMBO J. 22, 2156–2166 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by grants from the FIRB program of the “Ministero dell'Istruzione, Universita' e Ricerca,” Italy and from the “Fondazione CRTrieste” of Trieste, Italy. The authors are indebted to H. Masai (Tokyo Metropolitan Institute of Medical Science) for helpful discussion and to A. Dutta (University of Virginia), K. Helin (Biotech Research and Innovation Centre and Centre for Epigenetics), M. Benkirane (Institut de G–énétique Humaine), J. Pines (Wellcome Trust/Cancer Research UK Gurdon Institute) and H. Masai for the gift of reagents. The authors are grateful to V. Liverani for excellent technical support and to S. Kerbavcic for superb editorial assistance.

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  1. Roberta Paolinelli

    Present address: Present address: IFOM-Institute of Molecular Oncology-Foundation, Milan, Italy.,

  2. Roberta Paolinelli and Ramiro Mendoza-Maldonado: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular Biology Laboratory, Scuola Normale Superiore, AREA della Ricerca del CNR, Pisa, Italy

    Roberta Paolinelli & Anna Cereseto

  2. Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

    Roberta Paolinelli, Ramiro Mendoza-Maldonado & Mauro Giacca

  3. Department of Biomedicine, Faculty of Medicine, University of Trieste, Italy

    Mauro Giacca

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Contributions

All experiments were performed by R.P. and R.M.-M.; A.C. took part in the design of the initial CDC6 acetylation experiments; M.G. supervised the work and wrote the manuscript.

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Correspondence to Mauro Giacca.

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Paolinelli, R., Mendoza-Maldonado, R., Cereseto, A. et al. Acetylation by GCN5 regulates CDC6 phosphorylation in the S phase of the cell cycle. Nat Struct Mol Biol 16, 412–420 (2009). https://doi.org/10.1038/nsmb.1583

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