Threonine-11, phosphorylated by Rad3 and atm in vitro, is required for activation of fission yeast checkpoint kinase Cds1.
Journal: 2001/June - Molecular and Cellular Biology
ISSN: 0270-7306
Abstract:
Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T(11)Q(12). Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1(-) null mutant. Cds1(T11A) was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.
Relations:
Content
Citations
(27)
References
(44)
Drugs
(3)
Chemicals
(6)
Genes
(2)
Organisms
(1)
Processes
(2)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Mol Cell Biol 21(10): 3398-3404

Threonine-11, Phosphorylated by Rad3 and ATM In Vitro, Is Required for Activation of Fission Yeast Checkpoint Kinase Cds1

Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
Corresponding author. Mailing address: Department of Molecular Biology, MB3, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-2823. Fax: (858) 784-2265. E-mail: ude.sppircs@llessurp.
Received 2000 Oct 23; Revisions requested 2000 Dec 4; Accepted 2001 Feb 20.

Abstract

Fission yeast Cds1 is phosphorylated and activated when DNA replication is interrupted by nucleotide starvation or DNA damage. Cds1 enforces the S-M checkpoint that couples mitosis (M) to the completion of DNA synthesis (S). Cds1 also controls replicational stress tolerance mechanisms. Cds1 is regulated by a group of proteins that includes Rad3, a kinase related to human checkpoint kinase ATM (ataxia telangiectasia mutated). ATM phosphorylates serine or threonine followed by glutamine (SQ or TQ). Here we show that in vitro, Rad3 and ATM phosphorylate the N-terminal domain of Cds1 at the motif T11Q12. Substitution of threonine-11 with alanine (T11A) abolished Cds1 activation that occurs when DNA replication is inhibited by hydroxyurea (HU) treatment. The cds1-T11A mutant was profoundly sensitive to HU, although not quite as sensitive as a cds1 null mutant. Cds1 was unable to enforce the S-M checkpoint. These results strongly suggest that Rad3-dependent phosphorylation of Cds1 at threonine-11 is required for Cds1 activation and function.

Abstract

Inheritance of complete and accurate copies of the genome is the singular goal of cell division. Precise genome duplication is an intrinsically difficult process that can be strained further by external agents that interfere with DNA replication or damage DNA. Genome surveillance mechanisms exist to cope with these problems (16, 21). These systems serve two primary purposes. One purpose is to prevent mitosis when DNA replication is interrupted or DNA is damaged. These cell cycle checkpoints actively couple the onset mitosis to the completion of DNA replication and repair. The other purpose of genome surveillance mechanisms is to regulate various repair and replication systems that help cells survive replicational stress and DNA damage.

The fission yeast Schizosaccharomyces pombe has served as a valuable model system for the discovery and investigation of genome integrity checkpoint mechanisms (35). Genetic studies of fission yeast have uncovered a group of genes that are required for arresting cell division when DNA is damaged or when replication is inhibited with the drug hydroxyurea (HU). The products of these checkpoint RAD genes include Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1 (13). Other proteins such as Cut5 (also known as Rad4) and Rfc3, which are necessary for DNA replication, are also important for both the DNA replication (S-M) and G2-M DNA damage checkpoints (38, 40). The most intriguing checkpoint protein may be Rad3, a very large protein (2,386 amino acids) that is related to phosphatidylinositol kinases (PIKs) (6). Other PIK-like proteins include human ATM (ataxia telangiectasia mutated), ATR (ATM and Rad3 related) and DNA-dependent protein kinase (20, 39, 43). Although related to phosphatidylinositol 3-kinases, these enzymes function as protein kinases in vivo. In common with fission yeast Rad3, ATM is required for arrest in G2 phase of the cell cycle in response to DNA damage caused by ionizing radiation and for slowed replication of damaged DNA (16, 24, 37). ATM is thought to control G2 arrest in part by activating Cds1 (also known as Chk2) (7, 8, 26), the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases. ATR has been implicated in the checkpoint response to UV damage and the inhibition of DNA replication (11, 43). Recently, Chk1 phosphorylation was found to be ATR dependent, suggesting that Chk1 is regulated by ATR (19, 25).

Rad3 and the other checkpoint Rad proteins control two downstream protein kinases in fission yeast. When DNA is damaged during G2 phase, Chk1 becomes phosphorylated in a Rad3-dependent manner (42). Chk1 prevents the onset of mitosis by regulation of Cdc25 and Mik1, two proteins that control the inhibitory phosphorylation of the cyclin-dependent kinase Cdc2 (2, 17, 18, 34, 36). The significance of Chk1 phosphorylation is uncertain, but it correlates with the requirement for Chk1 to arrest cell division in response to DNA damage. It is unknown if Chk1 is a direct physiological substrate of Rad3.

Fission yeast Cds1 becomes phosphorylated and activated by a Rad3-dependent mechanism when DNA is damaged during S phase or when DNA replication is interrupted with HU or mutations of several essential genes (9, 24). Cds1 enforces the S-M checkpoint by regulating Cdc25 and Mik1 (9, 17). In cds1 mutants treated with HU, the onset of mitosis is prevented by Chk1, but these cells are inviable (9, 10, 24). This fact demonstrates that Cds1 has replicational stress recovery functions that are distinct from its cell cycle checkpoint activity. How Cds1 is regulated is unknown.

Fission yeast Cds1 and its homologs are recognizable by similar kinase domains, an N-terminal Ser-Gln/Thr-Gln (SQ/TQ) cluster domain, and a forkhead-associated (FHA) domain (8, 26). SQ and TQ sequences are the preferred sites of phosphorylation by ATM in p53, c-Abl, Brca1, and Nbs1 (3, 4, 12, 14, 23). FHA domains are believed to act as protein-protein interaction domains and in some instances can bind to phosphorylated partners (22, 41). Budding yeast Rad53 is unique in possessing a second C-terminal FHA domain (1).

Several findings link Rad3 to Cds1. As mentioned above, Rad3 is necessary for phosphorylation and activation of Cds1 in vivo (9, 24). Active Cds1 associates with overproduced Rad3 in vivo (30). Furthermore, Cds1 associates with Rad26 when both proteins are overproduced, and Rad26 forms a protein complex with Rad3 (15, 24). These findings suggested that Rad3 might directly activate Cds1 in vivo. Experiments designed to test this hypothesis are described in this report. We show that Rad3 and human ATM phosphorylate fission yeast Cds1 at threonine-11. Threonine-11 forms part of a conserved TQ motif. We report that threonine-11 is crucially important for Cds1 activation and function in vivo. These studies provide strong support for the model that Rad3 directly controls Cds1 activity by phosphorylating Cds1 at threonine-11.

ACKNOWLEDGMENTS

We are grateful to Teresa Wang for the gift of anti-Cds1 antibody, H. Murakami and H. Okayama for the gift of plasmid pAL-cds1 and the cds1::ura4 strain, and Beth Baber-Furnari for plasmid pREP1-GST-Rad3. Antonia Lopez-Girona made helpful comments and suggestions. Members of the Scripps Cell Cycle Groups provided support and encouragement.

K.T. was supported by The Naito Foundation. This work was funded by NIH grants awarded to C.H.G. and P.R.

ACKNOWLEDGMENTS

REFERENCES

REFERENCES

References

  • 1. Allen J B, Zhou Z, Siede W, Friedberg E C, Elledge S JThe SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev. 1994;8:2401–2415.[PubMed][Google Scholar]
  • 2. Baber-Furnari B A, Rhind N, Boddy M N, Shanahan P, Lopez-Girona A, Russell PRegulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint. Mol Biol Cell. 2000;11:1–11.[Google Scholar]
  • 3. Banin S, Moyal L, Shieh S, Taya Y, Anderson C W, Chessa L, Smorodinsky N I, Prives C, Reiss Y, Shiloh Y, Ziv YEnhanced phosphorylation of p53 by ATM in response to DNA damage. Science. 1998;281:1674–1677.[PubMed][Google Scholar]
  • 4. Baskaran R, Wood L D, Whitaker L L, Canman C E, Morgan S E, Xu Y, Barlow C, Baltimore D, Wynshaw-Boris A, Kastan M B, Wang J YAtaxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation. Nature. 1997;387:516–519.[PubMed][Google Scholar]
  • 5. Benes V, Hostomsky Z, Arnold L, Paces VM13 and pUC vectors with new unique restriction sites for cloning. Gene. 1993;130:151–152.[PubMed][Google Scholar]
  • 6. Bentley N J, Holtzman D A, Flaggs G, Keegan K S, DeMaggio A, Ford J C, Hoekstra M, Carr A MThe Schizosaccharomyces pombe rad3 checkpoint gene. EMBO J. 1996;15:6641–6651.[Google Scholar]
  • 7. Blasina A, Price B D, Turenne G A, McGowan C HCaffeine inhibits the checkpoint kinase ATM. Curr Biol. 1999;9:1135–1138.[PubMed][Google Scholar]
  • 8. Blasina A, Van de Weyer I, Laus M C, Luyten W H M L, Parker A E, McGowan C HA human homolog of the checkpoint kinase Cds1 directly inhibits Cdc25. Curr Biol. 1999;9:1–10.[PubMed][Google Scholar]
  • 9. Boddy M N, Furnari B, Mondesert O, Russell PReplication checkpoint enforced by kinases Cds1 and Chk1. Science. 1998;280:909–912.[PubMed][Google Scholar]
  • 10. Brondello J M, Boddy M N, Furnari B, Russell PBasis for the checkpoint signal specificity that regulates Chk1 and Cds1 protein kinases. Mol Cell Biol. 1999;19:4262–4269.[Google Scholar]
  • 11. Brown E J, Baltimore DATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 2000;14:397–402.[Google Scholar]
  • 12. Canman C E, Lim D S, Cimprich K A, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan M B, Siliciano J DActivation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science. 1998;281:1677–1679.[PubMed][Google Scholar]
  • 13. Carr A MAnalysis of fission yeast DNA structure checkpoints. Microbiology. 1998;144:5–11.[PubMed][Google Scholar]
  • 14. Cortez D, Wang Y, Qin J, Elledge S JRequirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. Science. 1999;286:1162–1166.[PubMed][Google Scholar]
  • 15. Edwards R J, Bentley N J, Carr A MA Rad3-Rad26 complex responds to DNA damage independently of other checkpoint proteins. Nat Cell Biol. 1999;1:393–398.[PubMed][Google Scholar]
  • 16. Elledge S JCell cycle checkpoints: preventing an identity crisis. Science. 1996;274:1664–1672.[PubMed][Google Scholar]
  • 17. Furnari B, Blasina A, Boddy M N, McGowan C H, Russell PCdc25 inhibited in vivo and in vitro by checkpoint kinases Cds1 and Chk1. Mol Biol Cell. 1999;10:833–845.[Google Scholar]
  • 18. Furnari B, Rhind N, Russell PCdc25 mitotic inducer targeted by chk1 DNA damage checkpoint kinase. Science. 1997;277:1495–1497.[PubMed][Google Scholar]
  • 19. Guo Z, Kumagai A, Wang S X, Dunphy W GRequirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev. 2000;14:2745–2756.[Google Scholar]
  • 20. Hartley K O, Gell D, Smith G C, Zhang H, Divecha N, Connelly M A, Admon A, Lees-Miller S P, Anderson C W, Jackson S PDNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995;82:849–856.[PubMed][Google Scholar]
  • 21. Hartwell L H, Weinert T ACheckpoints: controls that ensure the order of cell cycle events. Science. 1989;246:629–634.[PubMed][Google Scholar]
  • 22. Hofmann K, Bucher PThe FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors. Trends Biochem Sci. 1995;20:347–349.[PubMed][Google Scholar]
  • 23. Lim D S, Kim S T, Xu B, Maser R S, Lin J, Petrini J H, Kastan M BATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway. Nature. 2000;404:613–617.[PubMed][Google Scholar]
  • 24. Lindsay H D, Griffiths D J, Edwards R J, Christensen P U, Murray J M, Osman F, Walworth N, Carr A MS-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe. Genes Dev. 1998;12:382–395.[Google Scholar]
  • 25. Liu Q, Guntuku S, Cui X S, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower L A, Elledge S JChk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint. Genes Dev. 2000;14:1448–1459.[Google Scholar]
  • 26. Matsuoka S, Huang M, Elledge S JLinkage of ATM to cell cycle regulation by the Chk2 protein kinase. Science. 1998;282:1893–1897.[PubMed][Google Scholar]
  • 27. Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, Elledge S JAtaxia telangiectasia-mutated phosphorylates chk2 in vivo and in vitro. Proc Natl Acad Sci USA. 2000;97:10389–10394.[Google Scholar]
  • 28. Melchionna R, Chen X-B, Blasina A, McGowan C HThreonine 68 is required for radiation-induced phosphorylation and activation of Cds1. Nat Cell Biol. 2000;10:762–765.[PubMed][Google Scholar]
  • 29. Moreno S, Klar A, Nurse PMolecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823.[PubMed][Google Scholar]
  • 30. Moser B A, Brondello J-M, Baber-Furnari B, Russell PMechanism of caffeine-induced checkpoint override in fission yeast. Mol Cell Biol. 2000;20:4288–4294.[Google Scholar]
  • 31. Murakami H, Okayama HA kinase from fission yeast responsible for blocking mitosis in S phase. Nature. 1995;374:817–819.[PubMed][Google Scholar]
  • 32. Oishi I, Sugiyama S, Otani H, Yamamura H, Nishida Y, Minami YA novel Drosophila nuclear protein serine/threonine kinase expressed in the germline during its establishment. Mech Dev. 1998;71:49–63.[PubMed][Google Scholar]
  • 33. O'Neill T, Dwyer A J, Ziv Y, Chan D W, Lees-Miller S P, Abraham R H, Lai J H, Hill D, Shiloh Y, Cantley L C, Rathbun G AUtilization of oriented peptide libraries to identify substrate motifs selected by ATM. J Biol Chem. 2000;275:22719–22727.[PubMed][Google Scholar]
  • 34. Rhind N, Furnari B, Russell PCdc2 tyrosine phosphorylation is required for the DNA damage checkpoint in fission yeast. Genes Dev. 1997;11:504–511.[PubMed][Google Scholar]
  • 35. Rhind N, Russell PMitotic DNA damage and replication checkpoints in yeast. Curr Opin Cell Biol. 1998;10:749–758.[Google Scholar]
  • 36. Rhind N, Russell PRoles of the mitotic inhibitors Wee1 and Mik1 in the G2 DNA damage and replication checkpoints. Mol Cell Biol. 2001;21:1499–1508.[Google Scholar]
  • 37. Rhind N, Russell PThe Schizosaccharomyces pombe S-phase checkpoint differentiates between different types of DNA damage. Genetics. 1998;149:1729–1737.[Google Scholar]
  • 38. Saka Y, Yanagida MFission yeast cut5, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4Cell. 1993;74:383–393.[PubMed][Google Scholar]
  • 39. Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, Tagle D A, Smith S, Uziel T, Sfez S, et al A single ataxia telangiectasia gene with a product similar to PI-3 kinase. Science. 1995;268:1749–1753.[PubMed][Google Scholar]
  • 40. Shimada M, Okuzaki D, Tanaka S, Tougan T, Tamai K K, Shimoda C, Nojima HReplication factor C3 of Schizosaccharomyces pombe, a small subunit of replication factor C complex, plays a role in both replication and damage checkpoints. Mol Biol Cell. 1999;10:3991–4003.[Google Scholar]
  • 41. Sun Z, Hsiao J, Fay D S, Stern D FRad53 FHA domain associated with phosphorylated Rad9 in the DNA damage checkpoint. Science. 1998;281:272–274.[PubMed][Google Scholar]
  • 42. Walworth N C, Bernards Rrad-dependent response of the chk1-encoded protein kinase at the DNA damage checkpoint. Science. 1996;271:353–356.[PubMed][Google Scholar]
  • 43. Wright J A, Keegan K S, Herendeen D R, Bentley N J, Carr A M, Hoekstra M F, Concannon PProtein kinase mutants of human ATR increase sensitivity to UV and ionizing radiation and abrogate cell cycle checkpoint control. Proc Natl Acad Sci USA. 1998;95:7445–7450.[Google Scholar]
  • 44. Zeng Y, Forbes K C, Wu Z, Moreno S, Piwnica-Worms H, Enoch TReplication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature. 1998;395:507–510.[PubMed][Google Scholar]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.