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The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression

Nature Cell Biology volume 6, pages 138–145 (2004)Cite this article

Abstract

The conserved checkpoint kinases Chk1 and Rad53–Dun1 block the metaphase to anaphase transition by the phosphorylation and stabilization of securin, and block the mitotic exit network regulated by the Bfa1–Bub2 complex. However, both chk1 and rad53 mutants are able to exit from mitosis and initiate a new cell cycle, suggesting that both pathways have supporting functions in restraining anaphase and in blocking the inactivation of mitotic cyclin–Cdk1 complexes. Here we find that the cyclic-AMP-dependent protein kinase (PKA) pathway supports Chk1 in the regulation of mitosis by targeting the mitotic inducer Cdc20. Cdc20 is phosphorylated on PKA consensus sites after DNA damage, and this phosphorylation requires the Atr orthologue Mec1 and the PKA catalytic subunits Tpk1 and Tpk2. We show that the inactivation of PKA or expression of phosphorylation-defective Cdc20 proteins accelerates securin and Clb2 destruction in chk1 mutants and is sufficient to remove most of the DNA damage-induced delay. Mutation of the Cdc20 phosphorylation sites permitted the interaction of Cdc20 with Clb2 under conditions that should halt cell cycle progression. These data show that PKA pathways regulate mitotic progression through Cdc20 and support the DNA damage checkpoint pathways in regulating the destruction of Clb2 and securin.

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Figure 1: Deletion of TPK1 or TPK2 enhances the checkpoint defect of chk1-Δ cells.
Figure 2: Cdc20 is phosphorylated in response to DNA damage, interacts with Tpk1/Tpk2 and is phosphorylated by Tpk2 in vitro.
Figure 3: Cdc20 is phosphorylated on Ser 52 and Ser 88 in response to DNA damage.
Figure 4: The DNA-damage-induced phosphorylation of Cdc20 is defective in cells lacking Tpk1, Tpk2 or Mec1, and overexpression of TPK1 or TPK2 restored Clb2 stability and checkpoint delay to mec1-21 cells.
Figure 5: Phosphorylation-defective Cdc20 mutated proteins interact with Clb2 in the presence of a DNA damage signal, and their expression accelerates the checkpoint defect in chk1-Δ cells and suppresses a temperature-sensitive mutant of the APC component Cdc23.

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Acknowledgements

We thank J. Heitman, D. Kellogg, T. Stuckenberg, D. Robbins, U. Surana, E. Pereira and members of the Sanchez laboratory for reading this manuscript and/or for helpful discussions; S. Elledge, D. Koshland, O. Cohen-Fix, A. Murray, U. Surana and J. Heitman for plasmids and strains; K. Jiang, J. Poisson and I. Houston for generating constructs and strains; B. Russell for help with immunoprecipitations; F. Kanai and L. C. Cantley for information about Chk1 phosphorylation motifs; T. Sicheritz-Ponten and N. Blom for their assistance in predicting PKA phosphorylation sites; and X. Pan for his assistance with PKA reagents and kinase assays. This work was supported in part by grants from the Ohio Cancer Research Associates, NIH/NCI RO1 CA84463, The Department of Defense DAMD 17-01-1-020, and from the Pew Scholars program in the Biomedical Sciences to Y.S., and by grant P30 ES06096 from the National Institute of Environmental Health Sciences, NIH. B.J.W. was supported by an M.D./Ph.D. Scholar award from the University of Cincinnati Physician Scientist Training Program and is an Illick Fellow of the Albert J. Ryan Foundation. Y.S. is a Pew Scholar in the Biomedical Sciences.

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Authors and Affiliations

  1. Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, 45267-0524, OH, USA

    Jennifer S. Searle, Kaila L. Schollaert & Yolanda Sanchez

  2. Division of Molecular Cardiovascular Biology, Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, 45267-0524, OH, USA

    Benjamin J. Wilkins

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Correspondence to Yolanda Sanchez.

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Searle, J., Schollaert, K., Wilkins, B. et al. The DNA damage checkpoint and PKA pathways converge on APC substrates and Cdc20 to regulate mitotic progression. Nat Cell Biol 6, 138–145 (2004). https://doi.org/10.1038/ncb1092

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