Advertisement

Using the Semi-synthetic Epitope System to Identify Direct Substrates of the Meiosis-Specific Budding Yeast Kinase, Mek1

  • Hsiao-Chi Lo
  • Nancy M. HollingsworthEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 745)

Abstract

Recent studies have shown that the meiosis-specific kinase, Mek1, plays a key role in promoting recombination between homologous chromosomes during meiosis in budding yeast by suppressing recombination between sister chromatids, as well as playing a role in the meiotic recombination checkpoint. Understanding how Mek1 regulates recombination requires the identification of direct substrates of the kinase. We have applied the semi-synthetic epitope method developed by Shokat and colleagues to Mek1. This method uses an analog-sensitive version of Mek1, GST-Mek1-as, in conjunction with an ATPγS analog, for kinase assays that detect only those proteins that are directly phosphorylated by Mek1. This method may be applicable to any kinase for which an analog-sensitive version is available. In addition, it provides a non-radioactive alternative for kinase assays with wild-type kinases.

Key words

Meiosis Mek1 kinase assays Rad54 semi-synthetic epitope yeast 

Notes

Acknowledgments

We thank Jasmina Allen, Kevan Shokat, and Beatrice Wang for help with ideas and reagents in the early development of this protocol. Patrick Sung generously provided bacterially purified Rad54 protein. Aaron Neiman provided helpful comments on the manuscript. This work was supported by an NIH grant to N. M. H. (R01 GM50717).

References

  1. 1.
    Benjamin, K.R., Zhang, C., Shokat, K.M., and Herskowitz, I. (2003) Control of landmark events in meiosis by the CDK Cdc28 and the meiosis-specific kinase Ime2. Genes Dev 17, 1524–1539.PubMedCrossRefGoogle Scholar
  2. 2.
    Lee, B.H., and Amon, A. (2003) Role of Polo-like kinase CDC5 in programming meiosis I chromosome segregation. Science 300, 482–486.PubMedCrossRefGoogle Scholar
  3. 3.
    Matos, J., Lipp, J.J., Bogdanova, A., Guillot, S., Okaz, E., Junqueira, M., Shevchenko, A., and Zachariae, W. (2008) Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I. Cell 135, 662–678.PubMedCrossRefGoogle Scholar
  4. 4.
    Pak, J., and Segall, J. (2002) Regulation of the premiddle and middle phases of expression of the NDT80 gene during sporulation of Saccharomyces cerevisiae. Mol Cell Biol 22, 6417–6429.PubMedCrossRefGoogle Scholar
  5. 5.
    Sourirajan, A., and Lichten, M. (2008) Polo-like kinase Cdc5 drives exit from pachytene during budding yeast meiosis. Genes Dev 22, 2627–2632.PubMedCrossRefGoogle Scholar
  6. 6.
    Wan, L., de los Santos, T., Zhang, C., Shokat, K., and Hollingsworth, N.M. (2004) Mek1 kinase activity functions downstream of RED1 in the regulation of meiotic DSB repair in budding yeast. Mol Biol Cell 15, 11–23.PubMedCrossRefGoogle Scholar
  7. 7.
    Wan, L., Niu, H., Futcher, B., Zhang, C., Shokat, K.M., Boulton, S.J., and Hollingsworth, N.M. (2008) Cdc28-Clb5 (CDK-S) and Cdc7-Dbf4 (DDK) collaborate to initiate meiotic recombination in yeast. Genes Dev 22, 386–397.PubMedCrossRefGoogle Scholar
  8. 8.
    Ahmed, N.T., Bungard, D., Shin, M.E., Moore, M., and Winter, E. (2009) The Ime2 protein kinase enhances the disassociation of the sum1 repressor from middle meiotic promoters. Mol Cell Biol 29, 4352–4362.PubMedCrossRefGoogle Scholar
  9. 9.
    Henderson, K.A., Kee, K., Maleki, S., Santini, P., and Keeney, S. (2006) Cyclin-dependent kinase directly regulates initiation of meiotic recombination. Cell 125, 1321–1332.PubMedCrossRefGoogle Scholar
  10. 10.
    Bishop, A.C., Buzko, O., and Shokat, K.M. (2001) Magic bullets for protein kinases. Trends Cell Biol 11, 167–172.PubMedCrossRefGoogle Scholar
  11. 11.
    Bishop, A.C., Ubersax, J.A., Petsch, D.T., Matheos, D.P., Gray, N.S., Blethrow, J., Shimizu, E., Tsien, J.Z., Schultz, P.G., Rose, M.D., Wood, J.L., Morgan, D.O., and Shokat, K.M. (2000) A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401.PubMedCrossRefGoogle Scholar
  12. 12.
    Ubersax, J.A., Woodbury, E.L., Quang, P.N., Paraz, M., Blethrow, J.D., Shah, K., Shokat, K.M., and Morgan, D.O. (2003) Targets of the cyclin-dependent kinase Cdk1. Nature 425, 859–864.PubMedCrossRefGoogle Scholar
  13. 13.
    Allen, J.A., Li, M., Brinkworth, C.S., Paulson, J.L., Wang, D., Hubner, A., Chou, W.-H., Davis, R.J., Burlingame, A.L., Messing, R.O., Katayama, C.D., Hedrick, S.M., and Shokat, K.M. (2007) A semisynthetic epitope for kinase substrates. Nat Methods 4, 511–516.PubMedCrossRefGoogle Scholar
  14. 14.
    Niu, H., Wan, L., Busygina, V., Kwon, Y., Allen, J.A., Li, X., Kunz, R.C., Kubota, K., Wang, B., Sung, P., Shokat, K.M., Gygi, S.P., and Hollingsworth, N.M. (2009) Regulation of meiotic recombination via Mek1-mediated Rad54 phosphorylation. Mol Cell 36, 393–404.PubMedCrossRefGoogle Scholar
  15. 15.
    Xu, L., Weiner, B.M., and Kleckner, N. (1997) Meiotic cells monitor the status of the interhomolog recombination complex. Genes Dev 11, 106–118.PubMedCrossRefGoogle Scholar
  16. 16.
    Wan, L., Zhang, C., Shokat, K.M., and Hollingsworth, N.M. (2006) Chemical inactivation of Cdc7 kinase in budding yeast results in a reversible arrest that allows efficient cell synchronization prior to meiotic recombination. Genetics 174, 1667–1774.CrossRefGoogle Scholar
  17. 17.
    Niu, H., Li, X., Job, E., Park, C., Moazed, D., Gygi, S.P., and Hollingsworth, N.M. (2007) Mek1 kinase is regulated to suppress double-strand break repair between sister chromatids during budding yeast meiosis. Mol Cell Biol 27, 5456–5467.PubMedCrossRefGoogle Scholar
  18. 18.
    Bishop, D.K., Park, D., Xu, L., and Kleckner, N. (1992) DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation and cell cycle progression. Cell 69, 439–456.PubMedCrossRefGoogle Scholar
  19. 19.
    Padmore, R., Cao, L., and Kleckner, N.R. (1991) Temporal comparison of recombination and synaptonemal complex formation during meiosis in Saccharomyces cerevisiae. Cell 66, 1239–1256.PubMedCrossRefGoogle Scholar
  20. 20.
    Blethrow, J.D., Zhang, C., Shokat, K.M., and Weiss, E.L. (2004) Design and use of analog-sensitive kinases. Curr Protoc Mol Biol Chapter 18, Unit 18 11.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Biochemistry and Cell BiologyStony Brook UniversityNew YorkUSA

Personalised recommendations