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The Chemoselective One-Step Alkylation and Isolation of Thiophosphorylated Cdk2 Substrates in the Presence of Native Cysteine

  1. Dr. Sarah E. Lee1,†,
  2. Dr. Lucy M. Elphick2,†,
  3. Dr. Holger B. Kramer3,†,
  4. Dr. Alexandra M. E. Jones4,
  5. Dr. Emma S. Child2,
  6. Dr. Alexandra A. Anderson2,
  7. Dr. Laurent Bonnac1,
  8. Dr. Natsuko Suwaki2,
  9. Dr. Benedikt M. Kessler3,
  10. Prof. Dr. Véronique Gouverneur1,†,
  11. Dr. David J. Mann2,†,*

Article first published online: 8 FEB 2011

DOI: 10.1002/cbic.201000528

Issue

ChemBioChem

ChemBioChem

Volume 12, Issue 4, pages 633–640, March 7, 2011

Additional Information

How to Cite

Lee, S. E., Elphick, L. M., Kramer, H. B., Jones, A. M. E., Child, E. S., Anderson, A. A., Bonnac, L., Suwaki, N., Kessler, B. M., Gouverneur, V. and Mann, D. J. (2011), The Chemoselective One-Step Alkylation and Isolation of Thiophosphorylated Cdk2 Substrates in the Presence of Native Cysteine. ChemBioChem, 12: 633–640. doi: 10.1002/cbic.201000528

Author Information

  1. 1

    Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA (UK)

  2. 2

    Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)

  3. 3

    The Henry Wellcome Building for Molecular Physiology, Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (UK)

  4. 4

    The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UH (UK)

  1. †

    These authors contributed equally to this work.

*Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)

Publication History

  1. Issue published online: 25 FEB 2011
  2. Article first published online: 8 FEB 2011
  3. Manuscript Received: 3 SEP 2010

Funded by

  • Cancer Research UK
  • BBSRC

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Keywords:

  • ATP;
  • cdk2;
  • chemical genetics;
  • cyclin;
  • kinases;
  • substrate isolation;
  • thiophosphorylation

Abstract

The elucidation of signalling pathways relies heavily upon the identification of protein kinase substrates. Recent investigations have demonstrated the efficacy of chemical genetics using ATP analogues and modified protein kinases for specific substrate labelling. Here we combine N6-(cyclohexyl)ATPγS with an analogue-sensitive cdk2 variant to thiophosphorylate its substrates and demonstrate a pH-dependent, chemoselective, one-step alkylation to facilitate the detection or isolation of thiophosphorylated peptides.

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