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

Authors

  • Dr. Sarah E. Lee,

    1. Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA (UK)
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    • These authors contributed equally to this work.

  • Dr. Lucy M. Elphick,

    1. Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)
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    • These authors contributed equally to this work.

  • Dr. Holger B. Kramer,

    1. The Henry Wellcome Building for Molecular Physiology, Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (UK)
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    • These authors contributed equally to this work.

  • Dr. Alexandra M. E. Jones,

    1. The Sainsbury Laboratory, Norwich Research Park, Colney Lane, Norwich NR4 7UH (UK)
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  • Dr. Emma S. Child,

    1. Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)
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  • Dr. Alexandra A. Anderson,

    1. Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)
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  • Dr. Laurent Bonnac,

    1. Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA (UK)
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  • Dr. Natsuko Suwaki,

    1. Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)
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  • Dr. Benedikt M. Kessler,

    1. The Henry Wellcome Building for Molecular Physiology, Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN (UK)
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  • Prof. Dr. Véronique Gouverneur,

    1. Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA (UK)
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    • These authors contributed equally to this work.

  • Dr. David J. Mann

    Corresponding author
    1. Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)
    • Imperial College London, Cell Cycle Laboratory, Division of Cell and Molecular Biology, South Kensington, London SW7 2AZ (UK)
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    • These authors contributed equally to this work.


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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