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

Sarah E. Lee, Lucy M. Elphick, Holger B. Kramer, Alexandra M.E. Jones, Emma S. Child, Alexandra A. Anderson, Laurent Bonnac, Natsuko Suwaki, Benedikt M. Kessler, Véronique Gouverneur, David J. Mann

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 N 6-(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. It's a kinase magic: The identification of protein substrates is hampered by the large number of kinases active in cells at a given time. Here we use a chemical genetic approach to specifically label the substrates of an analogue-sensitive kinase with thiophosphate and use pH-dependent S-alkylation to selectively recover thiophosphorylated peptides.

Original languageEnglish (US)
Pages (from-to)633-640
Number of pages8
JournalChemBioChem
Volume12
Issue number4
DOIs
StatePublished - Mar 7 2011
Externally publishedYes

Keywords

  • ATP
  • Cdk2
  • Chemical genetics
  • Cyclin
  • Kinases
  • Substrate isolation
  • Thiophosphorylation

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