A quantitative comparison of wild-type and gatekeeper mutant Cdk2 for chemical genetic studies with ATP analogues

Lucy M. Elphick, Sarah E. Lee, Emma S. Child, Aarathi Prasad, Cristina Pignocchi, Sébastian Thibaudeau, Alexandra A. Anderson, Laurent Bonnac, Véronique Gouverneur, David J. Mann

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Chemical genetic studies with enlarged ATP binding sites and unnatural ATP analogues have been applied to protein kinases for characterisation and substrate identification. Although this system is becoming widely used, there are limited data available about the kinetic profile of the modified system. Here we describe a detailed comparison of the wild-type cdk2 and the mutant gatekeeper kinase to assess the relative efficiencies of these kinases with ATP and unnatural ATP analogues. Our data demonstrate that mutation of the kinase alters neither the substrate specificity nor the phosphorylation site specificity. We find comparable KM/Vmax values for mutant cdk2 and wild-type kinase. Furthermore, F80G cdk2 is efficiently able to compensate for a defective cdk in a biological setting.

Original languageEnglish (US)
Pages (from-to)1519-1526
Number of pages8
JournalChemBioChem
Volume10
Issue number9
DOIs
StatePublished - Jun 15 2009
Externally publishedYes

Keywords

  • Chemical genetics
  • Cyclin
  • Kinases
  • Nucleotides
  • Phosphorylation

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