Current Protocols in Molecular Biology

  • Blethrow J
  • Zhang C
  • Shokat K
 et al. 
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Abstract

Many protein kinases can be engineered to accept analogs of ATP that are not efficiently used by wild-type kinases. These engineered kinases, which are referred to as "analog-sensitive" or "-as" alleles, are also often sensitive to protein kinase inhibitor variants that do not block the activity of nonmutant kinases. Selective in vitro use of radiolabeled ATP analogs by -as kinases can be exploited to identify the direct phosphorylation targets of individual kinases in complex extracts. In organisms in which it is practical to replace wild-type kinase genes with engineered alleles, the in vivo activity of a -as kinase can be reversibly blocked with an allele-specific inhibitor. Thus, analog-sensitive kinases can be effective tools for discovery of the cellular functions and phosphorylation targets of individual enzymes. A theoretical background for the design and use of these alleles is discussed, as are strategies for construction of candidate -as alleles of any kinase.

Author-supplied keywords

  • Adenosine Triphosphate
  • Adenosine Triphosphate: analogs & derivatives
  • Alleles
  • Protein Engineering
  • Protein Kinase Inhibitors
  • Protein Kinase Inhibitors: chemical synthesis
  • Protein Kinases
  • Protein Kinases: chemistry
  • Pyrazoles
  • Pyrazoles: chemical synthesis
  • Pyrimidines
  • Pyrimidines: chemical synthesis
  • Yeasts
  • Yeasts: drug effects

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Authors

  • Justin Blethrow

  • Chao Zhang

  • Kevan M Shokat

  • Eric L Weiss

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