Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe

Nat Protoc. 2007;2(11):2996-3000. doi: 10.1038/nprot.2007.447.

Abstract

Reversible protein phosphorylation is a major regulatory mechanism in a cell. A chemical-genetic strategy to conditionally inactivate protein kinases has been developed recently. Mutating a single residue in the ATP-binding pocket confers sensitivity to small-molecule inhibitors. The inhibitor can only bind to the mutant kinase and not to any other wild-type kinase, allowing specific inactivation of the modified kinase. Here, we describe a protocol to construct conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe. This protocol can be completed in about 3 weeks and should be applicable to other organisms as well.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Mutagenesis
  • Phosphorylation
  • Protein Engineering / methods*
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinases / chemistry
  • Protein Kinases / genetics*
  • Schizosaccharomyces / enzymology*
  • Schizosaccharomyces pombe Proteins / antagonists & inhibitors
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / genetics*

Substances

  • Protein Kinase Inhibitors
  • Schizosaccharomyces pombe Proteins
  • Protein Kinases
  • hhp1 protein, S pombe
  • hhp2 protein, S pombe