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.
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Acknowledgements
This work was supported by the Austrian Science Fund (P18955-B03). L.C. was a recipient of EMBO and FEBS short-term fellowships. We thank Mark Petronczki and Maria Siomos for helpful discussions and comments on the manuscript.
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Supplementary Table 1
S. pombe protein kinases with the corresponding gate-keeper residue. (DOC 48 kb)
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Gregan, J., Zhang, C., Rumpf, C. et al. Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe. Nat Protoc 2, 2996–3000 (2007). https://doi.org/10.1038/nprot.2007.447
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DOI: https://doi.org/10.1038/nprot.2007.447
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