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

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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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Figure 1: Strategy for sensitizing protein kinases to small-molecule inhibitors.
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Figure 4: Sensitivity of cells expressing Hhp1-as to various inhibitors.


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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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Correspondence to Juraj Gregan.

Supplementary information

Supplementary Table 1

S. pombe protein kinases with the corresponding gate-keeper residue. (DOC 48 kb)

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