Abstract
Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry1. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases2. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants3. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.
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Acknowledgements
We thank B. Schulman and E. Harlow for early characterization of mutant cyclin-dependent kinases; C. Co, V. Nguyen and J. Li for help with flow cytometry; D. Toczyski for help with the Coulter counter; A. Su for help with transcript array analysis; S. Jaspersen, C. Carroll, C. Takizawa, E. Weiss, S. Biggins, A. Szidon, A. Rudner, D. Kahne and A. Murray for helpful advice and reagents; E. O'Shea and R. Deshaies for strains and reagents. This work was supported by funding from the National Institute of Allergy and Immunology (K.M.S.), GlaxoWellcome (K.M.S. and J.L.W.), National Institute of General Medical Sciences (D.O.M.), National Institutes of Health (J.L.W.), Bristol-Myers Squibb (J.L.W.), Yamanouchi (J.L.W.) and a predoctoral fellowship from the National Science Foundation (J.A.U.). K.M.S. is a Pew, Searle, and a Sloan Foundation Scholar.
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Bishop, A., Ubersax, J., Petsch, D. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase . Nature 407, 395–401 (2000). https://doi.org/10.1038/35030148
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DOI: https://doi.org/10.1038/35030148
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