title: A chemical switch for inhibitor-sensitive alleles of any protein kinase.
abstract: Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. 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 kinases. 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 mutants. 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.
authors.affiliation: Department of Chemistry, Princeton University, New Jersey 08544, USA.
authors.forename: A C
authors.initials: AC
authors.lastname: Bishop
authors.forename: J A
authors.initials: JA
authors.lastname: Ubersax
authors.forename: D T
authors.initials: DT
authors.lastname: Petsch
authors.forename: D P
authors.initials: DP
authors.lastname: Matheos
authors.forename: N S
authors.initials: NS
authors.lastname: Gray
authors.forename: J
authors.initials: J
authors.lastname: Blethrow
authors.forename: E
authors.initials: E
authors.lastname: Shimizu
authors.forename: J Z
authors.initials: JZ
authors.lastname: Tsien
authors.forename: P G
authors.initials: PG
authors.lastname: Schultz
authors.forename: M D
authors.initials: MD
authors.lastname: Rose
authors.forename: J L
authors.initials: JL
authors.lastname: Wood
authors.forename: D O
authors.initials: DO
authors.lastname: Morgan
authors.forename: K M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D017489:CDC28 Protein Kinase, S cerevisiae / Q000037:antagonists & inhibitors / Q000235:genetics; D002227:Carbazoles / Q000494:pharmacology; D002453:Cell Cycle; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors; D015870:Gene Expression; D006801:Humans; D026121:Indole Alkaloids; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000494:pharmacology; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
chemical_list: D002227:Carbazoles; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D026121:Indole Alkaloids; D047428:Protein Kinase Inhibitors; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; C073882:phosphoprotein phosphatase inhibitor 1; C049985:staurosporine aglycone; D011494:Protein Kinases; D017489:CDC28 Protein Kinase, S cerevisiae; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases