Construction of conditional analog-sensitive kinase alleles in the fission yeast Schizosaccharomyces pombe.
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.
Gregan
Juraj
J
Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Dr. Bohr-Gasse 1, 1030 Vienna, Austria. juraj.gregan@univie.ac.at
Zhang
Chao
C
Rumpf
Cornelia
C
Cipak
Lubos
L
Li
Zhang
Z
Uluocak
Pelin
P
Nasmyth
Kim
K
Shokat
Kevan M
KM
D000483:Alleles; D016296:Mutagenesis; D010766:Phosphorylation; D015202:Protein Engineering / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000737:chemistry; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics*; D012568:Schizosaccharomyces / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*
D047428:Protein Kinase Inhibitors; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; C088232:hhp1 protein, S pombe; C088231:hhp2 protein, S pombe