Analysis of protein tyrosine kinase inhibitors in recombinant yeast lacking the ERG6 gene.
Studies of small-molecule-protein interactions in yeast can be hindered by the limited permeability of yeast to small molecules. This diminished permeability is thought to be related to the unique sterol composition of fungal membranes, which are enriched in the steroid ergosterol. We report the construction of the novel Saccharomyces cerevisiae yeast strain DCY250, which is compatible with yeast two-hybrid-based systems and bears a targeted disruption of the ERG6 gene to ablate ergosterol biosynthesis and enhance permeability to small molecules. The small-molecule inhibitors of protein tyrosine kinases (PTKs) PP1, PP2, herbimycin A, and staurosporine were investigated with yeast tribrid systems that detect the activity of the PTKs v-Abl and v-Src. These tribrid systems function by expression of the PTK, a B42 activation domain fused to the phosphotyrosine-binding Grb2 SH2 domain, a DNA-bound LexA-GFP-(AAYANAA)(4) universal PTK substrate, and a lacZ reporter gene. Yeast genetic systems that lack functional ERG6 were found to be as much as 20-fold more sensitive to small-molecule inhibitors of PTKs than systems with ERG6, and these deficient systems may provide a useful platform for the discovery and analysis of small-molecule-protein interactions.
Clark
Daniel D
DD
Department of Chemistry, The Pennsylvania State University, 152 Davey Laboratory, University Park, PA 16802, USA.
Peterson
Blake R
BR
D000890:Anti-Infective Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000032:analysis*; D017353:Gene Deletion; D015151:Immunoblotting; D008780:Methyltransferases / Q000235:genetics*; D010539:Permeability; D010957:Plasmids / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D001616:beta-Galactosidase / Q000737:chemistry
D000890:Anti-Infective Agents; D004791:Enzyme Inhibitors; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase; D011505:Protein-Tyrosine Kinases; D001616:beta-Galactosidase