Sensitizing Plant Protein Kinases to Specific Inhibition by ATP-Competitive Molecules
The highly conserved nature of the protein kinase catalytic domain and the low permeability of plant cell membranes pose a challenge to the development of specific inhibitors that target individual protein kinases in vivo. Here, we describe a chemical-gen
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1. Introduction Members of the protein kinase superfamily regulate a wide variety of cellular processes and signal-transduction pathways in plants (e.g. 1–6). A major objective in the field of protein kinase research is to delineate the functional role of individual kinases. To accomplish this aim, strategies based on single knockout mutations and gene silencing are widely employed. However, such efforts may be compromised by lethality, in the case of essential genes, or by functional redundancy and cellular homeostasis resulting in no apparent phenotypic alterations. To overcome these limitations, a chemical-genetic approach was developed to sensitize protein kinases to specific inhibition
N. Dissmeyer and A. Schnittger (eds.), Plant Kinases: Methods and Protocols, Methods in Molecular Biology, vol. 779, DOI 10.1007/978-1-61779-264-9_10, © Springer Science+Business Media, LLC 2011
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by cell-permeable ATP-competitive molecules (7, 8). A functionally silent substitution of a conserved, bulky hydrophobic residue, termed the gatekeeper, to a small residue (alanine or glycine) generates a novel pocket in the kinase ATP-binding site not present in any wild-type kinase. The sensitized mutant can be selectively and potently inhibited by ATP-competitive molecules containing substituents that occupy this additional pocket. Importantly, wild-type kinases are not inhibited by such inhibitors due to lack of the novel pocket. The PP1(1-tert-Butyl-3-p-tolyl-1H-pyrazolo[3,4-d] pyrimidine-4-ylamine)-derived inhibitors employed in this approach were shown to be cell permeable in various systems, including plants, yeast, and mammalian cell cultures (8–11, 17). Combined with the relative ease of generating transgenic Arabidopsis plants harboring the sensitized allele, this approach allows for modulation of the activity of individual kinases in the plant and for delineation of their biological function in vivo.
2. Materials 2.1. Site-Directed Mutagenesis
1. Quikchange II site-directed mutagenesis kit (Stratagene).
2.2. Purification of GST-Fusion Protein
1. Terrific broth (TB) medium: 1.2% (w/v) tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 17 mM KH2PO4, and 72 mM K2HPO4. Add filter-sterilized phosphate salts after autoclaving. 2. 1 M Isopropyl-b-d-thiogalactopyranoside (IPTG) in water, store in aliquots at −20°C. 3. Phosphate buffered saline (PBS) (10×): 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4, adjust to pH 7.4 with HCl, autoclave. 4. 100 mM Phenylmethylsulfonyl fluoride (PMSF, Roche) in isopropanol. Aliquot and store at −20°C. Thaw and vortex before use. 5. 5 mg/mL aprotinin (Sigma) and 5 mg/mL leupeptin (both Sigma) in water, stored in aliquots at −20°C. 6. Glutathione agarose beads (Sigma), incubate with water at 4°C overnight. After swelling, wash the beads with 10 volumes of water and resuspend as 50% (w/v) slurry in water. Store at 4°C. 7. Elution buffer: 50 mM Tris–HCl (pH 8.0), 5 mM reduced glutathione (Sigma). 8. Bradford protein assay solution (Bio-Rad).
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