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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.
To inactivate essential proteins conditionally, temperature-sensitive alleles, regulatable promoters, degron alleles, conditionally active inteins as well as other strategies have been used1-7. However, these alleles are often leaky, require analysis under non-physiological conditions (high temperature) or their inactivation requires a long period of time. In addition, the molecular mechanism of temperature-sensitive protein inactivation is rarely understood. Small-molecule inhibitors selective for a target protein provide a valuable tool for conditional inactivation of proteins and provide several advantages over genetic approaches. Total as well as partial inhibition of the target protein can be achieved, depending on the amount of inhibitor added. The cell-permeable nature of these inhibitors allows reversible inhibition of the target protein both in vitro and in vivo. Rapid inactivation of the target protein by addition of the inhibitor leaves the cell with little time to adapt to or compensate for the missing protein activity. Small-molecule inhibitors do not typically alter the expression level of the target protein, nor do they disrupt protein complexes. In many cases, a small-molecule inhibitor and a genetic mutation can perturb a protein's activity in different ways. This may result in different phenotypes, often providing complementary information about the cellular function of a given kinase8. Small-molecule inhibitors can therefore reveal new biological functions of proteins that have already been studied genetically9.
Numerous small-molecule inhibitors have been discovered and employed in the study of various protein kinases. However, due to the large number of kinases in the genome and their highly conserved active sites, which these inhibitors target, many inhibitors suffer from poor selectivity10. Moreover, little is known about the specificity of these inhibitors against yeast kinases, as most of these inhibitors were developed and characterized against mammalian kinases. A chemical-genetic strategy for sensitizing protein kinases to small-molecule inhibitors has been developed recently11. A single residue in the ATP-binding pocket, termed the gate-keeper residue, controls sensitivity of protein kinases to designed small-molecule inhibitors. This gate-keeper residue is conserved as a bulky hydrophobic amino acid in the protein kinase superfamily. Mutation of the gate-keeper to a small residue (alanine or glycine) creates a novel pocket that can be uniquely targeted by inhibitors with properly enlarged substituents. The mutation thus confers inhibitor sensitivity but does not interfere with kinase function in the absence of inhibitor (Fig. 1). The ATP-binding pocket of kinases is so conserved that the inhibitor can only bind to the mutant kinase and not to any wild-type kinases due to steric clash of the enlarged substituent and the bulky wild-type gate-keeper residue, hence allowing specific inactivation of the mutant kinase. Importantly, the fact that only sensitized protein kinase is inhibited allows a critical control experiment to be performed in which wild-type cells are treated with the inhibitor, thus revealing any possible off-target effects of the inhibitor. Such a control experiment should be performed in parallel with every experiment involving sensitized protein kinase and the inhibitor. The gate-keeper residue can be easily identified from primary sequence alignments in many protein kinases from various kinase subfamilies (http://sequoia.ucsf.edu/ksd/) (see ref. 12). Thus, the same strategy can be used to construct conditional analog-sensitive alleles of a wide variety of protein kinases11,13-18. This technology can also be used for identification of kinase substrates and has multiple applications in the drug discovery process19-21. Moreover, this methodology of generating inhibitors that selectively target a sensitized enzyme is not limited to protein kinases. A similar approach has been successfully used to inhibit proteins from other families, including phosphatases, motor proteins and GTPases22-26.
Despite many advantages, the chemical-genetic strategy of protein kinase sensitization contains a number of limitations. The catalytic activity of the protein kinase may be diminished by mutating the gate-keeper residue. In such cases, a second-site suppressor mutation can be introduced to rescue the kinase activity27. Another limitation is that this approach is only applicable to organisms in which genetic techniques are available to knock out or inactivate the target kinase and introduce the sensitized kinase allele. Exquisite skills and lengthy procedures are necessitated to carry out gene replacement in high eukaryotes such as Caenorhabditis elegans, Drosophila melanogaster and Mus musculus, impeding chemical-genetic analysis of protein kinases from these organisms. However, such limitation does not apply to the fission yeast S. pombe, a single-celled fungus, which has been extensively studied and has served as an excellent model organism. Importantly, the genome of S. pombe has been sequenced28.
Here, we describe a detailed protocol for the construction of conditional analog-sensitive kinase alleles in S. pombe (Fig. 2). Hhp1 and Hhp2 are members of a highly conserved casein kinase 1 family involved in DNA repair in S. pombe. Mutants lacking both Hhp1 and Hhp2 proteins grow extremely slowly, which precludes careful analysis of the double mutant phenotype29,30. We therefore constructed an analog-sensitive hhp1 mutant and combined it with an hhp2 deletion mutant. This allowed us to grow sufficient amounts of cells in the absence of inhibitor and to harvest them after adding the inhibitor. In this way, we were able to analyze the mutant phenotype in cells with inactivated Hhp1 kinase and lacking the Hhp2 protein31. We mutated methionine 84 of hhp1 to a glycine residue (hhp1-as) and expressed it in an S. pombe strain lacking wild-type hhp1. The mutated gene hhp1-as complemented the phenotype caused by deleting the hhp1 gene and resulted in only a minor growth defect. Importantly, the hhp1-as mutant, but not a wild-type strain, was sensitive to the inhibitor 1-NM-PP1 (Figs. (Figs.33 and and4)4) (see ref. 31).
Prepare a midi-prep (Qiagen) of the cloning vector (pCloneHyg1).
Prepare 0.1 M lithium acetate in 1× TE buffer, pH 7.5.
Prepare 40% (wt/vol) PEG 3350 in 1× TE buffer, pH 7.5.
Make transformation-competent E. coli DH5alpha using CaCl2 method32. Alternatively, use commercially available competent E. coli cells.
Prepare standard 2× bacto-tryptone yeast (TY) medium for E. coli. For selection, add ampicillin (100 μg ml−1). For yeast cultivation, prepare standard YES medium supplemented with 0.15g liter−1 adenine and 0.1g liter−1 of each of uracil, l-histidine, l-lysine and l-leucine. For selection, add hygromycin B at 200 μg ml−1.
Dissolve oligonucleotide primers in TE buffer to 100 μM concentration. The oligonucleotides required to amplify the kinase gene are hhp1BHIterm2, ATATGGATCCGTATTATTAGCAAATGTACTAATAT and hhp1XhoIprom2, ATATCTCGAGAATATTATTAGATTTTGTATATAG. The mutagenic oligonucleotides required are h1AntisM84G, GGACCCAATAAATCCCCCACCAT AGCGTTG and h1M84G, CAACGCTATGGTGGGGGATTTATTGGGTCC. The oligonucleotides required to check correct integration are hhp1ATG-check2, ATCCACTGCCAATTTTACGACC and hhp1pchk2, ACAGCTTTTATTTTCGTCTGAG.
Contains 1× PCR buffer (containing 1.5 mM MgCl2, 0.2 mM of each dNTP mixture, 0.5 μM of each primer, 2 U per 100 μl Taq DNA polymerase and template DNA).
▲ CRITICAL STEP The promoter region must contain a unique restriction site that will be used to linearize the construct for integrating it to the genome (Step 9).
▲ CRITICAL STEP Ensure that the plasmid DNA template is isolated from a dam+E. coli strain (the majority of the commonly used E. coli strains are dam+).
The entire protocol can be completed in about 3 weeks.
Steps 1–4, cloning of the gene: 3 d
Steps 5–8, mutagenesis: 5 d
Steps 9-10, yeast transformation: 5 d
Step 11, testing functionality: 4 d
Steps 12–14, testing sensitivity: 4 d
Troubleshooting advice can be found in Table 1.
This protocol can be used to engineer protein kinases sensitive to cell-permeable inhibitors. Here we describe the construction of the first analog-sensitive allele of an S. pombe kinase (Fig. 4). As the gate-keeper residue can be identified in most protein kinases, we expect that our protocol will also work for other S. pombe protein kinases as well as other organisms. Analog-sensitive kinase alleles have been successfully created and studied in various organisms including the budding yeast Saccharomyces cerevisiae, mouse and human cells11,34,35.
This work was supported by the Austrian Science Fund (P18955-B03). L.C. was a recipient of EMBO and FEBS short-term fellowships. We thank Mark Petronczki and Maria Siomos for helpful discussions and comments on the manuscript.