A recent systematic deletion analysis of genes in the fission yeast S. pombe
revealed that 18 protein kinases are essential for viability.7,8
We created heterozygous diploid deletion strains for each of these 18 kinases and analyzed the haploid products after sporulation. We confirmed that 17 kinase genes are essential for viability. However, the sporulation of a heterozygous diploid deletion strain, followed by the dissection of the asci on rich medium, showed that one of the kinases, ppk28
, is not essential for viability (data not shown). This observation is consistent with previous reports showing that ppk28
is dispensable for normal cell growth.9
Of the 17 essential kinases, only one analog-sensitive allele (ark1 as3
was available when we started this project. Therefore, we decided to create conditional analog-sensitive alleles for 16 remaining protein kinases essential for viability ().
Functionality of analog-sensitive kinase mutants as determined by growth on YES plates
To create conditional analog-sensitive alleles, we mutated the gate-keeper residue within the kinase ATP-binding site to a glycine (as1) or an alanine (as2). The mutations were expressed in an S. pombe
strain lacking the wild-type allele of the corresponding kinase according to the protocol of Gregan et al.11
Out of the 16 kinases with a mutated gate-keeper residue, 13 remained functional as indicated by growth of haploid cells carrying the corresponding gate-keeper mutation in YES medium ( and and S1
). Tetrad analysis of heterozygous diploid strains showed that the remaining three mutants (cdc7-as, hsk1-as
) were not functional (). We also observed that two mutants (cdc2-as1
) were sensitive to elevated temperature (32°C), but they grew normally at a low temperature (25°C). The temperature sensitivity of cdc2-as1
has been described previously in references 12
. Thus, out of the 16 essential kinases, we constructed 13 functional alleles where the gate-keeper residue was replaced by a glycine or an alanine.
Figure 3 Sensitivity of cells expressing analog-sensitive kinase alleles to various inhibitors. Serial dilutions of wild-type cells or cells expressing indicated analog-sensitive kinase alleles were spotted on YES plates containing or lacking the indicated inhibitors (more ...)
To test the sensitivity of the 13 kinase mutants to inhibitors, we synthesized eight different inhibitors (ATP analogs) (). In addition to the mutants created in this study, we included the previously constructed ark1-as3
mutant in our analysis.10
Spot test on YES plates showed that eight mutants were highly sensitive to one or more of the tested inhibitors (≤10 µM ATP analog), and six mutants exhibited sensitivity only to higher concentrations of inhibitors ( and
). To further characterize the mutant phenotypes, we added an inhibitor to cultures exponentially growing in liquid YES medium and analyzed the effect of inhibitors on cell growth by counting cell number. Four hours after adding the inhibitor, we harvested cells and analyzed the morphology of the cells by microscopy and the cellular DNA content by flow cytometry (Fig. S1
). These analyses confirmed that all 13 kinase mutants were sensitive to at least one of the tested inhibitors, and the growth of the mutant cells was either inhibited (7 mutants) or completely blocked (6 mutants) in the presence of the inhibitor (Fig. S1
). Thus, we conclude that analog-sensitive alleles were constructed for 13 out of 16 of the essential kinases. Therefore, a chemical genetic strategy of conditional inactivation of protein kinases by creating mutants sensitive to ATP analogs is a viable option for studying S. pombe
protein kinases that are essential for cell growth and should encourage wider use of this strategy in yeast as well as other organisms.
Chemical structures of the inhibitors used in this study.
Sensitivity of analog-sensitive kinase mutants to various ATP analogs