Systematic deletion analysis of fission yeast protein kinases

Abstract

Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/kinome.

FIG. 1.

Strategy for deletion allele construction. (A) Cell containing a wild-type target gene. (B) Pair of primers containing flanking sequences of a target gene was used to synthesize a DNA fragment by PCR, using a template carrying a selective marker gene. The barcode embedded into the long PCR primers has not been discussed in this study. (C) Haploid cell containing a deletion allele. (D) Diploid cell containing a heterozygous deletion allele. The target gene is indicated in red, and the marker gene is in green (in this study, the marker gene is ura4⁺). wt, wild type; ups, upstream; dns, downstream; seq, sequence.

FIG. 2.

Unrooted phylogenic tree of the 106 protein kinases in S. pombe. Seventeen essential protein kinases are marked in red, and 31 kinases containing tyrosine phosphorylation signatures are underlined. Two regions of the phylogenic tree shaded blue indicate that the region is enriched with tyrosine kinase signatures. AGC, CaMK, and CMGC indicate protein kinase groups, and Polo, Casein I, Wee/Mik, Mekk/Ste11, Mek/Ste7, and Pak/Ste20 indicate protein kinase families that do not belong to the AGC, CaMK, and CMGC groups. The inset shows a schematic representation of protein kinase orthologs in S. pombe (Sp), S. cerevisiae (Sc), and human. One hundred six eukaryotic protein kinase catalytic-domain-containing proteins were selected in S. pombe, 119 in S. cerevisiae, and 491 in human. Analysis of orthologs showed that of 106 S. pombe protein kinases, 67 (25 plus 42) have orthologs in S. cerevisiae and 47 (42 plus 5) in human. Among these, 42 appeared to have orthologs in both S. cerevisiae and human. Numbers in parentheses indicate the numbers of nearest homologs.

FIG. 3.

Essential putative protein kinases. (A) Heterozygous deletion alleles in diploid mutant cells were verified by PCR using allele-specific primers as explained in the legend to Fig. 1. Wild-type (wt) alleles were confirmed by PCR using the target-specific primer pairs pA-pB for the 5′-end (head) joint and pC/pD for the 3′-end (tail) joint; deletion alleles were confirmed by PCR using primer pairs of target-specific pA-marker-specific pB-ura (head) and marker-specific pC-ura-target-specifc pD (tail). (B) Tetrad analysis confirmed that the putative protein kinases were essential for vegetative growth. (C) Free-spore assay of the mutants. DNA was stained using the fluorescent dye DAPI. Arrows and arrowheads indicate horse tail-like and condensed chromosomes, respectively. (D) Subcellular localization of GFP-tagged kinases in wild-type haploid cells. See the text for details.

FIG. 4.

Phenotypic assessment of nonessential protein kinase mutants. Approximately 5 μl of 10-fold serial dilutions of the 88 viable haploid h⁻ mutant cells (sck1⁻ mutant was not available) were spotted and grown under 17 different stress conditions with various dosages. Examples of cell growth under stress conditions such as high temperature (36°C), a microtubule poison (TBZ), and an agent that blocks DNA replication (HU) are shown. A mutant was described as hypersensitive to a stress condition when its growth was similar to that of wild-type cells at 2 (or >2) dilution factors lower. Mutant growth phenotypes under various conditions are individually summarized in Table 2.

FIG. 5.

Phenotypic clustering of hypersensitive mutants. Pearson correlation clustering of the growth pattern of the protein kinase mutants was performed to analyze 41 known and putative protein kinase mutants that exhibited hypersensitivity to at least one of the 17 stress conditions tested (see the text). Upon comparison of growth phenotypes under various conditions, 41 mutants were divided into eight groups with a minimal similarity of 50%. The 17 conditions used in the phenotypic assessment were separated into four clusters. rep., repair; MT, microtubule; Calc, calcofluor; BFA, brefeldin A; SDS, sodium dodecyl sulfate; Bleo, bleomycin; MM, minimum medium; Sorb, sorbitol.

FIG. 6.

Sporulation assay. Iodine staining of mutants under conditions promoting sporulation was performed. About 5 μl of 10-fold serial dilutions of 84 haploid h⁹⁰ mutant cells (5 h⁹⁰ mutants were not available) were tested for conjugation, meiosis, or sporulation. Mutants defective in conjugation, meiosis, or sporulation display negative staining with iodine vapor (I₂) and are indicated with “-.” Partially negative stainings are marked with “-/+.” Mutant cells which displayed the vegetative growth phenotype (Veg.) under at least one of the 17 conditions tested are indicated with “-” (see Fig. 5). A white crossed spot was a bacterial contaminant.

FIG. 7.

Expression signatures identify mutant phenotypes. Pearson correlation clustering of the expression signatures of the protein kinase mutants was performed. An expression profile of each mutant under optimal growth conditions was attained using oligonucleotide-based whole-genome DNA microarrays with a wild-type strain (ura4⁺) as a common reference. Eight hundred forty-five genes whose expression levels changed 1.6-fold or greater in at least one of the nine mutant profiles were selected for cluster analysis. Eight protein kinases are known to be involved in oxidative-stress responses (in red), cell wall integrity or polarized growth (in orange), and pheromone responses (in purple). A putative protein kinase, SPBC119.07, which showed growth defects in drug sensitivity assays similar to those of the kin1 mutant (Fig. 5) displayed an expression signature similar to that of the kin1 mutant. MT, microtubule.