Mutational analysis of the fission yeast p34cdc2 protein kinase gene.
The p34cdc2 protein serine-threonine kinase plays an essential role in the life cycle of fission yeast, being required for both the G1-S and G2-M transitions during mitotic growth, and also for the second meiotic nuclear division. Functional homologues of p34cdc2 (each ca. 60% identical to the fission yeast prototype) have been isolated from organisms as diverse as humans, insects and plants, and there is now considerable evidence supporting the view that fundamental aspects of the cell cycle controls uncovered in fission yeast will prove to be conserved in all eukaryotes. By comparing the amino acid sequences of fission yeast p34cdc2 with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34cdc2 function. Here the effects are described of mutating a number of these conserved residues. Twenty-three new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with tryptophan renders the resulting mutant protein p80cdc25-independent (while neither leucine, isoleucine nor valine has this effect) and that several of the amino acids within the highly conserved PSTAIRE region are not absolutely required for p34cdc2 function. Five acidic amino acids have also been mutated within p34cdc2, which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at three of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34cdc2 protein function. The results are discussed with reference to the predicted tertiary structure of the p34cdc2 enzyme.
MacNeill
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SA
Microbiology Unit, University of Oxford, UK.
Nurse
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D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic; D016203:CDC2 Protein Kinase / Q000235:genetics*; D002453:Cell Cycle / Q000235:genetics*; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D003545:Cysteine; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000235:genetics; D005434:Flow Cytometry; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000600:Amino Acids, Dicarboxylic; D016203:CDC2 Protein Kinase; D003545:Cysteine