Mutational analysis of the fission yeast p34(cdc2) protein kinase gene
1993
MacNeill, S.A. (Oxford Univ. (United Kingdom). Microbiology Unit. ICRF Cell Cycle Group.) | Nurse, P.
The p34(cdc2) 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 p34(cdc2) (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 p34(cdc2) with its higher eukaryotic counterparts it is possible to identify conserved residues that are likely to be centrally important for p34(cdc2) function. Here the effects are described of mutating a number of these conserved residues. 23 new mutant alleles have been constructed and tested. We show that replacing cysteine 67 with trypthophan renders the resulting mutant protein p80(cdc25) 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 p34(cdc2) function. Five acidic amino acids have also been mutated within p34(cdc2), which are invariant across the eukaryotic protein kinase family. Acid-to-base mutations at 3 of these residues resulted in a dominant-negative, cell cycle arrest phenotype while similar mutations at the other two simply abolished p34(cdc2) protein function. The results are discussed with reference to the predicted tertiary structure of the p34(cdc2) enzyme.
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