Dominant negative protein kinase mutations that confer a G1 arrest phenotype.

Abstract

The CDC28 gene of Saccharomyces cerevisiae encodes a protein kinase that is required for passage through the G1 phase of the cell cycle. We have used an inducible promoter fused to the CDC28 coding sequence to isolate conditionally dominant mutant alleles of CDC28. Overexpression of these dominant alleles causes arrest in the G1 phase of the cell cycle but permits the distinctive asymmetric growth that is characteristic of recessive temperature-sensitive cdc28 mutants. The dominant alleles encode products with no detectable protein kinase activity, and their phenotypic effects can be suppressed by simultaneous overproduction of the wild-type protein. DNA sequence analysis showed that the mutant site in at least one of the dominant alleles is in a residue that is highly conserved among protein kinases. These properties are best understood if the dominant mutation results in the catalytic inactivation of the protein kinase but still allows the binding of another component needed for CDC28 function. By this model, high levels of the mutant protein arrest cell division by denying the wild-type protein access to this other component. Suppressors that may encode this other component have been isolated on high-copy-number plasmids.

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