It is now well established that progression through the eukaryotic cell cycle is controlled by oscillations in the activity of cyclin- dependent kinases (CDKs). In many cases, however, the physiological substrate(s) of CDKs are unknown. The Saccharomyces cerevisiae PHO5 gene encodes a secreted acid phosphatase which is induced in response to phosphate starvation. The PHO5 gene is activated by the Pho4p transcription factor, which itself is negatively regulated through phosphorylation by the products of PHO80 and PHO85. Pho80p and Pho85p are homologous to cyclins and CDKs, respectively, and the Pho80p/Pho85p heterodimer satisfies the biochemical definition of a cyclin/CDK. In the present study, several reporter genes were expressed in S. cerevisiae from promoters which are activated by the transcription factor Pho4p, thereby generating yeast strains which exhibit quantifiable phenotypes that reflect the activity of a specific cyclin/CDK. Positive genetic selections for inhibition of cyclin/CDK function were characterized using the E. coli neo and yeast LEU2 genes. Chromosomal disruptions of the yeast PHO80 and PHO85 genes were constructed and conditions for complementation by plasmid-borne genes were defined. Complementation is achieved at very low levels of expression of both Pho80p and Pho85p. High-level expression of Pho80p results in aberrant PHO5 promoter regulation, characterized by failure to derepress in low-phosphate medium. Genes encoding hybrid CDKs in which regions of Pho85p were replaced with the homologous region of human Cdk2 were constructed, and tested for function in S. cerevisiae by complementation of the pho85 chromosomal gene disruption. Hybrid proteins in which more than two-thirds of the molecule were derived from human Cdk2 retained Pho85p function with respect to high-phosphate repression of the PHO5 promoter. The hybrid proteins require the PHO80 gene product for this function. A hybrid human-yeast CDK in which a single amino acid is deleted, within a nonapeptide sequence which is perfectly conserved in Pho85p and human Cdk2, retains full function. These results demonstrate that, within the context of the conserved structure of CDKs, considerable primary sequence variability can be introduced without loss of the cyclin-dependent function of the CDK.

• PMID: 9829836
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Journal Article | Research Support, U.S. Gov't, P.H.S.

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Bitter GA

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