FUNCTION OF HYBRID HUMAN YEAST CYCLIN-DEPENDENT KINASES IN SACCHAROMYCES-CEREVISIAE

It is now well established that progression through the eukaryotic cel l cycle is controlled by oscillations in the activity of cyclin- depen dent kinases (CDKs). In many cases, however, the physiological substra te(s) of CDKs are unknown. The Saccharomyces cerevisiae PHO5 gene enco des a secreted acid phosphatase which is induced in response to phosph ate starvation. The PHO5 gene is activated by the Pho4p transcription factor, which itself is negatively regulated through phosphorylation b y the products of PHO80 and PHO85. Pho80p and Pho85p are homologous to cyclins and CDKs, respectively, and the Pho80p/Pho85p heterodimer sat isfies the biochemical definition of a cyclin/CDK. In the present stud y, several reporter genes were expressed in S. cerevisiae from promote rs which are activated by the transcription factor Pho4p, thereby gene rating yeast strains which exhibit quantifiable phenotypes that reflec t the activity of a specific cyclin/CDK. Positive genetic selections f or inhibition of cyclin/CDK function were characterized using the E. c oli neo and yeast LEU2 genes. Chromosomal disruptions of the yeast PHO 80 and PHO85 genes were constructed and conditions for complementation by plasmid-borne genes were defined. Complementation is achieved at v ery low levels of expression of both Pho80p and Pho85p. High-level exp ression of Pho80p results in aberrant PHO5 promoter regulation, charac terized by failure to derepress in low-phosphate medium. Genes encodin g hybrid CDKs in which regions of Pho85p were replaced with the homolo gous region of human Cdk2 were constructed, and tested for function in S, cerevisiae by complementation of the pho85 chromosomal gene disrup tion. Hybrid proteins in which more than two-thirds of the molecule we re derived from human Cdk2 retained Pho85p function with respect to hi gh-phosphate repression of the PHO5 promoter. The hybrid proteins requ ire the PHO80 gene product for this function. A hybrid human-yeast CDK in which a single amino acid is deleted, within a nonapeptide sequenc e which is perfectly conserved in Pho85p and human Cdk2, retains full function. These results demonstrate that, within the context of the co nserved structure of CDKs, considerable primary sequence variability c an be introduced without loss of the cyclin-dependent function of the CDK.