Regulation by phosphorylation of Pho81p, a cyclin-dependent kinase inhibitor in Saccharomyces cerevisiae.
Cyclins, cyclin-dependent kinases (CDK) and their inhibitors play a critical role in many biological processes. In yeast, the ankyrin repeat protein Pho81p, by being an inhibitor of the Pho85p-Pho80p cyclin-dependent protein kinase complex, transcriptionally regulates the production of repressible acid phosphatase, encoded by the PHO5 gene. Recent studies in our laboratory showed that Pho81p is phosphorylated by the Pho80p-Pho85p CDK complex in vitro; and, to determine the significance of the phosphorylation, we used site-directed mutagenesis to alter the potential phosphorylation sites for this kinase complex. The resulting mutations were introduced into a yeast strain containing a deletion of PHO81 and the effect of the mutation on PHO5 expression was assayed. Results suggest that phosphorylation of particular residues within Pho81p is crucial for its activity as an inhibitor. Studies using a green fluorescent protein-Pho81p fusion and Western analysis indicate that the localization and half-life of the mutants are similar to wild-type Pho81 proteins. However, an in vivo binding assay indicates that the mutant Pho81p is deficient in binding to the Pho80p-Pho85p kinase complex. These findings support the observation that the mutant fails to inhibit kinase activity in low phosphate. These studies provide insight into the mechanism of regulation of CDK inhibitor activity.
Knight
Janine P
JP
Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, PA 19129, USA.
Daly
Thomas M
TM
Bergman
Lawrence W
LW
D000135:Acid Phosphatase / Q000378:metabolism; D003470:Culture Media; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors; D015966:Gene Expression Regulation, Fungal; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010957:Plasmids; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; C081227:PHO81 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases; D000135:Acid Phosphatase