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Dissertation
The regulation of Pho81p, a cyclin-dependent kinase inhibitor in Saccharomyces cerevisiae
Doctor of Philosophy (Ph.D.), Drexel University
Mar 2003
DOI:
https://doi.org/10.17918/00009111
Abstract
Cyclins, cyclin-dependent kinases (CDK) and their inhibitors play a critical role in many biological processes, including Eukaryotic stress response and cell division. In humans, the ankyrin-repeat-containing CDK inhibitor p16 INK4 functions as a tumor suppressor in the regulation of the Rb protein. In yeast, repressible acid phosphatase, encoded by the PHO5 gene, is transcriptionally regulated by another ankyrin-repeat protein, Pho81p. PHO5 is repressed in high phosphate growth media and de-repressed in low phosphate. The Pho80p-Pho85p cyclin-cyclin dependent kinase complex acts as a negative regulator to block PHO5 expression when phosphate levels are high. The PHO81 gene encodes a 134 kD inhibitor of the Pho80p-PHO85p complex. In high phosphate, Pho81p is inactive, resulting in an active CDK complex and thus no expression of PHO5 . In low phosphate, Pho81p is an active inhibitor of the Pho80p-Pho85p complex, resulting in expression of PHO5. However, the mechanism of regulation of Pho81p activity is unclear. Recent studies in our laboratory have shown that Pho81p is phosphorylated by the Pho80p-Pho85p CDK complex in-vitro, and Pho81p contains 9 potential CDK phosphorylation sites. To determine the significance of phosphorylation, we used site-directed mutagenesis to alter these potential phosphorylation sites. The resulting mutants 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 GFP-Pho81p fusion and western analysis indicate that the localization and half-life of the mutant is similar to wild type. However, an in-vivo binding assay indicates that 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 cyclin-dependent kinase inhibitor activity.
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Details
- Title
- The regulation of Pho81p, a cyclin-dependent kinase inhibitor in Saccharomyces cerevisiae
- Creators
- Janine Patricia Knight
- Contributors
- Lawrence W. Bergman (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- viii, 110 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- College of Medicine; Drexel University
- Other Identifier
- 991021888790604721