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Dissertation
Identification of multiple substrates of the PHO85 cyclin dependent protein kinase as key regulators of acid phosphatase expression and glycogen metabolism in the yeast Saccaromyces cerevisiae
Doctor of Philosophy (Ph.D.), Allegheny University of the Health Sciences
Dec 1996
DOI:
https://doi.org/10.17918/00009390
Abstract
In Saccharomyces cerevisiae, multiple signalling pathways allow the cell to respond to environmental variation and therefore are essential for the survival of the yeast. During nutrient stress, expression of acid phosphatase, encoded by PH05, is upregulated and functions to scavenge for inorganic phosphate required for cell growth. In addition, a hyperaccumulation of glycogen results to provide the cell an intracellular energy source in the event of long term starvation. The Pho85 cyclin dependent protein kinase regulates several metabolic pathways in the cell. Cyclin dependent protein kinases were initially identified as regulators of the cell cycle; however, in yeast Pho85 regulates not only the cell cycle, but also expression of acid phosphatase and glycogen metabolism. In the work described here, we further characterize Pho85 as a cyclin dependent protein kinase and identify key substrates of Pho85 involved in the mechanics of regulating PHO5 expression and glycogen biosynthesis. In the regulation of acid phosphatase expression, Pho85 associates with the Pho80 cyclin to form a Pho85-Pho80 kinase complex. During high phosphate conditions, this complex phosphorylates and inhibits the transcriptional activator Pho4. In addition, the Pho80 cyclin subunit is phosphorylated at Ser-234 and Ser-267 by Pho85. Phosphorylation of Ser-234 is required for a functional Pho85-Pho80 kinase complex, which represses PHO5 expression. We demonstrate that Pho81 is also phosphorylated by Pho85, and we localize the phosphorylation sites to the ankyrin repeat motifs within Pho81 that are essential for inhibiting the kinase activity of the Pho85-Pho80 complex. Disruption of Pho85 kinase activity results in a hyperaccumulation of glycogen. Glycogen synthase, Gsy2, is responsible for production of 90% of the glycogen in the cell. Gsy2 is regulated by reversible phosphorylation and allosteric activation. We identify Pho85 as a glycogen synthase kinase and show that phosphorylation by Pho85 inhibits glycogen synthase activity. By site directed mutagenesis, we identify Ser-654 as the Pho85 phosphorylation site. These results identify Pho85 as a key kinase in regulating several metabolic pathways, and they provide a mechanism for crosstalk between environmental stimuli and cell growth and development.
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Details
- Title
- Identification of multiple substrates of the PHO85 cyclin dependent protein kinase as key regulators of acid phosphatase expression and glycogen metabolism in the yeast Saccaromyces cerevisiae
- Creators
- Norman C. Waters
- Contributors
- Lawrence W. Bergman (Advisor) - Drexel University, Allegheny University of the Health Sciences (1996-1998)
- Awarding Institution
- Allegheny University of the Health Sciences
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Allegheny University of the Health Sciences; Philadelphia, Pennsylvania
- Number of pages
- x, 143 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Allegheny University of the Health Sciences (1996-1998); School of Medicine (1996-1998); Neoplastic Diseases [Historical]
- Other Identifier
- 991021888736404721