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Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation
1State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Abstract:
The induction of yeast Saccharomyces cerevisiae gene PHO5 expression is mediated by transcriptional factors PHO2 and PHO4. PHO4 protein has been reported to be phosphorylated and inactivated by a cyclin-CDK (cyclin-dependent kinase) complex, PHO80-PHO85. We report here that PHO2 can also be phosphorylated. A Ser-230 to Ala mutation in the consensus sequence (SPIK) recognized by cdc2/CDC28-related kinase in PHO2 protein led to complete loss of its ability to activate the transcription of PHO5 gene. Further investigation showed that the Pro-231 to Ser mutation inactivated PHO2 protein as well, whereas the Ser-230 to Asp mutation did not affect PHO2 activity. Since the PHO2 Asp-230 mutant mimics Ser-230-phosphorylated PHO2, we postulate that only phosphorylated PHO2 protein could activate the transcription of PHO5 gene. Two hybrid assays showed that yeast CDC28 could interact with PHO2. CDC28 immunoprecipitate derived from the YPH499 strain grown under low phosphate conditions phosphorylated GST-PHO2 in vitro. A phosphate switch regulates the transcriptional activation activity of PHO2, and mutations of the (SPIK) site affect the transcriptional activation activity of PHO2 and the interaction between PHO2 and PHO4. BIAcore(R) analysis indicated that the negative charge in residue 230 of PHO2 was sufficient to help PHO2 interact with PHO4 in vitro.
Insights
Phosphorylation of the PHO2 protein is crucial for activating Saccharomyces cerevisiae PHO5 gene expression. This phosphorylation, regulated by a phosphate switch and involving CDC28 kinase, impacts PHO2-PHO4 interactions.
Area of Science:
- Molecular Biology
- Yeast Genetics
- Transcriptional Regulation
Background:
- Gene expression in Saccharomyces cerevisiae is tightly regulated by various transcription factors.
- The PHO5 gene, involved in phosphate metabolism, is activated by PHO2 and PHO4 transcription factors.
- PHO4 activity is modulated by phosphorylation, typically leading to inactivation.
Purpose of the Study:
- To investigate the role of PHO2 phosphorylation in the regulation of PHO5 gene expression.
- To identify the specific kinase involved in PHO2 phosphorylation and its interaction partners.
- To elucidate how phosphorylation affects PHO2's transcriptional activity and its interaction with PHO4.
Main Methods:
- Site-directed mutagenesis of the PHO2 gene (Ser-230 to Ala, Pro-231 to Ser, Ser-230 to Asp).
- In vitro kinase assays using GST-PHO2 and CDC28 immunoprecipitates.
- Yeast two-hybrid assays to study protein-protein interactions.
- Surface plasmon resonance (BIAcore) analysis to quantify binding affinities.
Main Results:
- Mutations at the Ser-230 site of PHO2 abolished its transcriptional activation of PHO5.
- Phosphorylation of PHO2 by CDC28 kinase was demonstrated in vitro, particularly under low phosphate conditions.
- The phosphorylation status of PHO2, mimicked by the Asp-230 mutation, is essential for PHO5 activation.
- Mutations affecting the phosphorylation site altered PHO2's interaction with PHO4.
Conclusions:
- PHO2 phosphorylation is a critical regulatory step for PHO5 gene induction in yeast.
- A phosphate-sensitive switch involving CDC28 kinase regulates PHO2's transcriptional activity.
- Phosphorylation enhances the interaction between PHO2 and PHO4, facilitating transcriptional activation.