Regulation of the yeast transcriptional factor PHO2 activity by phosphorylation.
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.
Liu
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State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China.
Yang
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Yang
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J
Xia
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Ao
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D000135:Acid Phosphatase / Q000378:metabolism; D001665:Binding Sites; D015374:Biosensing Techniques; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D016384:Consensus Sequence / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D018398:Homeodomain Proteins; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D028061:Phosphate Transport Proteins; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D018398:Homeodomain Proteins; D026901:Membrane Transport Proteins; C051611:PHO2 protein, S cerevisiae; D028061:Phosphate Transport Proteins; D010710:Phosphates; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D012694:Serine; C055831:phosphate permease; D017489:CDC28 Protein Kinase, S cerevisiae; D000135:Acid Phosphatase