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Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes.

Springer M, Wykoff DD, Miller N, O'Shea EK - PLoS Biol. (2003)

Bottom Line: This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression.Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4.Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, USA.

ABSTRACT
A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

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A Strain Expressing Pho4SA14PA6 from a Low Copy Plasmid Differentially Expresses PHO5 and PHO84 in Intermediate-Phosphate Medium(A) Fluorescence microscopy of yeast cells containing Pho4SA14PA6–GFP grown in no, 100 μM, or 10 mM phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 from strains expressing Pho4SA14PA6 or Pho4WT1234PA6 from a low copy plasmid grown in no, 100 μM, or 10,000 μM phosphate medium.
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pbio.0000028-g005: A Strain Expressing Pho4SA14PA6 from a Low Copy Plasmid Differentially Expresses PHO5 and PHO84 in Intermediate-Phosphate Medium(A) Fluorescence microscopy of yeast cells containing Pho4SA14PA6–GFP grown in no, 100 μM, or 10 mM phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 from strains expressing Pho4SA14PA6 or Pho4WT1234PA6 from a low copy plasmid grown in no, 100 μM, or 10,000 μM phosphate medium.

Mentions: Site 6 phosphorylation correlates with differential gene expression in wild-type cells and is sufficient to cause differential gene expression in strains expressing Pho4 mutants, but this does not mean site 6 phosphorylation is necessary for differential gene induction. If site 6 phosphorylation is the sole mechanism to inhibit PHO5 expression in intermediate-phosphate conditions, a strain grown in intermediate-phosphate medium (where Pho4 is nuclear) expressing a mutant Pho4 that cannot be phosphorylated on site 6 should significantly induce PHO5. We therefore created a strain expressing a Pho4 mutant, Pho4WT1234PA6, that cannot be phosphorylated on site 6 but is able to bind Pho2. Fluorescence microscopy was used to confirm proper localization of this mutant (data not shown). In contrast to expectations, the mutant that prevents site 6 phosphorylation did not strongly influence the expression of PHO5 or PHO84 in intermediate-phosphate medium (Figure 5A). This suggests that a second mechanism that contributes to differential expression of PHO5 and PHO84 expression exists in intermediate-phosphate conditions. Because a Pho4 mutant that is refractory to phosphorylation, PHO4SA1234PA6 (see Figure 1C), induces all phosphate-responsive genes, we conclude that this second mechanism is likely to work through Pho4 and Pho80–Pho85.


Partially phosphorylated Pho4 activates transcription of a subset of phosphate-responsive genes.

Springer M, Wykoff DD, Miller N, O'Shea EK - PLoS Biol. (2003)

A Strain Expressing Pho4SA14PA6 from a Low Copy Plasmid Differentially Expresses PHO5 and PHO84 in Intermediate-Phosphate Medium(A) Fluorescence microscopy of yeast cells containing Pho4SA14PA6–GFP grown in no, 100 μM, or 10 mM phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 from strains expressing Pho4SA14PA6 or Pho4WT1234PA6 from a low copy plasmid grown in no, 100 μM, or 10,000 μM phosphate medium.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC261874&req=5

pbio.0000028-g005: A Strain Expressing Pho4SA14PA6 from a Low Copy Plasmid Differentially Expresses PHO5 and PHO84 in Intermediate-Phosphate Medium(A) Fluorescence microscopy of yeast cells containing Pho4SA14PA6–GFP grown in no, 100 μM, or 10 mM phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 from strains expressing Pho4SA14PA6 or Pho4WT1234PA6 from a low copy plasmid grown in no, 100 μM, or 10,000 μM phosphate medium.
Mentions: Site 6 phosphorylation correlates with differential gene expression in wild-type cells and is sufficient to cause differential gene expression in strains expressing Pho4 mutants, but this does not mean site 6 phosphorylation is necessary for differential gene induction. If site 6 phosphorylation is the sole mechanism to inhibit PHO5 expression in intermediate-phosphate conditions, a strain grown in intermediate-phosphate medium (where Pho4 is nuclear) expressing a mutant Pho4 that cannot be phosphorylated on site 6 should significantly induce PHO5. We therefore created a strain expressing a Pho4 mutant, Pho4WT1234PA6, that cannot be phosphorylated on site 6 but is able to bind Pho2. Fluorescence microscopy was used to confirm proper localization of this mutant (data not shown). In contrast to expectations, the mutant that prevents site 6 phosphorylation did not strongly influence the expression of PHO5 or PHO84 in intermediate-phosphate medium (Figure 5A). This suggests that a second mechanism that contributes to differential expression of PHO5 and PHO84 expression exists in intermediate-phosphate conditions. Because a Pho4 mutant that is refractory to phosphorylation, PHO4SA1234PA6 (see Figure 1C), induces all phosphate-responsive genes, we conclude that this second mechanism is likely to work through Pho4 and Pho80–Pho85.

Bottom Line: This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression.Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4.Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of California, San Francisco, USA.

ABSTRACT
A cell's ability to generate different responses to different levels of stimulus is an important component of an adaptive environmental response. Transcriptional responses are frequently controlled by transcription factors regulated by phosphorylation. We demonstrate that differential phosphorylation of the budding yeast transcription factor Pho4 contributes to differential gene expression. When yeast cells are grown in high-phosphate growth medium, Pho4 is phosphorylated on four critical residues by the cyclin-CDK complex Pho80-Pho85 and is inactivated. When yeast cells are starved for phosphate, Pho4 is dephosphorylated and fully active. In intermediate-phosphate conditions, a form of Pho4 preferentially phosphorylated on one of the four sites accumulates and activates transcription of a subset of phosphate-responsive genes. This Pho4 phosphoform binds differentially to phosphate-responsive promoters and helps to trigger differential gene expression. Our results demonstrate that three transcriptional outputs can be generated by a pathway whose regulation is controlled by one kinase, Pho80-Pho85, and one transcription factor, Pho4. Differential phosphorylation of Pho4 by Pho80-Pho85 produces phosphorylated forms of Pho4 that differ in their ability to activate transcription, contributing to multiple outputs.

Show MeSH
Related in: MedlinePlus