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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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Growth of Yeast Cells in Intermediate-Phosphate Medium Leads to Differential Phosphorylation of Pho4 and Differential Expression of PHO5 and PHO84(A) Fluorescence microscopy of yeast cells containing Pho4–GFP grown in no, 50 μM, 100 μM (intermediate [int]), 300 μM, or 10,000 μM (high) phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 in wild-type cells grown in medium containing different concentrations of phosphate.(C) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in wild-type cells grown for 1, 2, or 5 h in intermediate-phosphate medium.(D) Expression of genes in the phosphate-responsive cluster (Carroll et al. 2001) for a wild-type strain grown in intermediate- or no-phosphate medium compared to wild-type cells grown in high-phosphate medium. Cy5 and Cy3 samples are colored red and green, respectively. The percent of induction of each gene in intermediate-phosphate medium compared to its maximal induction in no-phosphate medium is presented on the right.(E) Analysis of Pho4 protein and phosphorylation by Western blotting for wild-type cells grown in no-, intermediate-, and high-phosphate medium. Samples were probed with phosphopeptide antibodies specific to sites 2, 3, and 6 of Pho4 and by a polyclonal antibody that recognizes Pho4.(F) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in a PHO4WT1234SD6 strain.
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pbio.0000028-g002: Growth of Yeast Cells in Intermediate-Phosphate Medium Leads to Differential Phosphorylation of Pho4 and Differential Expression of PHO5 and PHO84(A) Fluorescence microscopy of yeast cells containing Pho4–GFP grown in no, 50 μM, 100 μM (intermediate [int]), 300 μM, or 10,000 μM (high) phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 in wild-type cells grown in medium containing different concentrations of phosphate.(C) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in wild-type cells grown for 1, 2, or 5 h in intermediate-phosphate medium.(D) Expression of genes in the phosphate-responsive cluster (Carroll et al. 2001) for a wild-type strain grown in intermediate- or no-phosphate medium compared to wild-type cells grown in high-phosphate medium. Cy5 and Cy3 samples are colored red and green, respectively. The percent of induction of each gene in intermediate-phosphate medium compared to its maximal induction in no-phosphate medium is presented on the right.(E) Analysis of Pho4 protein and phosphorylation by Western blotting for wild-type cells grown in no-, intermediate-, and high-phosphate medium. Samples were probed with phosphopeptide antibodies specific to sites 2, 3, and 6 of Pho4 and by a polyclonal antibody that recognizes Pho4.(F) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in a PHO4WT1234SD6 strain.

Mentions: To test these predictions, we grew cells in medium containing different levels of phosphate (0, 50, 100, 300, and 10,000 μM) and monitored localization of a Pho4–GFP (green fluorescent protein) fusion protein and transcription of PHO5 and PHO84. We observed three different responses to different phosphate conditions. First, the high-phosphate response occurs at extracellular phosphate concentrations greater than 300 μM phosphate. Pho4 is cytoplasmic, and PHO5 and PHO84 are expressed at only basal levels under these conditions (Figure 2A and 2B). Second, the no-phosphate response occurs when there is no phosphate in the medium. Pho4 is nuclear, and PHO5 and PHO84 are expressed maximally under these conditions (Figure 2A and 2B). Third, the intermediate-phosphate response occurs at extracellular phosphate concentrations less than 100 μM phosphate and greater than no phosphate. Pho4 is nuclear, but under these conditions there is only approximately 10% of maximal expression of PHO5, while there is approximately 50% of maximal expression of PHO84 (Figure 2A and 2B).


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

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

Growth of Yeast Cells in Intermediate-Phosphate Medium Leads to Differential Phosphorylation of Pho4 and Differential Expression of PHO5 and PHO84(A) Fluorescence microscopy of yeast cells containing Pho4–GFP grown in no, 50 μM, 100 μM (intermediate [int]), 300 μM, or 10,000 μM (high) phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 in wild-type cells grown in medium containing different concentrations of phosphate.(C) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in wild-type cells grown for 1, 2, or 5 h in intermediate-phosphate medium.(D) Expression of genes in the phosphate-responsive cluster (Carroll et al. 2001) for a wild-type strain grown in intermediate- or no-phosphate medium compared to wild-type cells grown in high-phosphate medium. Cy5 and Cy3 samples are colored red and green, respectively. The percent of induction of each gene in intermediate-phosphate medium compared to its maximal induction in no-phosphate medium is presented on the right.(E) Analysis of Pho4 protein and phosphorylation by Western blotting for wild-type cells grown in no-, intermediate-, and high-phosphate medium. Samples were probed with phosphopeptide antibodies specific to sites 2, 3, and 6 of Pho4 and by a polyclonal antibody that recognizes Pho4.(F) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in a PHO4WT1234SD6 strain.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261874&req=5

pbio.0000028-g002: Growth of Yeast Cells in Intermediate-Phosphate Medium Leads to Differential Phosphorylation of Pho4 and Differential Expression of PHO5 and PHO84(A) Fluorescence microscopy of yeast cells containing Pho4–GFP grown in no, 50 μM, 100 μM (intermediate [int]), 300 μM, or 10,000 μM (high) phosphate medium.(B) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1 in wild-type cells grown in medium containing different concentrations of phosphate.(C) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in wild-type cells grown for 1, 2, or 5 h in intermediate-phosphate medium.(D) Expression of genes in the phosphate-responsive cluster (Carroll et al. 2001) for a wild-type strain grown in intermediate- or no-phosphate medium compared to wild-type cells grown in high-phosphate medium. Cy5 and Cy3 samples are colored red and green, respectively. The percent of induction of each gene in intermediate-phosphate medium compared to its maximal induction in no-phosphate medium is presented on the right.(E) Analysis of Pho4 protein and phosphorylation by Western blotting for wild-type cells grown in no-, intermediate-, and high-phosphate medium. Samples were probed with phosphopeptide antibodies specific to sites 2, 3, and 6 of Pho4 and by a polyclonal antibody that recognizes Pho4.(F) Quantitation of RNA levels by Northern blot analysis of PHO84, PHO5, and ACT1, in a PHO4WT1234SD6 strain.
Mentions: To test these predictions, we grew cells in medium containing different levels of phosphate (0, 50, 100, 300, and 10,000 μM) and monitored localization of a Pho4–GFP (green fluorescent protein) fusion protein and transcription of PHO5 and PHO84. We observed three different responses to different phosphate conditions. First, the high-phosphate response occurs at extracellular phosphate concentrations greater than 300 μM phosphate. Pho4 is cytoplasmic, and PHO5 and PHO84 are expressed at only basal levels under these conditions (Figure 2A and 2B). Second, the no-phosphate response occurs when there is no phosphate in the medium. Pho4 is nuclear, and PHO5 and PHO84 are expressed maximally under these conditions (Figure 2A and 2B). Third, the intermediate-phosphate response occurs at extracellular phosphate concentrations less than 100 μM phosphate and greater than no phosphate. Pho4 is nuclear, but under these conditions there is only approximately 10% of maximal expression of PHO5, while there is approximately 50% of maximal expression of PHO84 (Figure 2A and 2B).

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