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Roles of Phosphorylation Sites in Regulating Activity of the Transcription Factor Pho4

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Science  07 May 1999:
Vol. 284, Issue 5416, pp. 977-980
DOI: 10.1126/science.284.5416.977

Abstract

Transcription factors are often phosphorylated at multiple sites. Here it is shown that multiple phosphorylation sites on the budding yeast transcription factor Pho4 play distinct and separable roles in regulating the factor's activity. Phosphorylation of Pho4 at two sites promotes the factor's nuclear export and phosphorylation at a third site inhibits its nuclear import. Phosphorylation of a fourth site blocks the interaction of Pho4 with the transcription factor Pho2. Multiple phosphorylation sites provide overlapping and partially redundant layers of regulation that function to efficiently control the activity of Pho4.

Many signaling pathways rapidly and reversibly convert extracellular signals into changes in gene expression. Phosphorylation of a transcription factor, often at multiple sites, is a common mechanism for responding to signaling events (1). This modification can lead to changes in transcription factor concentration or activity in the nucleus (2). However, the role of multiple phosphorylation sites in regulating the activity of a protein is not well understood.

To study how multiple phosphorylation sites control protein activity, we focused on the regulation of Pho4, a transcription factor in budding yeast that activates expression of genes induced in response to phosphate starvation (3). When yeast cells are grown in phosphate-rich conditions, Pho4 is phosphorylated by the Pho80/Pho85 cyclin-cyclin–dependent kinase (CDK) complex (4) and exported to the cytoplasm (5), thereby terminating expression of phosphate-responsive genes. The kinase Pho80/Pho85 phosphorylates Pho4 on five Ser-Pro (SP) dipeptides, referred to as SP1, SP2, SP3, SP4, and SP6 (6). When yeast cells are starved for phosphate, the CDK inhibitor Pho81 inactivates Pho80/Pho85 (7), leading to the accumulation of unphosphorylated Pho4 in the nucleus (6) and the subsequent transcription of phosphate-responsive genes.

Addition of phosphate to a phosphate-starved culture causes rapid phosphorylation and nuclear export of Pho4 fused to the green fluorescent protein (5) (Fig. 1A). Export of Pho4 requires phosphorylation by Pho80/Pho85; Pho4 is localized to the nucleus and fully active transcriptionally in strains lacking Pho80 or Pho85 (6). Additionally, the nonphosphorylatable mutant Pho4SA12346 (containing Ser→Ala substitutions at the five sites of phosphorylation) is constitutively localized to the nucleus and partially active transcriptionally (6). To determine which of the five phosphorylation sites are required for the export of Pho4, we tested the ability of Pho4 mutants to be exported from the nucleus. Pho4SA1-GFP, Pho4SA4-GFP, and Pho4SA6-GFP, containing an individual Ser→Ala substitution at phosphorylation site 1, 4, or 6, had no defect in nuclear export (8). However, Pho4SA2-GFP and Pho4SA3-GFP, containing a Ser→Ala substitution at sites 2 and 3, respectively, could not be exported (Fig. 1A). Additionally, Pho4SA146-GFP, a mutant that can only be phosphorylated on sites 2 and 3, was exported from the nucleus upon addition of phosphate (Fig. 1A). Thus, phosphorylation of sites 2 and 3 is necessary and sufficient for nuclear export of Pho4.

Figure 1

Phosphorylation of sites 2 and 3 promotes nuclear export of Pho4. (A) Localization of wild-type Pho4-GFP, or the indicated Pho4 mutants fused to GFP, in cells grown in no phosphate medium. For the Feed Pi sample, phosphorylation and nuclear export of Pho4-GFP were triggered by addition of phosphate to a culture that had been grown in no phosphate medium (25). (B) Wild-type Pho4 and the indicated Pho4 mutants, joined to two IgG-binding z domains derived from protein A (Pho4-zz), were phosphorylated (+ATP) or mock phosphorylated (−ATP) in vitro, immobilized on IgG-Sepharose, and binding to Msn5-His6 was measured (26). (Top) The amount of bound Msn5-His6 was analyzed on a silver-stained SDS-PAGE (polyacrylamide gel electrophoresis) gel. (Bottom) The amount of immobilized Pho4-zz was analyzed on a Coomassie-stained SDS-PAGE gel.

Msn5, a member of the β-importin family of nuclear transport receptors, is the export receptor for Pho4 (5). In vitro, Msn5 and the small guanosine triphosphatase (GTPase) Ran (in its GTP-bound state) form a stable complex with phosphorylated Pho4, but not with unphosphorylated Pho4 (5). We examined whether phosphorylation of sites 2 and 3 is also required for an interaction with Msn5 in vitro. Pho4SA146and Pho4SA23 were tagged with two immunoglobulin G (IgG)–binding “z” domains derived from protein A (Pho4SA146-zz and Pho4SA23-zz), phosphorylated in vitro (9), and assayed for Msn5 binding in the presence of Gsp1Q71L, a yeast Ran mutant locked in the GTP-bound form. Pho4SA23-zz failed to interact with Msn5 in either its phosphorylated or unphosphorylated form (Fig. 1B). By contrast, Pho4SA146-zz interacted with Msn5 only when phosphorylated (Fig. 1B). Thus, phosphorylation of Pho4 at sites 2 and 3 is necessary and sufficient to promote binding to Msn5 (10).

Pse1, another member of the β-importin family of transport receptors, is the import receptor for Pho4 (11). Phosphorylation of Pho4 inhibits its interaction with Pse1. Because phosphorylation site 4 is contained within the nuclear localization signal (NLS) of Pho4 (11), phosphorylation of this site might inhibit the interaction between Pho4 and Pse1. Pho4SA4-zz (a mutant that can be phosphorylated on all sites except site 4) and Pho4SA1236-zz (a mutant that can only be phosphorylated on site 4) were phosphorylated in vitro and assayed for binding to Pse1. Phosphorylated Pho4SA4-zz bound to Pse1, whereas Pho4SA1236-zz failed to bind Pse1 when phosphorylated (Fig. 2A). Thus, phosphorylation of Pho4 at site 4 is necessary and sufficient to disrupt the association of Pho4 and Pse1.

Figure 2

Phosphorylation of site 4 inhibits nuclear import of Pho4. (A) Wild-type Pho4-zz and the indicated Pho4-zz mutants were phosphorylated (+ATP) or mock phosphorylated (−ATP) in vitro, immobilized on IgG-Sepharose, and binding to Pse1-His6 was measured (11). The amount of bound Pse1-His6 was analyzed on a silver-stained SDS-PAGE gel. The amount of immobilized Pho4-zz was analyzed on a Coomassie-stained SDS-PAGE gel. The band below Pse1-His6 is an NH2-terminally truncated form of the protein (indi- cated by the asterisk). (B) Expression of Pho4SA12346-GFP3 or Pho4SA1236SD4-GFP3 was induced, and localization was monitored by fluorescence microscopy (27).

We examined the role of phosphorylation of site 4 in regulating import of Pho4 in vivo. We used a mutant that cannot be exported, because export of Pho4 and a block in its import both lead to its cytoplasmic accumulation. Because phosphorylation of Pho4 by Pho80/Pho85 occurs in the nucleus (5), we attempted to mimic phosphorylation of site 4 by substituting Ser with Asp. Pho4SA1236SD4-zz (containing Ser→Ala substitutions at sites 1, 2, 3, and 6 and a Ser→Asp substitution at site 4) failed to bind Pse1 in vitro (Fig. 2A). To examine the effect of the Asp substitution on import of Pho4 in vivo, we induced expression of Pho4SA12346 and Pho4SA1236SD4 fused to three tandem copies of GFP (GFP3) (12) and monitored the localization of these proteins by fluorescence microscopy. One-and-one-half hours after induction, Pho4SA12346-GFP3 remained nuclear, whereas Pho4SA1236SD4-GFP3 was mainly cytoplasmic (13, 14) (Fig. 2B). Thus, phosphorylation at site 4 inhibits nuclear import of Pho4.

If control of nuclear localization is the only mechanism by which phosphorylation regulates the activity of Pho4, then Pho4 that is localized to the nucleus should activate transcription of phosphate-responsive genes in both high- and low-phosphate conditions. Therefore, we measured production of the secreted acid phosphatase Pho5 in a strain expressing Pho4SA1234, a mutant containing Ser→Ala substitutions at sites 1, 2, 3, and 4 (15) that was constitutively localized to the nucleus (Fig. 3A). Although expression of acid phosphatase was elevated in yeast expressing Pho4SA1234grown in phosphate-rich medium (16), it was further induced in response to phosphate starvation (Fig. 3B). Additionally, anmsn5Δ strain, in which Pho4 is constitutively localized to the nucleus because it cannot be exported, produces high levels of acid phosphatase when starved for phosphate (8), but not when grown in phosphate-rich medium (5). Thus, another mechanism, distinct from control of its localization, regulates the activity of Pho4.

Figure 3

Pho4 is regulated by a mechanism distinct from control of its nuclear localization. (A) Localization of the indicated Pho4 mutants fused to GFP in cells grown in no or high-phosphate medium (25). (B) Measurement of Pho5 acid phosphatase enzyme activity in either pho4Δ pho3Δ or pho4Δ pho80Δ pho3Δ yeast strains (24) transformed with a low-copy plasmid expressing the indicated Pho4 mutant (28). The pho4Δ pho3Δstrain expressing the indicated Pho4 mutant was grown in high- (black boxes) or low-phosphate (white boxes) medium and the pho4Δ pho80Δ pho3Δ strain was grown in high-phosphate medium (gray boxes). (C) Wild-type Pho4-zz and the indicated Pho4-zz mutants were phosphorylated (+ATP) or mock phosphorylated (−ATP) in vitro, immobilized on IgG-Sepharose, and binding to Pho2-His6 was measured (29). (Top) The amount of bound Pho2-His6was analyzed by SDS-PAGE followed by protein immunoblotting with anti-Pho2. (Bottom) The amount of immobilized Pho4-zz was analyzed on a Coomassie-stained SDS-PAGE gel.

The only site that can be phosphorylated in the Pho4SA1234 mutant is site 6. We constructed a mutant Pho4 that could not be phosphorylated on site 6 by making a Pro→Ala substitution in the Ser-Pro dipeptide corresponding to phosphorylation site 6 (Pho4PA6) (17). We did not use a Ser→Ala substitution to prevent phosphorylation of site 6 because the Pho4SA6mutant is not fully functional in activating transcription of acid phosphatase (18). Localization of Pho4PA6-GFP was regulated in response to phosphate levels (Fig. 3A), and Pho4PA6 was fully functional as a transcriptional activator (Fig. 3B). We combined the mutations that cause Pho4 to be constitutively localized to the nucleus with the Pro→Ala mutation at site 6 to create Pho4SA1234PA6 (Fig. 3A). In contrast to a strain expressing Pho4SA1234, a strain expressing Pho4SA1234PA6 produced acid phosphatase at nearly fully induced levels when grown in high-phosphate medium (19) (Fig. 3B). Additionally, a strain lacking the export receptor Msn5 and expressing Pho4PA6 produced high levels of acid phosphatase when grown in phosphate-rich medium (8). Thus, phosphorylation of site 6 provides an additional mode for regulating the activity of Pho4. These observations suggest that phosphorylation by Pho80/Pho85 is the primary mode of regulating Pho4 in response to phosphate availability (20).

Phosphorylation site 6 lies within a region of Pho4 involved in binding to the transcription factor Pho2 (21). Pho2 is required for transcription of PHO5 (3), interacts with Pho4, and binds cooperatively with Pho4 to thePHO5 promoter (22). To determine if phosphorylation of site 6 modulates the interaction between Pho4 and Pho2, we phosphorylated a Pho4-zz fusion protein in vitro and assayed for its binding to Pho2. Pho2 bound to unphosphorylated Pho4-zz, but not to phosphorylated Pho4-zz, indicating that phosphorylation of Pho4 inhibits its interaction with Pho2 (Fig. 3C). Pho4SA1234-zz, which can only be phosphorylated on site 6, bound to Pho2 when unphosphorylated, but not when phosphorylated (Fig. 3C). Additionally, Pho4PA6-zz, a mutant that can be phosphorylated on all sites except site 6, bound to Pho2 independent of its phosphorylation state (Fig. 3C). Thus, phosphorylation of site 6 is necessary and sufficient to inhibit interaction of Pho4 with Pho2 (23).

Regulation of nuclear localization and regulation of the interaction with Pho2 provide partially redundant levels of regulation to control the activity of Pho4; yeast expressing either Pho4PA6(regulated only by nuclear localization) or Pho4SA1234(regulated only by control of the interaction with Pho2) induce transcription of the acid phosphatase Pho5 in response to phosphate starvation (Fig. 3B). Although overlapping, both levels of regulation are required for complete repression of Pho5 expression, because acid phosphatase expression is not completely repressed in yeast expressing Pho4SA1234 or Pho4PA6 (Fig. 3B). Therefore, multiple phosphorylation sites may exist to ensure complete shutoff of transcription.

The phosphorylation events that modify Pho4 have unique and separable roles in regulating the protein's export, import, and ability to activate transcription in the nucleus (Fig. 4). Multiple levels of regulation cooperate to control Pho4 in a switchlike manner. Many transcription factors, CDK inhibitors, and other regulatory proteins are phosphorylated on multiple sites, but the role of these phosphorylation events is not well understood. Phosphorylation may provide multiple levels of control that are important for efficient regulation of proteins other than Pho4.

Figure 4

Phosphorylation events regulate Pho4 by distinct and separable mechanisms. Sites of phosphorylation consist of five Ser-Pro dipeptides labeled SP1, SP2, SP3, SP4, and SP6 (amino acids 100, 114, 128, 152, and 223) (6). The activation and DNA binding domains are indicated (30). Sites 2 and 3 regulate nuclear export, site 4 regulates import, and site 6 regulates the interaction with the transcription factor Pho2. We have not been able to determine a function for phosphorylation site 1 (15).

  • * To whom correspondence should be addressed. E-mail: oshea{at}biochem.ucsf.edu

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