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Sequential counteracting kinases restrict an asymmetric gene expression program to early G1.

Mazanka E, Weiss EL - Mol. Biol. Cell (2010)

Bottom Line: We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm.Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly.Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Gene expression is restricted to specific times in cell division and differentiation through close control of both activation and inactivation of transcription. In budding yeast, strict spatiotemporal regulation of the transcription factor Ace2 ensures that it acts only once in a cell's lifetime: at the M-to-G1 transition in newborn daughter cells. The Ndr/LATS family kinase Cbk1, functioning in a system similar to metazoan hippo signaling pathways, activates Ace2 and drives its accumulation in daughter cell nuclei, but the mechanism of this transcription factor's inactivation is unknown. We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm. Once exported, Ace2 cannot re-enter nuclei for the remainder of the cell cycle. Two separate mechanisms enforce Ace2's cytoplasmic sequestration: 1) phosphorylation of CDK consensus sites in Ace2 by the G1 CDKs Pho85 and Cdc28/CDK1 and 2) an unknown mechanism mediated by Pho85 that is independent of its kinase activity. Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly. Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

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Pho85 does not phosphorylate non-consensus motifs within Ace2. (A) Diagram of the ace2-AP allele, denoting positions of all 21 putative CDK consensus motifs ([S/T]-P), in which serines and threonines were changed to alanine. Sites with evidence for in vivo phosphorylation by mass spectrometry are noted, with an asterisk (*), indicating sites specifically identified, and dagger (†), indicating ambiguity of phosphorylation among sites within an examined peptide. (B) Ace2-HA and ace2-AP-HA substrates were immunoprecipitated from yeast cells and in vitro phosphorylated by bacterially expressed His-Pho85as/Pcl1 complex and γ-32P-ATP for 1 h. Protein levels are shown. (C) Quantification of assays shown in B. Phosphorylation was normalized to total protein, and background levels were subtracted. Graph shows average of three independent trials; error bars, SEM. Ace2-HA is efficiently phosphorylated, whereas ace2-AP-HA cannot be statistically significantly phosphorylated over background.
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Figure 5: Pho85 does not phosphorylate non-consensus motifs within Ace2. (A) Diagram of the ace2-AP allele, denoting positions of all 21 putative CDK consensus motifs ([S/T]-P), in which serines and threonines were changed to alanine. Sites with evidence for in vivo phosphorylation by mass spectrometry are noted, with an asterisk (*), indicating sites specifically identified, and dagger (†), indicating ambiguity of phosphorylation among sites within an examined peptide. (B) Ace2-HA and ace2-AP-HA substrates were immunoprecipitated from yeast cells and in vitro phosphorylated by bacterially expressed His-Pho85as/Pcl1 complex and γ-32P-ATP for 1 h. Protein levels are shown. (C) Quantification of assays shown in B. Phosphorylation was normalized to total protein, and background levels were subtracted. Graph shows average of three independent trials; error bars, SEM. Ace2-HA is efficiently phosphorylated, whereas ace2-AP-HA cannot be statistically significantly phosphorylated over background.

Mentions: Because simultaneous inhibition of both Pho85 and Cdc28 dramatically reduced Ace2's G1 cytoplasmic retention, we next sought to determine if phosphorylation at CDK consensus sites is similarly critical for inhibition of Ace2's G1 nuclear import. We changed Ace2's phosphoacceptor residues to alanine in all of its 21 potential CDK consensus motifs, an allele denoted ace2-AP (Figure 5A). HA-tagged ace2-AP showed no significant Cdc14-reversible shifting in mitotically synchronized cells, consistent with absence of phosphorylation that is normally removed by this phosphatase (Supplemental Figure 4C). Cells carrying the ace2-AP allele had no cell separation defects (Supplemental Figure 5), and the ace2-AP-GFP protein was normally segregated to the daughter cell nucleus (Supplemental Figure 5). Thus, CDK phosphorylation is not required for Ace2 transcriptional activity and asymmetry in normally cycling cells.


Sequential counteracting kinases restrict an asymmetric gene expression program to early G1.

Mazanka E, Weiss EL - Mol. Biol. Cell (2010)

Pho85 does not phosphorylate non-consensus motifs within Ace2. (A) Diagram of the ace2-AP allele, denoting positions of all 21 putative CDK consensus motifs ([S/T]-P), in which serines and threonines were changed to alanine. Sites with evidence for in vivo phosphorylation by mass spectrometry are noted, with an asterisk (*), indicating sites specifically identified, and dagger (†), indicating ambiguity of phosphorylation among sites within an examined peptide. (B) Ace2-HA and ace2-AP-HA substrates were immunoprecipitated from yeast cells and in vitro phosphorylated by bacterially expressed His-Pho85as/Pcl1 complex and γ-32P-ATP for 1 h. Protein levels are shown. (C) Quantification of assays shown in B. Phosphorylation was normalized to total protein, and background levels were subtracted. Graph shows average of three independent trials; error bars, SEM. Ace2-HA is efficiently phosphorylated, whereas ace2-AP-HA cannot be statistically significantly phosphorylated over background.
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Related In: Results  -  Collection

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Figure 5: Pho85 does not phosphorylate non-consensus motifs within Ace2. (A) Diagram of the ace2-AP allele, denoting positions of all 21 putative CDK consensus motifs ([S/T]-P), in which serines and threonines were changed to alanine. Sites with evidence for in vivo phosphorylation by mass spectrometry are noted, with an asterisk (*), indicating sites specifically identified, and dagger (†), indicating ambiguity of phosphorylation among sites within an examined peptide. (B) Ace2-HA and ace2-AP-HA substrates were immunoprecipitated from yeast cells and in vitro phosphorylated by bacterially expressed His-Pho85as/Pcl1 complex and γ-32P-ATP for 1 h. Protein levels are shown. (C) Quantification of assays shown in B. Phosphorylation was normalized to total protein, and background levels were subtracted. Graph shows average of three independent trials; error bars, SEM. Ace2-HA is efficiently phosphorylated, whereas ace2-AP-HA cannot be statistically significantly phosphorylated over background.
Mentions: Because simultaneous inhibition of both Pho85 and Cdc28 dramatically reduced Ace2's G1 cytoplasmic retention, we next sought to determine if phosphorylation at CDK consensus sites is similarly critical for inhibition of Ace2's G1 nuclear import. We changed Ace2's phosphoacceptor residues to alanine in all of its 21 potential CDK consensus motifs, an allele denoted ace2-AP (Figure 5A). HA-tagged ace2-AP showed no significant Cdc14-reversible shifting in mitotically synchronized cells, consistent with absence of phosphorylation that is normally removed by this phosphatase (Supplemental Figure 4C). Cells carrying the ace2-AP allele had no cell separation defects (Supplemental Figure 5), and the ace2-AP-GFP protein was normally segregated to the daughter cell nucleus (Supplemental Figure 5). Thus, CDK phosphorylation is not required for Ace2 transcriptional activity and asymmetry in normally cycling cells.

Bottom Line: We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm.Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly.Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Gene expression is restricted to specific times in cell division and differentiation through close control of both activation and inactivation of transcription. In budding yeast, strict spatiotemporal regulation of the transcription factor Ace2 ensures that it acts only once in a cell's lifetime: at the M-to-G1 transition in newborn daughter cells. The Ndr/LATS family kinase Cbk1, functioning in a system similar to metazoan hippo signaling pathways, activates Ace2 and drives its accumulation in daughter cell nuclei, but the mechanism of this transcription factor's inactivation is unknown. We found that Ace2's nuclear localization is maintained by continuous Cbk1 activity and that inhibition of the kinase leads to immediate loss of phosphorylation and export to the cytoplasm. Once exported, Ace2 cannot re-enter nuclei for the remainder of the cell cycle. Two separate mechanisms enforce Ace2's cytoplasmic sequestration: 1) phosphorylation of CDK consensus sites in Ace2 by the G1 CDKs Pho85 and Cdc28/CDK1 and 2) an unknown mechanism mediated by Pho85 that is independent of its kinase activity. Direct phosphorylation of CDK consensus sites is not necessary for Ace2's cytoplasmic retention, indicating that these mechanisms function redundantly. Overall, these findings show how sequential opposing kinases limit a daughter cell specific transcriptional program to a brief period during the cell cycle and suggest that CDKs may function as cytoplasmic sequestration factors.

Show MeSH