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Ndd1 turnover by SCF(Grr1) is inhibited by the DNA damage checkpoint in Saccharomyces cerevisiae.

Edenberg ER, Mark KG, Toczyski DP - PLoS Genet. (2015)

Bottom Line: Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle.We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1).While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1). In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.

No MeSH data available.


Related in: MedlinePlus

SCFGrr1 turns over Ndd1.A) GAL1p-Ndd1-Flag was induced with galactose in wildtype or cdc53-1 strains at 23°C for 5 hours. All strains were then shifted to 37°C for 2.5 hours to inactivate the temperature-sensitive allele before addition of cycloheximide (CHX). Turnover was then examined as in Fig 1B, except at 37°C. Cdc28 is shown as a loading control. B) GAL1p-Ndd1-Flag was induced with galactose in rgt1Δ or rgt1Δ grr1Δ strains for 2.5 hours. Protein turnover was followed as in [A]. C) Strains expressing NDD1p-Ndd1-13Myc with either an empty vector (pRS426) or pYES-Grr1ΔF-Flag (containing a galactose-inducible copy of Grr1-Flag lacking its F-box domain) were grown in galactose media overnight to double twice. Whole cell extracts (WCE) were prepared and immunoprecipitated with anti-Flag antibody, Western blotted and probed for Flag and Myc. D) Experiment was performed as in [B], expect that cells were pre-arrested in G1 using 10 μg/ml α–/ml im (αF) for 6 hours. grr1Δ cells do not arrest well, but FACS analysis shows strong enrichment of G1 peak. E) Experiment was performed as in [B] in wt or cdh1Δ strains.
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pgen.1005162.g003: SCFGrr1 turns over Ndd1.A) GAL1p-Ndd1-Flag was induced with galactose in wildtype or cdc53-1 strains at 23°C for 5 hours. All strains were then shifted to 37°C for 2.5 hours to inactivate the temperature-sensitive allele before addition of cycloheximide (CHX). Turnover was then examined as in Fig 1B, except at 37°C. Cdc28 is shown as a loading control. B) GAL1p-Ndd1-Flag was induced with galactose in rgt1Δ or rgt1Δ grr1Δ strains for 2.5 hours. Protein turnover was followed as in [A]. C) Strains expressing NDD1p-Ndd1-13Myc with either an empty vector (pRS426) or pYES-Grr1ΔF-Flag (containing a galactose-inducible copy of Grr1-Flag lacking its F-box domain) were grown in galactose media overnight to double twice. Whole cell extracts (WCE) were prepared and immunoprecipitated with anti-Flag antibody, Western blotted and probed for Flag and Myc. D) Experiment was performed as in [B], expect that cells were pre-arrested in G1 using 10 μg/ml α–/ml im (αF) for 6 hours. grr1Δ cells do not arrest well, but FACS analysis shows strong enrichment of G1 peak. E) Experiment was performed as in [B] in wt or cdh1Δ strains.

Mentions: We next wanted to know which E3 ligase targeted Ndd1 for degradation. Each member of the SCF family of E3 ligases contains the cullin Cdc53, Skp1, and the Ring finger subunit Rbx1, in addition to any one of several substrate adaptors called F-box proteins [21]. Budding yeast have 20 putative F-box proteins, most of which are thought to target a specific suite of substrates [22]. We found that Ndd1 was strongly stabilized at the non-permissive temperature in the temperature-sensitive mutant cdc53-1 (Fig 3A, quantification shown in S2C Fig), suggesting that Ndd1 is a substrate of an SCF ligase.


Ndd1 turnover by SCF(Grr1) is inhibited by the DNA damage checkpoint in Saccharomyces cerevisiae.

Edenberg ER, Mark KG, Toczyski DP - PLoS Genet. (2015)

SCFGrr1 turns over Ndd1.A) GAL1p-Ndd1-Flag was induced with galactose in wildtype or cdc53-1 strains at 23°C for 5 hours. All strains were then shifted to 37°C for 2.5 hours to inactivate the temperature-sensitive allele before addition of cycloheximide (CHX). Turnover was then examined as in Fig 1B, except at 37°C. Cdc28 is shown as a loading control. B) GAL1p-Ndd1-Flag was induced with galactose in rgt1Δ or rgt1Δ grr1Δ strains for 2.5 hours. Protein turnover was followed as in [A]. C) Strains expressing NDD1p-Ndd1-13Myc with either an empty vector (pRS426) or pYES-Grr1ΔF-Flag (containing a galactose-inducible copy of Grr1-Flag lacking its F-box domain) were grown in galactose media overnight to double twice. Whole cell extracts (WCE) were prepared and immunoprecipitated with anti-Flag antibody, Western blotted and probed for Flag and Myc. D) Experiment was performed as in [B], expect that cells were pre-arrested in G1 using 10 μg/ml α–/ml im (αF) for 6 hours. grr1Δ cells do not arrest well, but FACS analysis shows strong enrichment of G1 peak. E) Experiment was performed as in [B] in wt or cdh1Δ strains.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403921&req=5

pgen.1005162.g003: SCFGrr1 turns over Ndd1.A) GAL1p-Ndd1-Flag was induced with galactose in wildtype or cdc53-1 strains at 23°C for 5 hours. All strains were then shifted to 37°C for 2.5 hours to inactivate the temperature-sensitive allele before addition of cycloheximide (CHX). Turnover was then examined as in Fig 1B, except at 37°C. Cdc28 is shown as a loading control. B) GAL1p-Ndd1-Flag was induced with galactose in rgt1Δ or rgt1Δ grr1Δ strains for 2.5 hours. Protein turnover was followed as in [A]. C) Strains expressing NDD1p-Ndd1-13Myc with either an empty vector (pRS426) or pYES-Grr1ΔF-Flag (containing a galactose-inducible copy of Grr1-Flag lacking its F-box domain) were grown in galactose media overnight to double twice. Whole cell extracts (WCE) were prepared and immunoprecipitated with anti-Flag antibody, Western blotted and probed for Flag and Myc. D) Experiment was performed as in [B], expect that cells were pre-arrested in G1 using 10 μg/ml α–/ml im (αF) for 6 hours. grr1Δ cells do not arrest well, but FACS analysis shows strong enrichment of G1 peak. E) Experiment was performed as in [B] in wt or cdh1Δ strains.
Mentions: We next wanted to know which E3 ligase targeted Ndd1 for degradation. Each member of the SCF family of E3 ligases contains the cullin Cdc53, Skp1, and the Ring finger subunit Rbx1, in addition to any one of several substrate adaptors called F-box proteins [21]. Budding yeast have 20 putative F-box proteins, most of which are thought to target a specific suite of substrates [22]. We found that Ndd1 was strongly stabilized at the non-permissive temperature in the temperature-sensitive mutant cdc53-1 (Fig 3A, quantification shown in S2C Fig), suggesting that Ndd1 is a substrate of an SCF ligase.

Bottom Line: Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle.We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1).While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, United States of America.

ABSTRACT
In Saccharomyces cerevisiae, Ndd1 is the dedicated transcriptional activator of the mitotic gene cluster, which includes thirty-three genes that encode key mitotic regulators, making Ndd1 a hub for the control of mitosis. Previous work has shown that multiple kinases, including cyclin-dependent kinase (Cdk1), phosphorylate Ndd1 to regulate its activity during the cell cycle. Previously, we showed that Ndd1 was inhibited by phosphorylation in response to DNA damage. Here, we show that Ndd1 is also subject to regulation by protein turnover during the mitotic cell cycle: Ndd1 is unstable during an unperturbed cell cycle, but is strongly stabilized in response to DNA damage. We find that Ndd1 turnover in metaphase requires Cdk1 activity and the ubiquitin ligase SCF(Grr1). In response to DNA damage, Ndd1 stabilization requires the checkpoint kinases Mec1/Tel1 and Swe1, the S. cerevisiae homolog of the Wee1 kinase. In both humans and yeast, the checkpoint promotes Wee1-dependent inhibitory phosphorylation of Cdk1 following exposure to DNA damage. While this is critical for checkpoint-induced arrest in most organisms, this is not true in budding yeast, where the function of damage-induced inhibitory phosphorylation is less well understood. We propose that the DNA damage checkpoint stabilizes Ndd1 by inhibiting Cdk1, which we show is required for targeting Ndd1 for destruction.

No MeSH data available.


Related in: MedlinePlus