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Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks.

Kusumoto-Matsuo R, Ghosh D, Karmakar P, May A, Ramsden D, Bohr VA - Aging (Albany NY) (2014)

Bottom Line: While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm.Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type.Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA.

ABSTRACT
WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and -467 in WRN as major phosphorylation sites mediated by DNA-PK.In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.

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WRN but not phosphorylation mutant relocalizes to the nucleoli post etoposide exposure(A) AG11395 cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were incubated with 35 μM etposide for 3 hours. Cells were fixed and EGFP signals were visualized before and after incubation for another 24 hours in fresh medium. Representive images are shown. (B) The percent of cells containing WRN foci. At least 100 cells were scored at each time point. The average of three independent experiments with standard deviation is plotted. Asterisks (*) indicate significant difference between 0 h and 24 h (p<0.05). Plus (+) indicate significant difference between Wild type and mutants.
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Figure 5: WRN but not phosphorylation mutant relocalizes to the nucleoli post etoposide exposure(A) AG11395 cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were incubated with 35 μM etposide for 3 hours. Cells were fixed and EGFP signals were visualized before and after incubation for another 24 hours in fresh medium. Representive images are shown. (B) The percent of cells containing WRN foci. At least 100 cells were scored at each time point. The average of three independent experiments with standard deviation is plotted. Asterisks (*) indicate significant difference between 0 h and 24 h (p<0.05). Plus (+) indicate significant difference between Wild type and mutants.

Mentions: After the intensity of fluorescence of EGFP-WRN reached a plateau in the irradiated sites, it persisted at least for 4 hours after the laser irradiation[22]. Consistent with this, EGFP-WRN wild type and mutants persisted at the site of DSBs up to 1 hour in HeLa cells (data not shown). To address whether phosphorylation of WRN by DNA-PK affects its retention at the foci, we analyzed the accumulation of exogenous EGFP-WRN or the phosphorylation mutants in WS cells after 24 hours-recovery from exposure to etoposide, a DNA topoisomerase II inhibitor, which produces DSBs irrespective of DNA replication (Fig. 5A). All of the phosphorylation mutants as well as the wild type localized to nucleoli without etoposide exposure (Fig. 5A, undamaged). Consistent with a previous report [23], the wild type, as well as S440A, S467A, and S440A/S467Amutants formed foci in the nucleoplasm upon exposure to etoposide (Fig. 5A, 0 h). After the recovery, wild type WRN relocated to nucleoli, whereas many more foci of the phosphorylation mutants of WRN remained in the nucleoplasm (Fig. 5A, 24 h). The percentage of cells showing EGFP-WRN wild type foci in the nucleoplasm decreased to 20% after 24 hours-recovery from etoposide-induced damage, whereas for S440A 66% cells, for S467A 75% cells and for S440A/S467A 73% cells showed foci at the nucleoplasm after the recovery (Fig. 5B). These data suggested that the major phosphorylation sites, Ser-440 and −467, are required for the efficient relocalization of WRN to nucleoli.


Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks.

Kusumoto-Matsuo R, Ghosh D, Karmakar P, May A, Ramsden D, Bohr VA - Aging (Albany NY) (2014)

WRN but not phosphorylation mutant relocalizes to the nucleoli post etoposide exposure(A) AG11395 cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were incubated with 35 μM etposide for 3 hours. Cells were fixed and EGFP signals were visualized before and after incubation for another 24 hours in fresh medium. Representive images are shown. (B) The percent of cells containing WRN foci. At least 100 cells were scored at each time point. The average of three independent experiments with standard deviation is plotted. Asterisks (*) indicate significant difference between 0 h and 24 h (p<0.05). Plus (+) indicate significant difference between Wild type and mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927811&req=5

Figure 5: WRN but not phosphorylation mutant relocalizes to the nucleoli post etoposide exposure(A) AG11395 cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were incubated with 35 μM etposide for 3 hours. Cells were fixed and EGFP signals were visualized before and after incubation for another 24 hours in fresh medium. Representive images are shown. (B) The percent of cells containing WRN foci. At least 100 cells were scored at each time point. The average of three independent experiments with standard deviation is plotted. Asterisks (*) indicate significant difference between 0 h and 24 h (p<0.05). Plus (+) indicate significant difference between Wild type and mutants.
Mentions: After the intensity of fluorescence of EGFP-WRN reached a plateau in the irradiated sites, it persisted at least for 4 hours after the laser irradiation[22]. Consistent with this, EGFP-WRN wild type and mutants persisted at the site of DSBs up to 1 hour in HeLa cells (data not shown). To address whether phosphorylation of WRN by DNA-PK affects its retention at the foci, we analyzed the accumulation of exogenous EGFP-WRN or the phosphorylation mutants in WS cells after 24 hours-recovery from exposure to etoposide, a DNA topoisomerase II inhibitor, which produces DSBs irrespective of DNA replication (Fig. 5A). All of the phosphorylation mutants as well as the wild type localized to nucleoli without etoposide exposure (Fig. 5A, undamaged). Consistent with a previous report [23], the wild type, as well as S440A, S467A, and S440A/S467Amutants formed foci in the nucleoplasm upon exposure to etoposide (Fig. 5A, 0 h). After the recovery, wild type WRN relocated to nucleoli, whereas many more foci of the phosphorylation mutants of WRN remained in the nucleoplasm (Fig. 5A, 24 h). The percentage of cells showing EGFP-WRN wild type foci in the nucleoplasm decreased to 20% after 24 hours-recovery from etoposide-induced damage, whereas for S440A 66% cells, for S467A 75% cells and for S440A/S467A 73% cells showed foci at the nucleoplasm after the recovery (Fig. 5B). These data suggested that the major phosphorylation sites, Ser-440 and −467, are required for the efficient relocalization of WRN to nucleoli.

Bottom Line: While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm.Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type.Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA.

ABSTRACT
WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and -467 in WRN as major phosphorylation sites mediated by DNA-PK.In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.

Show MeSH
Related in: MedlinePlus