Limits...
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.

Show MeSH

Related in: MedlinePlus

Accumulation of WRN wild type and phosphorylation mutants at laser-induced DSBsHeLa cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were laser-irradiated at the sites indicated by arrows. Time-dependent accumulation of EGFP-WRN WT and mutants at the DSBs sites were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3927811&req=5

Figure 4: Accumulation of WRN wild type and phosphorylation mutants at laser-induced DSBsHeLa cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were laser-irradiated at the sites indicated by arrows. Time-dependent accumulation of EGFP-WRN WT and mutants at the DSBs sites were shown.

Mentions: To understand the biological significance of WRN phosphorylation at Ser-440 and −467, we analyzed WRN translocation within the nucleus using laser microirradiation and confocal microscopy. WRN localizes in nucleoli under normal conditions, and leaves the nucleoli to form foci in response to various stresses. Previously Lan et al. reported that GFP-WRN accumulated at sites of DSBs and the fluorescence at the sites reached a plateau within 3 min [22]. To set up conditions to produce DSBs by laser irradiation, we irradiated HeLa cells with the laser at several doses, and immunostained for γH2AX. We observed γH2AX foci at the site of irradiation using 14% intensity with a 435 nM laser (data not shown). HeLa cells were transfected with vectors to overexpress EGFP-WRN wild type, S440A, S467A or S440A/S467A mutants, and microirradiated at 14% intensity with the 435 nM laser to induce DSBs. EGFP-WRN wild type localized at nucleoli before irradiation, and accumulated at the sites of irradiation in a time-dependent manner. Within a second after irradiation WRN accumulated at the irradiated sites and fluorescence at the sites reached a plateau 30 seconds after irradiation (Fig. 4, upper panels). All of the phosphorylation mutants, EGFP-WRN S440A, S467 and S440A/S467A, also localized at nucleoli before irradiation, and accumulated at the irradiated site with the same kinetics as wild type. We confirmed that accumulation kinetics of the EGFP- WRN wild type and S467A was similar by quantifying and plotting of the fluorescence signals up to 60 seconds (supplementary Fig. S3). These results indicated that phosphorylation of WRN by DNA-PK is not required for its accumulation at the site of DSBs produced by laser irradiation.


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)

Accumulation of WRN wild type and phosphorylation mutants at laser-induced DSBsHeLa cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were laser-irradiated at the sites indicated by arrows. Time-dependent accumulation of EGFP-WRN WT and mutants at the DSBs sites were shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Accumulation of WRN wild type and phosphorylation mutants at laser-induced DSBsHeLa cells overexpressing either EGFP-WRN wild type (WT) or mutant (S440A, S467A or S440A/S467A) were laser-irradiated at the sites indicated by arrows. Time-dependent accumulation of EGFP-WRN WT and mutants at the DSBs sites were shown.
Mentions: To understand the biological significance of WRN phosphorylation at Ser-440 and −467, we analyzed WRN translocation within the nucleus using laser microirradiation and confocal microscopy. WRN localizes in nucleoli under normal conditions, and leaves the nucleoli to form foci in response to various stresses. Previously Lan et al. reported that GFP-WRN accumulated at sites of DSBs and the fluorescence at the sites reached a plateau within 3 min [22]. To set up conditions to produce DSBs by laser irradiation, we irradiated HeLa cells with the laser at several doses, and immunostained for γH2AX. We observed γH2AX foci at the site of irradiation using 14% intensity with a 435 nM laser (data not shown). HeLa cells were transfected with vectors to overexpress EGFP-WRN wild type, S440A, S467A or S440A/S467A mutants, and microirradiated at 14% intensity with the 435 nM laser to induce DSBs. EGFP-WRN wild type localized at nucleoli before irradiation, and accumulated at the sites of irradiation in a time-dependent manner. Within a second after irradiation WRN accumulated at the irradiated sites and fluorescence at the sites reached a plateau 30 seconds after irradiation (Fig. 4, upper panels). All of the phosphorylation mutants, EGFP-WRN S440A, S467 and S440A/S467A, also localized at nucleoli before irradiation, and accumulated at the irradiated site with the same kinetics as wild type. We confirmed that accumulation kinetics of the EGFP- WRN wild type and S467A was similar by quantifying and plotting of the fluorescence signals up to 60 seconds (supplementary Fig. S3). These results indicated that phosphorylation of WRN by DNA-PK is not required for its accumulation at the site of DSBs produced by laser irradiation.

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