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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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Mapping DNA-PK phosphorylation sites in WRN(A) Schematic representation of His- or GST-tagged WRN fragments used in in vitro phosphorylation assay. (B and C) In vitro phosphorylation assay. Purified His- or GST-tagged WRN fragments were incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Amido black staining is shown (B). The phosphorylation was visualized (C). Asterisk indicates the GST (500-946) fragment. Note that GST (239-499) migrated slower because of many acidic amino acids.
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Figure 1: Mapping DNA-PK phosphorylation sites in WRN(A) Schematic representation of His- or GST-tagged WRN fragments used in in vitro phosphorylation assay. (B and C) In vitro phosphorylation assay. Purified His- or GST-tagged WRN fragments were incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Amido black staining is shown (B). The phosphorylation was visualized (C). Asterisk indicates the GST (500-946) fragment. Note that GST (239-499) migrated slower because of many acidic amino acids.

Mentions: To map the region of WRN that is phosphorylated by DNA-PK, we first performed in vitro phosphorylation assays using a series of WRN fragments (Fig. 1). The WRN fragments are shown in Fig. 1A. These fragments were partially purified from E. coli using His- or GST-tags, and incubated with purified DNA-PKcs and Ku 70/80 in the presence of activated DNA and [γ-32P]ATP. The samples were subjected to SDS-PAGE and amido black staining, and the phosphorylation was visualized (Figs. 1B and 1C). GST itself was not phosphorylated by DNA-PK (Fig. 1C, lane 6). We found that the phosphorylation sites were located in the acidic region of WRN (239-499), and in the C-terminal domain of WRN (949-1432) (Fig. 1C, lanes 3 and 5). The signal from WRN (239-499) was much stronger than that of WRN (949-1432), suggesting that a major phosphorylation site or multiple phosphorylation sites are located in the acidic region. For fine mapping of WRN phosphorylation sites in the C-terminal domain, a truncated WRN (949-1236) was examined further, and since it was not phosphorylated, the minor phosphorylation site(s) were likely located in WRN (1237-1432) (supplementary Fig. S1).


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)

Mapping DNA-PK phosphorylation sites in WRN(A) Schematic representation of His- or GST-tagged WRN fragments used in in vitro phosphorylation assay. (B and C) In vitro phosphorylation assay. Purified His- or GST-tagged WRN fragments were incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Amido black staining is shown (B). The phosphorylation was visualized (C). Asterisk indicates the GST (500-946) fragment. Note that GST (239-499) migrated slower because of many acidic amino acids.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Mapping DNA-PK phosphorylation sites in WRN(A) Schematic representation of His- or GST-tagged WRN fragments used in in vitro phosphorylation assay. (B and C) In vitro phosphorylation assay. Purified His- or GST-tagged WRN fragments were incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Amido black staining is shown (B). The phosphorylation was visualized (C). Asterisk indicates the GST (500-946) fragment. Note that GST (239-499) migrated slower because of many acidic amino acids.
Mentions: To map the region of WRN that is phosphorylated by DNA-PK, we first performed in vitro phosphorylation assays using a series of WRN fragments (Fig. 1). The WRN fragments are shown in Fig. 1A. These fragments were partially purified from E. coli using His- or GST-tags, and incubated with purified DNA-PKcs and Ku 70/80 in the presence of activated DNA and [γ-32P]ATP. The samples were subjected to SDS-PAGE and amido black staining, and the phosphorylation was visualized (Figs. 1B and 1C). GST itself was not phosphorylated by DNA-PK (Fig. 1C, lane 6). We found that the phosphorylation sites were located in the acidic region of WRN (239-499), and in the C-terminal domain of WRN (949-1432) (Fig. 1C, lanes 3 and 5). The signal from WRN (239-499) was much stronger than that of WRN (949-1432), suggesting that a major phosphorylation site or multiple phosphorylation sites are located in the acidic region. For fine mapping of WRN phosphorylation sites in the C-terminal domain, a truncated WRN (949-1236) was examined further, and since it was not phosphorylated, the minor phosphorylation site(s) were likely located in WRN (1237-1432) (supplementary Fig. S1).

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