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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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Related in: MedlinePlus

In vitro phosphorylation at Ser-440 and −467 by DNA-PKGST-tagged WRN fragment (239-499) as schematically represented was used. Purified GST or GST-tagged fragment with or without Ala substitution at Ser-440 and/or −467 was incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Phosphorylation was visualized (upper panel). An immunoblot with anti-GST antibody is shown (lower panel).
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Figure 3: In vitro phosphorylation at Ser-440 and −467 by DNA-PKGST-tagged WRN fragment (239-499) as schematically represented was used. Purified GST or GST-tagged fragment with or without Ala substitution at Ser-440 and/or −467 was incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Phosphorylation was visualized (upper panel). An immunoblot with anti-GST antibody is shown (lower panel).

Mentions: Experiments were then performed to confirm that Ser-440 and −467 are phosphorylated in vitro. The GST-WRN (239-499) fragment with alanine substitution at Ser-440 and/or −467 was expressed in bacterial cells and partially purified. The mutated WRN fragment was utilized in in vitro phosphorylation assays (Fig. 3). The products were subjected to SDS-PAGE and transferred to a PVDF membrane. The fragment without mutation, but not the one with S440A and S467A substitution was phosphorylated by DNA-PK (Fig.3, lanes 4 and 8). Consistent with results in Fig. 2D, neither S440A nor S467A single mutant was phosphorylated (Fig. 3, lanes 6 and 10), suggesting that Ser-440 is required for phosphorylation of Ser-467 and that Ser-467 is required for phosphorylation of Ser-440. We also did not detect phosphorylation of the GST-WRN (239-499) fragment containing Ser-319, which is another residue phosphorylated by DNA-PK. The N-terminal region of WRN may be required for the phosphorylation, since WRN (1-333) was used for the Ser-319 phosphorylation experiments [7]. Taken together with the results from the in vivo phosphorylation assay, Ser-440 and −467 are major phosphorylation sites by DNA-PK both in in vitro and in vivo.


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)

In vitro phosphorylation at Ser-440 and −467 by DNA-PKGST-tagged WRN fragment (239-499) as schematically represented was used. Purified GST or GST-tagged fragment with or without Ala substitution at Ser-440 and/or −467 was incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Phosphorylation was visualized (upper panel). An immunoblot with anti-GST antibody is shown (lower panel).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: In vitro phosphorylation at Ser-440 and −467 by DNA-PKGST-tagged WRN fragment (239-499) as schematically represented was used. Purified GST or GST-tagged fragment with or without Ala substitution at Ser-440 and/or −467 was incubated with purified DNA-PKcs, Ku 70/86, and activated DNA in the presence of [γ-32P]ATP. Phosphorylation was visualized (upper panel). An immunoblot with anti-GST antibody is shown (lower panel).
Mentions: Experiments were then performed to confirm that Ser-440 and −467 are phosphorylated in vitro. The GST-WRN (239-499) fragment with alanine substitution at Ser-440 and/or −467 was expressed in bacterial cells and partially purified. The mutated WRN fragment was utilized in in vitro phosphorylation assays (Fig. 3). The products were subjected to SDS-PAGE and transferred to a PVDF membrane. The fragment without mutation, but not the one with S440A and S467A substitution was phosphorylated by DNA-PK (Fig.3, lanes 4 and 8). Consistent with results in Fig. 2D, neither S440A nor S467A single mutant was phosphorylated (Fig. 3, lanes 6 and 10), suggesting that Ser-440 is required for phosphorylation of Ser-467 and that Ser-467 is required for phosphorylation of Ser-440. We also did not detect phosphorylation of the GST-WRN (239-499) fragment containing Ser-319, which is another residue phosphorylated by DNA-PK. The N-terminal region of WRN may be required for the phosphorylation, since WRN (1-333) was used for the Ser-319 phosphorylation experiments [7]. Taken together with the results from the in vivo phosphorylation assay, Ser-440 and −467 are major phosphorylation sites by DNA-PK both in in vitro and in vivo.

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