Two serine phosphorylation sites in the C-terminus of Rad9 are critical for 9-1-1 binding to TopBP1 and activation of the DNA damage checkpoint response in HeLa cells

Satoshi Ueda, Yukimasa Takeishi, Eiji Ohashi, Toshiki Tsurimoto

研究成果: ジャーナルへの寄稿記事

7 引用 (Scopus)

抄録

A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70% and 20% by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9Rr) or Rad9Rr harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9Rr harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage.

元の言語英語
ページ(範囲)807-816
ページ数10
ジャーナルGenes to Cells
17
発行部数10
DOI
出版物ステータス出版済み - 10 1 2012

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HeLa Cells
Serine
DNA Damage
Phosphorylation
Small Interfering RNA
Hypersensitivity
Hydroxyurea
Methane
Proliferating Cell Nuclear Antigen
Alanine

All Science Journal Classification (ASJC) codes

  • Genetics
  • Cell Biology

これを引用

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abstract = "A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70{\%} and 20{\%} by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9Rr) or Rad9Rr harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9Rr harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage.",
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T1 - Two serine phosphorylation sites in the C-terminus of Rad9 are critical for 9-1-1 binding to TopBP1 and activation of the DNA damage checkpoint response in HeLa cells

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AU - Takeishi, Yukimasa

AU - Ohashi, Eiji

AU - Tsurimoto, Toshiki

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AB - A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70% and 20% by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9Rr) or Rad9Rr harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9Rr harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage.

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