National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Mol Cancer Res. 2010 Sep;8(9):1234-47. doi: 10.1158/1541-7786.MCR-10-0233. Epub 2010 Aug 18.
BLM helicase, the protein mutated in Bloom syndrome, is involved in signal transduction cascades after DNA damage. BLM is phosphorylated on multiple residues by different kinases either after stress induction or during mitosis. Here, we have provided evidence that both Chk1 and Chk2 phosphorylated the NH(2)-terminal 660 amino acids of BLM. An internal region within the DExH motif of BLM negatively regulated the Chk1/Chk2-dependent NH(2)-terminal phosphorylation event. Using in silico analysis involving the Chk1 structure and its known substrate specificity, we predicted that Chk1 should preferentially phosphorylate BLM on serine 646 (Ser(646)). The prediction was validated in vitro by phosphopeptide analysis on BLM mutants and in vivo by usage of a newly generated phosphospecific polyclonal antibody. We showed that the phosphorylation at Ser(646) on BLM was constitutive and decreased rapidly after exposure to DNA damage. This resulted in the diminished interaction of BLM with nucleolin and PML isoforms, and consequently decreased BLM accumulation in the nucleolus and PML nuclear bodies. Instead, BLM relocalized to the sites of DNA damage and bound with the damage sensor protein, Nbs1. Mutant analysis confirmed that the binding to nucleolin and PML isoforms required Ser(646) phosphorylation. These results indicated that Chk1-mediated phosphorylation on BLM at Ser(646) might be a determinant for regulating subnuclear localization and could act as a marker for the activation status of BLM in response to DNA damage.
BLM 解旋酶是布卢姆综合征中突变的蛋白，参与 DNA 损伤后的信号转导级联。BLM 可被不同的激酶在应激诱导后或有丝分裂过程中磷酸化多个残基。在这里，我们提供的证据表明 Chk1 和 Chk2 均可磷酸化 BLM 的 NH2-末端 660 个氨基酸。BLM 的 DExH 基序内的一个内部区域负调控 Chk1/Chk2 依赖性 NH2-末端磷酸化事件。通过涉及 Chk1 结构及其已知底物特异性的计算机分析，我们预测 Chk1 应优先在 BLM 的丝氨酸 646 上磷酸化（Ser646）。通过 BLM 突变体的体外磷酸肽分析和新生成的磷酸特异性多克隆抗体的体内验证验证了该预测。我们表明，BLM 上 Ser646 的磷酸化是组成性的，并且在暴露于 DNA 损伤后迅速降低。这导致 BLM 与核仁素和 PML 同种型的相互作用减弱，并且 BLM 在核仁中的积累和 PML 核体减少。相反，BLM 重新定位到 DNA 损伤部位，并与损伤传感器蛋白 Nbs1 结合。突变分析证实，与核仁素和 PML 同种型的结合需要 Ser646 磷酸化。这些结果表明，Chk1 在 BLM 上 Ser646 的磷酸化可能是调节亚核定位的决定因素，并可作为 BLM 对 DNA 损伤反应的激活状态的标志物。
