TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint.
TLK1B mediated phosphorylation of Rad9 regulates its nuclear/cytoplasmic localization and cell cycle checkpoint.
复制标题
TLK1B 介导的 Rad9 磷酸化调节其核/细胞质定位和细胞周期检查点。
DOI：
10.1186/s12867-016-0056-x
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发表时间：
2016-02-09
影响因子：
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通讯作者：
De Benedetti A
中科院分区：
文献类型：
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作者：
Awate S;De Benedetti A
Tousled like kinase 1B（TLK 1B）是DNA修复和细胞存活的关键。在DNA损伤时，Chk 1在S457处磷酸化TLK 1B，导致其瞬时抑制。一旦TLK 1B恢复其激酶活性，其在S328处磷酸化Rad 9。在这项工作中，我们研究了这一机制的意义，通过过表达突变体TLK 1B，其中抑制磷酸化位点被消除。这些表达TLK 1B抵抗DNA损伤的细胞显示出即使在存在羟基脲（HU）的情况下也发生的Rad 9 S328的组成性磷酸化，这导致延迟的检查点恢复。一种可能的解释是Rad 9的过早磷酸化导致其在停滞的复制叉处与9-1-1解离，导致它们的崩溃和S期检查点的延长激活。我们发现，在S328磷酸化的Rad 9的结果，它从染色质和重新分布到细胞质中的解离。这导致双链断裂形成，伴随ATM的活化和H2 AX的磷酸化。此外，Rad 9（S328 D）拟磷酸化突变体仅定位于细胞质，而不是染色质。另一个Rad 9拟磷酸化突变体（T355 D）也是由TLK 1磷酸化的位点，其定位正常。在用HU处理的表达突变体TLK 1B的细胞中，Rad 9与Hus 1和WRN的结合大大减少，再次表明其磷酸化导致其从停滞的分叉中过早释放。我们建议，通常情况下，TLK 1B的失活复制停滞和基因毒性应激功能，使9-1-1的保留在网站的损害或停滞叉。TLK 1B的合成伴随着基因毒素的诱导，在TLK 1B的再激活后，Rad 9在S328处过度磷酸化，导致其解离和一旦修复完成就发生的检查点的失活。本文的在线版本（doi：10.1186/s12867-016-0056-x）包含补充材料，可供授权用户使用。
The Tousled like kinase 1B (TLK1B) is critical for DNA repair and survival of cells. Upon DNA damage, Chk1 phosphorylates TLK1B at S457 leading to its transient inhibition. Once TLK1B regains its kinase activity it phosphorylates Rad9 at S328. In this work we investigated the significance of this mechanism by overexpressing mutant TLK1B in which the inhibitory phosphorylation site was eliminated. These cells expressing TLK1B resistant to DNA damage showed constitutive phosphorylation of Rad9 S328 that occurred even in the presence of hydroxyurea (HU), and this resulted in a delayed checkpoint recovery. One possible explanation was that premature phosphorylation of Rad9 caused its dissociation from 9-1-1 at stalled replication forks, resulting in their collapse and prolonged activation of the S-phase checkpoint. We found that phosphorylation of Rad9 at S328 results in its dissociation from chromatin and redistribution to the cytoplasm. This results in double stranded breaks formation with concomitant activation of ATM and phosphorylation of H2AX. Furthermore, a Rad9 (S328D) phosphomimic mutant was exclusively localized to the cytoplasm and not the chromatin. Another Rad9 phosphomimic mutant (T355D), which is also a site phosphorylated by TLK1, localized normally. In cells expressing the mutant TLK1B treated with HU, Rad9 association with Hus1 and WRN was greatly reduced, suggesting again that its phosphorylation causes its premature release from stalled forks. We propose that normally, the inactivation of TLK1B following replication arrest and genotoxic stress functions to allow the retention of 9-1-1 at the sites of damage or stalled forks. Following reactivation of TLK1B, whose synthesis is concomitantly induced by genotoxins, Rad9 is hyperphosphorylated at S328, resulting in its dissociation and inactivation of the checkpoint that occurs once repair is complete. The online version of this article (doi:10.1186/s12867-016-0056-x) contains supplementary material, which is available to authorized users.