Howard Hughes Medical Institute and Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Mol Biol Cell. 2012 Oct;23(20):4118-28. doi: 10.1091/mbc.E12-06-0475. Epub 2012 Aug 23.
The Cdc14 phosphatase family antagonizes Cdk1 phosphorylation and is important for mitotic exit. To access their substrates, Cdc14 phosphatases are released from nucleolar sequestration during mitosis. Clp1/Flp1, the Schizosaccharomyces pombe Cdc14 orthologue, and Cdc14B, a mammalian orthologue, also exit the nucleolus during interphase upon DNA replication stress or damage, respectively, implicating Cdc14 phosphatases in the response to genotoxic insults. However, a mechanistic understanding of Cdc14 phosphatase nucleolar release under these conditions is incomplete. We show here that relocalization of Clp1 during genotoxic stress is governed by complex phosphoregulation. Specifically, the Rad3 checkpoint effector kinases Cds1 and/or Chk1, the cell wall integrity mitogen-activated protein kinase Pmk1, and the cell cycle kinase Cdk1 directly phosphorylate Clp1 to promote genotoxic stress-induced nucleoplasmic accumulation. However, Cds1 and/or Chk1 phosphorylate RxxS sites preferentially upon hydroxyurea treatment, whereas Pmk1 and Cdk1 preferentially phosphorylate Clp1 TP sites upon H(2)O(2) treatment. Abolishing both Clp1 RxxS and TP phosphosites eliminates any genotoxic stress-induced redistribution. Reciprocally, preventing dephosphorylation of Clp1 TP sites shifts the distribution of the enzyme to the nucleoplasm constitutively. This work advances our understanding of pathways influencing Clp1 localization and may provide insight into mechanisms controlling Cdc14B phosphatases in higher eukaryotes.
Cdc14 磷酸酶家族与 Cdk1 的磷酸化作用相拮抗，对于有丝分裂退出至关重要。为了接近其底物，Cdc14 磷酸酶在有丝分裂期间从核仁隔离中释放出来。Clp1/Flp1（裂殖酵母 Cdc14 的直系同源物）和 Cdc14B（哺乳动物的直系同源物）也分别在 DNA 复制应激或损伤期间在间期离开核仁，这表明 Cdc14 磷酸酶参与了对遗传毒性损伤的反应。然而，对于这些条件下 Cdc14 磷酸酶核仁释放的机制理解尚不完全。我们在这里表明，Clp1 在遗传毒性应激下的重新定位受到复杂磷酸化调节的控制。具体而言，Rad3 检查点效应激酶 Cds1 和/或 Chk1、细胞壁完整性有丝分裂原激活蛋白激酶 Pmk1 和细胞周期激酶 Cdk1 直接磷酸化 Clp1，以促进遗传毒性应激诱导的核质积累。然而，Cds1 和/或 Chk1 在羟基脲处理时优先磷酸化 RxxS 位点，而 Pmk1 和 Cdk1 在 H2O2 处理时优先磷酸化 Clp1 的 TP 位点。消除 Clp1 的 RxxS 和 TP 磷酸化位点会消除任何遗传毒性应激诱导的重新分布。相反，防止 Clp1 TP 位点的去磷酸化会使酶的分布在组成型上转移到核质中。这项工作增进了我们对影响 Clp1 定位途径的理解，并可能为控制高等真核生物中 Cdc14B 磷酸酶的机制提供了一些见解。
