Bahassi El Mustapha, Myer David L, McKenney Richard J, Hennigan Robert F, Stambrook Peter J
Department of Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267, USA.
Mutat Res. 2006 Apr 11;596(1-2):166-76. doi: 10.1016/j.mrfmmm.2005.12.002. Epub 2006 Feb 14.
The tumor suppressor gene Chk2 encodes a serine/threonine kinase that signals DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 is phosphorylated on threonine 68 (T68) by ataxia-telangiectasia mutated (ATM) protein leading to its activation. We have previously shown that polo-like kinase 3 (Plk3), a protein involved in DNA damage checkpoint and M-phase functions, interacts with and phosphorylates Chk2. When Chk2 was immunoprecipitated from Daudi cells (Plk3-deficient), it had weak kinase activity towards Cdc25C compared with Chk2 derived from T47D cells (Plk3-expressing cells). This activity was restored by addition of recombinant Plk3 in a dose-dependent manner. Plk3 phosphorylates Chk2 at two residues, serine 62 (S62) and serine 73 (S73) in vitro, and this phosphorylation facilitates subsequent phosphorylation of Chk2 on T68 by ATM in response to DNA damage. When the Chk2 mutant construct GFP-Chk2 S73A (serine 73 mutated to alanine) is transfected into cells, it no longer associates with a large complex in vivo, and manifests a significant reduction in kinase activity. It is also inefficiently activated by ATM by phosphorylation at T68 and, in turn, is unable to phosphorylate the Cdc25C peptide 200-256, which contains the inhibitory S216 target phosphorylation residue. As a consequence, tyrosine 15 (Y15) on Cdc2 remains hypophosphorylated, and there is a loss of the G2/M checkpoint. These data describe a functional role for Plk3 in a pathway linking ATM, Plk3, Chk2, Cdc25C and Cdc2 in cellular response to DNA damage.
肿瘤抑制基因Chk2编码一种丝氨酸/苏氨酸激酶，该激酶将DNA损伤信号传递至细胞周期检查点。响应电离辐射时，Chk2在苏氨酸68（T68）位点被共济失调毛细血管扩张症突变（ATM）蛋白磷酸化，从而导致其激活。我们之前已经表明，参与DNA损伤检查点和M期功能的蛋白polo样激酶3（Plk3）与Chk2相互作用并使其磷酸化。当从Daudi细胞（Plk3缺陷型）中免疫沉淀Chk2时，与源自T47D细胞（表达Plk3的细胞）的Chk2相比，它对Cdc25C的激酶活性较弱。通过以剂量依赖的方式添加重组Plk3可恢复此活性。Plk3在体外使Chk2的两个位点丝氨酸62（S62）和丝氨酸73（S73）磷酸化，这种磷酸化促进了DNA损伤时ATM对Chk2的T68位点的后续磷酸化。当将Chk2突变体构建体GFP-Chk2 S73A（丝氨酸73突变为丙氨酸）转染到细胞中时，它在体内不再与大型复合物结合，并且激酶活性显著降低。它也不能被ATM有效地在T68位点磷酸化而激活，进而无法磷酸化包含抑制性S216靶标磷酸化残基的Cdc25C肽200 - 256。因此，Cdc2上的酪氨酸15（Y15）仍然处于低磷酸化状态，并且G2/M检查点功能丧失。这些数据描述了Plk3在细胞对DNA损伤反应中连接ATM、Plk3、Chk2、Cdc25C和Cdc2的信号通路中的功能作用。
