Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation
Checkpoint kinase 2 (Chk2) monomers or dimers phosphorylate Cdc25C after DNA damage regardless of threonine 68 phosphorylation
复制标题
DOI：
10.1074/jbc.m208321200
复制
发表时间：
2002-12-13
影响因子：
4.8
通讯作者：
Prives, C
中科院分区：
文献类型：
--
作者：
Ahn, J;Prives, C
我们已经从杆状病毒感染的昆虫细胞和未经处理或DNA损伤应激的人HCT116细胞中纯化并鉴定了人Chk2。来自非应激人类细胞的Chk2主要是单体，对其底物CDc25C的磷酸化不活跃。它在Thr-68处也是非磷酸化的，Thr-68是共济失调毛细血管扩张突变蛋白激酶的靶点。HCT116细胞用拟放射化合物新卡津抑素处理后，活性Chk2以稳定的Thr-68磷酸化二聚体和可相互转化的Thr-68非磷酸化单体和二聚体的形式存在。有趣的是，来自昆虫细胞的Chk2在所有标准测试中的表现都与来自新卡西宁处理的HCT116细胞的活性Chk2相似。根据斯托克斯半径和沉降系数值，Chk2单体和二聚体具有不对称的形状，而不是球状。Thr-68磷酸化和Thr-68非磷酸化形式的活性Chk2都能够磷酸化CDc25C。因此，尽管Thr-68的磷酸化可能是Chk2的初始齐聚和激活所必需的，但它不是维持二聚化或激酶活性所必需的。
We have purified and characterized human Chk2 both from baculovirus-infected insect cells and from either untreated or DNA damage-stressed human HCT116 cells. Chk2 from unstressed human cells is largely monomeric and inactive in phosphorylating its substrate, Cdc25C. It is also unphosphorylated at Thr-68, a site that is the target of the ataxia telangiectasia-mutated protein kinase. After treatment of HCT116 cells with a radiomimetic compound neocarzinostatin, active Chk2 exists as stable Thr-68-phosphorylated dimers as well as interconvertable Thr-68-unphosphorylated monomers and dimers. Interestingly, Chk2 from insect cells behaves by all criteria tested like active Chk2 from neocarzinostatin-treated HCT116 cells. Based on Stokes radius and sedimentation coefficient values, Chk2 monomers and dimers have asymmetric rather than globular shapes. Both Thr-68-phosphorylated and Thr-68-unphosphorylated forms of active Chk2 are capable of phosphorylating Cdc25C. Thus, although phosphorylation of Thr-68 may be required for initial oligomerization and activation of Chk2, it is not needed for maintenance of dimerization or kinase activity.