Li Zhe, Li Xueyan, Nai Shanshan, Geng Qizhi, Liao Ji, Xu Xingzhi, Li Jing
From the Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing 100048, China and.
From the Beijing Key Laboratory of DNA Damage Response and College of Life Sciences, Capital Normal University, Beijing 100048, China and
J Biol Chem. 2017 Dec 1;292(48):19548-19555. doi: 10.1074/jbc.M117.811646. Epub 2017 Oct 11.
Checkpoint kinase 1 (Chk1) is a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint, and cytokinesis. Despite Chk1's pivotal role in multiple cellular processes, many of its substrates remain elusive. Here, we identified linked β--acetylglucosamine (-GlcNAc)-transferase (OGT) as one of Chk1's substrates. We found that Chk1 interacts with and phosphorylates OGT at Ser-20, which not only stabilizes OGT, but also is required for cytokinesis. Phospho-specific antibodies of OGT-pSer-20 exhibited specific signals at the midbody of the cell, consistent with midbody localization of OGT as reported previously. Moreover, phospho-deficient OGT (S20A) cells attenuated cellular -GlcNAcylation levels and also reduced phosphorylation of Ser-71 in the cytoskeletal protein vimentin, a modification critical for severing vimentin filament during cytokinesis. Consequently, elongated vimentin bridges were observed in cells depleted of OGT via an sibased approach. Lastly, expression of plasmids resistant to si efficiently rescued the vimentin bridge phenotype, but the OGT-S20A rescue plasmids did not. Our results suggest a Chk1-OGT-vimentin pathway that regulates the intermediate filament network during cytokinesis.
关卡激酶1（Chk1）是一种在协调DNA复制、DNA损伤关卡、纺锤体组装关卡和胞质分裂过程中发挥重要作用的激酶。尽管Chk1在多个细胞过程中起着关键作用，但其许多底物仍不明确。在此，我们鉴定出连接β-N-乙酰葡糖胺（O-GlcNAc）转移酶（OGT）是Chk1的底物之一。我们发现Chk1与OGT相互作用并在Ser-20位点使其磷酸化，这不仅使OGT稳定，也是胞质分裂所必需的。OGT-pSer-20的磷酸化特异性抗体在细胞的中体处显示出特异性信号，这与之前报道的OGT在中体的定位一致。此外，磷酸化缺陷型OGT（S20A）细胞减弱了细胞的O-GlcNAcylation水平，还降低了细胞骨架蛋白波形蛋白中Ser-71的磷酸化，这种修饰对于在胞质分裂期间切断波形蛋白丝至关重要。因此，通过基于小干扰RNA的方法使OGT缺失的细胞中观察到波形蛋白桥延长。最后，对小干扰RNA有抗性的质粒表达有效地挽救了波形蛋白桥表型，但OGT-S20A挽救质粒则不能。我们的结果表明存在一条在胞质分裂期间调节中间丝网络的Chk1-OGT-波形蛋白通路。
