Watanabe Nobumoto, Sekine Tomomi, Takagi Masatoshi, Iwasaki Jun-ichi, Imamoto Naoko, Kawasaki Hisashi, Osada Hiroyuki
Chemical Biology Department, Antibiotics Laboratory, Advanced Science Institute, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan.
J Biol Chem. 2009 Jan 23;284(4):2344-53. doi: 10.1074/jbc.M805308200. Epub 2008 Nov 25.
Polo-like kinase 1 (Plk1) is one of the key regulators of mitotic cell division. In addition to an N-terminal protein kinase catalytic domain, Plk1 possesses a phosphopeptide binding domain named polo box domain (PBD) at its C terminus. PBD is postulated to be essential for Plk1 localization and substrate targeting. Here, we developed a high-throughput screening system to identify inhibitors of PBD-dependent binding and screened a chemical library. We isolated a benzotropolone-containing natural compound derived from nutgalls (purpurogallin (PPG)) that inhibited PBD-dependent binding in vitro and in vivo. PPG not only delayed the onset of mitosis but also prolonged the progression of mitosis in HeLa cells. Although apparently normal bipolar spindles were formed even in the presence of PPG, the perturbation of chromosome alignment at metaphase plates activated the spindle assembly checkpoint pathway. These results demonstrate the predominant role of PBD-dependent binding on smooth chromosome congression at metaphase.
Polo样激酶1（Plk1）是有丝分裂细胞分裂的关键调节因子之一。除了N端蛋白激酶催化结构域，Plk1在其C端还拥有一个名为polo框结构域（PBD）的磷酸肽结合结构域。据推测，PBD对于Plk1的定位和底物靶向至关重要。在此，我们开发了一种高通量筛选系统来鉴定PBD依赖性结合的抑制剂，并筛选了一个化学文库。我们分离出一种源自五倍子的含苯并托酚酮的天然化合物（紫铆因（PPG）），它在体外和体内均抑制PBD依赖性结合。PPG不仅延迟了有丝分裂的起始，还延长了HeLa细胞中有丝分裂的进程。尽管即使在存在PPG的情况下也能形成明显正常的双极纺锤体，但中期染色体在中期板上排列的扰动激活了纺锤体组装检查点途径。这些结果证明了PBD依赖性结合在中期染色体顺利排列中的主要作用。
