Niiya F, Tatsumoto T, Lee K S, Miki T
Laboratory of Cell Biology, National Cancer Institute, NIH Bldg. 37, 37 Convent Drive, Bethesda, MD 20892-4256, USA.
Oncogene. 2006 Feb 9;25(6):827-37. doi: 10.1038/sj.onc.1209124.
The epithelial cell transforming gene 2 (ECT2) protooncogene encodes a Rho exchange factor, and regulates cytokinesis. ECT2 is phosphorylated in G2/M phases, but its role in the biological function is not known. Here we show that two mitotic kinases, Cdk1 and polo-like kinase 1 (Plk1), phosphorylate ECT2 in vitro. We identified an in vitro Cdk1 phosphorylation site (T412) in ECT2, which comprises a consensus phosphospecific-binding module for the Plk1 polo-box domain (PBD). Endogenous ECT2 in mitotic cells strongly associated with Plk1 PBD, and this binding was inhibited by phosphatase treatment. A phosphorylation-deficient mutant form of ECT2, T412A, did not exhibit strong association with Plk1 PBD compared with wild-type (WT) ECT2. Moreover, ECT2 T412A, but not phosphomimic T412D, displayed a diminished accumulation of GTP-bound RhoA compared with WT ECT2, suggesting that phosphorylation of Thr-412 is critical for the catalytic activity of ECT2. Moreover, while overexpression of WT ECT2 or the T412D mutant caused cortical hyperactivity in U2OS cells during cell division, this activity was not observed in cells expressing ECT2 T412A. These results suggest that ECT2 is regulated by Cdk1 and Plk1 in concert.
上皮细胞转化基因2（ECT2）原癌基因编码一种Rho交换因子，并调节胞质分裂。ECT2在G2/M期被磷酸化，但其在生物学功能中的作用尚不清楚。在此，我们表明两种有丝分裂激酶，细胞周期蛋白依赖性激酶1（Cdk1）和polo样激酶1（Plk1），在体外使ECT2磷酸化。我们在ECT2中鉴定出一个体外Cdk1磷酸化位点（T412），其包含一个用于Plk1 polo盒结构域（PBD）的共有磷酸特异性结合模块。有丝分裂细胞中的内源性ECT2与Plk1 PBD强烈结合，并且这种结合被磷酸酶处理所抑制。与野生型（WT）ECT2相比，ECT2的磷酸化缺陷突变形式T412A与Plk1 PBD没有表现出强烈的结合。此外，与WT ECT2相比，ECT2 T412A而非磷酸模拟物T412D显示出结合GTP的RhoA积累减少，这表明Thr-412的磷酸化对于ECT2的催化活性至关重要。此外，虽然WT ECT2或T412D突变体的过表达在细胞分裂期间导致U2OS细胞中的皮质过度活跃，但在表达ECT2 T412A的细胞中未观察到这种活性。这些结果表明ECT2受Cdk1和Plk1协同调节。
