Department of Biochemistry and Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA.
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14633-8. doi: 10.1073/pnas.1006615107. Epub 2010 Aug 2.
Nuclear envelope breakdown (NEBD) is an essential step during the G2/M transition in higher eukaryotic cells. Increasing evidence supports the notion that both microtubules and microtubule-associated motor proteins are critical regulators of NEBD. Although it has been described that p150(Glued), the major component of the dynein/dynactin complex, localizes in the nuclear envelope during prophase, the exact role of p150(Glued) and its regulation during NEBD are largely elusive. Polo-like kinase 1 (Plk1), the best characterized Ser/Thr kinase, is involved in mitotic entry in several systems; however, the targets of Plk1 during NEBD are unknown. Herein, we show that in mammalian cells both Plk1 and p150(Glued) regulate NEBD and that Plk1 interacts with and phosphoryates p150(Glued) during NEBD at prophase. Using various approaches, we showed that Plk1 phosphorylates p150(Glued) at Ser-179 and that the pS179 epitope is generated at the nuclear envelope of prophase cells. Significantly, Plk1-mediated phosphorylation of p150(Glued) at Ser-179 positively regulates its accumulation at the nuclear envelope during prophase. Finally, we found that cells expressing the Plk1-unphosphorylatable mutant (p150(Glued)-S179A) arrest at G2, as indicated by reduced NEBD, increased levels of cyclin B and phospho-H3, but a decreased level of Cdc2 kinase activity. Taking these data together, we conclude that Plk1 phosphorylation of p150(Glued) might be one major pathway of NEBD regulation.
核膜崩解(NEBD)是高等真核细胞 G2/M 转换过程中的一个必要步骤。越来越多的证据支持这样一种观点,即微管和微管相关的马达蛋白都是 NEBD 的关键调节因子。虽然已经描述了 p150(Glued),即动力蛋白/dynactin 复合物的主要成分,在前期定位于核膜,但 p150(Glued)的确切作用及其在 NEBD 中的调节在很大程度上仍不清楚。Polo 样激酶 1(Plk1)是研究最为深入的 Ser/Thr 激酶,在多个系统中参与有丝分裂的进入;然而,Plk1 在 NEBD 期间的靶标尚不清楚。在此,我们发现在哺乳动物细胞中,Plk1 和 p150(Glued)都调节 NEBD,并且 Plk1 在前期与 p150(Glued)相互作用并磷酸化 p150(Glued)。通过各种方法,我们发现 Plk1 在 Ser-179 处磷酸化 p150(Glued),并且 pS179 表位在前期细胞的核膜上产生。重要的是,Plk1 介导的 p150(Glued)在 Ser-179 处的磷酸化正向调节其在前期的核膜积累。最后,我们发现表达 Plk1 不可磷酸化突变体(p150(Glued)-S179A)的细胞停滞在 G2 期,这表明 NEBD 减少,cyclin B 和磷酸化 H3 的水平增加,但 Cdc2 激酶活性降低。综上所述,我们得出结论,Plk1 对 p150(Glued)的磷酸化可能是 NEBD 调节的主要途径之一。