Golsteyn R M, Mundt K E, Fry A M, Nigg E A
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges.
J Cell Biol. 1995 Jun;129(6):1617-28. doi: 10.1083/jcb.129.6.1617.
Correct assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the duplicated genome to daughter cells. Protein phosphorylation has long been implicated in controlling spindle function and chromosome segregation, and genetic studies have identified several protein kinases and phosphatases that are likely to regulate these processes. In particular, mutations in the serine/threonine-specific Drosophila kinase polo, and the structurally related kinase Cdc5p of Saccharomyces cerevisae, result in abnormal mitotic and meiotic divisions. Here, we describe a detailed analysis of the cell cycle-dependent activity and subcellular localization of Plk1, a recently identified human protein kinase with extensive sequence similarity to both Drosophila polo and S. cerevisiae Cdc5p. With the aid of recombinant baculoviruses, we have established a reliable in vitro assay for Plk1 kinase activity. We show that the activity of human Plk1 is cell cycle regulated, Plk1 activity being low during interphase but high during mitosis. We further show, by immunofluorescent confocal laser scanning microscopy, that human Plk1 binds to components of the mitotic spindle at all stages of mitosis, but undergoes a striking redistribution as cells progress from metaphase to anaphase. Specifically, Plk1 associates with spindle poles up to metaphase, but relocalizes to the equatorial plane, where spindle microtubules overlap (the midzone), as cells go through anaphase. These results indicate that the association of Plk1 with the spindle is highly dynamic and that Plk1 may function at multiple stages of mitotic progression. Taken together, our data strengthen the notion that human Plk1 may represent a functional homolog of polo and Cdc5p, and they suggest that this kinase plays an important role in the dynamic function of the mitotic spindle during chromosome segregation.
在细胞分裂过程中，有丝分裂纺锤体的正确组装和功能对于将复制后的基因组准确分配到子细胞中至关重要。长期以来，蛋白质磷酸化一直被认为参与控制纺锤体功能和染色体分离，并且遗传学研究已经鉴定出几种可能调节这些过程的蛋白激酶和磷酸酶。特别是，果蝇丝氨酸/苏氨酸特异性激酶polo以及酿酒酵母中结构相关的激酶Cdc5p发生突变会导致有丝分裂和减数分裂异常。在此，我们描述了对Plk1（一种最近鉴定出的人类蛋白激酶，与果蝇polo和酿酒酵母Cdc5p具有广泛的序列相似性）的细胞周期依赖性活性和亚细胞定位的详细分析。借助重组杆状病毒，我们建立了一种可靠的体外检测Plk1激酶活性的方法。我们发现人类Plk1的活性受细胞周期调节，在间期活性较低，而在有丝分裂期间活性较高。我们还通过免疫荧光共聚焦激光扫描显微镜进一步表明，人类Plk1在有丝分裂的所有阶段都与有丝分裂纺锤体的成分结合，但随着细胞从中期进入后期，会发生显著的重新分布。具体而言，直到中期Plk1都与纺锤体极相关联，但随着细胞进入后期，它会重新定位到赤道平面，即纺锤体微管重叠的区域（中间区）。这些结果表明Plk1与纺锤体的关联是高度动态的，并且Plk1可能在有丝分裂进程的多个阶段发挥作用。综上所述，我们的数据强化了人类Plk1可能代表polo和Cdc5p功能同源物的观点，并表明该激酶在染色体分离过程中有丝分裂纺锤体的动态功能中起重要作用。
