Nigg E A, Blangy A, Lane H A
Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, Geneva 4, CH-1211, Switzerland.
Exp Cell Res. 1996 Dec 15;229(2):174-80. doi: 10.1006/excr.1996.0356.
During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly, cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure required for the segregation of sister chromatids. Many of the above events are controlled by reversible phosphorylation. Hence, our laboratory is interested in characterizing the kinases involved in promoting progression through mitosis and in identifying their relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2 kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation regulates the association of HsEg5 with the mitotic spindle and that the function of this plus-end directed motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in regulating progression through mitosis is Plk1 (polo-like kinase 1), the human homologue of the Drosophila gene product "polo." By antibody microinjection we have found that Plk1 is required for the functional maturation of centrosomes and hence for entry into mitosis. Furthermore, we found that microinjected anti-Plk1 antibodies caused a more severe block to cell cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the presence of functional centrosomes.
在有丝分裂过程中，脊椎动物细胞核的结构会发生深刻变化。在有丝分裂开始时，核膜解体，核纤层解聚，间期染色质浓缩成染色体。与此同时，细胞质微管会重新组织成有丝分裂纺锤体装置，这是姐妹染色单体分离所需的高度动态结构。上述许多事件都受可逆磷酸化作用控制。因此，我们实验室有兴趣表征参与促进有丝分裂进程的激酶，并确定它们的相关底物。负责调节进入有丝分裂的激酶中，突出的是Cdc2激酶，它是细胞周期蛋白依赖性激酶（Cdk）家族的首个成员。最近，我们发现Cdc2使HsEg5磷酸化，HsEg5是一种与中心体和纺锤体装置相关的人类驱动蛋白相关运动蛋白。我们的结果表明，磷酸化调节HsEg5与有丝分裂纺锤体的结合，并且这种正端定向运动蛋白的功能对于中心体分离和双极纺锤体形成至关重要。另一种与调节有丝分裂进程有关的激酶是Plk1（类polo激酶1），它是果蝇基因产物“polo”的人类同源物。通过抗体显微注射，我们发现Plk1是中心体功能成熟所必需的，因此也是进入有丝分裂所必需的。此外，我们发现显微注射抗Plk1抗体对二倍体成纤维细胞的细胞周期进程造成的阻滞比对永生化肿瘤细胞更严重。这一观察结果暗示存在一个将Cdc2激活与功能性中心体的存在联系起来的检查点。
