Polo-like Kinase Functions in Normal and Tumor Cells

描述（由申请人提供）：哺乳动物polo样激酶Plk 1是细胞分裂所必需的，显然在有丝分裂和胞质分裂的几个点都需要。由于它们在细胞分裂的几个步骤中起作用，因此必须用生化分析来补充M期polo样激酶的遗传数据。为了描述Plk 1如何执行其各种功能，需要表征其底物及其功能。我们有三个具体目标：i）肿瘤和正常细胞中Plk1的耗竭。当Plk1的表达被RNA干扰（RNAi）沉默时，肿瘤细胞经历p53非依赖性凋亡。我们建议确定导致Plk1缺失的肿瘤细胞中细胞死亡的分子事件。由于Plk 1可能被认为是癌症治疗中的潜在靶点，因此在这方面将正常细胞与肿瘤细胞进行比较是必要的。由于肿瘤细胞缺乏检查点控制，正常细胞可能对细胞死亡具有抗性。使用慢病毒表达靶向Plk1 mRNA中不同序列的各种RNAi，将使正常细胞中的Plk1表达沉默。我们将确定这是否会导致正常细胞发生凋亡，以及它是否依赖于p53。Plk1亚型将从表达靶向不同Plk1 mRNA序列的RNAi的细胞中选择，并检查中心体缺陷、纺锤体缺陷和非整倍性的发展，这些都与肿瘤发展相关。ii）Plk1底物。我们已经确定了几个Plk1基板，似乎有重要的功能，在细胞分裂和细胞活力所需的。我们建议使用RNAi来耗尽这些底物，并突变这些底物中的磷酸化位点，以更完整地了解它们的作用以及Plk1在细胞分裂中的作用。iii）MKLP相互作用蛋白。Plk1的底物之一是有丝分裂驱动蛋白样蛋白（MKLP 1），我们已经证明它对胞质分裂而不是核分裂是必不可少的。我们将使用以前成功的Plk1技术，以确定MKLP1相互作用的蛋白质，可能有助于MKLP1在胞质分裂的功能。我们将在体外和体内研究与野生型MKLP 1和Plk1磷酸化位点突变的MKLP 1相互作用的蛋白质。还将用RNAi方法研究可能促进胞质分裂的MKLP1相互作用蛋白的作用。

DESCRIPTION (provided by applicant): The mammalian polo-like kinase, Plk1, is essential for cell division, apparently required at several points in mitosis and cytokinesis. Because they act at several steps during cell division, genetic data on the M-phase polo-like kinases must be complemented with biochemical analysis. In order to describe how Plk1 executes its various functions, characterization of its substrates and their functions is required. We have three specific aims: i) Plk1 depletion in tumor and normal cells. When expression of Plk1 is silenced by RNA interference (RNAi), tumor cells undergo p53- independent apoptosis. We propose to determine the molecular events that lead to cell death in tumor cells depleted of Plk1. Because Plk1 may be regarded as a potential target in cancer therapy, comparison of normal cells to tumor cells in this regard is necessary. Normal cells may be resistant to cell death as a consequence of checkpoint controls that tumor cells lack. Plk1 expression in normal cells will be silenced using lentiviruses to express various RNAis that target different sequences in Plk1 mRNA. We shall determine if this causes normal cells to undergo apoptosis and whether or not it is p53-dependent. Plk1 hypomorphs will be selected from cells expressing RNAi targeted to different Plk1 mRNA sequences and examined for centrosome deficiencies, spindle defects, and development of aneuploidy, which are all relevant to tumor development, ii) Plk1 substrates. We have identified several Plk1 substrates that appear to have important functions in cell division and that are required for cell viability. We propose to use RNAi to deplete these substrates and to mutate phosphorylation sites in these substrates to develop a more complete picture of their role and that of Plk1 in cell division, iii) MKLP-interactive proteins. One Plk1 substrate is the mitotic kinesin-like protein (MKLP1), which we have shown is essential for cytokinesis but not karyokinesis. We will use techniques previously successful for Plk1 to identify MKLP1- interacting proteins that may contribute to MKLP1 functions during cytokinesis. We shall study interacting proteins in vitro and in vivo with wild type MKLP1 and MKLP1 with Plk1 phosphorylation site mutations. The role of MKLPl-interacting proteins that may promote cytokinesis will be also investigated with RNAi methods.