Polo-like Kinase Functions in Normal and Tumor Cells

描述(申请人提供)：哺乳动物的Polo样激酶，Plk1，对于细胞分裂是必不可少的，显然在有丝分裂和胞质分裂的几个点上都需要。因为它们在细胞分裂的几个步骤中起作用，所以关于M期Polo样激酶的遗传数据必须与生化分析相补充。为了描述Plk1如何执行其各种功能，需要对其底物及其功能进行表征。我们有三个特定的目标：i)肿瘤细胞和正常细胞中Plk1的缺失。当Plk1的表达被RNA干扰(RNAi)沉默时，肿瘤细胞经历P53非依赖性的凋亡。我们建议确定在Plk1缺失的肿瘤细胞中导致细胞死亡的分子事件。由于Plk1可能被认为是癌症治疗的潜在靶点，在这方面比较正常细胞和肿瘤细胞是必要的。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抵抗力。PLK1在正常细胞中的表达将通过慢病毒来表达不同的RNAi，这些RNAi针对Plk1 mRNA中的不同序列。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亚型将从表达针对不同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体的发展，这些都与肿瘤的发展有关，ii)Plk1底物。我们已经确定了几种Plk1底物，它们似乎在细胞分裂中具有重要功能，是细胞存活所必需的。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和Plk1在细胞分裂中的作用，iii)MKLP相互作用蛋白。一种Plk1底物是有丝分裂激动素样蛋白(MKLP1)，我们已经证明它是胞质分裂所必需的，但不是核分裂所必需的。我们将使用以前成功的Plk1技术来识别在细胞质分裂过程中可能有助于MKLP1功能的MKLP1相互作用蛋白。我们将在体外和体内研究与野生型MKLP1和带有Plk1磷酸化位点突变的MKLP1相互作用的蛋白质。可能促进胞质分裂的MKLPl相互作用蛋白的作用也将用RNAi方法进行研究。

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.