PPK: Radiation Response and Cell Proliferation

癌症的一个特征是失去了控制细胞增殖的调节机制。在被减弱或失去的控制中，有一种能力是在电离辐射和随之而来的DNA损伤或其他细胞损伤的情况下，阻止细胞周期进程。这些检查点包括G1/S边界、S期、G2/M过渡和有丝分裂纺锤体组装时的阻滞。Polo激酶是一个激酶家族，在蛋白质的羧基端保留一个进化上保守的氨基酸序列，称为Polo盒。它们参与细胞从G2晚期进入有丝分裂，通过有丝分裂和退出有丝分裂。在本应用中检测的polo激酶是Plk3（前命名为Prk），它是已知的三种哺乳动物polo激酶之一，其功能仍不明确。我们已经证明，该蛋白在整个细胞周期中相对恒定地表达，但发现其激酶活性波动。基于共免疫沉淀研究，它与两性相促进复合物（APC）相关，但与之相互作用的蛋白质尚不清楚。在电离辐射和DNA损伤下，它也会迅速磷酸化，这表明它在细胞周期阻滞或DNA修复中起作用，并且在细胞暴露于诺可达唑后，它会磷酸化，这表明它在纺锤体组装检查点中起作用。奇怪的是，与它的近亲Plk1不同，Plk3在转染后转化NIH 3T3细胞，Plk3在转染后导致哺乳动物细胞发生凋亡，并在细胞分裂期间阻止细胞。虽然过表达的Plk3与有丝分裂后的中间体相关，但内源性Plk3与之无关，这表明对Plk3过表达结果的解释可能无法准确反映Plk3的真实功能。本应用的直接目标是表征Plk3并阐明其在细胞调节和响应辐射损伤和其他损伤中的功能。具体来说，我们建议建立辐照和诺可达唑处理后Plk3的磷酸化模式，确定其激酶活性的底物和与之相互作用的其他蛋白质，并分离出突变的Plk3，这些突变体绕过细胞凋亡并允许转染后存活。利用这些信息和具有突变或无Plk3等位基因的小鼠胚胎成纤维细胞，我们提出阐明Plk3参与细胞增殖调控以及对辐射和随后的DNA损伤的反应的途径。

One hallmark of cancer is the loss of regulatory mechanisms that control cell proliferation. Among the controls that are attenuated or lost is the capacity to arrest cell cycle progression in response to ionizing radiation and consequent DNA damage or other insult to the cell. Such checkpoints include arrest at the G1/S boundary, during the S phase, during the G2/M transition, and at the point of mitotic spindle assembly. The Polo kinases are a family of kinases that retain an evolutionarily conserved amino acid sequence, designated the polo box, at the carboxy end of the protein. They participate in the transit of cells from late G2 into mitosis, through mitosis and exit from mitosis. The polo kinase examined in this application is Plk3 (former by designated Prk), one of three known mammalian polo kinases, whose function remains ill defined. We have shown that this protein is expressed relatively constantly throughout the cell cycle but find that its kinase activity fluctuates. Based on co-immunoprecipitation studies, it associates with the amphase promoting complex (APC), but the proteins with which it interacts are not known. It is also very rapidly phosphorylated in response to ionizing radiation and DNA damage, suggestive of a role in cell cycle arrest or DNA repair, and it becomes phosphorylated following exposure of cells to nocodazole, suggestive of a role in the spindle assembly checkpoint. Curiously, unlike its close relative Plk1, which transforms NIH 3T3 cells following transfection, Plk3 causes mammalian cells to undergo apoptosis following transfection and arrests cells during cytokinesis. Whereas overexpressed Plk3 associates with a post-mitotic midbody, endogenous Plk3 does not, suggesting that interpretation of results derived from Plk3 overexpression may not accurately reflect the Plk3 true function(s). The immediate goals of this application are to characterize Plk3 and to elucidate its function(s) in cellular regulation and in response radiation damage and other insult. Specifically we propose to establish phosphorylation patterns of Plk3 following irradiation and nocodazole treatment, identify substrates for its kinase activity and other proteins with which it interacts, and isolate mutant Plk3 that circumvent apoptosis and permit survival following transfection. Using this information and mouse embryo fibroblasts with mutant or null Plk3 alleles, we propose to elucidate the pathway(s) in which Plk3 participates in the regulation of cell proliferation and in response to radiation and consequent DNA damage.