PPK: Radiation Response and Cell Proliferation

癌症的一个标志是控制细胞增殖的调节机制的丧失。 在减弱或丧失的控制中，是响应于电离辐射和随后的DNA损伤或对细胞的其他损伤而阻止细胞周期进展的能力。 这些检查点包括在G1/S边界、S期、G2/M转换和有丝分裂纺锤体组装点的阻滞。 波罗激酶是在蛋白质的羧基端保留进化上保守的氨基酸序列（称为polo盒）的激酶家族。 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的转运。在本申请中检测的波罗激酶是Plk 3（以前称为Prk），三种已知的哺乳动物波罗激酶之一，其功能仍然不清楚。我们已经表明，这种蛋白质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。 基于免疫共沉淀研究，它与amphase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损伤时，它也非常迅速地磷酸化，提示在细胞周期阻滞或DNA修复中的作用，并且在细胞暴露于诺考达唑后，它变得磷酸化，提示在纺锤体组装检查点中的作用。 奇怪的是，与其近亲Plk 1不同，Plk 1在转染后转化NIH 3 T3细胞，Plk 3在转染后导致哺乳动物细胞凋亡，并在胞质分裂期间阻止细胞。 而过表达的Plk 3与有丝分裂后的中间体相关，内源性Plk 3不相关，这表明对来自Plk 3过表达的结果的解释可能不能准确地反映Plk 3的真实功能。 本申请的直接目标是表征Plk 3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，我们建议建立照射和诺考达唑治疗后的Plk 3的磷酸化模式，确定其激酶活性和其他蛋白质与它相互作用的底物，并分离突变体Plk 3，规避细胞凋亡，并允许生存转染后。 利用这些信息和小鼠胚胎成纤维细胞突变或无效Plk 3等位基因，我们建议阐明途径（S），其中Plk 3参与调节细胞增殖和响应辐射和随之而来的DNA损伤。

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.