Mechanisms of Rad9 Mediated Checkpoints and Apoptosis

描述（申请人提供）：RAD 9介导的细胞周期检查点和细胞凋亡的机制：细胞周期检查点在维持细胞周期中起关键作用。 通过抑制细胞周期的进展来保持基因组完整性，或 启动程序性细胞死亡的存在下受损的DNA或不完整的 DNA复制。对裂变酵母的研究表明， 检查点蛋白家族，包括Rad1、Rad3、Rad9、Rad17、Rad26和 Hus1在DNA损伤和复制的激活中起重要作用 检查站我们已经报道Rad9可以与Bcl-2和Bcl-xL相互作用， 通过位于Rad9蛋白的氨基末端的BH 3样区域， 并可促进哺乳动物细胞的凋亡。DNA损伤增强Rad9 磷酸化并诱导Rad9移动到核膜，在那里它 与Bcl-2共定位。此外，我们的初步数据表明， c-Abl介导的Y28上Rad9的磷酸化诱导增加的 Rad9与BcI-xL结合，并增强Rad9对细胞凋亡诱导的作用。 有趣的是，Rad9蛋白被过度磷酸化，并且某些形式似乎 是细胞周期依赖性的。此外，我们最近发现，Hus1形成一个 蛋白复合物与PCNA在人皮肤Flow2000成纤维细胞当DNA 被破坏或复制被抑制。Flow2000细胞暴露于8戈伊 电离辐射（诱导G2/M期阻滞但不诱导凋亡）或羟基脲 引发了胡希从细胞质到细胞核的易位， 与PCNA和Rad9共定位。这种核移位和复合体 PCNA与细胞周期的变化密切相关 分布在辐射暴露的反应。本提案的目的是 验证Rad9是DNA完整性的重要调节剂的假设 检查点路径决定细胞是否应该暂时延迟 细胞周期进程或DNA损伤后死亡及损伤诱导复合物 一组离散的细胞蛋白质的形成在 Rad9函数。

DESCRIPTION (PROVIDED BY APPLICANT):MECHANISMS OF RAD9-MEDIATED CHECKPOINTS AND APOPTOSIS: Cell-cycle checkpoints play a critical role in the maintenance of genomic integrity by inhibiting progression through the cell cycle or initiating programmed cell death in the presence of damaged DNA or incomplete DNA replication. Studies in fission yeast implicate that members of the Rad family of checkpoint proteins including Radi, Rad3, Rad9, Radl7, Rad26, and Hus1 play important roles in the activation of DNA damage and replication checkpoints. We have reported that Rad9 can interact with Bcl-2 and Bcl-xL through a BH3-like region located in the amino terminus of the Rad9 protein, and can promote apoptosis in mammalian cells. DNA damage enhances Rad9 phosphorylation and induces Rad9 to move to the nuclear envelope, where it colocalizes with Bcl-2. In addition, our preliminary data indicate that c-Abl-mediated phosphorylation of Rad9 on Y28 induces increased association of Rad9 with BcI-xL and enhances the effect of Rad9 on apoptosis induction. Interestingly, the Rad9 protein is hyperphosphorylated and some form appears to be cell cycle-dependent. Moreover, we have recently found that Hus1 forms a protein complex with PCNA in human skin Flow2000 fibroblasts when DNA is damaged or replication is inhibited. Exposure of Flow2000 cells to 8 Gy ionizing radiation (that induces G2/M-arrest but not apoptosis) or hydroxyurea triggered translocation of Husi from the cytosol to the nucleus, where it colocalized with PCNA and Rad9. This nuclear translocation and the complex formation of Husl with PCNA correlate closely with changes in cell cycle distribution in response to radiation exposure. The goal of this proposal is to test the hypothesis that Rad9 is an important modulator of the DNA integrity checkpoint pathway determining whether a cell should transiently delay cell-cycle progression or die after DNA damage and that damage induced complex formation with a discrete set of cellular proteins plays a critical role in Rad9 function.