BUBR1 in the Spindle Checkpoint and Tumor Suppression

描述（由申请人提供）：主轴检查点延迟 中期到后期的进程，直到所有的染色体都正常 附着在有丝分裂纺锤体上检查点功能的丧失通常 导致遗传不稳定，使细胞容易发生恶性肿瘤。 转型为了了解功能失活的分子基础， PI的实验室， 已经集中于两种蛋白激酶hBUB 1和hBUBR 1的作用（42;53;81）， 在结肠直肠癌细胞中检测到其突变（4）。最近的研究 提示hBUBR 1是纺锤体检查点重要组成部分 信号通路，它的目标是后期促进复合物（APC） 通过与p55 CDC/hCdc 2 O的相互作用。具体而言，尽管hBUBR 1是 在间期期间在各种细胞系中表达为120-kDa实体， 诺考达唑（Noc）阻滞的有丝分裂细胞含有不同的hBUBR 1种类， 在SDS-聚丙烯酰胺凝胶上显示出降低的迁移率。磷酸酶处理 使有丝分裂hBUBR 1种类的迁移率恢复到 间期状态，表明hBUBR 1的迁移率降低是由于 磷酸化。酵母双杂交，谷胱甘肽S-转移酶下拉，和 免疫沉淀分析显示p55 CDC与hBUBR 1相互作用。hBUBR 1 p55 CDC在体外也被磷酸化。此外，APC组分CDC 16和 Noc处理的细胞的裂解物中存在的CDC 27与hBUBR 1亲和力相互作用 树脂.最后，我们已经表明，应激活化蛋白激酶p38 HeLa细胞中noc与hBUBR 1相互作用及noc诱导hBUBR 1磷酸化 细胞通过用p38的特异性抑制剂预处理而被阻断。根据 已知的生物化学和生物活性的hBUBR 1，因此，我们 假设p38对hBUBR 1激活诱导其与 p55 CDC和随后在纺锤体检查点期间对APC的抑制 激活，并且由于结构异常导致hBUBR 1功能丧失 可能导致非整倍体和癌症。为了验证这个假设，我们将（i） 研究小鼠BUBR 1的显性负突变体是否促进 （ii）确定p38是否是转基因小鼠中的直接肿瘤发生; 在纺锤体检查点激活期间hBUBR 1的上游激活剂;（iii）定义 与hBUBR 1相互作用的p55 CDC结构域;（iv）检测hBUBR 1是否 hBUBR 1和CDK 1-细胞周期蛋白B复合物是否 在不同位点磷酸化p55 CDC;和（v）评估hBUBR 1 通过磷酸化CDC 16和CDC 27抑制APC活性。的长期目标 这个项目是为了确定蛋白质磷酸化 调节纺锤体检查点，以及放松管制如何导致 癌

DESCRIPTION (provided by applicant): The spindle checkpoint delays the progression of metaphase to anaphase until all chromosomes are properly attached to the mitotic spindle. A loss of the checkpoint function often results in genetic instability, which predisposes cells to malignant transformation. To understand the molecular basis of functional inactivation of this surveillance mechanism in human cancer development, the PI's laboratory has focused on the roles of two protein kinases, hBUB1 and hBUBR1 (42;53;81), mutations of which are detected in colorectal cancer cells (4). Recent studies suggest that hBUBR1 is an important component of the spindle checkpoint signaling pathway, and that it targets the anaphase-promoting complex (APC) through interaction with p55CDC/hCdc2O. Specifically, whereas hBUBR1 is expressed as a 120-kDa entity in various cell lines during interphase, nocodazole (Noc)-arrested mitotic cells contain a distinct hBUBR1 species that exhibits a reduced mobility on SDS-polyacrylamide gels. Phosphatase treatment restored the mobility of the mitotic hBUBR1 species to that characteristic of the interphase state, indicating that the decrease in mobility of hBUBR1 is due to phosphorylation. Yeast two-hybrid, glutathione S-transferase pull-down, and immunoprecipitation analyses revealed that p55CDC interacts with hBUBR1. hBUBR1 also phosphorylated p55CDC in vitro. Furthermore, the APC components CDC16 and CDC27 present in lysates of Noc-treated cells interact with hBUBR1 affinity resins. Finally, we have shown that the stress-activated protein kinase p38 interacts with hBUBR1 and that Noc-induced phosphorylation of hBUBR1 in HeLa cells is blocked by pretreatment with a specific inhibitor of p38. On the basis of the known biochemical and biological activities of hBUBR1, we therefore hypothesize that activation of hBUBR1 by p38 induces its interaction with p55CDC and subsequent inhibition of the APC during spindle checkpoint activation, and that a loss of hBUBR1 function due to structural abnormalities may result in aneuploidy and cancer. To test this hypothesis, we will (i) investigate whether a dominant negative mutant of mouse BUBR1 promotes tumorigenesis in transgenic mice; (ii) determine whether p38 is an immediate upstream activator of hBUBR1 during spindle checkpoint activation; (iii) define the domain of p55CDC that interacts with hBUBR1; (iv) examine whether hBUBR1 phosphorylates p5SCDC in vivo and whether hBUBR1 and the CDK1-cyclin B complex phosphorylate p55CDC on different sites; and (v) evaluate whether hBUBR1 inhibits APC activity by phosphorylating CDC16 and CDC27. The long-term goal of this project is to determine the mechanism by which protein phosphorylation regulates the spindle checkpoint and how deregulation of it may result in cancer.