BUBR1 in the Spindle Checkpoint and Tumor Suppression

描述(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。检查点功能的丢失通常 导致遗传不稳定，这使细胞容易发生恶性 转型。了解细胞功能失活的分子基础 这种监测机制在人类癌症的发展中，PI的实验室 重点研究了两种蛋白激酶hBUB1和hBUBR1(42；53；81)的作用， 在结直肠癌细胞中检测到其突变(4)。最新研究 建议hBUBR1是磁盘轴检查点的重要组件 信号通路，并以后期促进复合体(APC)为靶点 通过与p55CDC/hCdc2O的相互作用，可使p55CDC/hCdc2O/p55CDC/hCdc2O细胞发生相互作用。具体来说，鉴于hBUBR1是 在间期不同细胞系中表达为120 kDa实体， 诺可达唑(Noc)抑制的有丝分裂细胞包含一种独特的hBUBR1物种，该物种 在十二烷基硫酸钠-聚丙烯酰胺凝胶上表现出较低的流动性。磷酸酶处理 将有丝分裂的hBUBR1物种的迁移率恢复到 间期状态，表明hBUBR1的迁移率下降是由于 到磷酸化。酵母双杂交，谷胱甘肽S转移酶下拉，和 免疫沉淀分析表明p55CDC与hBUBR1相互作用。HBUBR1 体外对p55CDC也有磷酸化作用。此外，APC组件CDC16和 Noc处理细胞裂解产物中CDC27与hBUBR1亲和力的相互作用 树脂。最后，我们已经证明了应激激活蛋白激酶p38 与hBUBR1相互作用及Noc诱导HeLa中hBUBR1磷酸化 细胞被p38的特异性抑制物预处理所阻断。在此基础上 在已知的hBUBR1的生化和生物学活性中，我们因此 假设p38激活hBUBR1诱导其与 P55CDC和随后在主轴检查点期间对APC的抑制 激活，以及hBUBR1功能因结构异常而丧失 可能导致非整倍体和癌症。为了检验这一假设，我们将(I) 研究小鼠BUBR1的显性负性突变体是否促进 转基因小鼠的肿瘤发生；(Ii)确定p38是否是即刻的 在磁盘轴检查点激活期间hBUBR1的上游激活器；(Iii)定义 P55CDC的结构域与hBUBR1相互作用；(Iv)检查hBUBR1是否 在体内磷酸化p5SCDC以及hBUBR1和CDK1-Cyclin B复合体 在不同的位点上磷酸化p55CDC；和(V)评估hBUBR1 通过使CDC16和CDC27磷酸化来抑制APC活性。的长期目标是 这个项目是为了确定蛋白质磷酸化的机制。 管理磁盘轴检查点以及解除对其的控制可能导致的结果 癌症。

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.