Regulation of the Mitotic Checkpoint by Gsk3

项目摘要 癌细胞中染色体数量的变化可能会对细胞的增殖、细胞死亡、 以及抗药性。绝大多数肿瘤是非整倍体的，但相关机制却不是。 完全知道。有丝分裂检查点确保了对子代细胞的平等染色体分离，但仍然 在许多人类肿瘤来源的细胞系中发挥作用。更有可能的是，在强度或保真度方面的细微缺陷 检查点可能是非整倍体的基础。癌细胞是由生长因子通路的错误调节和 相关的信号转导机制。我们检测到了对信号转导激酶的需求 GSK3位于有丝分裂检查点。GSK3是重新招募检查点蛋白MAD2和BubR1所必需的 动点和有丝分裂检查点复合体(MCC)的形成，使细胞保持有丝分裂，直到 染色体正确地附着在纺锤体上。我们还观察到GSK3不影响有丝分裂检查点 在缺乏脱乙酰酶的细胞中，SIRT2表明可能的靶标。GSK3的活动被一些 生长因子途径，其中WNT途径是研究最好的例子之一。WNT激活 在某些实验系统中增强非整倍体。鉴于WNT信号在许多癌症中升高 细胞，GSK3可能提供了这一途径，有丝分裂检查点的强度和非整倍体之间的联系。我们 提出了三个具体的目标来确定GSK3调节有丝分裂检查点和 WNT信号、生长因子信号、GSK3活性与有丝分裂调控之间的联系。目标1。 GSK3对MCC的调控。抑制GSK3破坏由MAD2、CDC20、BubR1和 Bub3，减少MAD2和BubR1的运动中枢。我们计划分析动粒定位 其他关键检查点蛋白，并确定GSK3是否调节 MCC.这些实验将有助于确定GSK3调节有丝分裂的机制。 检查站设备。目的2.GSK3在有丝分裂检查点的近端靶点。这个 GSK3的动粒效应可能离直接的GSK3底物有几步之遥。SIRT2是一种潜在的 检查点响应中GSK3的目标。我们将分析SIRT2的特定突变体的影响并分析 SIRT2脱乙酰酶活性来确定该蛋白是否作用于GSK3下游。Axin2和 腺瘤性息肉病结肠也调节有丝分裂检查点，并与GSK3相互作用。其他内容 实验将确定这些蛋白质是否与GSK3对有丝分裂的影响有关。目标3. GSK3上游调控因子对有丝分裂的调控。有丝分裂检查点的调控 通过GSK3潜在地将许多在癌症中上调的信号转导通路与核心过程联系起来 控制染色体稳定性。为了研究这种联系，WNT信令将被中断，随后 有丝分裂检查点的分析。我们还将确定Akt，另一个调节 GSK3可以改变有丝分裂检查点的强度。

Project Summary Changes in chromosome number in cancer cells can have drastic effects on proliferation, cell death, and drug resistance. The vast majority of tumors are aneuploid, yet the mechanisms responsible are not completely known. The mitotic checkpoint ensures equal chromosome segregation to daughter cells, but still functions in many human tumor-derived cell lines. More likely, subtle defects in the strength or fidelity of the checkpoint may underlie aneuploidy. Cancer cells are defined by mis-regulation of growth factor pathways and associated signal transduction machinery. We have detected a requirement for the signal transducing kinase Gsk3 in the mitotic checkpoint. Gsk3 is required for the recruitment of checkpoint proteins Mad2 and BubR1 to kinetochores and for formation of the mitotic checkpoint complex (MCC) that keeps cells in mitosis until chromosomes properly attach to the spindle. We also observed that Gsk3 did not affect the mitotic checkpoint in cells lacking the deacetylase SIRT2 indicating a possible target. Gsk3 activity is silenced by a number of growth factor pathways, with the WNT pathway being one of the best studied examples. WNT activation enhances aneuploidy in some experimental systems. Given that WNT signaling is elevated in many cancer cells, Gsk3 may provide a link between this pathway, the strength of the mitotic checkpoint and aneuploidy. We propose three specific aims to determine the mechanism by which Gsk3 regulates the mitotic checkpoint and the linkage between WNT signaling, growth factor signaling, Gsk3 activity and mitotic regulation. AIM 1. REGULATION OF MCC BY GSK3. Inhibiting Gsk3 disrupts the MCC composed of Mad2, Cdc20, BubR1 and Bub3 and reduces Mad2 and BubR1 at kinetochores. We plan to analyze the kinetochore localization of additional key checkpoint proteins and to determine whether Gsk3 regulates assembly or disassembly of the MCC. These experiments will help to determine the mechanism by which Gsk3 regulates the mitotic checkpoint apparatus. AIM 2. PROXIMAL TARGETS OF GSK3 IN THE MITOTIC CHECKPOINT. The kinetochore effects of Gsk3 may be several steps away from direct Gsk3 substrates. SIRT2 is one potential target of Gsk3 in the checkpoint response. We will analyze effects of specific mutants of SIRT2 and analyze SIRT2 deacetylase activity to determine whether this protein acts downstream of Gsk3. Axin2 and Adenomatous polyposis coli also regulate the mitotic checkpoint and interact with Gsk3. Additional experiments will determine whether these proteins are responsible for the effects of Gsk3 on mitosis. AIM 3. MODULATION OF MITOSIS BY UPSTREAM REGULATORS OF GSK3. Regulation of the mitotic checkpoint by Gsk3 potentially links many signal transduction pathways upregulated in cancer with a core process controlling chromosome stability. To investigate this linkage, WNT signaling will be disrupted followed by analysis of the mitotic checkpoint. We will also determine whether Akt, another signal transducer that regulates Gsk3 can modify the strength of the mitotic checkpoint.