Regulation of the Mitotic Checkpoint by Gsk3

项目摘要 癌细胞中染色体数目的变化可以对增殖、细胞死亡、 和耐药性。绝大多数肿瘤是非整倍体，但其机制并非如此。 完全知道。有丝分裂检查点确保染色体平等地分离到子细胞，但仍然 在许多人肿瘤衍生细胞系中发挥作用。更有可能的是，在强度或保真度的细微缺陷， 检查点可能是非整倍体的基础。癌细胞由生长因子途径的错误调节定义， 相关的信号转导机制。我们已经检测到信号转导激酶的需要， 有丝分裂检查点中的GSK3。Gsk3是募集检查点蛋白Mad2和BubR1所必需的， 有丝分裂检查点复合物（MCC）的形成，使细胞保持有丝分裂， 染色体正确地附着在纺锤体上。我们还观察到Gsk 3不影响有丝分裂检查点 在缺乏脱乙酰基酶SIRT2的细胞中，这表明可能的靶点。GSK3的活性被许多 生长因子途径，WNT途径是研究得最好的例子之一。WNT活化 在某些实验系统中增强了非整倍性。鉴于WNT信号在许多癌症中升高， 细胞，Gsk 3可能提供了这一途径之间的联系，有丝分裂检查点和非整倍性的强度。我们 提出了三个具体目标，以确定Gsk 3调节有丝分裂检查点的机制， WNT信号传导、生长因子信号传导、Gsk 3活性与有丝分裂调控之间的联系。AIM 1. GSK对MCC的监管3.抑制Gsk3破坏了由Mad2、Cdc20、BubR1和Cdc20组成的MCC。 Bub3和减少Mad2和BubR1在着丝粒。我们计划分析 另外的关键检查点蛋白，并确定Gsk3是否调节细胞的组装或拆卸。 MCC。这些实验将有助于确定Gsk 3调节有丝分裂的机制。 检查点装置AIM 2. GSK 3在有丝分裂的ERK点的近端靶点。的 Gsk 3的动粒效应可能与直接的Gsk 3底物有几步之遥。SIRT2是一种潜在的 在检查点响应中的Gsk3的目标。我们将分析SIRT2特定突变体的影响，并分析 SIRT2脱乙酰酶活性，以确定该蛋白质是否在Gsk3下游起作用。Axin2和 结肠腺瘤性息肉病也调节有丝分裂检查点并与Gsk 3相互作用。额外 实验将确定这些蛋白质是否负责GSK 3对有丝分裂的影响。AIM 3. 通过GSK 3的上游调节子调节有丝分裂。有丝分裂检查点的调节 通过Gsk3可能将癌症中上调的许多信号转导途径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信号传导将被破坏，然后 有丝分裂检查点的分析。我们还将确定Akt，另一种调节 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.