Roles of MyoGEF in cytokinesis

描述（由申请人提供）：我们的长期目标是了解调节哺乳动物细胞胞质分裂的分子机制，并利用这些知识开发新的癌症治疗策略。胞质分裂的功能失调可导致不受控制的细胞增殖，这是癌症的一个关键特征。胞质分裂的完整性依赖于收缩环组装的定位和时间的精确协调调节。一种流行的模型是来自中心轴的信号确定收缩环组件的定位和定时。Polo-like kinase 1（Plk 1）和aurora kinase B（aurora B）是两种重要的有丝分裂激酶，定位于纺锤体，与小GT3信号协同调节收缩环组装。小的GTP酶蛋白，如RhoA，Rac 1和Cdc 42，被鸟嘌呤核苷酸交换因子（GEF）激活，并被GTP酶激活蛋白（GAP）灭活。到目前为止，至少有三个GEF，即ECT 2，GEF-H1和MyoGEF，涉及调节胞质分裂。然而，目前还不清楚为什么细胞需要三个GEF的胞质分裂，以及它们如何合作，以调节赤道RhoA的激活和定位。这项资助申请的总体目标是了解MyoGEF如何调节胞质分裂，以及为什么哺乳动物细胞需要一个以上的GEF进行胞质分裂。我们的中心假设是Plk 1和极光B协调招募MyoGEF的中央纺锤体和卵裂沟，其中MyoGEF与ECT 2和/或GEF-H1合作，以调节RhoA激活和定位在卵裂沟。我们提出了两个目标来测试我们的中心假设，通过使用生物化学，细胞生物学和分子生物学的组合，与哺乳动物细胞作为模型。在具体目标#1中，我们将阐明Plk 1和aurora B如何与MyoGEF协调调节胞质分裂。这一目标将测试我们的工作假设，在后期，极光B创建一个Plk 1对接网站在MyoGEF，并允许Plk 1绑定和磷酸化MyoGEF，反过来促进招聘MyoGEF的中央纺锤体，从而有助于调节赤道RhoA激活和肌球蛋白收缩环组装。在具体目标#2中，我们将确定为什么哺乳动物细胞需要一个以上的GEF进行胞质分裂。这一目标将检验我们的工作假设：1）MyoGEF、ECT 2和/或GEF-H1冗余地调节某些细胞中的胞质分裂; 2）MyoGEF、ECT 2和/或GEF-H1参与细胞类型特异性的胞质分裂调节; 3）MyoGEF、ECT 2和GEF-H1协调赤道RhoA激活和定位的调节。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the molecular mechanism(s) that regulate(s) cytokinesis in mammalian cells, and to use this knowledge to develop novel cancer therapeutic strategies. Dysfunctional regulation of cytokinesis can lead to uncontrolled cell proliferation, a key characteristic of cancer. The integrity of cytokinesis relies on the precise coordinated regulation of the positioning and timing of contractile ring assembly. One of the prevalent models is that signals from the central spindle determine the positioning and timing of contractile ring assembly. Polo-like kinase 1 (Plk1) and aurora kinase B (aurora B) are two important mitotic kinases, which localize to the central spindle and coordinate with small GTPase signaling to regulate contractile ring assembly. The small GTPase proteins, such as RhoA, Rac1, and Cdc42, are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Thus far, at least three GEFs, i.e. ECT2, GEF-H1, and MyoGEF, are implicated in regulating cytokinesis. However, it is still not clear why cells need three GEFs for cytokinesis and how they cooperate to regulate equatorial RhoA activation and localization. The overall objective of this grant application is to understand how MyoGEF regulates cytokinesis and why mammalian cells need more than one GEF for cytokinesis. Our central hypothesis is that Plk1 and aurora B coordinate to recruit MyoGEF to the central spindle and cleavage furrow, where MyoGEF cooperates with ECT2 and/or GEF-H1 to regulate RhoA activation and localization at the cleavage furrow. We propose two aims to test our central hypothesis, by using a combination of biochemistry, cell biology, and molecular biology, with mammalian cells as a model. In Specific Aim #1, we will elucidate how Plk1 and aurora B coordinate with MyoGEF in regulating cytokinesis. This aim will test our working hypothesis that, during anaphase, aurora B creates a Plk1 docking site in MyoGEF and allows Plk1 to bind and phosphorylate MyoGEF, in turn promoting the recruitments of MyoGEF to the central spindle, thus contributing to the regulation of equatorial RhoA activation and myosin contractile ring assembly. In Specific Aim #2, we will determine why mammalian cells need more than one GEF for cytokinesis. This aim will test our working hypothesis: 1) MyoGEF, ECT2, and/or GEF-H1 redundantly regulate cytokinesis in certain cells; 2) MyoGEF, ECT2, and/or GEF-H1 are implicated in cell type-specific regulation of cytokinesis; 3) MyoGEF, ECT2, and GEF-H1 coordinate the regulation of equatorial RhoA activation and localization.