Defining how Abelson Kinase Regulates Cell Adhesion and Actin Dynamics

项目总结 细胞自组装成器官的能力是非同寻常的，需要细胞之间的紧密协调 机械调节黏附和细胞骨架。这种协调能力的缺陷会导致疾病 从发育缺陷到癌症；因此，了解 实现了细胞黏附和肌动蛋白细胞骨架的协调。这一过程的一个调节者是Abelson 酪氨酸激酶(Abl)，它连接受体、小接头蛋白和细胞骨架调节因子。ABL激酶 是关键的癌基因和发育调节因子。目前存在三种ABL函数模型：1)ABL 磷酸化蛋白质靶标，从而改变它们的功能并影响细胞行为，2)Abl直接 通过其保守的C末端肌动蛋白结合域(FABD)调节细胞骨架动力学，以及3)Abl 作为组装多蛋白质信号复合体的支架。然而，在这个过程中每个角色的重要性 形态发生仍未得到测试。为了区分这些模型，我们测试了缺乏突变的abl蛋白。 激酶活性，F-肌动蛋白结合，或参与蛋白质相互作用的基序。我们的初步数据显示 不同的突变体对从背部闭合到根尖收缩的不同形态发生事件有不同的影响 中枢神经系统轴突生长。值得注意的是，虽然完整的abl活性需要有激酶活性，但缺乏激酶的突变体 活性或FABD在形态发生过程中保持着重要的功能。相反，一个保守的PXXP主题在 在形态发生的某些方面，连接区对Abl的功能比两个激酶都更重要。 活动和FABD。我们还确定了PXXP主题的候选合作伙伴，并发现他们会影响 过程与受Abl影响的过程相似。因此，我们假设ABL作为一台强大的监管机器发挥作用 在形态发生过程中，其作用机制的不同方面(例如，激酶活性，FABD结构域， PXXP有约束力的伙伴等)对某些生物过程具有不同的重要性。我们将通过以下途径对此进行测试 具体目标如下：(1)确定abl的特定功能域如何对其有不同的贡献。 以果蝇背部闭合(DC)为模型，研究PXXP在形态发生过程中的作用； 结合伙伴与Abl一起调节DC过程中的动态细胞行为。具体来说，我们将使用 结合生化、遗传和定量细胞生物学方法来定义ABL的不同之处 作用机制有助于其塑造细胞动力学，组织细胞之间的功能联系 肌动蛋白细胞骨架和细胞黏附，以及塑造多个下游肌动蛋白之间的调控相互作用 监管机构，包括Ena、Dia和Abi。同时，我们将确定Abl及其PXXP绑定伙伴是如何 (ABI或Crk)调节胚胎形态发生过程中的动态细胞行为。这项研究将揭示Abl 及其相互作用的伙伴在发育过程中调节细胞黏附和肌动蛋白动态，提供 关于Abl如何在人类发育和疾病期间调节动态细胞行为的新线索。

PROJECT SUMMARY The ability of cells to self-assemble into organs is extraordinary and requires tight coordination between the cell machinery modulating adhesion and the cytoskeleton. Defects in this coordination contribute to diseases ranging from developmental defects to cancer; thus it is critical to understand the mechanisms by which coordination of cell adhesion and the actin cytoskeleton is achieved. One regulator of this process is Abelson tyrosine kinase (Abl), which links receptors, small adaptor proteins, and cytoskeletal regulators. Abl kinases are key oncogenes and developmental regulators. Three models for Abl function currently exist: 1) Abl phosphorylates protein targets, thus altering their function and influencing cell behavior, 2) Abl directly modulates cytoskeletal dynamics through its conserved C-terminal actin binding domain (FABD), and 3) Abl acts as a scaffold to assemble multi-protein signaling complexes. However, the importance of each role during morphogenesis remains untested. To distinguish between these models, we tested mutant Abl proteins lacking kinase activity, F-actin binding, or motifs involved in protein interactions. Our preliminary data reveal that different mutants differentially affect distinct morphogenetic events from dorsal closure to apical constriction to CNS axon outgrowth. Strikingly, while kinase activity is required for full Abl activity, mutants lacking kinase activity or the FABD retain significant function during morphogenesis. Instead, a conserved PXXP motif within the linker region is more essential for Abl function during some aspects of morphogenesis than both kinase activity and the FABD. We also have identified candidate partners for the PXXP motif and found they affect processes similar to those affected by Abl. Thus, we hypothesize Abl functions as a robust regulatory machine during morphogenesis, with different aspects of its mechanisms of action (e.g., kinase activity, FABD domain, PXXP binding partners, etc.) being differentially important for certain biological processes. We will test this via the following specific aims: (1) Determine how specific functional domains of Abl differentially contribute to its function during morphogenesis using Drosophila dorsal closure (DC) as a model; (2) Determine how PXXP- binding partners work with Abl to regulate dynamic cell behaviors during DC. Specifically, we will use a combination of biochemical, genetic, and quantitative cell biological approaches to define how Abl's different mechanisms of action contribute to its ability to shape cell dynamics, organize functional linkages between the actin cytoskeleton and cell adhesions, and to shape regulatory interactions between multiple downstream actin regulators, including Ena, Dia, and Abi. In parallel, we will determine how Abl and its PXXP-binding partners (Abi or Crk) regulate dynamic cell behaviors during embryonic morphogenesis. This study will reveal how Abl and its interacting partners regulate cell adhesion and actin dynamics in a developmental context, providing new clues as to how Abl works to regulate dynamic cell behaviors during human development and disease.