Defining how Abelson Kinase Regulates Cell Adhesion and Actin Dynamics

定义 Abelson 激酶如何调节细胞粘附和肌动蛋白动力学

• 批准号: 9407705
• 负责人: Andrew J Spracklen
• 金额: $ 5.92万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9407705
• 关键词: Actins; Adaptor Signaling Protein; Address; Adhesions; Affect; Apical; Axon; Binding; Biochemical; Biochemical Genetics; Biological; Biological Process; C-terminal; Cell Adhesion; Cell Shape; Cell-Cell Adhesion; Cells; Complex; Cytoskeleton; Data; Defect; Development; Disease; Dorsal; Drosophila genus; Embryo; Embryonic Development; Event; F-Actin; Genetic; Goals; Human Development; Image; Individual; Lead; Link; Malignant Neoplasms; Mammals; Microscopy; Modeling; Morphogenesis; Neuraxis; Oncogenes; Oncogenic; Organ; PXXP Motif; Pattern; Phosphotransferases; Play; Process; Protein Tyrosine Kinase; Proteins; RNA Interference; Regulation; Resolution; Role; Shapes; Signal Transduction; Solid Neoplasm; Systems Development; Testing; Work; cell behavior; cell motility; constriction; human disease; insight; leukemia; mutant; novel; receptor; scaffold

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.

项目摘要 细胞自我组装成器官的能力是非凡的，需要细胞之间的紧密协调。 机械调节粘附和细胞骨架。这种协调的缺陷会导致疾病 从发育缺陷到癌症;因此，了解其机制至关重要， 实现了细胞粘附和肌动蛋白细胞骨架的协调。这个过程的一个监管者是Abelson 酪氨酸激酶（Abl），其连接受体、小衔接蛋白和细胞骨架调节剂。Abl激酶类 是关键的致癌基因和发育调节因子。目前存在三种Abl功能模型：1）Abl 磷酸化蛋白质靶点，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-末端肌动蛋白结合结构域（FABD）调节细胞骨架动力学，和3）Abl 充当组装多蛋白质信号传导复合物的支架。然而，每个角色的重要性 形态发生尚未经过测试。为了区分这些模型，我们测试了缺乏 激酶活性、F-肌动蛋白结合或蛋白质相互作用中涉及的基序。我们的初步数据显示， 不同的突变体差异影响不同的形态发生事件，从背部关闭顶端收缩， 中枢神经系统轴突生长。引人注目的是，虽然激酶活性是完整Abl活性所必需的，但缺乏激酶活性的突变体是不稳定的。 活性或FABD在形态发生期间保留显著功能。相反，一个保守的PXXP基序， 在形态发生某些方面，接头区比两种激酶对Abl功能更重要 活动和FABD。我们还确定了PXXP基序的候选伙伴，并发现它们影响 因此，我们假设Abl功能是一个强大的调节机器， 在形态发生期间，其作用机制的不同方面（例如，激酶活性，FABD结构域， PXXP结合伴侣等）对某些生物过程的重要性不同。我们将通过 （1）确定Abl的特定功能结构域如何差异性地贡献其功能， 使用果蝇背侧闭合（DC）作为模型，在形态发生过程中发挥作用;（2）确定PXXP- 结合配偶体与Abl一起调节DC期间的动态细胞行为。具体来说，我们将使用 结合生物化学，遗传学和定量细胞生物学方法来确定Abl的不同 作用机制有助于其塑造细胞动力学的能力，组织细胞之间的功能联系， 肌动蛋白细胞骨架和细胞粘附，并形成多个下游肌动蛋白之间的调节相互作用， 监管机构，包括Ena，Dia和Abi。同时，我们将确定Abl和它的PXXP结合伙伴 (Abi或Crk）调节胚胎形态发生期间的动态细胞行为。这项研究将揭示Abl 及其相互作用的伙伴调节细胞粘附和肌动蛋白动力学在发展的背景下，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。