The mechanism of Arg kinase activation by integrin B1

整合素B1激活Arg激酶的机制

• 批准号: 8282334
• 负责人: Titus Jonathon Boggon
• 金额: $ 32.33万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8282334
• 关键词: Adhesions; Adolescent; Affinity; Atrophic; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biological Assay; Biological Models; Brain; Calorimetry; Cell Surface Receptors; Cells; Chromosomal translocation; Complex; Cyclins; Cytoplasmic Tail; Data; Dendrites; Dendritic Spines; Development; Disease; Down-Regulation; Event; Extracellular Matrix; Fibroblasts; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Genetic; Growth Factor Receptors; Guanosine Triphosphate Phosphohydrolases; Hippocampus (Brain); Human; Immune system; Integrin Binding; Integrins; Lead; Lobe; Malignant Neoplasms; Measures; Mediating; Modeling; Molecular; Mus; Mutagenesis; N-terminal; Nervous System Physiology; Nervous system structure; Neurons; Peptides; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protein Binding; Protein Tyrosine Kinase; Psychological reinforcement; Receptor Signaling; Recruitment Activity; Relative (related person); Resolution; Role; Series; Signal Transduction; Solid Neoplasm; Structure; Surface; Synapses; Systems Development; Tail; Testing; Titrations; Work; X-Ray Crystallography; adhesion receptor; base; cancer therapy; design; excitatory neuron; immune function; in vivo; leukemia; mutant; novel; novel strategies; protein protein interaction; receptor; receptor binding; reconstitution; scaffold; src Homology Region 2 Domain; tumor progression

DESCRIPTION (provided by applicant): Integrin adhesion receptor signaling through the Abl and Arg nonreceptor tyrosine kinases activates several major cytoskeletal effectors to coordinate changes in cytoskeletal structure. Abl/Arg-mediated signaling events are essential for nervous and immune system development and function, while inappropriately elevated Abl/Arg signaling is associated with several cancers. Despite these critical physiological roles, the mechanisms by which integrins activate Abl and Arg are poorly understood. Based on extensive preliminary evidence, we hypothesize a multistage model for integrin ¿1 activation of Arg kinase activity that we will test in three Aims. Our first aim is to identify and characterize he interfaces that mediate initial Arg recruitment to the integrin ¿1 tail. We find that a sequence in the integrin ¿1 tail not known to interact with other integrin-binding proteins binds directly to te isolated Arg kinase domain and activates Arg kinase activity. We will identify the interfaces on integrin ¿1 and Arg that mediate these interactions and use mutagenesis and binding assays to determine the contributions of specific residues to this interaction. We have expressed, purified and obtained crystals of the Arg kinase domain. We will now use X-ray crystallography to determine the structure of the Arg kinase domain in complex with integrin ¿1 tail peptides. Our second aim is to investigate the dual roles that the Arg SH2 domain plays in both retainment and reinforcement of Arg kinase activation. Arg can phosphorylate integrin ¿1 tail directly, thereby creating a binding site for the Arg SH2 domain, which we hypothesize retains Arg and promotes optimal kinase activation. We will use biochemistry and X-ray crystallography to elucidate an atomic resolution understanding of how the Arg SH2 domain and kinase domains bind coordinately to the phosphorylated integrin ¿1 tail. As part of this work, we will test the hypothesis that coordinated binding to the phosphorylated integrin ¿1 tail promotes "cyclin-like" Arg SH2 domain binding to the Arg kinase domain N-terminal lobe to reinforce kinase activation. Our third aim is to investigate the role of the recruitment, retainment, and reinforcement model in regulating Arg signaling and its control of dendritic spine and dendrite stability in vivo. Interin signaling through Arg and its substrate p190RhoGAP negatively regulates the RhoA GTPase, a major antagonist of synapse, dendritic spine, and dendrite stability. Genetic studies suggest that integrin ¿1 interacts functionally with Arg to mediate dendritic spine and dendrite stabilization i the adolescent mouse brain. We will generate integrin ¿1 and Arg mutants deficient in interactions that mediate Arg kinase recruitment, retainment, or reinforcement, and reconstitute them into integrin ¿1- or Arg-deficient fibroblasts and neurons. We will employ biochemical, FRET, and cell-based assays to determine how these interfaces contribute to integrin ¿1: Arg interactions and Arg- mediated signaling in fibroblasts and neurons, and control of dendritic spine and dendrite stability in neurons. PUBLIC HEALTH RELEVANCE: The Abl and Arg nonreceptor tyrosine kinases regulate the development and function of the immune and nervous sytstems. Chromosomal translocations involving the Abl and Arg nonreceptor tyrosine kinases have long been known to cause leukemia in humans and increased activation of native Abl and Arg may be associated with cancer progression of solid tumors, but how these kinases are activated by upstream receptors is unclear. We have found that direct binding of the intracellular tail of the integrin receptor ¿1 subunit can activate the Arg tyrosine kinase. We propose to determine the mechanism and structural basis for integrin ¿1 activation of Arg. We anticipate these studies will lead to new approaches to target Abl and Arg signaling in the treatment of cancer and other diseases.

描述（由适用提供）：通过ABL和ARG非受体酪氨酸激酶通过ABL和ARG的整合素粘合受体信号传导激活了几种主要的细胞骨架效应，以协调细胞骨架结构的变化。 ABL/ARG介导的信号传导事件对于神经和免疫系统的发展和功能至关重要，而ABL/ARG信号不当升高与多种癌症有关。尽管有这些关键的生理作用，但对整联蛋白激活ABL和ARG的机制知之甚少。基于广泛的初步证据，我们假设一个整合素的多阶段模型»ARG激酶活性的激活，我们将以三个目标进行测试。我们的第一个目的是识别和表征他将媒体介绍到整合素»1尾的媒体介绍的界面。我们发现在 整联蛋白�1尾巴与其他整合素结合蛋白相互作用的尾巴直接与孤立的ARG激酶结构域结合并激活ARG激酶活性。我们将确定整联蛋白上的接口，并介导这些相互作用并使用诱变和结合测定来确定特定抗性对这种相互作用的贡献。我们已经表达，纯化和获得的ARG激酶结构域的晶体。现在，我们将使用X射线晶体学来确定与整联蛋白»1尾肽中ARG激酶结构域的结构。我们的第二个目的是研究ARG SH2结构域在ARG激酶激活的保留和增强中起着的双重作用。 ARG可以直接磷酸化整联蛋白»1尾部，从而为ARG SH2结构域创建一个结合位点，我们假设该结构域保留ARG并促进最佳激酶激活。我们将使用生物化学和X射线晶体学来阐明对ARG SH2结构域和激酶结构域如何与磷酸化的整联蛋白»1尾的共同结合的原子分辨率理解。作为这项工作的一部分，我们将测试以下假设：与磷酸化整联蛋白的协调结合»1尾促进了“细胞周期蛋白样” ARG SH2结构域与ARG激酶结构域N末端叶的结合以增强激酶激活。我们的第三个目的是研究募集，保留和强化模型在调节ARG信号传导中的作用及其对体内树突状脊柱和树突稳定性的控制。通过ARG及其底物P190RHOGAP的信号传导对RhoA GTPase进行负调节，RhoA GTPase是突触，树突状脊柱和树突稳定性的主要拮抗剂。遗传学研究表明，整联蛋白»1与ARD在功能上相互作用，介导了树突状脊柱和树突稳定I青少年小鼠脑。我们将产生整联蛋白�1，并在介导ARG激酶募集，保留或增强的相互作用中缺乏ARG突变体，并将其重新构成整合素»1-或ARG缺乏的成纤维细胞和神经元。我们将采用生化，FRET和基于细胞的测定法来确定这些界面如何对整合素€1：ARG相互作用和在成纤维细胞和神经元中的ARG相互作用和ARG介导的信号传导，以及对神经元中树突状脊柱和树突状稳定性的控制。 公共卫生相关性：ABL和ARG非受体酪氨酸激酶调节免疫和神经系统的发展和功能。长期以来，涉及ABL和ARG非受体酪氨酸激酶的染色体易位已知会引起人类白血病，而天然ABL和ARG的激活可能与实体瘤的癌症进展有关，但是这些激酶是如何被上游受体激活的。我们发现整联蛋白受体的细胞内尾巴的直接结合„ 1 亚基可以激活ARG酪氨酸激酶。我们建议确定整联蛋白的机制和结构基础»ARG的激活。我们预计这些研究将导致靶向ABL和ARG信号的新方法，以治疗癌症和其他疾病。