The mechanism of Arg kinase activation by integrin B1

描述（由申请人提供）：通过Abl和Arg非受体酪氨酸激酶的整合素粘附受体信号传导激活几种主要的细胞骨架效应物，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发育和功能是必不可少的，而不适当地升高的Abl/Arg信号传导与几种癌症相关。尽管有这些重要的生理作用，整合素激活Abl和Arg的机制知之甚少。基于广泛的初步证据，我们假设整合素1激活Arg激酶活性的多阶段模型，我们将在三个目标中进行测试。我们的第一个目标是确定和表征介导初始Arg募集到整合素1尾部的界面。我们发现， 整合素1尾不知道与其他整合素结合蛋白的相互作用直接结合到分离的精氨酸激酶结构域和激活精氨酸激酶活性。我们将确定介导这些相互作用的整合素1和精氨酸的界面，并使用诱变和结合试验来确定特定残基对这种相互作用的贡献。我们已经表达，纯化和获得晶体的精氨酸激酶结构域。我们现在将使用X射线晶体学来确定与整合素1尾肽复合的Arg激酶结构域的结构。我们的第二个目的是研究Arg SH 2结构域在保留和增强Arg激酶激活中所起的双重作用。Arg可以直接磷酸化整联蛋白1尾，从而为Arg SH 2结构域创造一个结合位点，我们假设它保留了Arg并促进了最佳的激酶活化。我们将使用生物化学和X-射线晶体学来阐明Arg SH 2结构域和激酶结构域如何协调结合磷酸化整联蛋白1尾的原子分辨率理解。作为这项工作的一部分，我们将测试的假设，协调结合磷酸化整联蛋白1尾巴促进“细胞周期蛋白样”精氨酸SH 2域结合精氨酸激酶域N-末端叶，以加强激酶激活。我们的第三个目的是研究的招聘，保留和强化模型中的作用，调节精氨酸信号和树突棘和树突的稳定性在体内的控制。通过Arg和其底物p190 RhoGAP的Interin信号传导负调节RhoA GTf 3，其是突触、树突棘和树突稳定性的主要拮抗剂。遗传学研究表明，整合素1在功能上与精氨酸相互作用，以介导青春期小鼠大脑中树突棘和树突的稳定。我们将产生整合素。1和Arg突变体缺乏相互作用，介导Arg激酶募集，保留或加强，并将其重组为整合素1或Arg缺陷的成纤维细胞和神经元。我们将采用生物化学，FRET和基于细胞的测定来确定这些界面如何有助于成纤维细胞和神经元中的整合素1：Arg相互作用和Arg介导的信号传导，以及神经元中树突棘和树突稳定性的控制。

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.