Substrate specificity of nonreceptor tyrosine kinases

非受体酪氨酸激酶的底物特异性

• 批准号: 7849943
• 负责人: W Todd MILLER
• 金额: $ 22.08万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7849943
• 关键词: Active Sites; Adaptor Signaling Protein; Address; Affinity; Animals; Antineoplastic Agents; Apoptosis; Binding; Binding Sites; Biochemical; Biological; Breast Cancer Cell; Calorimetry; Catalytic Domain; Cells; Collaborations; Data; Differentiation and Growth; ERBB2 gene; Environment; Enzymes; Eukaryotic Cell; Evolution; Family; Focal Adhesions; Genome; Goals; Heteronuclear NMR; Human; Hydrogen; Immunoprecipitation; In Vitro; Lead; Length; Ligands; Malignant Neoplasms; Mammalian Cell; Mammary Tumorigenesis; Maps; Mass Spectrum Analysis; Modeling; Mus; Organism; Peptides; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Phosphotyrosine; Polymers; Positioning Attribute; Protein Tyrosine Kinase; Proteins; Proteomics; Reaction; Regulation; Role; SH3 Domains; Signal Pathway; Signal Transduction; Signaling Protein; Site; Structure; Substrate Domain; Substrate Specificity; Testing; Titrations; Variant; base; cell transformation; design; human BCAR1 protein; human protein tyrosine kinase brk; in vivo; insight; kinase inhibitor; malignant breast neoplasm; migration; mutant; overexpression; public health relevance; research study; src Homology Region 2 Domain; src-Family Kinases; stable cell line; tumor

DESCRIPTION (provided by applicant): Tyrosine kinases are important regulators of growth, differentiation, and apoptosis in eukaryotic cells. Inappropriate activation of tyrosine kinases often occurs in human cancers. The goal of this project is to understand the contributions of the catalytic and noncatalytic domains of nonreceptor tyrosine kinases (NRTKs) in substrate recognition and regulation. These studies could provide a basis for the design of molecules to interfere with the recognition of specific substrates by NRTKs. In Aim 1, we will investigate the hypothesis that the identity, arrangement, and placement of the noncatalytic domains govern Src kinase substrate specificity. First, we will carry out experiments with peptide and protein substrates, and mutant forms of Src in which the domains have been rearranged. Second, we have shown that Src substrate specificity can be reengineered by replacing the natural SH3 and SH2 domains with heterologous PDZ domains. We will study substrate recognition by these chimeric PDZ-kinases, and construct artificial signaling pathways by PDZ targeting. Third, we will characterize tyrosine kinases with unusual domain combinations from the unicellular organism Monosiga brevicollis. Aim 2 focuses on the processive phosphorylation of the Cas adaptor protein; we hypothesize that processive phosphorylation is critical for downstream signaling. We will test the importance of the Src SH2 and SH3 domains on processive phosphorylation, cell transformation, and migration. To gain insight into the biological purpose of processive phosphorylation, we will study Cas mutants in which the substrate domain has been replaced by arrays of polymerized phosphorylation motifs. We will use heteronuclear NMR to determine the structure of a Cas peptide bound in the active site of Src. Aim 3 focuses on Brk (breast tumor kinase), a NRTK related to Src. In collaboration with Dr. Senthil Muthuswamy, we recently showed that Brk and ErbB2 are co-amplified and co-overexpressed in human breast cancers. We will study the biochemical basis for the interaction between ErbB2 and Brk. In particular, we will test the hypothesis that autophosphorylation sites on ErbB2 bind to the SH2 domain of Brk, releasing auto inhibition. To test the model that the C-terminus of Brk interacts with the enzyme's SH2 domain, we will use hydrogen-exchange mass spectrometry (HXMS). We will carry out proteomic experiments to identify proteins that interact with Brk in cells co-expressing Brk and ErbB2. These studies may provide a framework for strategies to block the activity of Brk in ErbB2-positive breast cancers. PUBLIC HEALTH RELEVANCE: Tyrosine kinases are enzymes that enable normal mammalian cells to respond to signals from their environment that cause the cells to grow and divide. In many forms of cancer, however, tyrosine kinases such as Src or Brk (breast tumor kinase) are inappropriately active. This project investigates the mechanisms by which Src and Brk recognize cellular proteins; the experiments will provide new information that could be used to develop specific kinase inhibitors as anticancer agents.

描述（由申请人提供）：酪氨酸激酶是真核细胞生长，分化和凋亡的重要调节剂。酪氨酸激酶的不当激活通常发生在人类癌症中。该项目的目的是了解非受体酪氨酸激酶（NRTKS）在底物识别和调节中的催化和非催化结构域的贡献。这些研究可以为设计分子的设计提供基础，以干扰NRTK对特定底物的识别。在AIM 1中，我们将研究以下假设：非催化结构域的身份，排列和位置控制SRC激酶底物特异性。首先，我们将使用肽和蛋白质底物进行实验，以及已重新排列域的SRC突变形式。其次，我们已经表明，可以通过用异源PDZ域代替天然SH3和SH2域来重新设计SRC底物特异性。我们将研究这些嵌合PDZ-激酶的底物识别，并通过PDZ靶向构建人工信号通路。第三，我们将表征来自单细胞生物Monosiga Brevicollis的异常域组合的酪氨酸激酶。 AIM 2专注于CAS适配器蛋白的磷酸化；我们假设过程磷酸化对于下游信号传导至关重要。我们将测试SRC SH2和SH3结构域对过程磷酸化，细胞转化和迁移的重要性。为了深入了解过程磷酸化的生物学目的，我们将研究CAS突变体，其中底物结构域已被聚合磷酸化基序替代。我们将使用异核NMR确定在SRC活性位点结合的CAS肽的结构。 AIM 3专注于BRK（乳腺肿瘤激酶），这是与SRC相关的NRTK。与Senthil Muthuswamy博士合作，我们最近表明BRK和ERBB2在人类乳腺癌中共同扩增和共存。我们将研究ERBB2和BRK之间相互作用的生化基础。特别是，我们将检验以下假设：ERBB2上的自磷酸化位点与BRK的SH2结构域结合，从而释放自动抑制。为了测试BRK的C端与酶的SH2结构域相互作用的模型，我们将使用氢交换质谱法（HXMS）。我们将进行蛋白质组学实验，以鉴定与BRK和ERBB2共同表达的细胞中与BRK相互作用的蛋白质。这些研究可能为阻止BRK在ERBB2阳性乳腺癌中的活性提供框架。公共卫生相关性：酪氨酸激酶是使正常哺乳动物细胞从其环境中响应的信号，导致细胞生长和分裂的酶。然而，在许多形式的癌症中，酪氨酸激酶（如SRC或BRK（乳腺肿瘤激酶））不当活跃。该项目研究了SRC和BRK识别细胞蛋白的机制。这些实验将提供可用于开发特定激酶抑制剂作为抗癌剂的新信息。