Investigation of the catalytic and non-catalytic roles of Src family kinases in o

Src 家族激酶在 o 中的催化和非催化作用的研究

• 批准号: 8771265
• 负责人: Stephen Leonard
• 金额: $ 1.92万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2014-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8771265
• 关键词: Accounting; Active Sites; Adopted; Adoption; Affect; Binding; Binding Sites; Biochemical; Biological Assay; Cancer Biology; Catalytic Domain; Cells; Chemicals; Complex; DNA Binding; Development; Drug Design; Embryo; Enzymes; Event; Family; Fibroblasts; Fluorescence Polarization; Future; Gatekeeping; Goals; Health; Integrins; Investigation; Knockout Mice; Ligand Binding; Ligands; Malignant Neoplasms; Molecular; Molecular Conformation; Mutation; Neoplasm Metastasis; Oncogenic; Outcome; PTK2 gene; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Predisposition; Protein Kinase; Proteins; Proteolysis; Regulation; Research; Role; SH3 Domains; Scaffolding Protein; Series; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; System; Technology; Therapeutic; cell motility; chemical genetics; complex biological systems; design; drug development; drug discovery; inhibitor/antagonist; kinase inhibitor; member; mutant; protein function; protein protein interaction; scaffold; small molecule; src Homology Region 2 Domain; src-Family Kinases; therapeutic development; tool; tumor

DESCRIPTION (provided by applicant): Disregulation of Src family kinases (SFKs) plays an important role in tumor development, specifically metastasis. Current therapeutic development targets the enzymatic activity of these signaling molecules. However, this may not completely block the aberrant activities of these oncogenic proteins. A recent unbiased screen of kinase mutations in cancers revealed a large number of inactivating mutations indicating important non-catalytic functions of kinases in cancer biology. Accumulating evidence suggests that non-catalytic roles of protein kinases such as scaffolding, subcellular targeting or DNA binding, are essential and in some cases sufficient for function. The long term goal of our study is to gain better understanding of the non-catalytic functions of SFK members which will guide a better therapeutic approach to cancers involving SFK signaling pathways. In addition to catalytic activity, kinases can act as scaffolding molecules via protein-protein interactions through SH2 and SH3 domains. Accessibility of these domains can be regulated through conformational changes which are stabilized by ligand binding in the ATP-binding site. In this proposal, the availability of the SH2 and SH3 domains of SFKs for protein-protein interactions and accessibility for post- translational modifications in multiple ATP-binding site conformations will be determined using two sets of biochemical assays. First, fluorescence polarization, limited proteolysis and pull-down assays will be used to probe the accessibility of the SH2 and SH3 domains to inter-molecular binding partners. Second, a series of enzymatic assays with regulatory kinases and phosphatases will investigate the susceptibility of inhibitor- bound SFKs to post-translational modifications. In order to study the effects of varying SH2 and SH3 domain accessibility to inter-molecular interactions in a complex biological system, a chemical genetic approach using orthogonal kinase ligands capable of stabilizing specific ATP-binding site conformations of drug-sensitized Src family kinases. Finally, the effects of stabilizing distinct SFK ATP-binding site conformations on Src/FAK complex formation, phosphorylation of complex substrates, and rescue of cell motility will be investigated in SFK-knockout mouse embryonic fibroblasts expressing drug-sensitized Src or Fyn gatekeeper mutants. The ability to modulate protein-protein interactions outside of the ATP binding site with ATP-competitive inhibitors will allow greater control of kinase function and has the potential to selectively inhibt specific signaling pathways. This study should guide the future therapeutic design of SFK inhibitors in aberrant signaling networks by developing inhibitors which selectively or cooperatively inactivate catalytic and non-catalytic functions. Furthermore, the technology developed in this proposal will be widely applicable to other kinase families, providing a powerful tool in understanding kinase function in oncogenic systems.

描述（由申请人提供）：脱离SRC家族激酶（SFK）在肿瘤发育，特别是转移中起重要作用。当前的治疗发育靶向这些信号分子的酶活性。但是，这可能无法完全阻止这些致癌蛋白的异常活性。最近在癌症中的激酶突变的无偏筛网表明，大量的灭活突变表明激酶在癌症生物学中的重要非催化功能。积累的证据表明，蛋白激酶（例如脚手架，亚细胞靶向或DNA结合）的非催化作用是必不可少的，在某些情况下是足够的功能。我们研究的长期目标是更好地了解SFK成员的非催化功能，这将指导涉及SFK信号通路的癌症更好的治疗方法。除催化活性外，激酶还可以通过SH2和SH3结构域通过蛋白质 - 蛋白质相互作用充当支架分子。这些结构域的可访问性可以通过构象变化来调节，构象变化通过ATP结合位点的配体结合稳定。在此提案中，SFK的SH2和SH3域的可用性用于蛋白质 - 蛋白质相互作用以及多个ATP结合位点构象的转化后修饰的可访问性 使用两组生化测定法确定。首先，将使用荧光极化，有限的蛋白水解和下拉测定法来探测SH2和SH3结构域对分子间结合伴侣的可访问性。其次，一系列具有调节激酶和磷酸酶的酶试验将研究抑制剂结合的SFK对翻译后修饰的敏感性。为了研究复杂的生物系统中不同SH2和SH3结构域的可及性对分子间相互作用的影响，一种使用正交激酶配体的化学遗传方法，能够稳定药物敏化的SRC家族激酶的特定ATP结合位点构象。最后，将在SFK-KNOCKOUT小鼠胚胎成纤维细胞中研究表达药物敏感的SRC或Fyn守门员突变体的SFK-KNOCKOUT小鼠胚胎成纤维细胞中稳定不同SFK ATP结合位点构象对SRC/FAK复合物形成，复杂底物的磷酸化以及挽救细胞运动的影响的影响。在ATP结合位点外调节蛋白质蛋白相互作用与ATP竞争力抑制剂之外的能力将允许更好地控制激酶功能，并有可能选择性地抑制特定的信号传导途径。这项研究应通过开发有选择性或合作失活的催化和非催化功能的抑制剂来指导异常信号网络中SFK抑制剂的未来治疗设计。此外，该提案中开发的技术将广泛适用于其他激酶家庭，提供强大的功能 了解激酶功能在致癌系统中的工具。