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

描述（由申请人提供）：Src家族激酶（SFKs）的失调在肿瘤发展，特别是转移中起重要作用。目前的治疗发展目标是这些信号分子的酶活性。然而，这可能无法完全阻断这些致癌蛋白的异常活性。最近对癌症中激酶突变的无偏筛选揭示了大量失活突变，表明激酶在癌症生物学中的重要非催化功能。越来越多的证据表明，蛋白激酶的非催化作用，如支架，亚细胞靶向或DNA结合，是必不可少的，在某些情况下足以发挥作用。我们研究的长期目标是更好地了解SFK成员的非催化功能，这将指导更好地治疗涉及SFK信号通路的癌症。除了催化活性之外，激酶可以通过SH 2和SH 3结构域经由蛋白质-蛋白质相互作用充当支架分子。这些结构域的可接近性可以通过构象变化来调节，所述构象变化通过ATP结合位点中的配体结合来稳定。在该提议中，SFK的SH 2和SH 3结构域对于蛋白质-蛋白质相互作用的可用性和在多个ATP结合位点构象中对翻译后修饰的可及性将 可以使用两组生化测定来确定。首先，将使用荧光偏振、有限蛋白水解和下拉测定来探测SH 2和SH 3结构域对分子间结合伴侣的可及性。第二，一系列用调节激酶和磷酸酶的酶促测定将研究抑制剂结合的SFK对翻译后修饰的敏感性。为了研究在复杂的生物系统中不同的SH 2和SH 3结构域可及性对分子间相互作用的影响，使用能够稳定药物致敏的Src家族激酶的特异性ATP结合位点构象的正交激酶配体的化学遗传学方法。最后，稳定不同的SFK ATP结合位点构象的影响Src/FAK复合物的形成，磷酸化的复杂的底物，和救援的细胞运动将在SFK基因敲除小鼠胚胎成纤维细胞表达药物致敏的Src或Fyn看门突变体。用ATP竞争性抑制剂调节ATP结合位点之外的蛋白质-蛋白质相互作用的能力将允许更好地控制激酶功能，并且具有选择性阻断特异性信号传导途径的潜力。本研究将指导未来SFK抑制剂在异常信号网络中的治疗设计，通过开发选择性或协同性地抑制催化和非催化功能的抑制剂。此外，该提案中开发的技术将广泛适用于其他激酶家族，提供强大的 了解致癌系统中激酶功能的工具。

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.