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

描述(由申请者提供)：Src家族激酶(SFK)的失调在肿瘤的发展，特别是转移中起着重要的作用。目前的治疗发展针对的是这些信号分子的酶活性。然而，这并不能完全阻断这些致癌蛋白的异常活性。最近对癌症中的激酶突变进行的一项无偏见的筛查显示，大量的失活突变表明了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，这将指导涉及SFK信号通路的癌症更好的治疗方法。除了催化活性，激酶还可以通过SH2和SH3结构域的蛋白质-蛋白质相互作用起到支架分子的作用。这些结构域的可及性可以通过构象变化来调节，构象变化由ATP结合位点上的配体结合来稳定。在这项建议中，SFK的SH2和SH3结构域可用于蛋白质-蛋白质相互作用，以及可用于多个ATP结合位点构象的翻译后修饰 使用两套生化分析方法进行测定。首先，荧光偏振、有限蛋白分解和下拉实验将被用来探测SH2和SH3结构域与分子间结合伙伴的可及性。其次，一系列的调节激酶和磷酸酶的酶分析将研究抑制物结合的SFK对翻译后修饰的敏感性。为了研究复杂生物系统中不同SH2和SH3结构域的可及性对分子间相互作用的影响，提出了一种使用能够稳定药物敏化的Src家族激酶的特定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结合位点构象对表达药物敏感的Src或Fyn门卫突变体的SFK基因敲除小鼠胚胎成纤维细胞中的Src/FAK复合体的形成、复合底物的磷酸化以及细胞运动的挽救的影响将被研究。用ATP竞争性抑制剂调节ATP结合部位外的蛋白质-蛋白质相互作用的能力将允许更好地控制激酶的功能，并有可能选择性地抑制特定的信号通路。这项研究应该通过开发选择性或协同失活催化和非催化功能的抑制剂来指导未来SFK抑制剂在异常信号网络中的治疗设计。此外，本方案中开发的技术将广泛适用于其他激酶家族，提供了强大的 了解致癌系统中的激酶功能的工具。

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.