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，特别是转移中起着重要作用。目前的治疗发展目标是这些信号分子的酶活性。然而，这可能不能完全阻断这些致癌蛋白的异常活动。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具有重要的非催化功能。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，这将指导更好的治疗涉及SFK信号通路的癌症的方法。除了催化活性外，激酶还可以通过SH2和SH3结构域通过蛋白-蛋白相互作用充当脚手架分子。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结合位点的配体结合来稳定。在本研究中，sfk的SH2和SH3结构域在蛋白-蛋白相互作用中的可用性以及在多个atp结合位点构象中翻译后修饰的可及性将被进一步研究

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.