Conformational Control of Protein Kinases

蛋白激酶的构象控制 抽象的。激酶信号往往过于简单，只关注下游磷酸化网络 由蛋白激酶（PKs）催化。这种传统的激酶信号转导方法忽略了非催化的 这些功能对于健康细胞和疾病细胞中的细胞信号传导至关重要。许多PK包含监管 域（例如，SH2和SH3结构域），其与激酶串联以及独立地起作用 催化域PKs的非催化功能通常依赖于蛋白质-蛋白质相互作用 在激酶的多结构域结构和相互作用蛋白（通常是其他激酶或支架）之间 蛋白质）。此外，这些蛋白质-蛋白质相互作用由蛋白质构象的大的变化来调节。 蛋白激酶的四级结构。有证据表明，蛋白激酶的非催化功能， 然而，由于缺乏遗传和基因调控，这些功能对细胞信号传导至关重要， 化学工具来研究它们。尽管非催化激酶信号的作用正在出现，但 了解激酶构象对信号通路的重要性一直受到缺乏工具的阻碍 以评估和调节PKs的整体构象。 在之前的资助期间，我们开发了一种“选择性蛋白水解”方法来表征 蛋白激酶的四级构象。这种方法很快被学术界和 然而，该方法依赖于嗜热菌蛋白酶切割SH2-KD接头的能力， 在激酶内，并且不是所有的激酶都具有这种带有适当识别序列的接头。在这里，我们建议 一个基于细胞的生物传感器，报告激酶构象。使用我们的激酶构象生物传感器，我们 建议研究蛋白质构象在单点突变中的作用，这些突变导致对临床 激酶抑制剂。除了提供关于耐药突变的构象影响的信息外， 所提出的研究将为理解Abl，BTK， BRAF和EGFR激酶。最后，我们建议研究变构和ATP位点抑制剂的相互作用， 开发Abl和c-Src激酶的多价激酶抑制剂。多价抑制剂将由以下物质构建： 匹配构象变构和ATP位点抑制剂。这项提议将开发新的生物测定方法来研究 蛋白激酶构象，以更好地了解蛋白激酶的耐药性，并开发 研究构象在激酶信号传导中的作用的新型抑制剂。

Conformational Control of Protein Kinases Abstract. Kinase signaling is often oversimplified by focusing only on the downstream phosphorylation networks catalyzed by protein kinases (PKs). This traditional approach to kinase signaling ignores the noncatalytic functions that are critical for cellular signaling in both healthy and disease cells. Many PKs contain regulatory domains (e.g., SH2 and SH3 domains) that function both in tandem with, as well as independent to, the kinase catalytic domain. The noncatalytic functions of PKs are typically dependent on protein–protein interactions between the kinase’s multi-domain architecture and an interacting protein (often other kinases or scaffolding proteins). Moreover, these protein–protein interactions are regulated by large conformational changes to the quaternary structure of the protein kinase. There is evidence that noncatalytic functions of protein kinases are essential to cell signaling, however, these functions remain underappreciated due to a lack of genetic and chemical tools available to study them. Despite the emerging role of noncatalytic kinase signaling, efforts to understand the importance of kinase conformation on signaling pathways have been hampered by a lack of tools to assess and modulate the global conformation of PKs. During the previously funded period, we developed a ‘selective proteolysis’ methodology to characterize the quaternary conformation of protein kinases. This method was quickly adopted by academics and the pharmaceutical industry; however, the method relies on the ability of thermolysin to cleave the SH2-KD linker within a kinase and not all kinases have this linker with the appropriate recognition sequence. Here, we propose a cell-based biosensor that reports on kinase conformation. Using our kinase conformation biosensors, we propose to study the role of protein conformation in single-point mutations that lead to resistance to clinical kinase inhibitors. In addition to providing information on the conformational impact of drug resistant mutations, the proposed studies will provide critical information to understand the conformational regulation of Abl, BTK, BRAF, and EGFR kinases. Finally, we propose to study the interplay of allosteric and ATP-site inhibitors and to develop multivalent kinase inhibitors for Abl and c-Src kinases. The multivalent inhibitors will be constructed from matched-conformation allosteric and ATP-site inhibitors. This proposal will develop novel bioassays to study protein kinase conformation in cellulo, to better understand drug resistance in protein kinases, and to develop novel inhibitors to study the role of conformation in kinase signaling.