Conformational Control of Protein Kinases

蛋白激酶的构象控制 抽象的。由于只关注下游的磷酸化网络，激酶信号经常被过度简化。 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 对健康细胞和疾病细胞的细胞信号都至关重要的功能。许多PKs包含监管 结构域(例如，SH2和SH3结构域)，这些结构域既与该激酶协同工作，又独立于该激酶工作 催化域。蛋白激酶的非催化功能通常依赖于蛋白质与蛋白质的相互作用。 在该蛋白的多域结构和相互作用的蛋白(通常是其他蛋白或支架)之间 蛋白质)。此外，这些蛋白质之间的相互作用受到大的构象变化的调节。 蛋白激酶的四级结构。有证据表明，蛋白激酶的非催化功能是 然而，对于细胞信号来说，这些功能仍然没有得到充分认识，因为缺乏遗传和 可用于研究它们的化学工具。尽管非催化激酶信号转导的作用正在显现，但努力 了解激酶构象在信号通路上的重要性因缺乏工具而受阻 评估和调节PKS的全球构象。 在之前的资助期间，我们开发了一种‘选择性蛋白分解’方法来表征 蛋白激酶的四级构象。这种方法很快就被学术界和 制药行业；然而，该方法依赖于热裂解酶切割SH2-KD连接子的能力 并不是所有的激酶都有这个具有适当识别序列的连接物。在这里，我们建议 一种以细胞为基础的生物传感器，报告激酶的构象。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导致临床耐药的单点突变中的作用 这是一种激酶抑制剂。除了提供有关耐药突变的构象影响的信息外， 拟议的研究将为理解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，并 开发Abl和c-Src激酶的多价激酶抑制剂。多价抑制剂将由 匹配构象变构和三磷酸腺苷位点抑制剂。这项提议将开发新的生物检测方法来研究 蛋白激酶在细胞中的构象，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转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.