Signaling by growth factor receptors with intracellular pseudokinase domains

描述(申请人提供)：受体酪氨酸激酶(RTK)是许多用于癌症治疗的治疗药物的靶标。尤其是抑制RTK细胞内催化活性的小分子酪氨酸激酶抑制剂(TKIs)在临床上取得了显著的成功。例子包括厄洛替尼和吉非替尼(抑制EGF受体)、伊马替尼(抑制Kit)和舒尼替尼(抑制几个RTK)。人类蛋白质组中的58个RTK中的许多现在都与癌症和其他疾病有关--要么是作为致癌驱动因素，要么是与现有靶向治疗的耐药机制有关。因此，正在努力将已成功用于EGFR和Kit的方法推广到其他RTK。这一建议集中在RTK的一个子集上，这些RTK被作为潜在的TKI靶点而被忽视，因为序列分析表明它们包含非活性的激酶结构域。六种RTK(ErbB3、CCK4/PTK7、RyK、EphB6、EphA10和SuRTK106/STYK1/NOK)的胞内结构域都含有所谓的“假激酶”结构域。这些结构域类似于正常的酪氨酸激酶结构域，但关键基序的序列发生了变化，被认为使它们具有催化不活性。此外，两个RTK(Ror1和Ror2)的几个催化基序中有不寻常的序列。总而言之，我们把它们称为‘RTK假蛋白激酶’。在人类蛋白质组的518个蛋白激酶中，有10%属于这一类。已有研究提出，RTK假蛋白通过调节变构变化和/或控制信号分子组装的支架功能来激活信号通路。几项生化研究已经报告了缺乏激酶活性的情况。然而，当组装在膜表面以模拟其在激活的RTK中的寡聚作用时，我们最近证明了几种RTK假激酶的激动域确实具有显著的催化活性。在这个提议中，我们计划验证这样的假设，即RTK假蛋白激酶只是不寻常的激酶，但仍然使用它们的催化活性来进行跨膜信号传递。我们的方法结合了结构和酶学分析与细胞信号和激酶抑制剂的作用的研究。我们的具体目标是：1.验证人RTK中的假激酶域具有激酶活性的假设，确定其范围、结构基础以及对ATP竞争性抑制剂的抑制敏感性。2.验证一种假说，即RTK假蛋白激酶的活性是其在细胞内的信号活性所必需的，并且它们可以被ATP竞争性抑制剂所抑制。解决这些问题将为RTK假蛋白激酶的信号机制提供重要的新线索，这些信号都与人类癌症有关。如果我们的假设是正确的，这些发现将为开发一套针对长期被忽视的靶点的新的TKI奠定基础，这些靶点将具有非常显著的临床影响。如果假设是错误的，我们的研究将为探索(和抑制)这些受体的独特信号机制提供有价值的结构和生化框架。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases (RTKs) are the targets of numerous therapeutic agents used in cancer treatment. In particular, small molecule tyrosine kinase inhibitors (TKIs) that inhibit the intracellular catalytic activity of RTKs have had significant success in the clinic. Examples include erlotinib and gefitinib (which inhibit the EGF receptor), imatinib (which inhibits Kit), and sunitinib (which inhibits several RTKs). Many of the 58 RTKs in the human proteome have now been implicated in cancer and other diseases - either as oncogenic drivers or in mechanisms of resistance to existing targeted therapies. Accordingly, efforts are underway to extend approaches that have been successful for EGFR and Kit, for example, to other RTKs. This proposal focuses on a subset of RTKs that have been neglected as potential TKI targets because sequence analysis suggests that they contain inactive kinase domains. The intracellular domains of six RTKs (ErbB3, CCK4/PTK7, Ryk, EphB6, EphA10 and SuRTK106/STYK1/NOK) contain so-called 'pseudokinase' domains. These resemble normal tyrosine kinase domains but have sequence alterations in key motifs that are thought to render them catalytically inactive. In addition, two RTKs (Ror1 and Ror2) have unusual sequences in several of their catalytic motifs. Collectively, we term these the 'RTK pseudokinases'. 10% of the 518 protein kinases in the human proteome fall into this category. It has been proposed that RTK pseudokinases activate signaling pathways through regulated allosteric changes and/or scaffolding functions that control assembly of signaling molecules. Several biochemical studies have reported an absence of kinase activity. However, when assembled on membrane surfaces to mimic their oligomerization in an activated RTK, we have recently shown that the kinase domains of several RTK pseudokinases do have significant catalytic activity. In this proposal, we plan to test the hypothesis that the RTK pseudokinases are simply unusual kinases, but still use their catalytic activity for transmembrane signaling. Our approaches combine structural and enzymological analyses with studies of cell signaling and the effects of kinase inhibitors. Our Specific Aims are: 1. to test the hypothesis that pseudokinase domains in human RTKs have kinase activity, to determine its extent, its structural basis, and susceptibility to inhibition by ATP-competitive inhibitors. 2. To test the hypothesis that kinase activity of RTK pseudokinases is required for their signaling activity in cells, and that they can be inhibited with ATP-competitive inhibitors. Addressing these questions will shed important new light on the mechanism of signaling by RTK pseudokinases, which have all been associated with human cancer. If our hypothesis is correct, the findings will lay the foundation for developing a new set of TKIs against long-neglected targets that would have very significant clinical impact. If the hypothesis is incorrect, our studies will provide valuable structural and biochemical frameworks for exploring (and inhibiting) the unique signaling mechanisms of these receptors.