Signaling by growth factor receptors with intracellular pseudokinase domains

描述（由申请人提供）：受体酪氨酸激酶（rtk）是用于癌症治疗的许多治疗剂的靶标。特别是，抑制rtk细胞内催化活性的小分子酪氨酸激酶抑制剂（TKIs）在临床中取得了显著的成功。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（抑制几种rtk）。人类蛋白质组中58种rtk中的许多现已与癌症和其他疾病有关-要么作为致癌驱动因素，要么作为对现有靶向治疗的抗性机制。因此，人们正在努力将EGFR和Kit的成功方法扩展到其他rtk。这一建议的重点是被忽视的rtk的一个子集，作为潜在的TKI靶点，因为序列分析表明它们含有非活性的激酶结构域。六种rtk （ErbB3, CCK4/PTK7, Ryk, EphB6， EphA10和SuRTK106/STYK1/NOK）的细胞内结构域包含所谓的“假激酶”结构域。这些类似于正常酪氨酸激酶结构域，但在关键基序上有序列改变，这被认为使它们具有催化活性。此外，两个rtk （Ror1和Ror2）在其几个催化基序中具有不寻常的序列。我们将这些统称为“RTK假激酶”。人类蛋白质组中518种蛋白激酶中有10%属于这一类。有人提出RTK假激酶通过调节变构变化和/或控制信号分子组装的支架功能激活信号通路。一些生化研究已经报道了激酶活性的缺失。然而，当组装在膜表面以模拟它们在活化的RTK中的寡聚化时，我们最近表明，几种RTK假激酶的激酶结构域确实具有显著的催化活性。在这项提议中，我们计划验证RTK假激酶只是不寻常的激酶的假设，但仍然使用它们的跨膜信号传导的催化活性。我们的方法将结构和酶学分析与细胞信号传导和激酶抑制剂作用的研究相结合。我们的具体目标是：1；为了验证人类RTKs中假激酶结构域具有激酶活性的假设，以确定其程度、结构基础以及对atp竞争性抑制剂的抑制敏感性。2. 为了验证RTK假激酶的激酶活性是其在细胞中的信号活动所必需的，并且它们可以被atp竞争抑制剂抑制的假设。解决这些问题将为RTK假激酶的信号传导机制提供重要的新线索，这些假激酶都与人类癌症有关。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目标的新tki奠定基础，这将具有非常重要的临床影响。如果假设不正确，我们的研究将为探索（和抑制）这些受体独特的信号传导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. PUBLIC HEALTH RELEVANCE: Successful completion of our proposed research should lead to the generation of a new set of tyrosine kinase inhibitors (TKIs) that target receptors called the RTK pseudokinases, which are all implicated in human cancer. RTK pseudokinases have been neglected as therapeutic targets for agents analogous to erlotinib, gefitinib and lapatinib (which target EGFR); because sequence analyses suggest that they do not possess catalytic activity like EGFR. We find that several RTK pseudokinases do in fact have this catalytic activity, and will explore its importance while devising approaches to inhibit this new set of targets in cancer therapy.