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）是癌症治疗中使用的许多治疗剂的靶标。特别地，抑制RTK的细胞内催化活性的小分子酪氨酸激酶抑制剂（TKI）在临床上取得了显著的成功。实例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）。人类蛋白质组中的58种RTK中的许多现在已经与癌症和其他疾病有关-无论是作为致癌驱动因素还是对现有靶向治疗的耐药机制。因此，正在努力将已经成功用于EGFR和Kit的方法扩展到例如其他RTK。 该提案侧重于RTK的一个子集，这些RTK被忽视为潜在的TKI靶点，因为序列分析表明它们含有无活性的激酶结构域。六种RTK（ErbB 3、CCK 4/PTK 7、Ryk、EphB 6、EphA 10和SuRTK 106/STYK 1/NOK）的胞内结构域包含所谓的“假激酶”结构域。这些类似于正常的酪氨酸激酶结构域，但在关键基序中有序列改变，被认为使它们无催化活性。此外，两种RTK（Ror 1和Ror 2）在其催化基序中具有不寻常的序列。总的来说，我们将这些称为“RTK假激酶”。人类蛋白质组中的518种蛋白激酶中有10%属于这一类。已经提出RTK假激酶通过控制信号分子组装的受调节的变构变化和/或支架功能来激活信号传导途径。一些生物化学研究报告了激酶活性的缺失。然而，当组装在膜表面上，以模拟其在活化的RTK寡聚化，我们最近已经表明，激酶结构域的几个RTK假激酶有显着的催化活性。在这个建议中，我们计划测试的假设，RTK假激酶只是不寻常的激酶，但仍然使用其催化活性的跨膜信号。我们的方法将联合收割机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。测试人RTK中的假激酶结构域具有激酶活性的假设，以确定其程度、结构基础和对ATP竞争性抑制剂抑制的敏感性。2.为了检验RTK假激酶的激酶活性是其在细胞中的信号传导活性所必需的，并且它们可以被ATP竞争性抑制剂抑制的假设。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靶点的新TKI奠定基础，这些靶点将具有非常重要的临床影响。如果假设是不正确的，我们的研究将为探索（和抑制）这些受体的独特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.