Role of the membrane environment in the activation and dysregulation of JAK2

膜环境在 JAK2 激活和失调中的作用

• 批准号: 326558201
• 负责人: Professor Dr. Jacob Piehler
• 金额: --
• 依托单位: Arbeitsgruppe Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/326558201?language=en
• 关键词: Role; membrane; environment; activation; dysregulation

Proliferation and differentiation of blood cells is largely regulated by signaling via class I cytokine receptors, which prominently involves the Janus family tyrosine kinase JAK2. Dysregulation of JAK2 by constitutively activating mutations is a major cause of various types of both, myeloid and lymphoid leukemia. The molecular mechanism of JAK2 activation and its dysregulation, however, has remained unclear. In the first funding period, we have uncovered important mechanistic principles of JAK2 activation by paradigmatic homodimeric class I cytokine receptors for erythropoietin (EpoR), thrombopoietin (TpoR) and growth hormone (GHR). Quantitative receptor dimerization assays based on dual-color single molecule imaging, revealed ligand-induced receptor dimerization as key principle of receptor activation, yet identified weak intrinsic receptor dimerization propensity involving interactions between the JAK2 pseudokinase (PK) domains of and the receptor trans- and juxtamembrane (TM/JM) regions. Strikingly, several, but not all constitutively activating mutations in the JAK2 PK domain initiated ligand-independent dimerization. Based on this data, we generated by MD simulations structural models of JAK2 within homodimeric cytokine receptors in the plasma membrane. These models confirmed the relevance of an intermolecular JAK2 PK-PK interaction, which destabilizes the negative-regulatory intramolecular interaction with the tyrosine kinase (TK) domain. This model also explains different types of constitutively activating mutations in the JAK2 PK domain: “mode I” mutations stabilizing intermolecular PK-PK interactions and thus dimerizing the receptor; and “model II” mutations destabilizing the intramolecular JAK2 PK-TK interaction, thus directly releasing kinase activity. Furthermore, we could confirm the functional relevance of a membrane interaction site in the JAK2 FERM/SH2 (FS) domain predicted by our structural model. The aim for the second funding period is to consolidate and validate this model with the additional focus of unraveling the role of the membrane environment in regulating receptor assembly and activation. To this end, we will pinpoint the mechanistic bases of different modes of oncogenic dysregulation using new assays to probe activity and conformational organization of JAK2. We will explore the relevance of the receptor transmembrane domain in dimerization, activation and dysregulation exploiting single molecule FRET for detecting transient interaction in the plasma membrane of living cells. Furthermore, we will pinpoint the putative membrane binding site of JAK2 by mutagenesis in conjunction with live cell binding assays and identify its functional relevance for different types of oncogenic mutations. These live cell analyses will be complemented by reconstituting the receptor TM/JM domain bound to the JAK2 FS domain in polymer-supported membrane to pinpoint the role of membrane composition in JAK2 binding and receptor dimerization.

血细胞的增殖和分化在很大程度上由经由I类细胞因子受体的信号传导调节，其主要涉及Janus家族酪氨酸激酶JAK2。JAK2通过组成性激活突变的失调是各种类型的骨髓性和淋巴性白血病的主要原因。然而，JAK2激活及其失调的分子机制仍不清楚。在第一个资助期内，我们已经发现了JAK2激活的重要机制原则，通过范例同型二聚体I类细胞因子受体促红细胞生成素（EpoR），促血小板生成素（TpoR）和生长激素（GHR）。基于双色单分子成像的定量受体二聚化测定揭示了配体诱导的受体二聚化是受体激活的关键原理，但发现了弱的内在受体二聚化倾向，涉及JAK 2假激酶（PK）结构域与受体之间的相互作用跨膜和近膜（TM/JM）区域。引人注目的是，JAK2 PK结构域中的几个但不是全部组成型激活突变启动了配体非依赖性二聚化。基于这些数据，我们通过MD模拟在质膜中的同源二聚体细胞因子受体内产生JAK2的结构模型。这些模型证实了分子间JAK2 PK-PK相互作用的相关性，其使与酪氨酸激酶（TK）结构域的负调控分子内相互作用不稳定。该模型还解释了JAK2 PK结构域中不同类型的组成型激活突变："模式I"突变稳定分子间PK-PK相互作用，从而使受体二聚化;"模式II"突变使分子内JAK2 PK-TK相互作用不稳定，从而直接释放激酶活性。此外，我们可以确认我们的结构模型预测的JAK2 FERM/SH2（FS）结构域中的膜相互作用位点的功能相关性。第二个资助期的目的是巩固和验证这一模型，并重点阐明膜环境在调节受体组装和激活中的作用。为此，我们将使用新的检测方法来探测JAK2的活性和构象组织，以确定不同模式的致癌失调的机制基础。我们将探讨受体跨膜结构域的相关性，在二聚化，激活和失调利用单分子FRET检测瞬时相互作用在活细胞的质膜。此外，我们将通过诱变结合活细胞结合试验来确定JAK2的假定膜结合位点，并确定其与不同类型致癌突变的功能相关性。这些活细胞分析将通过在聚合物支持的膜中重建与JAK2 FS结构域结合的受体TM/JM结构域来补充，以查明膜组成在JAK2结合和受体二聚化中的作用。