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

点击翻译按钮获取中文摘要

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.