Characterisation of the contribution of the kinase and RNase activities of Ire1α to activation of apoptotic JNK signalling

Abstract

The unfolded protein response (UPR) is a highly conserved mechanism by which all eukaryotes respond to endoplasmic reticulum (ER) stress. In higher eukaryotes this response is mediated by three ER transmembrane stress sensors: activating transcription factor 6 (ATF6/), PKR-like ER kinase (PERK) and inositol requiring 1 (IRE1/). IRE1 is the most highly conserved of the three ER stress sensors and is also the only sensor to mediated UPR signalling via two different enzymatic domains.
It is currently believed that during prolonged ER stress, the RNase domain of IRE1α provides cytoprotection via XBP1 splicing, whilst the kinase domain initiates proapoptotic JNK signalling via interaction with the adaptor protein TRAF2. However, characterising how these domains contribute to cell fate decisions is complicated by the fact that traditional models use ER stress mimetic drugs, which activate all three branches of the UPR and thus make it difficult to attribute downstream events to individual effectors. Therefore, the aim of the research presented in this thesis was to produce a model that allowed isolated activation of IRE1α in order to determine the contribution of its kinase and RNase activities to proapoptotic JNK signalling, without input from other upstream effectors.
Using the Fv2E-IRE1α system, the data presented in this thesis provides novel insight into the mechanism by which IRE1α instigates proapoptotic JNK signalling by suggesting that a functional kinase domain is not required for IRE1α to interact with TRAF2 and that endoribonuclease function may be required for downstream JNK activation in humans. Furthermore, evidence is also provided to suggest that, whilst kinase activity is not required for interaction with TRAF2, it is required for downstream JNK activation. This gives rise to the possibility that, contrary to current knowledge, the IRE1α kinase domain has the capacity to phosphorylate proteins other than IRE1α.