Poster abstracts
Poster number 162 submitted by Swapnil Mukherjee
Dissecting the activation mechanism of the bifunctional kinase RNase IRE1 with orthogonal control over oligomerization and phosphorylation
Swapnil Mukherjee (Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University), Vladislav Belyy (Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University)
Abstract:
The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum is known as ER stress, a condition relevant in healthy development as well as several metabolic and neurological disorders. Inositol-requiring enzyme 1 (IRE1) is an essential ER membrane-localized bifunctional kinase/RNase receptor that senses ER stress. When activated, mammalian IRE1 initiates a nonconventional cytosolic splicing reaction, which results in the production of the transcription factor X-box binding protein (XBP1s) and constitutes a key step in ER stress alleviation. While existing studies in live cells have shown that the activation of IRE1 relies on some form of oligomerization, the sequence of oligomeric transitions that leads to its activation is still unknown. We engineered an in vitro fusion protein system to study the consequences of oligomerization and phosphorylation on the activity of IRE1. We used in vitro RNA cleavage as a readout to study how the activity of IRE1 is modulated by different forms of IRE1. We find that the RNase activity of IRE1 increases to varying degrees upon dimerization as well as phosphorylation but a combination of both lead to much higher levels of RNase activity. This indicates that the transition from RNase inactive to active IRE1 is accompanied by both phosphorylation and dimerization. We also show that dimerization leads to autophosphorylation of IRE1. However, previous studies have demonstrated that IRE1 exists as dimers even in its inactive state in the cell, leading us to believe that oligomerization might lead to yet higher levels of autophosphorylation. To assess this hypothesis we engineered an additional set of IRE1 fusion proteins that can be assembled into larger oligomers. Using this system, we noticed that dimers are more capable of autophosphorylation as well as more likely to be autophosphorylated when assembled into larger oligomers. Overall, our studies lead to a model of IRE1 activation whereby dephosphorylated dimers transiently oligomerize, which leads to trans-autophosphorylation and the subsequent formation of RNase-active phosphorylated dimers or oligomers.
Keywords: RNase, Phosphorylation, ER Stress