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 bi functional 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 the activation of IRE1 has been shown to rely on 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 robustly increases upon dimerization as well as phosphorylation. However, we do not see any further increase in RNase activity upon tetramerization, suggesting that dimerization of IRE1 is sufficient for inducing XBP1 mRNA cleavage. However, initial results suggest that tetramerization leads to higher autophosphorylation activity, likely due to dimers coming together in a phospho-competent conformation. Interestingly, we also notice that pre-incubation with physiological concentrations of ADP inhibits the RNase activity of dephosphorylated IRE1 dimers to a greater degree than their phosphorylated counterparts. 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 active phosphorylated dimers.
Mukherjee et al. (Sun,) studied this question.