Phosphorylation and Oligomerization are Key Steps in the Activation of the Endoplasmic Reticulum Stress Sensor IRE1α

Core Concepts

Dimerization of IRE1α is not sufficient for its phosphorylation and activation. Higher-order oligomerization is necessary to trigger cross-phosphorylation and unleash the full endonuclease activity of IRE1α.

Abstract

The study investigates the stepwise activation of the endoplasmic reticulum (ER) stress sensor IRE1α. The authors find that dimerization of IRE1α, while necessary, is not sufficient for its phosphorylation and full enzymatic activation. Key insights: IRE1α phosphorylation and RNase activity correlate with the intensity of ER stress. Under mild stress, IRE1α is only mildly activated, while unresolved stress leads to sustained high levels of phosphorylation and RNase activity. Using a panel of IRE1α mutants, the authors show that phosphorylation does not occur within isolated IRE1α dimers. Rather, it requires collisions between dimers or formation of higher-order oligomers. The dimerizable IRE1α chimera (Dim-IRE1α) is only phosphorylated and active when expressed at very high levels, suggesting that reaching a critical concentration of IRE1α dimers is key for triggering cross-phosphorylation. Phosphorylation and nucleotide-binding stabilize the active, oligomeric state of IRE1α, further enhancing its RNase activity. Stable clustering of IRE1α is not necessary for XBP1 splicing, but may modulate the potency and duration of the response. The authors propose a model where the stepwise activation of IRE1α, from dispersed dimers to concentrated oligomers, allows cells to fine-tune the ER stress response, preventing excessive activation under mild stress conditions while unleashing full-blown IRE1α activity only upon intense and unresolved ER stress.

Stats

The fraction of phosphorylated IRE1α increases when ERAD is blocked by kifunensine, particularly during the late phases of the ER stress response. Preventing phosphorylation of IRE1α (AAA mutant) abolishes its RNase activity even at high expression levels. The phosphomimetic Dim-DDD IRE1α mutant is active at much lower expression levels compared to the non-phosphorylated Dim-IRE1α.

Quotes

"Reaching a critical concentration of IRE1α dimers in the ER membrane is a key event." "Formation of stable IRE1α clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1α to phosphatases."

Key Insights Distilled From

Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

by Bakunts,A., ... at www.biorxiv.org 12-19-2023

https://www.biorxiv.org/content/10.1101/2023.12.18.572154v1

Deeper Inquiries

How do cells regulate the local concentration of IRE1α dimers to control the threshold for full activation?

Cells regulate the local concentration of IRE1α dimers through a series of steps to control the threshold for full activation. Initially, under basal unstressed conditions, IRE1α exists mostly in a monomeric state. When the accumulation of misfolded proteins in the ER lumen occurs, IRE1 dimers are formed. However, these dimers alone are not sufficient for full activation. Encounters between IRE1α dimers allow for trans-phosphorylation, a crucial step in triggering endonuclease activity. This trans-phosphorylation event is essential for the activation of IRE1α. As the intensity and duration of ER stress increase, IRE1α clusters are formed, leading to maximal phosphorylation and enzymatic activity. These clusters help to increase the local concentration of dimers, segregating them away from phosphatases and unleashing pathways like regulated IRE1-dependent decay (RIDD) and proapoptotic pathways. The formation of these supramolecular complexes further enhances and prolongs IRE1α activation, eventually driving cells into apoptosis. Therefore, by congregating a sufficient number of IRE1α molecules in a restricted area, cells can achieve full activation of the protein.

What are the potential physiological or pathological implications of dysregulated IRE1α oligomerization and phosphorylation?

Dysregulated IRE1α oligomerization and phosphorylation can have significant physiological and pathological implications. In physiological conditions, proper regulation of IRE1α activation is crucial for maintaining ER homeostasis and cell survival. However, dysregulation of IRE1α activation, such as excessive oligomerization and phosphorylation, can lead to aberrant activation of downstream pathways, including RIDD and proapoptotic signaling. This dysregulation can result in excessive cell death, disrupting tissue homeostasis and contributing to the pathogenesis of various diseases. In pathological conditions, dysregulated IRE1α activation has been implicated in several diseases, including cancer and diabetes. In cancer, aberrant activation of IRE1α can promote tumor cell survival and growth by inducing pro-survival pathways. On the other hand, in diabetes, dysregulated IRE1α activation can lead to beta cell death and impaired insulin production, contributing to the progression of the disease. Therefore, understanding and controlling the balance of IRE1α oligomerization and phosphorylation is crucial for maintaining cellular homeostasis and preventing disease development.

Could the insights from this study on IRE1α activation be extended to understand the regulation of other ER stress sensors, such as PERK and ATF6?

The insights gained from the study on IRE1α activation could potentially be extended to understand the regulation of other ER stress sensors, such as PERK and ATF6. These ER stress sensors also play critical roles in the unfolded protein response (UPR) and are involved in maintaining ER homeostasis. Like IRE1α, PERK and ATF6 undergo activation processes that involve dimerization, oligomerization, and phosphorylation. By studying the mechanisms of activation of IRE1α, researchers can gain valuable insights into the general principles of ER stress sensor activation. For example, the importance of higher-order oligomerization for full activation, as observed in IRE1α, may also apply to PERK and ATF6. Understanding how these sensors regulate their local concentrations, interact with other proteins, and undergo post-translational modifications can provide a broader understanding of UPR signaling pathways. Overall, while each ER stress sensor has unique features and functions, the fundamental principles of activation and regulation may be shared among them. By applying the knowledge gained from studying IRE1α activation, researchers can potentially uncover new insights into the regulation of PERK and ATF6 in response to ER stress.