Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.

Alexandra Papaioannou,Rémy Pedeux,Alice Blondel,Albert C Koong,Luc Negroni,Federica Centonze,Gabriella Svensson,Michel L Tremblay,Claudio Hetz,Leif A Eriksson,Eric Chevet,Marion Maurel,Sayyed Jalil Mahdizadeh,Matías Gonzalez-Quiroz,Alice Metais,Ensieh Zare

doi:10.26508/lsa.202201379

Abstract

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

Highlights

The imbalance between the cellular demand to fold secretory and transmembrane proteins and the ER capacity to achieve this function can result in the accumulation of improperly folded proteins in this compartment, a situation known as ER stress (Almanza et al, 2018)
We observed the significantly reduced mRtcB-Flag tyrosine phosphorylation when cells were treated with the tyrosine kinase inhibitor dasatinib, which targets c-ABL in a specific manner and which may affect the activity of other tyrosine kinases such as SRC (Fig 2D and E)
Because our observations suggested that the RtcB Y306-dependent interaction could control the XBP1 mRNA splicing activity of the inositol-requiring enzyme 1 alpha (IRE1)/RtcB complex, we reconstituted in vitro the splicing reaction using in vitro transcribed XBP1 mRNA, recombinant IRE1 cytosolic domain whose optimal concentration was empirically determined (Fig S7F) and RtcB immunoprecipitated from cells

Summary

Introduction

The imbalance between the cellular demand to fold secretory and transmembrane proteins and the ER capacity to achieve this function can result in the accumulation of improperly folded proteins in this compartment, a situation known as ER stress (Almanza et al, 2018). The activation of the ER stress sensors inositol-requiring enzyme 1 alpha (IRE1), activating transcription factor 6 alpha (ATF6α), and protein kinase RNA (PKR)–like ER kinase (PERK) aims to restore ER homeostasis and is known as the adaptive unfolded protein response (UPR). When the stress cannot be resolved, the UPR triggers cell death (McGrath et al, 2021), which is referred to as terminal UPR. The mechanisms controlling the switch between adaptive (aUPR) and terminal (tUPR) UPR remain incompletely characterized. Similar to the other sensors, IRE1 is an ER transmembrane protein activated after dissociation from binding immunoglobin protein and/or direct binding to improperly folded proteins (Karagoz et al, 2017)

Objectives

Methods

Results

Conclusion