Regulation of PERK Signaling and Leukemic Cell Survival by a Novel Cytosolic Isoform of the UPR Regulator GRP78/BiP

Min Ni,Shiuan Wey,Amy S Lee,Peter Baumeister,Hui Zhou,Michael Polymenis

doi:10.1371/journal.pone.0006868

Abstract

The unfolded protein response (UPR) is an evolutionarily conserved mechanism to allow cells to adapt to stress targeting the endoplasmic reticulum (ER). Induction of ER chaperone GRP78/BiP increases protein folding capacity; as such it represents a major survival arm of UPR. Considering the central importance of the UPR in regulating cell survival and death, evidence is emerging that cells evolve feedback regulatory pathways to modulate the key UPR executors, however, the precise mechanisms remain to be elucidated. Here, we report the fortuitous discovery of GRP78va, a novel isoform of GRP78 generated by alternative splicing (retention of intron 1) and alternative translation initiation. Bioinformatic and biochemical analyses revealed that expression of GRP78va is enhanced by ER stress and is notably elevated in human leukemic cells and leukemia patients. In contrast to the canonical GRP78 which is primarily an ER lumenal protein, GRP78va is devoid of the ER signaling peptide and is cytosolic. Through specific knockdown of endogenous GRP78va by siRNA without affecting canonical GRP78, we showed that GRP78va promotes cell survival under ER stress. We further demonstrated that GRP78va has the ability to regulate PERK signaling and that GRP78va is able to interact with and antagonize PERK inhibitor P58IPK. Our study describes the discovery of GRP78va, a novel cytosolic isoform of GRP78/BiP, and the first characterization of the modulation of UPR signaling via alternative splicing of nuclear pre-mRNA. Our study further reveals a novel survival mechanism in leukemic cells and other cell types where GRP78va is expressed.

Highlights

Major advances have been made during the past decade in the understanding of how cells respond to impairment or saturation of the protein maturation machinery of the endoplasmic reticulum (ER)
We determined that the fortuitous production of the novel PCR band could be due to the high sequence homology between the p2a primer complementary sequence at the exon 1/2 junction and the intron 1/exon 2 junction sequence (Figure S1D), which led to the amplification of both the canonical Grp78 and the intron 1 retention variant form
A large number of apoptotic regulators undergo alternative splicing, suggesting that this is a crucial mechanism for survival and death decisions [24]

Summary

Introduction

Major advances have been made during the past decade in the understanding of how cells respond to impairment or saturation of the protein maturation machinery of the endoplasmic reticulum (ER). The initiation of UPR is primarily regulated by the ER-transmembrane sensor proteins PERK, IRE1 and ATF6 [1,2]. Along with activated ATF6 and IRE1/XBP-1, ATF4 activates transcription of ER chaperones and folding enzymes, improving the protein processing capacity and alleviating protein aggregation in the ER. GRP78 binds to PERK, IRE1 and ATF6 and maintains them in an inactive form [10,11]. When unfolded proteins pull GRP78 away from them, these pathways are activated, triggering the UPR, and if the stress is too severe, apoptosis. As a key UPR regulator and target, GRP78 is postulated to play pivotal roles in pathological processes, the precise mechanisms remain to be determined [15,16,17]

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Results

Conclusion