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Bypass of Activation Loop Phosphorylation by Aspartate 836 in Activation of the Endoribonuclease Activity of Ire1

Lookup NU author(s): Karen Baty, Dr Achim Treumann

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2017 American Society for Microbiology. The bifunctional protein kinase-endoribonuclease Ire1 initiates splicing of the mRNA for the transcription factor Hac1 when unfolded proteins accumulate in the endoplasmic reticulum. Activation of Saccharomyces cerevisiae Ire1 coincides with autophosphorylation of its activation loop at S840, S841, T844, and S850. Mass spectrometric analysis of Ire1 expressed in Escherichia coli identified S837 as another potential phosphorylation site in vivo. Mutation of all five potential phosphorylation sites in the activation loop decreased, but did not completely abolish, splicing of HAC1 mRNA, induction of KAR2 and PDI1 mRNAs, and expression of a β-galactosidase reporter activated by Hac1i. Phosphorylation site mutants survive low levels of endoplasmic reticulum stress better than IRE1 deletions strains. In vivo clustering and inactivation of Ire1 are not affected by phosphorylation site mutants. Mutation of D836 to alanine in the activation loop of phosphorylation site mutants nearly completely abolished HAC1 splicing, induction of KAR2, PDI1, and β-galactosidase reporters, and survival of ER stress, but it had no effect on clustering of Ire1. By itself, the D836A mutation does not confer a phenotype. These data argue that D836 can partially substitute for activation loop phosphorylation in activation of the endoribonuclease domain of Ire1.

Publication metadata

Author(s): Armstrong MC, Sestak S, Ali AA, Sagini HAM, Brown M, Baty K, Treumann A, Schroder M

Publication type: Article

Publication status: Published

Journal: Molecular and Cellular Biology

Year: 2017

Volume: 37

Issue: 16

Print publication date: 01/08/2017

Online publication date: 30/05/2017

Acceptance date: 22/05/2017

Date deposited: 09/08/2017

ISSN (print): 0270-7306

ISSN (electronic): 1098-5549

Publisher: American Society for Microbiology

URL: https://doi.org/10.1128/MCB.00655-16

DOI: 10.1128/MCB.00655-16

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