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Mol Cell Biol. 2017 Jul 28;37(16). pii: e00655-16. doi: 10.1128/MCB.00655-16. Print 2017 Aug 15.

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

Author information

1
Durham University, Department of Biosciences, Durham, United Kingdom.
2
Biophysical Sciences Institute, Durham University, Durham, United Kingdom.
3
North East England Stem Cell Institute (NESCI), Life Bioscience Centre, International Centre for Life, Newcastle upon Tyne, United Kingdom.
4
Slovak Academy of Sciences, Institute of Chemistry, Department of Glycobiology, Bratislava, Slovakia.
5
Molecular Biology Department, National Research Center, Cairo, Egypt.
6
NEPAF, Newcastle upon Tyne, United Kingdom.
7
Durham University, Department of Biosciences, Durham, United Kingdom martin.schroeder@durham.ac.uk.

Abstract

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.

KEYWORDS:

Ire1; endoplasmic reticulum; phosphorylation; phosphorylation-independent RD kinase; protein kinase; stress response; unfolded protein response

PMID:
28559428
PMCID:
PMC5533888
DOI:
10.1128/MCB.00655-16
[Indexed for MEDLINE]
Free PMC Article

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