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

Michael C Armstrong,Karen Baty,Achim Treumann,Martin Schröder,Max Brown,Ahmed A Ali,Sergej Šestak,Hanan A M Sagini

doi:10.1128/mcb.00655-16

Michael C Armstrong, Karen Baty + Show 6 more

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https://doi.org/10.1128/mcb.00655-16

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Abstract

ABSTRACTThe 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.

Highlights

Introduction of theD836A mutation nearly completely eliminated expression of ␤-galactosidase from unfolded protein response element (UPRE)-lacZ reporters when introduced into S840A-S841A- and Q-A-Ire1 (Fig. 9)
To identify autophosphorylation sites in the activation segment of Ire1, we expressed the cytosolic portion of Ire1 starting at Q556 as an N-terminal glutathione S-transferase fusion protein in Escherichia coli, purified it by affinity chromatography on glutathione-Sepharose beads, and analyzed tryptic digests by tandem mass spectrometry (MS/MS)
The data presented here show that Ire1 can transduce a partial endoplasmic reticulum (ER) stress signal in the absence of phosphorylation in its activation loop (Fig. 3 and 4)

Summary

Introduction

D836A mutation nearly completely eliminated expression of ␤-galactosidase from UPRE-lacZ reporters when introduced into S840A-S841A- and Q-A-Ire (Fig. 9). Introduction of the D836A mutation into P-A-Ire further decreased expression of ␤-galactosidase 2 h after induction of ER stress with 2 mM DTT (from 11.2 Ϯ 1.6 U/g to 5.6 Ϯ 1.4 U/g) to levels very close to and statistically undistinguishable from levels seen in IRE1 deletion strains exposed to ER stress for 2 h (2.7 Ϯ 0.4 U/g), but this decrease did not reach statistical significance. Differences in expression levels of the ␤-galactosidase reporter in IRE1 deletion cells or cells expressing D836A-S840AS841A-, D836A-Q-A-, or D836A-P-A-Ire were not statistically significant (Fig. 7). The D836A mutation, did not affect expression of ␤-galactosidase when introduced into WT Ire (Fig. 9).

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