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

• Published in: Molecular and cellular biology Vol. 37; no. 16
• Main Authors: Armstrong, Michael C., Šestak, Sergej, Ali, Ahmed A., Sagini, Hanan A. M., Brown, Max, Baty, Karen, Treumann, Achim, Schröder, Martin
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 01.08.2017; American Society for Microbiology
• Subjects: Amino Acid Sequence; Aspartic Acid - metabolism; Cell Survival; endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum Stress - genetics; Enzyme Activation; Epistasis, Genetic; Genes, Reporter; Green Fluorescent Proteins - metabolism; Ire1; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - metabolism; Mutant Proteins - chemistry; Mutant Proteins - metabolism; Mutation - genetics; Phosphorylation; phosphorylation-independent RD kinase; protein kinase; Protein Phosphatase 2 - metabolism; Protein Serine-Threonine Kinases - chemistry; Protein Serine-Threonine Kinases - metabolism; RNA Splicing - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - metabolism; Sequence Alignment; stress response; Structure-Activity Relationship; unfolded protein response; unfolded protein response; stress response; Ire1; phosphorylation-independent RD kinase; phosphorylation; endoplasmic reticulum; protein kinase
• ISSN: 1098-5549; 0270-7306; 1098-5549
• DOI: 10.1128/MCB.00655-16

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 Hac1 i . 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.