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

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 <i>Saccharomyces cerevisiae</i> Ire1 coincides with autophosphorylation of its activation loop at S840, S841, T844, and S850. Mass spectrometric analysis of Ire1 expressed in <i>Escherichia coli</i> identified S837 as another potential phosphorylation site <i>in vivo</i> Mutation of all five potential phosphorylation sites in the activation loop decreased, but did not completely abolish, splicing of <i>HAC1</i> mRNA, induction of <i>KAR2</i> and <i>PDI1</i> mRNAs, and expression of a β-galactosidase reporter activated by Hac1ⁱ Phosphorylation site mutants survive low levels of endoplasmic reticulum stress better than <i>IRE1</i> deletions strains. <i>In vivo</i> 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 <i>HAC1</i> splicing, induction of <i>KAR2</i>, <i>PDI1</i>, 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.