The role of phosphorylation of Ire1 in its activation loop in regulation of its RNase activity

Ire1 is a protein kinase endoribonuclease (RNase) and a resident protein of the endoplasmic reticulum (ER) of Saccharomyces cerevisiae and a homologue to the Ire1a ER protein found in humans. Ire1 activates splicing of the mRNA of the unfolded protein response (UPR) gene HAC1. This splicing is a response to the accumulation of unfolded or misfolded proteins in the ER lumen. Splicing of HAC1 mRNA results in the translation of the Hac1 protein Hac1i which contains a bZIP transcription factor which promotes the expression of UPR-associated genes which ultimately leads to the alleviation of ER stress. The activation of Ire1 was previously thought to be dependent on phosphorylation within the Ire1 activation loop (a-loop). Here it is shown that in “phospho-dead” mutants, some level of splicing and UPR-activity is retained and that the aspartic acid residue (D836) within the a-loop allows for this retention. Furthermore, it was confirmed that mutation of D836 to alanine completely eliminates HAC1 mRNA splicing. This work suggests that phosphorylation of the a-loop is critical but not essential to RNase activation and the UPR.