title: An ire1-phk1 chimera reveals a dispensable role of autokinase activity in endoplasmic reticulum stress response.
abstract: The endoplasmic reticulum transmembrane receptor Ire1 senses over-accumulation of unfolded proteins in the endoplasmic reticulum and initiates the unfolded protein response (UPR). The cytoplasmic portion of Ire1 has a protein kinase domain (KD) and a kinase extension nuclease (KEN) domain that cleaves an mRNA for encoding the Hac1 transcription factor needed to express UPR genes. During this UPR signaling, Ire1 proteins self-assemble into an oligomer of dimers, which essentially requires autophosphorylation of a constituent activation loop in the KD. However, it is not clear how dimerization, autophosphorylation, and KEN domain function are precisely coordinated. In this study, we uncoupled the KD and KEN domain functions, by removing the activation loop along with an extended region that we called the auto-inhibitory region (AIR), or by swapping the activation loop with a homologous loop from phosphorylase kinase 1 (Ire1(PHK)). Both Ire1(ΔAIR) and Ire1(PHK) activated the UPR even when either protein contained a mutation (D797A) that abolished the ability of Ire1 KD to transfer phosphates to the AIR. Neither protein functioned when containing mutations in key ATP binding residues (E746A and N749A) or in residues that disrupted Ire1 dimer interface (W426A or R697D). We interpret these results as evidence supporting the notion that the primary function of the kinase domain is to autophosphorylate the AIR in order to relieve auto-inhibition and that ADP acts as a switch to activate the KEN domain-catalyzed HAC1 mRNA cleavage.
authors.affiliation: Department of Biological Sciences, UW-Milwaukee, Milwaukee, WI, USA.
authors.forename: M Amin-ul
authors.initials: MA
authors.lastname: Mannan
authors.forename: William R
authors.initials: WR
authors.lastname: Shadrick
authors.forename: Gabriel
authors.initials: G
authors.lastname: Biener
authors.forename: Byung-Sik
authors.initials: BS
authors.lastname: Shin
authors.forename: Ashish
authors.initials: A
authors.lastname: Anshu
authors.forename: Valerica
authors.initials: V
authors.lastname: Raicu
authors.forename: David N
authors.initials: DN
authors.lastname: Frick
authors.forename: Madhusudan
authors.initials: M
authors.lastname: Dey
mesh_terms: D059865:Endoplasmic Reticulum Stress; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010764:Phosphorylase Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D010764:Phosphorylase Kinase; C082863:IRE1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases