Faculty Collaboration Database

An ire1-phk1 chimera reveals a dispensable role of autokinase activity in endoplasmic reticulum stress response. J Mol Biol 2013 Jun 26;425(12):2083-99

Date

04/02/2013

Pubmed ID

23541589

DOI

10.1016/j.jmb.2013.02.036

Scopus ID

2-s2.0-84878233437 (requires institutional sign-in at Scopus site)   16 Citations

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.

Author List

Mannan MA, Shadrick WR, Biener G, Shin BS, Anshu A, Raicu V, Frick DN, Dey M

Authors

Madhusudan Dey PhD Assistant Professor in the Biological Sciences department at University of Wisconsin - Milwaukee
David N. Frick PhD Associate Professor in the Chimistry & Biochemistry department at University of Wisconsin - Milwaukee

MESH terms used to index this publication - Major topics in bold

Endoplasmic Reticulum Stress
Membrane Glycoproteins
Models, Biological
Phosphorylase Kinase
Phosphorylation
Protein Multimerization
Protein Processing, Post-Translational
Protein Structure, Tertiary
Recombinant Fusion Proteins
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins