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. 2012 Dec;8(12):982-9.
doi: 10.1038/nchembio.1094. Epub 2012 Oct 21.

Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors

Likun Wang  1 , B Gayani K Perera, Sanjay B Hari, Barun Bhhatarai, Bradley J Backes, Markus A Seeliger, Stephan C Schürer, Scott A Oakes, Feroz R Papa, Dustin J Maly
Affiliations

Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors

Likun Wang et al. Nat Chem Biol. 2012 Dec.

Abstract

Under endoplasmic reticulum stress, unfolded protein accumulation leads to activation of the endoplasmic reticulum transmembrane kinase/endoRNase (RNase) IRE1α. IRE1α oligomerizes, autophosphorylates and initiates splicing of XBP1 mRNA, thus triggering the unfolded protein response (UPR). Here we show that IRE1α's kinase-controlled RNase can be regulated in two distinct modes with kinase inhibitors: one class of ligands occupies IRE1α's kinase ATP-binding site to activate RNase-mediated XBP1 mRNA splicing even without upstream endoplasmic reticulum stress, whereas a second class can inhibit the RNase through the same ATP-binding site, even under endoplasmic reticulum stress. Thus, alternative kinase conformations stabilized by distinct classes of ATP-competitive inhibitors can cause allosteric switching of IRE1α's RNase--either on or off. As dysregulation of the UPR has been implicated in a variety of cell degenerative and neoplastic disorders, small-molecule control over IRE1α should advance efforts to understand the UPR's role in pathophysiology and to develop drugs for endoplasmic reticulum stress-related diseases.

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Figures

Figure 1
Figure 1. Interaction of ATP-competitive inhibitors with the bifunctional kinase/RNase, IRE1α
(a) Proposed binding modes of type I and type II kinase inhibitors with the ATP-binding pocket of IRE1α. Left panel shows the contacts the type I inhibitor APY29 forms with yeast IRE1α (PDB code 3SDJ). The right panel shows the proposed contacts a type II inhibitor 1 forms with IRE1α based on the co-crystal structure of the same inhibitor bound to Src (PDB code 3EL8) (also see Supplementary Fig. 3). (b) XBP1 RNA minisubstrate assay used for screening IRE1α modulators. The recombinant human IRE1α—IRE1α*—used in the assay spans residues 469–977, which includes the cytosolic kinase and RNase domains. Cleavage of the 5’FAM-3’BHQ-labeled XBP1 minisubstrate by IRE1α* results in FRET-dequenching. (c) Endpoint fluorescence of IRE1α* catalyzed cleavage reaction of XBP1 minisubstrate in the presence of varying concentrations of inhibitors, or DMSO. STF-083010 is an imine-based compound that covalently inhibits the RNase domain. Relative fluorescence intensity is scaled to the signal observed with IRE1α* (1.0), or without IRE1α* (0). (mean ± SD, n = 3). (d) Structures of the type II kinase inhibitors used in this study.
Figure 2
Figure 2. APY29 and 3 divergently modulate the RNase activity and oligomerization state of IRE1α*
(a) Inhibition of IRE1α* autophosphorylation in vitro by APY29 and 3. Normalized autophosphorylation levels and IC50 values for both compounds are shown. (b) λ-PPase treatment of IRE1α* produces dephosphorylated IRE1α* (dP-IRE1α*). Immunoblots using anti-IRE1α and anti-phospho IRE1α antibodies are shown. (c) RNase activities of IRE1α* and dP-IRE1 α* under varying concentrations of APY29 or 3 per the assay of Figure 1b. EC50 values were determined by fitting normalized fluorescence intensities (mean ± SD, n = 3). (d) Urea PAGE of XBP1 mini-substrate cleavage by IRE1α* and dP-IRE1α* with and without 3 or APY29. (e) RNase competition assays between APY29 and 3. The red line shows IRE1α* RNase activity under fixed 3 and varying APY29 concentrations. The black line shows IRE1α* RNase activity under fixed APY29 and varying 3 concentrations. The blue line shows IRE1α* RNase activity under fixed STF-083010 and varying APY29 concentrations (mean ± SD, n = 3).
Figure 3
Figure 3. Characterization of 3's interaction with the ATP-binding site of IRE1α
Results of the ICAT footprinting experiments with IRE1α*. Alkylation rates were measured in the presence of DMSO (black), APY29 (blue) (20 µM), or 3 (red) (20 µM) (mean ± SD, n = 3). (a) Alkylation rate of Cys572. (b) Alkylation rate of Cys645. (c) Alkylation rate of Cys715. (d) A molecular model of 3's interaction with the ATP-binding site of IRE1α (grey). IRE1α is in the DFG-out inactive conformation. The imidazopyrazine ring of 3 occupies the adenine pocket and the 3-trifluoromethylurea occupies the DFG-out pocket. No favorable poses for 3 bound to the DFG-in conformation of IRE1α could be determined.
Figure 4
Figure 4. APY29 and 3 differentially affect the oligomerization state of IRE1α*
(a) Left panels shows immunoblots of IRE1α* after treatment with the crosslinker DSS (250 µM). Increasing concentrations of IRE1α* were incubated with DMSO, APY29 (200 µM) or 3 (200 µM). The right panel shows quantitation of the ratios of oligomeric to monomeric IRE1α* (b) Model of how type I and type II kinase inhibitors affect the RNase activities and oligomeric states of IRE1α* and dP-IRE1α*.
Figure 5
Figure 5. Chemical-genetic modulation of IRE1α kinase and RNase activity in vivo
(a) Anti-total and anti-phospho IRE1α immunoblots of T-Rex 293 cells expressing “holed” IRE1αI642A under Doxycycline (Dox) control. Cells were pre-treated for 1 hr with 3 at indicated concentrations, then induced with Dox (1 µM) for 8 hrs. Plots show normalized phosphorylation levels and ratios of spliced XBP1 mRNA under varying concentrations 3 (mean ± SD, n >= 3). (b) Quantification of the XBP1 cDNA amplicons from the cells described in (a). EtBr-stained agarose gels are shown in Supplementary Figure 16 (c). Competition between the “bumped” kinase inhibitor 1NM-PP1 and 3 against IRE1αI642A. T-Rex 293 cells expressing IRE1αI642A were pre-treated for 1 hr with 3 (1 µM) ± varying concentrations 1NM-PP1 before Dox induction (1 µM) for 8 hrs. Quantification show ratios of spliced XBP1 mRNA as a function of 3 and 1NM-PP1 concentrations. [XBP1S = spliced XBP1; XBP1U = unspliced XBP1]
Figure 6
Figure 6. Divergent modulation of endogenous IRE1α RNase activity under ER stress with types I and II kinase inhibitors
(a) Quantification of EtBr-stained agarose gel of XBP1 complementary DNA (cDNA) amplicons from INS-1 cells pre-treated for 1 hr with 3 or APY29 at indicated concentrations, followed by Tg (6 nM) for 4 hrs. Ratios of XBP1S over (XBP1S + XBP1U) are plotted (mean ± SD, n = 3). Gels are shown in Supplemental Figure 19. (b) Anti-total and anti-phospho IRE1α immunoblots using extracts from INS-1 cells pre-treated for 1 hr with 3, sunitinib or STF-083010 at indicated concentrations, followed by Tg (6 nM) for 2 hrs. (c) Quantification of EtBr-stained agarose gel of XBP1 complementary DNA (cDNA) amplicons from the INS-1 cells described in (b). Gels are shown in Supplemental Figure 20. (d) EtBr-stained agarose gel of XBP1 cDNA amplicons from INS-1 cells pre-treated for 1 hr with 4 at indicated concentrations, followed by Tg (6 nM) for 4 hrs. (e) Model of how type I kinase inhibitors (APY29 or sunitinib), type II kinase inhibitors (3), and RNase inhibitors (STF-083010) modulate the enzymatic activities of WT IRE1α. APY29 inhibits IRE1α trans-autophosphorylation but promotes oligomerization and activates the RNase domain. STF-083010 inhibits the RNase activity of IRE1α but does not affect kinase activity or the overall oligomerization state. 3 inhibits both the kinase and RNase domains of IRE1α and stabilizes the monomeric form. [Please note that these cartoons are not meant to differentiate between the relative orientations of monomer subunits in IRE1α.]
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