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. 1998 Jun 15;12(12):1812-24.
doi: 10.1101/gad.12.12.1812.

A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells

W Tirasophon  1 , A A Welihinda, R J Kaufman
Affiliations

A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells

W Tirasophon et al. Genes Dev. .

Abstract

Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP. We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiae Ire1p, a proximal sensor for this signal transduction pathway in yeast. The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L. However, the luminal domain has extensively diverged from the yeast gene product. hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5' splice site of yeast HAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p. Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex. Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity. Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation. These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells.

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Figures

Figure 1
Figure 1
Structure and amino acid sequence analysis of hIre1p. (A) Alignment and restriction map of overlapping complementary DNAs encoding human Ire1p. RH3 was the primary probe used to screen a human fetal liver cDNA library to obtain cDNA clones 3-1-1, 3-1.2, 8-1, 9-1,13-1, and 17-1. F14 was a 5′ RACE–PCR product amplified from RNA isolated from the human hepatoma cell line HepG2. The open bar represents the predicted ORF coding for hIre1p. (B) Domain organization of hIre1p. (Solid box) Potential signal sequence; (formula image) potential N-linked glycosylation site; (TM) a putative transmembrane region; (Linker) a region having no homology to known proteins; (S/T kinase) catalytic domain of Ser/Thr protein kinase; (RNase L) a domain having high homology to 2-5 oligo-(A)–dependent RNase. Percent identity to the corresponding domains of S. cerevisiae and C. elegans is indicated. (C) (H.s.) Amino acid sequence alignment of human Ire1p, (S.c.) S. cerevisiae Ire1p and (C.e.) its putative homologous protein from C. elegans. (Open boxes) The identical sequence; (shaded boxes) conserved residues; (dashes) gaps between residues to obtain maximum matching. Numbers are the position of the last amino acid. (▿) Potential signal peptide cleavage site; (•) invariant residues in protein kinase domain; (*) invariant Lys599 residue in kinase subdomain II. The glutamine rich cluster (I) and the serine rich cluster (II) in the linker region are also identified.
Figure 1
Figure 1
Structure and amino acid sequence analysis of hIre1p. (A) Alignment and restriction map of overlapping complementary DNAs encoding human Ire1p. RH3 was the primary probe used to screen a human fetal liver cDNA library to obtain cDNA clones 3-1-1, 3-1.2, 8-1, 9-1,13-1, and 17-1. F14 was a 5′ RACE–PCR product amplified from RNA isolated from the human hepatoma cell line HepG2. The open bar represents the predicted ORF coding for hIre1p. (B) Domain organization of hIre1p. (Solid box) Potential signal sequence; (formula image) potential N-linked glycosylation site; (TM) a putative transmembrane region; (Linker) a region having no homology to known proteins; (S/T kinase) catalytic domain of Ser/Thr protein kinase; (RNase L) a domain having high homology to 2-5 oligo-(A)–dependent RNase. Percent identity to the corresponding domains of S. cerevisiae and C. elegans is indicated. (C) (H.s.) Amino acid sequence alignment of human Ire1p, (S.c.) S. cerevisiae Ire1p and (C.e.) its putative homologous protein from C. elegans. (Open boxes) The identical sequence; (shaded boxes) conserved residues; (dashes) gaps between residues to obtain maximum matching. Numbers are the position of the last amino acid. (▿) Potential signal peptide cleavage site; (•) invariant residues in protein kinase domain; (*) invariant Lys599 residue in kinase subdomain II. The glutamine rich cluster (I) and the serine rich cluster (II) in the linker region are also identified.
Figure 1
Figure 1
Structure and amino acid sequence analysis of hIre1p. (A) Alignment and restriction map of overlapping complementary DNAs encoding human Ire1p. RH3 was the primary probe used to screen a human fetal liver cDNA library to obtain cDNA clones 3-1-1, 3-1.2, 8-1, 9-1,13-1, and 17-1. F14 was a 5′ RACE–PCR product amplified from RNA isolated from the human hepatoma cell line HepG2. The open bar represents the predicted ORF coding for hIre1p. (B) Domain organization of hIre1p. (Solid box) Potential signal sequence; (formula image) potential N-linked glycosylation site; (TM) a putative transmembrane region; (Linker) a region having no homology to known proteins; (S/T kinase) catalytic domain of Ser/Thr protein kinase; (RNase L) a domain having high homology to 2-5 oligo-(A)–dependent RNase. Percent identity to the corresponding domains of S. cerevisiae and C. elegans is indicated. (C) (H.s.) Amino acid sequence alignment of human Ire1p, (S.c.) S. cerevisiae Ire1p and (C.e.) its putative homologous protein from C. elegans. (Open boxes) The identical sequence; (shaded boxes) conserved residues; (dashes) gaps between residues to obtain maximum matching. Numbers are the position of the last amino acid. (▿) Potential signal peptide cleavage site; (•) invariant residues in protein kinase domain; (*) invariant Lys599 residue in kinase subdomain II. The glutamine rich cluster (I) and the serine rich cluster (II) in the linker region are also identified.
Figure 2
Figure 2
hIRE1 is ubiquitously expressed in human tissues. Northern blot analysis of poly(A)+ RNA isolated from various human tissues (Clontech) by hybridization with 32P-labeled-cDNA probes corresponding to the hIRE1 luminal domain, the hIRE1 cytoplasmic domain, or human β-actin cDNA. Exposure of the Ire1 autoradiograph was 24-fold longer than that of β-actin.
Figure 3
Figure 3
Overexpression of hIre1p in transiently transfected COS-1 monkey cells. (A) hIre1p expression in transfected COS-1 cells. COS-1 cells were transiently transfected with or without expression plasmids encoding wild-type hIre1p (pED–hIRE1) or its kinase defective mutant (pED–hIRE1 K599A). Transfected cells were pulse labeled with [35S]-methionine and cysteine for 15 min. Cell extracts were prepared and equal amounts were immunoprecipitated with α-hIre1p antibodies and analyzed by SDS-PAGE and autoradiography. (B) Expression of eIF-2α in cotransfected cells. COS-1 cells were mock transfected (lane 1) or cotransfected with pED–eIF-2α in the presence of pED (lane 2), pED–hIRE1 (lane 3), or pED–hIRE1 K599A (lane 4). The cells were pulse-labeled with [35S]methionine and cysteine for 15 min. Cell extracts were prepared and equal cpm of radiolabeled protein were analyzed directly by SDS-PAGE and autoradiography. (C) Functional hIre1p limits accumulation of hIRE1 mRNA. Total RNA was isolated from COS-1 cells transfected with pED, pED–hIRE1, or pED–hIRE1 K599A plasmid and treated in the presence (+) or absence (−) of cycloheximide. RNA samples (10 μg) were resolved in a formaldehyde–agarose gel, blotted onto nylon membrane, and hybridized with 32P-labeled hIRE1 cDNA probe. (Arrow) The hIRE1 transcript. (D) hIre1p has intrinsic kinase activity. Wild-type or K599A mutant hIre1p was immunoprecipitated from transiently transfected COS-1 cells. (Mock) Cells that did not receive plasmid DNA. The proteins were incubated in kinase buffer in the presence of [γ-32P]ATP at 30°C for 40 min. The proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. (Top) Incorporation of [32P]phosphate into hIre1p determined by autoradiography; (bottom panel) the Ire1p protein level determined by estern blot analysis by use of α-hIre1p antibodies and alkaline phosphatase staining. The amount of K599A mutant hIre1p loaded onto the gel is one-third the amount of the immunoprecipitated proteins loaded for lanes 1 and 2. Therefore, the amount of steady-state K599A mutant hIre1p is ∼10-fold greater than the wild-type hIre1p.
Figure 3
Figure 3
Overexpression of hIre1p in transiently transfected COS-1 monkey cells. (A) hIre1p expression in transfected COS-1 cells. COS-1 cells were transiently transfected with or without expression plasmids encoding wild-type hIre1p (pED–hIRE1) or its kinase defective mutant (pED–hIRE1 K599A). Transfected cells were pulse labeled with [35S]-methionine and cysteine for 15 min. Cell extracts were prepared and equal amounts were immunoprecipitated with α-hIre1p antibodies and analyzed by SDS-PAGE and autoradiography. (B) Expression of eIF-2α in cotransfected cells. COS-1 cells were mock transfected (lane 1) or cotransfected with pED–eIF-2α in the presence of pED (lane 2), pED–hIRE1 (lane 3), or pED–hIRE1 K599A (lane 4). The cells were pulse-labeled with [35S]methionine and cysteine for 15 min. Cell extracts were prepared and equal cpm of radiolabeled protein were analyzed directly by SDS-PAGE and autoradiography. (C) Functional hIre1p limits accumulation of hIRE1 mRNA. Total RNA was isolated from COS-1 cells transfected with pED, pED–hIRE1, or pED–hIRE1 K599A plasmid and treated in the presence (+) or absence (−) of cycloheximide. RNA samples (10 μg) were resolved in a formaldehyde–agarose gel, blotted onto nylon membrane, and hybridized with 32P-labeled hIRE1 cDNA probe. (Arrow) The hIRE1 transcript. (D) hIre1p has intrinsic kinase activity. Wild-type or K599A mutant hIre1p was immunoprecipitated from transiently transfected COS-1 cells. (Mock) Cells that did not receive plasmid DNA. The proteins were incubated in kinase buffer in the presence of [γ-32P]ATP at 30°C for 40 min. The proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. (Top) Incorporation of [32P]phosphate into hIre1p determined by autoradiography; (bottom panel) the Ire1p protein level determined by estern blot analysis by use of α-hIre1p antibodies and alkaline phosphatase staining. The amount of K599A mutant hIre1p loaded onto the gel is one-third the amount of the immunoprecipitated proteins loaded for lanes 1 and 2. Therefore, the amount of steady-state K599A mutant hIre1p is ∼10-fold greater than the wild-type hIre1p.
Figure 3
Figure 3
Overexpression of hIre1p in transiently transfected COS-1 monkey cells. (A) hIre1p expression in transfected COS-1 cells. COS-1 cells were transiently transfected with or without expression plasmids encoding wild-type hIre1p (pED–hIRE1) or its kinase defective mutant (pED–hIRE1 K599A). Transfected cells were pulse labeled with [35S]-methionine and cysteine for 15 min. Cell extracts were prepared and equal amounts were immunoprecipitated with α-hIre1p antibodies and analyzed by SDS-PAGE and autoradiography. (B) Expression of eIF-2α in cotransfected cells. COS-1 cells were mock transfected (lane 1) or cotransfected with pED–eIF-2α in the presence of pED (lane 2), pED–hIRE1 (lane 3), or pED–hIRE1 K599A (lane 4). The cells were pulse-labeled with [35S]methionine and cysteine for 15 min. Cell extracts were prepared and equal cpm of radiolabeled protein were analyzed directly by SDS-PAGE and autoradiography. (C) Functional hIre1p limits accumulation of hIRE1 mRNA. Total RNA was isolated from COS-1 cells transfected with pED, pED–hIRE1, or pED–hIRE1 K599A plasmid and treated in the presence (+) or absence (−) of cycloheximide. RNA samples (10 μg) were resolved in a formaldehyde–agarose gel, blotted onto nylon membrane, and hybridized with 32P-labeled hIRE1 cDNA probe. (Arrow) The hIRE1 transcript. (D) hIre1p has intrinsic kinase activity. Wild-type or K599A mutant hIre1p was immunoprecipitated from transiently transfected COS-1 cells. (Mock) Cells that did not receive plasmid DNA. The proteins were incubated in kinase buffer in the presence of [γ-32P]ATP at 30°C for 40 min. The proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. (Top) Incorporation of [32P]phosphate into hIre1p determined by autoradiography; (bottom panel) the Ire1p protein level determined by estern blot analysis by use of α-hIre1p antibodies and alkaline phosphatase staining. The amount of K599A mutant hIre1p loaded onto the gel is one-third the amount of the immunoprecipitated proteins loaded for lanes 1 and 2. Therefore, the amount of steady-state K599A mutant hIre1p is ∼10-fold greater than the wild-type hIre1p.
Figure 3
Figure 3
Overexpression of hIre1p in transiently transfected COS-1 monkey cells. (A) hIre1p expression in transfected COS-1 cells. COS-1 cells were transiently transfected with or without expression plasmids encoding wild-type hIre1p (pED–hIRE1) or its kinase defective mutant (pED–hIRE1 K599A). Transfected cells were pulse labeled with [35S]-methionine and cysteine for 15 min. Cell extracts were prepared and equal amounts were immunoprecipitated with α-hIre1p antibodies and analyzed by SDS-PAGE and autoradiography. (B) Expression of eIF-2α in cotransfected cells. COS-1 cells were mock transfected (lane 1) or cotransfected with pED–eIF-2α in the presence of pED (lane 2), pED–hIRE1 (lane 3), or pED–hIRE1 K599A (lane 4). The cells were pulse-labeled with [35S]methionine and cysteine for 15 min. Cell extracts were prepared and equal cpm of radiolabeled protein were analyzed directly by SDS-PAGE and autoradiography. (C) Functional hIre1p limits accumulation of hIRE1 mRNA. Total RNA was isolated from COS-1 cells transfected with pED, pED–hIRE1, or pED–hIRE1 K599A plasmid and treated in the presence (+) or absence (−) of cycloheximide. RNA samples (10 μg) were resolved in a formaldehyde–agarose gel, blotted onto nylon membrane, and hybridized with 32P-labeled hIRE1 cDNA probe. (Arrow) The hIRE1 transcript. (D) hIre1p has intrinsic kinase activity. Wild-type or K599A mutant hIre1p was immunoprecipitated from transiently transfected COS-1 cells. (Mock) Cells that did not receive plasmid DNA. The proteins were incubated in kinase buffer in the presence of [γ-32P]ATP at 30°C for 40 min. The proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. (Top) Incorporation of [32P]phosphate into hIre1p determined by autoradiography; (bottom panel) the Ire1p protein level determined by estern blot analysis by use of α-hIre1p antibodies and alkaline phosphatase staining. The amount of K599A mutant hIre1p loaded onto the gel is one-third the amount of the immunoprecipitated proteins loaded for lanes 1 and 2. Therefore, the amount of steady-state K599A mutant hIre1p is ∼10-fold greater than the wild-type hIre1p.
Figure 4
Figure 4
hIre1p is a site-specific endoribonuclease. (A) In vitro cleavage of yeast HAC1 mRNA by hIre1p. An in vitro-transcribed 32P-labeled HAC1 mRNA was incubated with E. coli-expressed GST or GST–Ire1p adsorbed to glutathione beads or with COS-1 cell-expressed hIre1p or hIre1p K599A protein adsorbed to protein A–Sepharose beads. After the indicated period of time, the cleavage products were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel. Schemes on the left depict the predicted cleavage products. Numbers at right indicate predicted base pair size of RNA products expected based on yeast HAC1 mRNA cleavage by yeast Ire1p (Sidrauski and Walter 1997). (B) hIre1p cleaves yeast HAC1 mRNA at residue G661. The HAC1 RNA cleavage site was mapped using in vitro-transcribed HAC1mRNA after incubation with GST,GST–Ire1p, hIre1p, or hIre1p K599A as described in A. The products were reverse transcribed with Superscript II Reverse Transcriptase (Bethesda Research Labs) by use of oligonucleotide primer complementary to the intron of HAC1 RNA. Sequencing ladders on the left represent HAC1 DNA sequence determined with the same primer. (Arrow) Position of primer extended products.
Figure 4
Figure 4
hIre1p is a site-specific endoribonuclease. (A) In vitro cleavage of yeast HAC1 mRNA by hIre1p. An in vitro-transcribed 32P-labeled HAC1 mRNA was incubated with E. coli-expressed GST or GST–Ire1p adsorbed to glutathione beads or with COS-1 cell-expressed hIre1p or hIre1p K599A protein adsorbed to protein A–Sepharose beads. After the indicated period of time, the cleavage products were analyzed by electrophoresis on a 5% denaturing polyacrylamide gel. Schemes on the left depict the predicted cleavage products. Numbers at right indicate predicted base pair size of RNA products expected based on yeast HAC1 mRNA cleavage by yeast Ire1p (Sidrauski and Walter 1997). (B) hIre1p cleaves yeast HAC1 mRNA at residue G661. The HAC1 RNA cleavage site was mapped using in vitro-transcribed HAC1mRNA after incubation with GST,GST–Ire1p, hIre1p, or hIre1p K599A as described in A. The products were reverse transcribed with Superscript II Reverse Transcriptase (Bethesda Research Labs) by use of oligonucleotide primer complementary to the intron of HAC1 RNA. Sequencing ladders on the left represent HAC1 DNA sequence determined with the same primer. (Arrow) Position of primer extended products.
Figure 5
Figure 5
hIre1p contains high-mannose core oligosaccharides. Transfected COS-1 cells that overexpress hIre1p were pulse labeled with [35S]methionine and cysteine for 15 min in the presence (lane 2) or absence (lane 1) of tunicamycin and cell extracts were prepared. In parallel, cells pulse labeled 15 min in the absence of tunicamycin were incubated 3 hr in medium containing excess unlabeled methionine and cysteine before harvesting cell extracts. The 35Slabeled hIre1p was immunoprecipitated from cell extracts and analyzed by SDS-PAGE. Prior to SDS-PAGE, immunoprecipitated samples were incubated in the absence (lanes 1–3) or presence (lane 4) of endoglycosidase H.
Figure 6
Figure 6
Confocal laser scanning fluorescence microscopy of hIre1p expressed in COS-1 cells. The subcellular localization of hIre1p in transfected COS-1 cells was determined by immunofluorescence with mouse α-hIre1p. (A) COS-1 cells transfected with wild-type, or (B) K599A mutant IRE1 expression plasmids were double labeled with mouse α-hIre1p and rabbit α-GRP94. (C) COS-1 cells transfected with wild-type IRE1 expression plasmid were double labeled with mouse α-hIre1p and guinea pig α-RanGAP1. Secondary antibodies used were either rhodamine-conjugated goat α-rabbit or rhodamine-conjugated goat α-guinea pig (red) in the presence of fluorescein-conjugated goat α-mouse (green). The images were merged where colocalization is shown in yellow. Cells were viewed and digitally photographed with a Bio-Rad confocal fluorescence microscope. Bar, 25 μm.
Figure 6
Figure 6
Confocal laser scanning fluorescence microscopy of hIre1p expressed in COS-1 cells. The subcellular localization of hIre1p in transfected COS-1 cells was determined by immunofluorescence with mouse α-hIre1p. (A) COS-1 cells transfected with wild-type, or (B) K599A mutant IRE1 expression plasmids were double labeled with mouse α-hIre1p and rabbit α-GRP94. (C) COS-1 cells transfected with wild-type IRE1 expression plasmid were double labeled with mouse α-hIre1p and guinea pig α-RanGAP1. Secondary antibodies used were either rhodamine-conjugated goat α-rabbit or rhodamine-conjugated goat α-guinea pig (red) in the presence of fluorescein-conjugated goat α-mouse (green). The images were merged where colocalization is shown in yellow. Cells were viewed and digitally photographed with a Bio-Rad confocal fluorescence microscope. Bar, 25 μm.
Figure 6
Figure 6
Confocal laser scanning fluorescence microscopy of hIre1p expressed in COS-1 cells. The subcellular localization of hIre1p in transfected COS-1 cells was determined by immunofluorescence with mouse α-hIre1p. (A) COS-1 cells transfected with wild-type, or (B) K599A mutant IRE1 expression plasmids were double labeled with mouse α-hIre1p and rabbit α-GRP94. (C) COS-1 cells transfected with wild-type IRE1 expression plasmid were double labeled with mouse α-hIre1p and guinea pig α-RanGAP1. Secondary antibodies used were either rhodamine-conjugated goat α-rabbit or rhodamine-conjugated goat α-guinea pig (red) in the presence of fluorescein-conjugated goat α-mouse (green). The images were merged where colocalization is shown in yellow. Cells were viewed and digitally photographed with a Bio-Rad confocal fluorescence microscope. Bar, 25 μm.
Figure 7
Figure 7
hIre1p-dependent induction of UPR in mammalian cells. The activation of the unfolded protein response was measured by cotransfection of COS-1 cells with a luciferase reporter plasmid under the control of the rat BiP promoter, RSV–β-gal and either pED–hIRE1 or pED–hIRE1 K599A plasmid DNAs. At 60 hr post-transfection, the cells were treated with 10 μg/ml tunicamycin for 6 hr. The luciferase activity was determined from triplicate independent transfection experiments and was normalized to β-galactosidase activity to correct for transfection efficiency.
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