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Vol. 10, Issue 11, 3787-3799, November 1999
HSP Research Institute, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan
Submitted June 7, 1999; Accepted September 8, 1999| |
ABSTRACT |
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The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.
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INTRODUCTION |
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The endoplasmic reticulum (ER) provides the optimal environment
for proper folding and assembly of newly synthesized proteins destined
for the secretory pathway (Gething and Sambrook, 1992
; Helenius
et al., 1992
; Huppa and Ploegh, 1998
). Eukaryotic cells from
yeast to human have developed specific and tight communication systems
between the ER and cell nucleus to gear the folding capacity in the ER
to the requirements within the ER. Thus, accumulation of unfolded
proteins under so-called "ER stress" conditions leads to induction
of transcription of genes encoding molecular chaperones and folding
enzymes localized in the ER (Lee, 1987
; Kozutsumi et al.,
1988
; Normington et al., 1989
; Rose et al.,
1989
). This transcriptional induction process coupled with
intracellular signaling from the ER to the nucleus is called the
unfolded protein response (UPR) (McMillan et al., 1994
;
Shamu et al., 1994
).
The most proximal event in the UPR is the sensing step of ER
stress, which is considered to be mediated by Ire1p/Ern1p, a transmembrane protein kinase in the ER originally isolated by genetic
screening in Saccharomyces cerevisiae (Cox et
al., 1993
; Mori et al., 1993
). Although the detailed
mechanism is unknown, Ire1p is activated via oligomerization and
autophosphorylation upon accumulation of unfolded proteins in the ER
(Shamu and Walter, 1996
; Welihinda and Kaufman, 1996
). Ire1p is likely
to be negatively regulated by Ptc2p, a serine/threonine-specific
protein phosphatase (Welihinda et al., 1998
). Recent cloning
of the mammalian homologue of yeast Ire1p revealed the presence of at
least two different molecular species in mammalian cells (Tirasophon
et al., 1998
; Wang et al., 1998
). Overexpression
of either type of mammalian Ire1p constitutively activated
transcription of ER chaperone genes as in the case of yeast Ire1p,
suggesting similarity in the sensing system between yeast and mammals.
Interestingly, mammalian ER contains one more transmembrane protein
kinase called PEK/PERK, the lumenal domain of which shows
significant homology to that of Ire1p (Shi et al.,
1998
; Harding et al., 1999
). PEK/PERK is capable of
phosphorylating the
subunit of eukaryotic translation initiation
factor 2 and thus appears to be responsible for ER stress-induced
translational attenuation (Prostko et al., 1992
; reviewed by
Kaufman, 1999
).
The most distal step in the UPR is the transcription step that depends
on the specific interaction of a trans-acting factor with a
cis-acting element present in the promoter regions of UPR target genes. In S. cerevisiae, the cis-acting
unfolded protein response element (UPRE) was originally identified as a
sequence necessary and sufficient for induction of the KAR2
gene encoding the yeast homologue of a major ER chaperone, GRP78/BiP
(Mori et al., 1992
; Kohno et al., 1993
). We
recently showed that KAR2 UPRE contains an E box-like
partially palindromic sequence separated by a spacer of one nucleotide
(CAGCGTG) that is essential for its function
(Mori et al., 1996
). Subsequent characterization of UPRE
sequences responsible for induction of other ER stress-responsive genes
led us to propose that this unique feature of UPRE explains why only a
specific set of ER proteins are induced in yeast UPR (Mori et
al., 1998
).
In contrast to yeast UPR, multiple cis-acting elements
appeared to be involved in the mammalian UPR (Wooden et al.,
1991
). Recently, however, we and others independently found that a
unique sequence with a consensus of CCAAT-N9-CCACG is commonly present in promoter regions of almost all ER stress-responsive mammalian genes
(Yoshida et al., 1998
; Roy and Lee, 1999
). This novel
cis-acting element was designated the ER stress response
element (ERSE). By extensive mutational analysis, we demonstrated that
ERSE is necessary and sufficient for induction of at least three major ER chaperones (GRP78, GRP94, and calreticulin) (Yoshida et
al., 1998
). The CCAAT portion of ERSE was confirmed to be a
binding site for the general transcription factor
NF-Y/CBF as shown previously (Roy and Lee, 1995
; Roy
et al., 1996
). Thus, we proposed that the CCACG portion of
ERSE exactly 9 bp downstream of the CCAAT portion provides specificity
of the mammalian UPR (Yoshida et al., 1998
). The CCACG may
correspond to a half site of the palindromic sequence found in the
yeast UPRE.
In yeast, Hac1p/Ern4p was identified as the UPR-specific transcription
factor (Cox and Walter, 1996
; Mori et al., 1996
; Nikawa et al., 1996
). Hac1p contains a basic leucine zipper (bZIP)
motif and specifically binds to the E box-like palindromic sequence in
UPRE. Yeast cells lacking Hac1p show the same phenotype as those
lacking Ire1p; cells are unable to induce transcription of ER chaperone
genes in response to ER stress. Subsequent analysis conducted in
Walter's and our laboratories revealed an unexpected connection
between the most proximal event in the ER and the most distal event in
the nucleus, namely the unconventional splicing system for
HAC1 mRNA. HAC1 mRNA is constitutively expressed,
but its translation is tightly blocked because of the presence of an
intron of 252 nucleotides within the Hac1p-coding region. The HAC1 intron is specifically removed by a splicing event that
is activated by signaling from the ER. Spliced mRNA is translated, and
the Hac1p thus synthesized activates transcription of UPR target genes
through the UPRE (Cox and Walter, 1996
; Chapman and Walter, 1997
;
Kawahara et al., 1997
, 1998
). Enzymes responsible for this
unconventional mRNA splicing were identified by Walter and
colleagues. The splicing reaction is initiated by Ire1p, a sensor molecule of ER stress by itself; both 5'- and 3' splice sites
are cleaved by the action of the C-terminal endonuclease domain of
Ire1p, which is postulated to be activated upon ER stress (Sidrauski
and Walter, 1997
). The splicing reaction is completed by Rlg1p
(Sidrauski et al., 1996
), a tRNA ligase located in the nucleus (Clark and Abelson, 1987
). This unique system allows yeast cells to produce transcription factor Hac1p only when they sense ER
stress and require increased amounts of ER chaperones to cope with
unfolded proteins accumulated in the ER (reviewed by Sidrauski et
al., 1998
).
Recently, we screened for putative human ERSE-binding proteins and
obtained the bZIP protein ATF6 as a candidate (Yoshida et
al., 1998
). The amino acid sequence of the ATF6 basic region shows
significant similarity with that of yeast Hac1p. In contrast to Hac1p,
however, ATF6 is constitutively expressed as a 90-kDa protein, and ATF6
mRNA is not spliced in response to ER stress (Zhu et al.,
1997
; Yoshida et al., 1998
). Here, we analyzed the mechanism
of activation of ATF6 by ER stress and revealed an important step that
connects the event in the ER with that in the nucleus.
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MATERIALS AND METHODS |
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Plasmid Construction
Recombinant DNA techniques were performed according to standard
procedures (Sambrook et al., 1989
). The ATF6 expression
plasmid pCGN-ATF6 (Zhu et al., 1997
) was kindly provided by
Dr. R. Prywes (Columbia University, New York, NY). This plasmid
contains the full-length ATF6 cDNA at the XbaI site of the
mammalian expression vector pCGN and thus expresses ATF6 protein tagged
with the influenza virus hemagglutinin (HA) epitope at the N
terminus under the control of the cytomegalovirus promoter
[referred to here as pCGN-ATF6 (670)]. pCGN-ATF6 (402), pCGN-ATF6
(373), and pCGN-ATF6 (366) encoding truncated forms of ATF6 were
constructed by PCR-mediated amplification of the region corresponding
to amino acids 1-402, 1-373, and 1-366 of ATF6, respectively,
together with a stop codon (TAG) followed by insertion of the resultant
fragments into the XbaI site of pCGN after their sequences
had been confirmed.
Cell Culture and Transient Transfection
HeLa cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Cells were maintained in a 5% CO2 incubator at 37°C. Transfection
was carried out by the standard calcium phosphate method (Sambrook
et al., 1989
) as described previously (Yoshida et
al., 1998
) with some modifications. Briefly, HeLa cells plated at
~10% confluency 1 d before transfection were incubated with
calcium phosphate-DNA complex for 16 h at 37°C. After washing
with PBS, cells were cultured in fresh medium for 24 h
before extraction for immunoblotting or fixation for immunofluorescence analysis unless otherwise specified. Transfection efficiency depended on the amount of DNA used: usually ~5% with 1 µg of DNA and ~50% with 10 µg of DNA. To induce UPR, cells were treated with 2 µg/ml tunicamycin (Sigma, St. Louis, MO), 300 nM thapsigargin (Sigma), or 1 mM dithiothreitol for various periods as indicated.
Purification of Anti-ATF6 Antibody
Rabbit anti-B03N antiserum raised against the N-terminal region
of ATF6 (amino acids 6-307) fused to Escherichia coli
maltose-binding protein (Yoshida et al., 1998
) was purified
by removing materials bound to CH-Sepharose 4B (Amersham Pharmacia
Biotech, Buckinghamshire, UK) on which soluble proteins extracted from
E. coli cells producing large amounts of maltose-binding
protein from the plasmid pMAL-c2 (New England BioLabs, Beverly, MA) had
been immobilized. The flow-through fraction highly specific to ATF6
(see Figure 1) was used as anti-ATF6 antibody.
Immunoblotting
Immunoblotting analysis was carried out
according to the standard procedure (Sambrook et al., 1989
)
as described previously (Yoshida et al., 1998
) using an
enhanced chemiluminescence Western blotting detection system kit
(Amersham Pharmacia Biotech). Mouse anti-KDEL monoclonal antibody
(clone 10C3), mouse anti-HSP70 monoclonal antibody (clone C92F3A-5),
and rabbit antisera against the N or C terminus of calnexin were
obtained from StressGen Biotechnologies (Victoria, British Columbia,
Canada). Rabbit anti-HA epitope polyclonal antibody (Y-11) and goat
anti-lamin B polyclonal antibody (M-20) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA).
Pulse-Chase Analysis of ATF6
HeLa cells cultured in 60-mm dishes until ~80% confluency
were incubated for 20 min in methionine- and cysteine-free Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, antibiotics, and 10% dialyzed fetal calf serum. Cells were then pulse labeled for
30 min using 0.5 mCi (18.5 MBq)/plate
EXPRE35S35S protein
labeling mix (DuPont, Wilmington, DE) dissolved in 1 ml of the above
medium. To chase 35S-labeled proteins, cells were
washed with fresh complete medium three times and incubated in fresh
complete medium for various periods. After removal of medium, cells
were lysed by incubation for 10 min on ice in 0.1 ml of lysis buffer
(1× Tris-buffered saline containing 1% SDS, 1 mM PMSF, and 5 µg/ml
leupeptin). The lysates were boiled for 5 min and, after addition of
0.4 ml of dilution buffer (1× Tris-buffered saline containing 2%
Triton X-100), clarified by centrifugation at 15,000 × g for 10 min. The supernatant was incubated with 50 µl of
normal rabbit serum (Sigma) for 3 h at 4°C, and the mixture was
rotated overnight at 4°C after addition of 50 µl of 50% protein
A-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech). The mixture was
clarified by brief centrifugation, and 10 µl of anti-ATF6 antibody
were added to the resulting supernatant. After standing for 3 h at
4°C, 50 µl of 50% protein A-Sepharose 4 Fast Flow were added, and
the mixture was rotated for 2 h at 4°C. The resin was then
washed as described by Franzusoff et al. (1991)
, and the
immunoprecipitates were subjected to SDS-PAGE.
Indirect Immunofluorescence
HeLa cells were grown directly on slide glasses in 90-mm dishes. Cells were fixed with PLP solution (2% p-formaldehyde, 10 mM sodium metaperiodate, and 75 mM L-lysine in PBS) for 10 min at room temperature, permeabilized with ice-cold acetone for 10 min, and then reacted with various primary antibodies for 1 h at 37°C. Rabbit anti-ATF6 antibody was diluted by 50-fold with 1% BSA-TBST solution (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20, and 1% BSA) before use. Rabbit anti-HA epitope antibody and mouse anti-KDEL antibody were diluted with 1% BSA-TBST solution to concentrations of 4 and 20 µg/ml, respectively. Primary antibodies were visualized by incubation for 1 h at 37°C with either 10 µg/ml FITC-conjugated goat anti-rabbit immunoglobulin G antibody (Vector Laboratories, Burlingame, CA) or 50 µg/ml rhodamine-conjugated goat anti-mouse immunoglobulin G antibody (ICN Pharmaceuticals, Aurora, OH). The cells were counterstained with 0.3 µg/ml DAPI in PBS-glycerol (1:9). Photographs were taken using a 465- to 495-nm excitation filter and a 515- to 555-nm emission filter for fluorescein isothiocyanate and a 525- to 540-nm excitation filter and a 605- to 655-nm emission filter for rhodamine to avoid mutual interference.
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RESULTS |
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ER Stress Induces Processing of ATF6
We and others have shown that ATF6 is constitutively expressed in
HeLa cells as a 90-kDa protein (p90ATF6) (Zhu et al., 1997
; Yoshida et al., 1998
). Interestingly, however, we found that
the p90ATF6 level decreased, and instead a new band of 50 kDa (p50ATF6) appeared in ER-stressed cells before the induction of GRP78, a major
target protein of the mammalian UPR (Yoshida et al., 1998
). As the anti-B03N antiserum used in the previous study reacted with
multiple proteins present in HeLa cell extracts, we purified it as
described in MATERIALS AND METHODS. The purified antibody recognized
p90ATF6 almost exclusively on immunoblotting of
extracts of unstressed HeLa cells (Figure
1A, lane 1). We thus used this as
anti-ATF6 antibody, which should recognize the N-terminal region of
ATF6 (amino acids 6-307, indicated by the thick line in Figure 4B).
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We reexamined the time course of the conversion of p90ATF6 to p50ATF6
using the purified antibody in HeLa cells treated with thapsigargin,
which causes ER stress by inhibiting the ER
Ca2+-ATPase (Li et al., 1993
).
Consistent with our previous report (Yoshida et al., 1998
),
p90ATF6 level decreased, and p50ATF6 appeared from 1 h after
thapsigargin treatment, whereas an increase in the level of GRP78 was
detected from 4 h (Figure 1A). To examine whether p90ATF6 was
directly converted to p50ATF6 upon ER stress, we performed pulse-chase
experiments (Figure 1B). ATF6 was immunoprecipitated from HeLa cells
pulse labeled for 30 min with [35S]methionine
and [35S]cysteine and then chased for various
periods. In the absence of thapsigargin in the chase medium, p90ATF6
level decreased with a half-life of ~2 h, and no band corresponding
to p50ATF6 was detected (Figure 1B, lanes 1-5). In the presence
of thapsigargin, p50ATF6 appeared after the chase (Figure 1B, lanes
6-10), suggesting that ATF6 is synthesized as a precursor protein
(p90ATF6) in unstressed cells and processed into a mature form
(p50ATF6) in response to ER stress.
To further confirm whether ER stress did not induce de novo synthesis
of p50ATF6, we examined the effects of blocking new protein synthesis
on the conversion of p90ATF6 to p50ATF6. It was recently shown that
PERK, a transmembrane protein kinase involved in ER stress-induced
translational attenuation, was activated by thapsigargin treatment of
COS-1 cells even in the absence of protein synthesis (Harding et
al., 1999
). In marked contrast, p50ATF6 was not produced when HeLa
cells (Figure 2, lanes 1-3) or COS-1
cells (our unpublished observation) were pretreated for 30 min with
cycloheximide, an inhibitor of protein synthesis, before thapsigargin
treatment. The reason for the difference is currently unknown.
Importantly, however, we found that the conversion of p90ATF6 to
p50ATF6 could occur in the absence of protein synthesis under a certain
condition such as dithiothreitol treatment, which disrupts disulfide
bonding and thus malfolds proteins in the ER directly (Figure 2, lanes
4-6). All of the above results strongly indicate that the appearance
of p50ATF6 in ER-stressed cells is due to proteolytic cleavage of
preexisting p90ATF6.
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We also reexamined the time course of the processing of ATF6 in HeLa
cells treated with tunicamycin, which causes ER stress by inhibiting
N-glycosylation of newly synthesized proteins (Lee, 1987
; Kozutsumi
et al., 1988
). Consistent with our previous report (Yoshida
et al., 1998
), p90ATF6 level decreased, and p50ATF6 appeared from 2 h after tunicamycin treatment, whereas an increase in GRP78 level was detected from 6 h (Figure
3). Although the processing of ATF6 was
significantly slower in tunicamycin-treated cells than that in
thapsigargin-treated cells, appearance of p50ATF6 accompanied the
increase in level of GRP78 mRNA (Figure 3), suggesting that the
proteolytic conversion of p90ATF6 to p50ATF6 is a key regulatory step
in mammalian UPR.
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ATF6 Contains a Hydrophobic Stretch That Anchors in the ER Membrane
The experiments shown in Figures 1-3 revealed the production of a
novel band migrating slightly faster than p90ATF6 in addition to
p50ATF6 in tunicamycin-treated cells but not in thapsigargin- or
dithiothreitol-treated cells, suggesting that p90ATF6 might be
glycosylated and thus associated with the ER. In fact, when unstressed
HeLa cells were examined by indirect immunofluorescence analysis
(Figure 4A), fine reticular structures
surrounding the nucleus were observed using purified anti-ATF6
antibody, and the staining pattern was very similar to that obtained
with anti-KDEL antibody, which recognizes two major ER chaperones
(GRP78 and GRP94) in HeLa cells. Furthermore, hydropathy analysis
identified a hydrophobic stretch of 21 amino acids (amino acids
378-398) near the center of the ATF6 molecule, which is long enough to span the membrane once (Figure 4B). These results suggested that p90ATF6 is a transmembrane protein in the ER.
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p90ATF6 Is a Type II Transmembrane Glycoprotein in the ER
We conducted fractionation experiments using untreated HeLa cells
and those treated with tunicamycin for 4 h as starting materials to monitor recovery of p90ATF6 and p50ATF6, respectively (Figure 5). After the first low-speed
centrifugation of the homogenate, p50ATF6 was recovered exclusively in
the nuclear pellet similarly to the nuclear protein lamin B (Figure 5,
lane 4), whereas the majority of p90ATF6 remained in the supernatant
(Figure 5, lane 5). After the second high-speed centrifugation, all
p90ATF6 was recovered in the membrane fraction (Figure 5, lane 9). The
distribution pattern of p90ATF6 was almost identical to that of
calnexin, a transmembrane-type chaperone localized in the ER (Wada
et al., 1991
), but completely different from those of lamin
B (Moir et al., 1995
) and the cytosolic protein HSP70 (Welch
and Suhan, 1986
), revealing that p90ATF6 is indeed associated with
membranes. It should be noted that p90ATF6 remaining in
tunicamycin-treated cells as well as a protein band migrating slightly
faster than p90ATF6 (lane 2) were distributed in each fraction
similarly to p90ATF6 in untreated cells.
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To determine whether p90ATF6 is a peripheral or integral membrane
protein, differential solubilization experiments were carried out
(Figure 6A). The supernatant fraction
obtained after low-speed centrifugation of untreated HeLa cell
homogenate was treated with various reagents and then centrifuged at
high speed. Neither p90ATF6 nor calnexin was released from membranes by
0.5 M NaCl or 0.1 M Na2CO3,
pH 11, treatment, which should extract peripheral membrane proteins. In
contrast, both proteins were released into the supernatant by
detergents such as 1% SDS and 1% sodium deoxycholate, whereas only
calnexin was extracted by 1% Triton X-100; Triton X-100 may cause
aggregation of p90ATF6 as in the case of yeast Ire1p (Mori et
al., 1993
). Thus, p90ATF6 is an integral membrane protein.
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Next, the orientation of p90ATF6 in membranes was examined by trypsin treatment (Figure 6B). Calnexin, a type I transmembrane protein, was used as a control and digestion of calnexin was monitored using two different antibodies specific to either the N-terminal region present in the ER lumen (Calnexin-N) or the C-terminal region located in the cytoplasm (Calnexin-C). At the concentration of trypsin that decreased the amount of full-length calnexin, anti-Calnexin-N antibody (Figure 6B, lanes 7 and 8) but not anti-Calnexin-C antibody (Figure 6B, lanes 11 and 12) detected a fragment the size of which matched that of the N-terminal region of calnexin, indicating that proteins or segments in the lumen were protected from digestion by trypsin as expected. Under these conditions, p90ATF6 disappeared at the lowest concentration of trypsin used, and no fragments of ~50 kDa were detected by anti-ATF6 antibody raised against the N-terminal region of ATF6 (Figure 6B, lane 2). These results strongly indicated that the N-terminal domain of ATF6 containing the bZIP region is oriented facing the cytoplasm.
Examination of the amino acid sequence of the lumenal domain of ATF6 indicated the presence of three potential glycosylation sites as schematically presented in Figure 4B. To determine whether p90ATF6 is actually glycosylated, unstressed HeLa cell extracts were denatured and treated with endoglycosidase H followed by immunoblotting analysis (Figure 6C). p90ATF6 treated with endoglycosidase H (Figure 6C, lane 3) migrated faster than untreated p90ATF6 (Figure 6C, lane 2) but migrated at the same position as in vitro-translated full-length ATF6 (Figure 6C, lane 1), whereas the mobility of calnexin was unaffected by endoglycosidase H treatment (Figure 6C, lanes 4 and 5); calnexin contains no potential glycosylation sites. The sensitivity of the carbohydrate moiety to digestion with endoglycosidase H implied that p90ATF6 stays in the ER and is not subjected to retrograde transport from the Golgi apparatus.
We further determined the localization of p90ATF6 after transfection. pCGN-ATF6 (670) expressed full-length ATF6 tagged with the HA epitope at the N terminus (see MATERIALS AND METHODS). Immunofluorescence analysis of transfected HeLa cells using anti-HA epitope antibody revealed that expression of ATF6 (670) was restricted to perinuclear structures, which were also stained with anti-KDEL antibody (Figure 6D, compare a with b and c), demonstrating that p90ATF6 was localized in the ER even when overexpressed. Taken together, we concluded that p90ATF6 is a type II transmembrane glycoprotein localized in the ER.
p50ATF6 Is a Soluble Nuclear Protein
In contrast to the tight association of p90ATF6 with ER membranes,
p50ATF6 detected in ER-stressed cells was recovered exclusively in the
nuclear pellet fraction (Figure 5, lane 4). As a small portion of
p90ATF6 was also recovered in the same fraction (Figure 5, lane 3), we
further extracted the nuclear pellet with high salt buffer (Figure
7A). As a result, more than half of
p50ATF6 was released into the supernatant (Figure 7A, compare lane 2 with lane 4), whereas all p90ATF6 remained in the pellet (Figure 7A, compare lane 1 with lane 3). Furthermore, when HeLa cells were extracted by repeated freezing and thawing (Figure 7B), all p50ATF6 was
recovered in the supernatant (Figure 7B, compare lane 2 with lane 4),
whereas all p90ATF6 was recovered in the pellet (Figure 7B, compare
lane 1 with lane 3). From these results, we concluded that p50ATF6 is a
soluble nuclear protein in marked contrast to p90ATF6.
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Nonetheless, we could not determine the localization of endogenous
p50ATF6 by immunofluorescence analysis. We reproducibly observed
decreased staining of the ER with anti-ATF6 antibody 4 h after
tunicamycin treatment but hardly detected increased staining of the
nucleus, probably because the small amounts of p50ATF6 produced
diffused out in the nucleus, the volume of which was much larger than
that of the ER (see Figure 4A). Because of this technical problem, it
was also not possible to visualize the change in localization of ATF6
from the ER to the nucleus after tunicamycin or thapsigargin treatment
of HeLa cells transfected with pCGN-ATF6 (670) and thus overproducing
full-length ATF6. It should be noted that in thapsigargin-treated cells
the amounts of p90ATF6 were much greater than those of p50ATF6 at all
time points (Figure 1A). Similarly, the total amounts of p90ATF6 plus unglycosylated p90ATF6 were much greater than those of p50ATF6 in
tunicamycin-treated cells at all time points (Figure 3). To circumvent
this difficulty, we overexpressed various ATF6 mutants and examined
their localization in transiently transfected HeLa cells (Figure
8).
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The N-terminal Fragment of ATF6 Representing p50ATF6 Translocates into the Nucleus
The above results indicated that ER stress triggers processing of ATF6, which not only decreases its molecular mass from 90 to 50 kDa but also alters its subcellular localization. We hypothesized that such dual outcomes could result from proteolysis of ATF6 in the C-terminal region; p50ATF6 reacts with anti-ATF6 antibody raised against the N-terminal region of ATF6. We therefore constructed various C-terminal deletion mutants and examined the localization of expressed proteins (see schematic structures depicted in Figure 8A). The boundaries of three functional domains should be noted; the basic region (R308-Y330), the leucine zipper (L334-X6-A-X6-L-X6-L-X6-V-X6-L369), and the transmembrane domain (V378-L398). Thus, ATF6 (402) lacked the majority of the C-terminal lumenal domain but retained the transmembrane domain, whereas two mutants, ATF6 (373) and ATF6 (366), lacked both lumenal and transmembrane domains. ATF6 (373) contained the entire bZIP region. ATF6 (366) contained the entire basic region and majority of the leucine zipper region. All mutant proteins were tagged with the HA epitope at the N terminus (Figure 8A).
Before localization analysis, we examined by immunoblotting whether these mutant forms of ATF6 were actually expressed in transfected HeLa cells (Figure 8B). Simultaneously, this analysis provided useful information on the cleavage site in p90ATF6. The doublet protein bands detected at ~50 kDa in cells transfected with pCGN-ATF6 (670) (Figure 8B, lane 2) served as a molecular size marker for p50ATF6; these proteins were considered to represent p50ATF6, which was produced by constitutively activated proteolysis of the HA-tagged ATF6 (670) overexpressed in transfected cells (see DISCUSSION for explanation). The faster-migrating band might have lost the HA tag. Comparison of the mobilities of various C-terminal deletion mutants on SDS-PAGE revealed that the size of p50ATF6 was close to those of ATF6 (373) and ATF6 (366) (Figure 8B, lanes 4 and 5, respectively), suggesting that p90ATF6 may be cleaved between the bZIP and transmembrane domains to produce p50ATF6 when the cellular UPR was activated. We thus used ATF6 (373) and ATF6 (366) as representatives of p50ATF6 in the following experiments.
Immunofluorescence analysis showed that ATF6 (402) was localized in the ER (Figure 8C, compare a with b and c), indicating that the C-terminal lumenal domain is dispensable for the association with ER membranes. In marked contrast, ATF6 mutants lacking both lumenal and transmembrane domains entered the nucleus; the nucleus was clearly stained with anti-HA epitope antibody in cells overexpressing ATF6 (373) and ATF6 (366) (Figure 8C, compare d and g with e, f, h, and i). These results demonstrated that the localization of ATF6 is determined by the presence or absence of the transmembrane domain in the molecule and strongly suggested that the ATF6 molecule released from ER membranes (p50ATF6) can translocate into the nucleus.
ATF6 Mutants Representing p50ATF6 Enhance the Levels of Endogenous GRP78 by Activating Transcription
We finally examined the effects of overexpression of ATF6 mutants
on the levels of endogenous UPR targets at both mRNA and protein levels
(Figure 9). The level of GRP78 mRNA was
slightly enhanced by overexpression of full-length ATF6, ATF6 (670),
compared with the vector control (Figure 9, compare lane 2 with lane
1), although ATF6 (670) was primarily localized in the ER (Figure 6D;
see DISCUSSION for explanation). Most importantly, the levels of both
GRP78 mRNA and GRP78 were markedly enhanced by overexpression of ATF6
(373) and ATF6 (366) (Figure 9, lanes 3 and 4), both of which were
localized in the nucleus (Figure 8C). We concluded that these two ATF6
mutants representing p50ATF6 directly activated transcription of the
GRP78 genes in the nucleus, leading to enhanced levels of GRP78 in the
ER.
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DISCUSSION |
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|
|
|---|
Eukaryotic cells possess multiple intracellular signaling pathways
from the ER to the nucleus, each modulating gene expression in response
to changes in or surrounding the ER (reviewed by Pahl and Baeuerle,
1997
). One of these, the UPR, deals with homeostasis of the folding
capacity in the ER. Without this system, both yeast cells (Cox et
al., 1993
; Mori et al., 1993
, 1996
; Nikawa et
al., 1996
) and mammalian cells (Li and Lee, 1991
; Little and Lee,
1995
; Morris et al., 1997
; Liu et al., 1998
) are
unable to survive under ER stress conditions that cause continuous
accumulation of unfolded proteins in the ER.
In this study, we analyzed the mechanism of activation of ATF6 by ER
stress, which we recently isolated as a candidate for a mammalian
UPR-specific transcription factor. We showed that constitutively
expressed 90-kDa protein (p90ATF6) is directly converted to a 50-kDa
protein (p50ATF6) specifically in ER-stressed cells before the
induction of GRP78, a major target protein of the mammalian UPR
(Figures 1-3). The results presented here revealed the most important
consequence of this conversion, that is, alteration of subcellular
localization (see our current model depicted in Figure
10). Fractionation (Figure 5),
differential solubilization (Figure 6A), topological studies (Figure
6B), glycosylation analysis (Figure 6C), and immunoflurescence analysis
(Figures 4A and 6D) clearly demonstrated that p90ATF6 is a type II
transmembrane glycoprotein embedded in the ER membrane. In contrast,
p50ATF6 is a soluble nuclear protein (Figures 5 and 7). When
overexpressed, ATF6 mutants representing p50ATF6 were accumulated in
the nucleus (Figure 8) and constitutively activated transcription of
the GRP78 gene, leading to enhanced levels of GRP78 in the ER (Figure
9). We thus propose that p50ATF6 represents an active and mature form
of the mammalian transcription factor ATF6, and its production is
mediated by ER stress-induced proteolysis of p90ATF6, which is
synthesized as a precursor protein embedded in the ER membrane. Upon ER
stress, p50ATF6 is released from the ER membrane, allowing it to enter the nucleus. In the nucleus, p50ATF6 containing a bZIP domain activates
transcription of ER chaperone genes such as GRP78 through ERSE in
collaboration with the general transcription factor NF-Y; we recently
found that ATF6 recognizes ERSE and directly interacts with NF-Y (our
unpublished observation).
|
Interestingly, p90ATF6 appears to be turned over fairly quickly; its
half-life within the cell is ~2 h (Figure 1). This rapid turnover
rate allowed the cells to restore p90ATF6 at 16 h after thapsigargin treatment (Figure 1A). Similarly, p90ATF6 was restored at
16 h after tunicamycin treatment, although it was unglycosylated (Figure 3). Therefore, p90ATF6 itself might serve as a sensor molecule
of ER stress; under the conditions that cause accumulation of unfolded
proteins in the ER, p90ATF6 would not be able to fold properly, and
this may somehow activate proteolytic processing of p90ATF6, resulting
in production of p50ATF6. In this connection, it is noteworthy that
overexpression of full-length ATF6, ATF6 (670), constitutively
activated transcription of the GRP78 gene, albeit only slightly (Figure
9). We reasoned that overproduction of ATF6 (670), a transmembrane
protein in the ER, is sensed as ER stress by the cell probably because
normal levels of ER chaperones are insufficient for proper folding of
exogenous proteins expressed at high levels. As a result, portions of
endogenous p90ATF6 and exogenous ATF6 (670) are subjected to
proteolytic processing constitutively, resulting in enhanced
transcription of ER chaperone genes by constitutively produced p50ATF6
through ERSE in the nucleus. Indeed, p50ATF6-like doublet protein bands
were detected in extracts of cells transfected with pCGN-ATF6 (670)
(Figure 8B, lane 2). This also explains why various promoters of ER
chaperone genes were constitutively activated when full-length ATF6 was
overexpressed in HeLa cells (Yoshida et al., 1998
).
We showed here that the mammalian UPR uses a system very similar to
that previously identified for cholesterol homeostasis, another
well-investigated intracellular signaling pathway from the ER to the
nucleus (Yokoyama et al., 1993
; Wang et al.,
1994
; Sakai et al., 1996
, reviewed by Brown and Goldstein,
1997
). Cholesterol metabolism is primarily regulated at the level of
transcription, and sterol regulatory element binding proteins (SREBPs)
mediate transcriptional activation of genes involved in cholesterol
biosynthesis as well as receptor-mediated endocytosis of
cholesterol-containing lipoproteins from plasma. Under normal
conditions, SREBPs are bound to membranes of the ER and nuclear
envelope through two hydrophobic stretches separated by a spacer of 31 amino acids and located around the center of the molecule. Depletion of
sterols from culture medium activates proteolytic cleavage of SREBPs at the two sites in a sequential manner, allowing entrance of the N-terminal fragment into the nucleus. This released N-terminal fragment
contains all of the functional domains necessary for active
transcription factors such as DNA-binding (basic helix-loop-helix), dimerization (leucine zipper), and transactivation (acid blob) domains
and thus enhances transcription of target genes in the nucleus.
In contrast to SREBPs, the precise cleavage site of ATF6 is as
yet unknown. The region from amino acid 378 to 398 functions as a
transmembrane domain because it is the only hydrophobic segment found
in ATF6 (Figure 4B). The calculated molecular weight of the N-terminal
region (1-377) is 41,161 and is thus significantly smaller than the
size of p50ATF6 estimated from the mobility on SDS-PAGE. However, yeast
Hac1p behaves as a 41-kDa protein on SDS-PAGE despite its calculated
molecular weight of 26,903, presumably because of high contents of
basic amino acids (Kawahara et al., 1997
). Similarly,
full-length ATF6 behaves as a 90-kDa protein on SDS-PAGE despite its
calculated molecular weight of 74,597 (Zhu et al., 1997
;
Yoshida et al., 1998
). Comparison of the mobilities of
various C-terminal deletion mutants with that of p50ATF6 on SDS-PAGE
(Figure 8B) suggested that ATF6 is cleaved between the bZIP and
transmembrane domains, although we cannot exclude the possibility that
ATF6 is cleaved within the transmembrane domain, similarly to cleavage
at site 2 in SREBP2 (Sakai et al., 1996
), or cleaved around
the boundary between the cytoplasmic and transmembrane domains,
similarly to Notch-1 (Chan and Jan, 1998
; Schroeter et al.,
1998
). We are currently carrying out mutational analysis to determine
the precise cleavage site. Such information will be useful for
identification of the protease(s) responsible for proteolysis of ATF6.
We have identified a key regulatory step that connects events in the ER
with those in the nucleus in mammalian UPR, and our observations have
raised an important question: i.e., whether ER stress-induced
proteolysis of ATF6 is regulated by mammalian Ire1p, a putative sensor
molecule of ER stress identified recently (Tirasophon et
al., 1998
; Wang et al., 1998
). The detection of endonuclease activity in the C-terminal tail region of human Ire1p suggests the presence of a mammalian mRNA splicing system similar to
that for yeast HAC1 mRNA (Tirasophon et al.,
1998
), but ATF6 mRNA is not spliced (Yoshida et al., 1998
).
This raises the question of whether there are any substrates of such a
mammalian splicing system, and if so, what is the consequence of such
mRNA splicing. In yeast, transcriptional induction of ER chaperones is
coupled to phospholipid biosynthesis; yeast cells lacking either Ire1p or Hac1p require exogenous inositol for growth (Nikawa and
Yamashita, 1992
; Cox et al., 1993
; Mori et al.,
1993
, 1996
; Nikawa et al., 1996
; Sidrauski et
al., 1996
). It has been proposed that yeast UPR coordinates the
synthesis of ER chaperones and ER membranes via the Ire1p-Hac1p
pathway (Cox et al., 1997
). This provides a basis for one
intriguing speculation that in mammalian cells ER membrane biosynthesis
may be controlled differently from transcriptional regulation of ER
chaperone genes. Thus, the mammalian mRNA splicing system may be
specialized to adjust the production of phospholipids according to the
requirements within the ER. On the other hand, mammalian Ire1p may
regulate the synthesis of ER chaperones by phosphorylating a putative
protease(s), which activates ATF6. Answers to these important questions
will further extend our understanding of the molecular mechanism of the
mammalian UPR.
| |
ACKNOWLEDGMENTS |
|---|
We thank Masako Nakayama, Mayumi Ueda, Hideaki Kanazawa, Rika Takahashi, Seiji Takahara, and Tomoko Yoshifusa for technical assistance.
| |
FOOTNOTES |
|---|
* Corresponding author. E-mail address: kazumori{at}hsp.co.jp.
| |
ABBREVIATIONS |
|---|
Abbreviations used: bZIP, basic leucine zipper; ER, endoplasmic reticulum; ERSE, ER stress response element; HA, influenza virus hemagglutinin; SREBP, sterol regulatory element binding protein; UPR, unfolded protein response; UPRE, unfolded protein response element.
| |
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||||
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E. Karaskov, C. Scott, L. Zhang, T. Teodoro, M. Ravazzola, and A. Volchuk Chronic Palmitate But Not Oleate Exposure Induces Endoplasmic Reticulum Stress, Which May Contribute to INS-1 Pancreatic {beta}-Cell Apoptosis Endocrinology, July 1, 2006; 147(7): 3398 - 3407. [Abstract] [Full Text] [PDF] |
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C. Koumenis and B. G. Wouters "Translating" Tumor Hypoxia: Unfolded Protein Response (UPR)-Dependent and UPR-Independent Pathways Mol. Cancer Res., July 1, 2006; 4(7): 423 - 436. [Abstract] [Full Text] [PDF] |
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J. B. DuRose, A. B. Tam, and M. Niwa Intrinsic Capacities of Molecular Sensors of the Unfolded Protein Response to Sense Alternate Forms of Endoplasmic Reticulum Stress Mol. Biol. Cell, July 1, 2006; 17(7): 3095 - 3107. [Abstract] [Full Text] [PDF] |
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G. Donati, C. Imbriano, and R. Mantovani Dynamic recruitment of transcription factors and epigenetic changes on the ER stress response gene promoters Nucleic Acids Res., June 6, 2006; 34(10): 3116 - 3127. [Abstract] [Full Text] [PDF] |
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C. Bellodi, K. Kindle, F. Bernassola, D. Dinsdale, A. Cossarizza, G. Melino, D. Heery, and P. Salomoni Cytoplasmic Function of Mutant Promyelocytic Leukemia (PML) and PML-Retinoic Acid Receptor-{alpha} J. Biol. Chem., May 19, 2006; 281(20): 14465 - 14473. [Abstract] [Full Text] [PDF] |
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J. J. Martindale, R. Fernandez, D. Thuerauf, R. Whittaker, N. Gude, M. A. Sussman, and C. C. Glembotski Endoplasmic Reticulum Stress Gene Induction and Protection From Ischemia/Reperfusion Injury in the Hearts of Transgenic Mice With a Tamoxifen-Regulated Form of ATF6 Circ. Res., May 12, 2006; 98(9): 1186 - 1193. [Abstract] [Full Text] [PDF] |
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J. L. Vogel and T. M. Kristie Site-specific proteolysis of the transcriptional coactivator HCF-1 can regulate its interaction with protein cofactors PNAS, May 2, 2006; 103(18): 6817 - 6822. [Abstract] [Full Text] [PDF] |
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H. Kikuchi, G. Almer, S. Yamashita, C. Guegan, M. Nagai, Z. Xu, A. A. Sosunov, G. M. McKhann II, and S. Przedborski Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model PNAS, April 11, 2006; 103(15): 6025 - 6030. [Abstract] [Full Text] [PDF] |
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Y. Gu, X. Luo, S. Basu, H. Fujioka, and N. Singh Cell-specific metabolism and pathogenesis of transmembrane prion protein. Mol. Cell. Biol., April 1, 2006; 26(7): 2697 - 2715. [Abstract] [Full Text] [PDF] |
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K. Zhang and R. J. Kaufman The unfolded protein response: A stress signaling pathway critical for health and disease Neurology, January 24, 2006; 66(1_suppl_1): S102 - S109. [Abstract] [Full Text] [PDF] |
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Y. Kimata, Y. Ishiwata-Kimata, S. Yamada, and K. Kohno Yeast unfolded protein response pathway regulates expression of genes for anti-oxidative stress and for cell surface proteins Genes Cells, January 1, 2006; 11(1): 59 - 69. [Abstract] [Full Text] [PDF] |
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J. Stirling and P. O'Hare CREB4, a Transmembrane bZip Transcription Factor and Potential New Substrate for Regulation and Cleavage by S1P Mol. Biol. Cell, January 1, 2006; 17(1): 413 - 426. [Abstract] [Full Text] [PDF] |
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R. B. Hamanaka, B. S. Bennett, S. B. Cullinan, and J. A. Diehl PERK and GCN2 Contribute to eIF2{alpha} Phosphorylation and Cell Cycle Arrest after Activation of the Unfolded Protein Response Pathway Mol. Biol. Cell, December 1, 2005; 16(12): 5493 - 5501. [Abstract] [Full Text] [PDF] |
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Y. Wu, H. Zhang, Y. Dong, Y.-M. Park, and C. Ip Endoplasmic Reticulum Stress Signal Mediators Are Targets of Selenium Action Cancer Res., October 1, 2005; 65(19): 9073 - 9079. [Abstract] [Full Text] [PDF] |
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Z.-M. Huang, T. Tan, H. Yoshida, K. Mori, Y. Ma, and T. S. B. Yen Activation of Hepatitis B Virus S Promoter by a Cell Type-Restricted IRE1-Dependent Pathway Induced by Endoplasmic Reticulum Stress Mol. Cell. Biol., September 1, 2005; 25(17): 7522 - 7533. [Abstract] [Full Text] [PDF] |
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K. Tsuchimochi, N. Yagishita, S. Yamasaki, T. Amano, Y. Kato, K.-i. Kawahara, S. Aratani, H. Fujita, F. Ji, A. Sugiura, et al. Identification of a Crucial Site for Synoviolin Expression Mol. Cell. Biol., August 15, 2005; 25(16): 7344 - 7356. [Abstract] [Full Text] [PDF] |
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M. J. Turner, D. P. Sowders, M. L. DeLay, R. Mohapatra, S. Bai, J. A. Smith, J. R. Brandewie, J. D. Taurog, and R. A. Colbert HLA-B27 Misfolding in Transgenic Rats Is Associated with Activation of the Unfolded Protein Response J. Immunol., August 15, 2005; 175(4): 2438 - 2448. [Abstract] [Full Text] [PDF] |
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U. K. Misra and S. V. Pizzo Up-regulation of GRP78 and antiapoptotic signaling in murine peritoneal macrophages exposed to insulin J. Leukoc. Biol., July 1, 2005; 78(1): 187 - 194. [Abstract] [Full Text] [PDF] |
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M. Thomas, Z. Yu, N. Dadgar, S. Varambally, J. Yu, A. M. Chinnaiyan, and A. P. Lieberman The Unfolded Protein Response Modulates Toxicity of the Expanded Glutamine Androgen Receptor J. Biol. Chem., June 3, 2005; 280(22): 21264 - 21271. [Abstract] [Full Text] [PDF] |
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P. Baumeister, S. Luo, W. C. Skarnes, G. Sui, E. Seto, Y. Shi, and A. S. Lee Endoplasmic Reticulum Stress Induction of the Grp78/BiP Promoter: Activating Mechanisms Mediated by YY1 and Its Interactive Chromatin Modifiers Mol. Cell. Biol., June 1, 2005; 25(11): 4529 - 4540. [Abstract] [Full Text] [PDF] |
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I. Nagamori, N. Yabuta, T. Fujii, H. Tanaka, K. Yomogida, Y. Nishimune, and H. Nojima Tisp40, a spermatid specific bZip transcription factor, functions by binding to the unfolded protein response element via the Rip pathway Genes Cells, June 1, 2005; 10(6): 575 - 594. [Abstract] [Full Text] [PDF] |
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M. Abdelrahim, S. Liu, and S. Safe Induction of Endoplasmic Reticulum-induced Stress Genes in Panc-1 Pancreatic Cancer Cells Is Dependent on Sp Proteins J. Biol. Chem., April 22, 2005; 280(16): 16508 - 16513. [Abstract] [Full Text] [PDF] |
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Y. Iwata and N. Koizumi An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants PNAS, April 5, 2005; 102(14): 5280 - 5285. [Abstract] [Full Text] [PDF] |
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K.-T. Chin, H.-J. Zhou, C.-M. Wong, J. M.-F. Lee, C.-P. Chan, B.-Q. Qiang, J.-G. Yuan, I. O.-l. Ng, and D.-Y. Jin The liver-enriched transcription factor CREB-H is a growth suppressor protein underexpressed in hepatocellular carcinoma Nucleic Acids Res., March 30, 2005; 33(6): 1859 - 1873. [Abstract] [Full Text] [PDF] |
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J. Ramos-Castaneda, Y.-n. Park, M. Liu, K. Hauser, H. Rudolph, G. E. Shull, M. F. Jonkman, K. Mori, S. Ikeda, H. Ogawa, et al. Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum (ER)-associated Degradation and Sensitivity to ER Stress J. Biol. Chem., March 11, 2005; 280(10): 9467 - 9473. [Abstract] [Full Text] [PDF] |
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W. Vandenberghe, R. A. Nicoll, and D. S. Bredt Interaction with the Unfolded Protein Response Reveals a Role for Stargazin in Biosynthetic AMPA Receptor Transport J. Neurosci., February 2, 2005; 25(5): 1095 - 1102. [Abstract] [Full Text] [PDF] |
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J. Shen, E. L. Snapp, J. Lippincott-Schwartz, and R. Prywes Stable Binding of ATF6 to BiP in the Endoplasmic Reticulum Stress Response Mol. Cell. Biol., February 1, 2005; 25(3): 921 - 932. [Abstract] [Full Text] [PDF] |
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A. J. Kim, Y. Shi, R. C. Austin, and G. H. Werstuck Valproate protects cells from ER stress-induced lipid accumulation and apoptosis by inhibiting glycogen synthase kinase-3 J. Cell Sci., January 1, 2005; 118(1): 89 - 99. [Abstract] [Full Text] [PDF] |
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S. M. Weber, K. T. Chambers, K. G. Bensch, A. L. Scarim, and J. A. Corbett PPAR{gamma} ligands induce ER stress in pancreatic {beta}-cells: ER stress activation results in attenuation of cytokine signaling Am J Physiol Endocrinol Metab, December 1, 2004; 287(6): E1171 - E1177. [Abstract] [Full Text] [PDF] |
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Y. Kimata, D. Oikawa, Y. Shimizu, Y. Ishiwata-Kimata, and K. Kohno A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1 J. Cell Biol., November 8, 2004; 167(3): 445 - 456. [Abstract] [Full Text] [PDF] |
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J.-H. Hung, I.-J. Su, H.-Y. Lei, H.-C. Wang, W.-C. Lin, W.-T. Chang, W. Huang, W.-C. Chang, Y.-S. Chang, C.-C. Chen, et al. Endoplasmic Reticulum Stress Stimulates the Expression of Cyclooxygenase-2 through Activation of NF-{kappa}B and pp38 Mitogen-activated Protein Kinase J. Biol. Chem., November 5, 2004; 279(45): 46384 - 46392. [Abstract] [Full Text] [PDF] |
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D. Ito, J. R. Walker, C. S. Thompson, I. Moroz, W. Lin, M. L. Veselits, A. M. Hakim, A. A. Fienberg, and G. Thinakaran Characterization of Stanniocalcin 2, a Novel Target of the Mammalian Unfolded Protein Response with Cytoprotective Properties Mol. Cell. Biol., November 1, 2004; 24(21): 9456 - 9469. [Abstract] [Full Text] [PDF] |
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J. N. Lee and J. Ye Proteolytic Activation of Sterol Regulatory Element-binding Protein Induced by Cellular Stress through Depletion of Insig-1 J. Biol. Chem., October 22, 2004; 279(43): 45257 - 45265. [Abstract] [Full Text] [PDF] |
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R. Sriburi, S. Jackowski, K. Mori, and J. W. Brewer XBP1: a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum J. Cell Biol., October 11, 2004; 167(1): 35 - 41. [Abstract] [Full Text] [PDF] |
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J. Shen and R. Prywes Dependence of Site-2 Protease Cleavage of ATF6 on Prior Site-1 Protease Digestion Is Determined by the Size of the Luminal Domain of ATF6 J. Biol. Chem., October 8, 2004; 279(41): 43046 - 43051. [Abstract] [Full Text] [PDF] |
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H.-R. Park, A. Tomida, S. Sato, Y. Tsukumo, J. Yun, T. Yamori, Y. Hayakawa, T. Tsuruo, and K. Shin-ya Effect on Tumor Cells of Blocking Survival Response to Glucose Deprivation J Natl Cancer Inst, September 1, 2004; 96(17): 1300 - 1310. [Abstract] [Full Text] [PDF] |
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K. Yamamoto, H. Yoshida, K. Kokame, R. J. Kaufman, and K. Mori Differential Contributions of ATF6 and XBP1 to the Activation of Endoplasmic Reticulum Stress-Responsive cis-Acting Elements ERSE, UPRE and ERSE-II J. Biochem., September 1, 2004; 136(3): 343 - 350. [Abstract] [Full Text] [PDF] |
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D. T. Nguyen, S. Kebache, A. Fazel, H. N. Wong, S. Jenna, A. Emadali, E.-h. Lee, J. J.M. Bergeron, R. J. Kaufman, L. Larose, et al. Nck-dependent Activation of Extracellular Signal-regulated Kinase-1 and Regulation of Cell Survival during Endoplasmic Reticulum Stress Mol. Biol. Cell, September 1, 2004; 15(9): 4248 - 4260. [Abstract] [Full Text] [PDF] |
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C. Chen, E. E. Dudenhausen, Y.-X. Pan, C. Zhong, and M. S. Kilberg Human CCAAT/Enhancer-binding Protein {beta} Gene Expression Is Activated by Endoplasmic Reticulum Stress through an Unfolded Protein Response Element Downstream of the Protein Coding Sequence J. Biol. Chem., July 2, 2004; 279(27): 27948 - 27956. [Abstract] [Full Text] [PDF] |
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K. Zhang and R. J. Kaufman Signaling the Unfolded Protein Response from the Endoplasmic Reticulum J. Biol. Chem., June 18, 2004; 279(25): 25935 - 25938. [Full Text] [PDF] |
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S. Nadanaka, H. Yoshida, F. Kano, M. Murata, and K. Mori Activation of Mammalian Unfolded Protein Response Is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum Mol. Biol. Cell, June 1, 2004; 15(6): 2537 - 2548. [Abstract] [Full Text] [PDF] |
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