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Originally published In Press as doi:10.1074/jbc.M300923200 on June 2, 2003

J. Biol. Chem., Vol. 278, Issue 33, 31024-31032, August 15, 2003
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A Serine Protease Inhibitor Prevents Endoplasmic Reticulum Stress-induced Cleavage but Not Transport of the Membrane-bound Transcription Factor ATF6*

Tetsuya Okada {ddagger} §, Kyosuke Haze ¶, Satomi Nadanaka {ddagger} §, Hiderou Yoshida {ddagger} ||, Nabil G. Seidah **, Yuko Hirano {ddagger}{ddagger}, Ryuichiro Sato {ddagger}{ddagger}, Manabu Negishi {ddagger} and Kazutoshi Mori {ddagger} § §§

From the {ddagger}Graduate School of Biostudies, Kyoto University, Kyoto 606-8304, Japan, §CREST, Japan Science and Technology Corporation, Saitama 332-0012, Japan, the HSP Research Institute, Kyoto Research Park, Kyoto 600-8813, Japan, ||PRESTO, Japan Science and Technology Corporation, Saitama 332-0012, Japan, the **Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W1R7, Canada, and the {ddagger}{ddagger}Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

Received for publication, January 28, 2003 , and in revised form, May 19, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6{alpha} and ATF6{beta}), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355–1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6{alpha} and ATF6{beta}, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6{alpha} and ATF6{beta} by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The endoplasmic reticulum (ER)1 is now known to dispatch various signals to the nucleus in response to perturbation in or around the ER, such as the depletion of intracellular sterols (1) or the accumulation of unfolded proteins in the ER (2–5). These intracellular signaling pathways from the ER to the nucleus culminate in the activation of transcription of appropriate genes whose products are required to cope with the disturbance, leading to the maintenance of homeostasis.

Sterol depletion activates certain basic helix-loop-helix-leucine zipper-type transcription factors, the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2), by regulated intramembrane proteolysis (RIP). Thus, the precursor forms of SREBPs synthesized as transmembrane proteins in the ER are cleaved in response to sterol depletion so that the soluble N-terminal fragments of SREBPs released from the membrane (mature forms) enter the nucleus and activate the transcription of genes involved in cholesterol biosynthesis as well as receptor-mediated endocytosis of cholesterol-containing lipoproteins from plasma (6–9).

Cells cope with unfolded proteins accumulated in the ER under ER stress conditions primarily by transient attenuation of translation and by transcriptional induction of genes encoding ER-resident molecular chaperones (BiP/GRP78, GRP94 etc.) and folding enzymes (protein-disulfide isomerase, peptidyl-prolyl cis-trans isomerase, etc.), leading to augmenting the folding capacity in the ER. These processes are collectively termed the unfolded protein response (UPR) (2–5). ER chaperones commonly contain in their promoter regions a unique cis-acting element designated as the ER stress response element (ERSE), whose consensus sequence is CCAAT-N9-CCACG; the ERSE is necessary and sufficient for the transcriptional induction of ER chaperone genes (10, 11). As the general transcription factor NF-Y (CBF) constitutively occupies the CCAAT part of the ERSE (12), the binding of ER stress response factor(s) to the ERSE requires a component that is capable of binding to the CCACG part of the ERSE and that is specifically activated during the UPR. We previously identified the basic leucine zipper proteins ATF6{alpha} (encoded by the ATF6 gene) and ATF6{beta} (encoded by the G13/cAMP response element-binding protein-related protein gene) as CCACG-binding proteins (10, 13). Both ATF6{alpha} and ATF6{beta} are constitutively synthesized as type II transmembrane glycoproteins that are anchored in the ER and are activated by proteolysis in response to ER stress (13, 14). The N-terminal fragments thereby released from the membrane (mature forms of ATF6{alpha} and ATF6{beta}) enter the nucleus and activate the transcription of their target genes via direct binding to the CCACG part of the ERSE in a manner dependent on the binding of NF-Y to the CCAAT part (15–17).

In this study, we used a pharmacological approach to gain insight into the mechanism of activation of ATF6{alpha} and ATF6{beta} by ER stress, and found that the serine protease inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) inhibits ER stress-induced proteolysis of both ATF6{alpha} and ATF6{beta}, resulting in inhibition of transcriptional induction of ATF6-target genes by ER stress. While this was in progress, Ye et al. (18) showed by transient transfection experiments that upon ER stress ATF6{alpha} is cleaved sequentially by the Site-1 protease (S1P) and Site-2 protease (S2P), which are known to process SREBP sequentially in response to sterol depletion. S1P, also called subtilisin kexin isozyme-1 (19), is a membrane-anchored serine protease with its active site facing the lumenal side, whereas S2P is an unusually hydrophobic zinc metalloprotease spanning the membrane at multiple times (9). Thus, two intracellular signaling systems from the ER to the nucleus (the SREBP and ATF6 pathways) share molecular machinery that connects events in the ER to those in the nucleus. This observation led to the prediction that ATF6 would need to relocate from the ER to the Golgi apparatus to be cleaved, similarly to SREBP, as S1P is localized in the Golgi apparatus (8). It was recently demonstrated by transient transfection experiments that this is the case (20, 21). We show here for the first time that endogenous ATF6 was translocated from the ER to the nucleus via the Golgi apparatus in response to ER stress and that AEBSF blocked the nuclear translocation of ATF6 without affecting its movement from the ER to the Golgi apparatus. Furthermore, we provide evidence suggesting that AEBSF inhibited S1P directly.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Protease Inhibitors—HeLa cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin sulfate) in a 5% CO2, 95% air incubator at 37 °C. Various protease inhibitors were purchased from Calbiochem (San Diego, CA) and dissolved in H2O or dimethyl sulfoxide according to the manufacturer's instructions.

Construction of Plasmids and Transfection—Recombinant DNA techniques were performed according to standard procedures (22). To express the wild-type (WT) or enzymatically inactive mutant version (H249A) of human S1P/subtilisin kexin isozyme-1 in the ER forcedly, a series of plasmids were constructed as follows. A double-stranded oligonucleotide coding for the ER retention signal KDEL flanked by the 5' XhoI site and 3' stop codon and ApaI sites (5'-TCGAGAAAGACGAACTCTGAGGGCC-3' plus 5'-CTCAGAGTTCGTCTTTC-3') was inserted between the XhoI and ApaI sites of pcDNA3.1(+) (Invitrogen) to create pcDNA-KDEL-stop. A double-stranded oligonucleotide encoding the c-Myc epitope tag (EQKLISEEDL) flanked by 5' BamHI and 3' XhoI sites (5'-GATCCGAGCAGAAGCTGATCTCAGAGGAGGACCTGC-3' plus 5'-TCGAGCAGGTCCTCCTCTGAGATCAGCTTCTGCTCG-3') was inserted between the BamHI and XhoI sites of pcDNA-KDEL-stop to create pcDNA-c-Myc-KDEL-stop. The amino acid region 1–997 of the WT or H249A mutant of human S1P/subtilisin kexin isozyme-1 (23) was amplified by PCR together with the 5' NheI site (set in the 5'-untranslated region) and 3' BamHI site (set immediately after the amino acid 997), and inserted between the NheI and BamHI sites of pcDNA-c-Myc-KDEL-stop to create pcDNA-S1P({Delta}TMD)-KDEL(WT) or pcDNA-S1P({Delta}TMD)-KDEL(H249A) after their sequences had been confirmed. HeLa cells (1 x 106 cells) were transfected with these plasmids using Cell Line Nucleofector Kit R (Amaxa, Cologne, Germany) according to the manufacturer's instruction.

Immunoblotting—ATF6{alpha} and ATF6{beta} were detected with anti-ATF6{alpha} (14) and anti-ATF6{beta} (13) antibodies, respectively. Mouse anti-KDEL monoclonal antibody (clone 10C3) was obtained from StressGen Biotechnologies (Victoria, British Columbia, Canada). Mouse anti-c-Myc epitope monoclonal antibody (clone 9E10) and rabbit anti-ATF4 (cAMP-response element-binding protein 2) polyclonal antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HeLa cells cultured until ~70% confluency were treated in the experiments as described in the text. Whole cell extracts were prepared as described previously (24) and analyzed by the standard procedure (22) using an enhanced chemiluminescence Western blotting detection system kit (Amersham Biosciences). Chemiluminescence was detected using an LAS-1000plus LuminoImage analyzer (Fuji Film, New York). PERK was first immunoprecipitated from HeLa cells with anti-PERK antibody (a kind gift of Dr. David Ron, New York University School of Medicine) and then detected by immunoblotting with the same antibody as described previously (25).

To study the processing of SREBP-2 in response to sterol depletion, HeLa cells were cultured for 2 days in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine, antibiotics, 5% lipoprotein-deficient serum (Sigma), 50 µM pravastatin (hydroxymethylglutaryl-CoA reductase inhibitor), and 50 µM sodium mevalonate. Nuclear extracts were prepared as described (26) and examined by immunoblotting using a polyclonal antibody (RS004) against amino acids 1–481 of human SREBP-2 (27).

Northern Blot Hybridization—Total RNA was extracted by the acid guanidinium-phenol-chloroform method using ISOGEN (Nippon Gene, Tokyo, Japan). Ten-microgram aliquots of RNA were subjected to electrophoresis using 1.2% agarose gels containing 2.2 M formaldehyde and analyzed by standard Northern blotting procedures (22) using an Alk-Phos direct labeling kit (Amersham Bioscience). Chemiluminescence was detected using an LAS-1000plus LuminoImage analyzer.

Immunofluorescence—HeLa cells were grown on Lab-tek chamber slides (Nalge Nunc International K.K., Rochester, NY). The cells were washed with phosphate-buffered saline one time, fixed with 3.7% formaldehyde for 10 min, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. They were then incubated for 1 h at 37 °C in 1% bovine serum albumin and 0.05% Tween 20 in phosphate-buffered saline with rabbit anti-ATF6{alpha} antibody (1:50 dilution) or mouse anti-GM130 antibody (1:50 dilution) obtained from Cell Signaling Technology (Beverly, MA). Primary antibodies bound to the cells were visualized by further incubation of the cells for 1 h at 37 °C with 20 µg/ml fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G antibody (ICN Pharmaceuticals, Inc., Costa Mesa, CA) or 20 µg/ml rhodamine-conjugated goat anti-mouse immunoglobulin G antibody (ICN Pharmaceuticals, Inc.), followed by confocal microscopy performed using an MRC-1024 laser scanning confocal imaging system (Bio-Rad).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of ER Stress-induced Proteolysis of ATF6{alpha} and ATF6{beta} by AEBSF—We employed various types of protease inhibitors to determine the nature of the protease responsible for the conversion of ATF6{alpha} and ATF6{beta} from the precursor forms (designated pATF6{alpha}(P) and pATF6{beta}(P), respectively) to the mature forms (designated pATF6{alpha}(N) and pATF6{beta}(N), respectively) in response to ER stress. HeLa cells were pretreated for 1 h with each of the 13 chemicals tested at the concentrations recommended by the manufacturer, and then treated for 2 h with 300 nM thapsigargin, which induces ER stress by inhibiting ER Ca2+-ATPase (2). As a result, only AEBSF, a serine protease inhibitor, was found to block the production of pATF6{alpha}(N) and pATF6{beta}(N) (Fig. 1, lane 3). The inhibitory effect of AEBSF was dependent on its dose, and pretreatment with 300 µM AEBSF was required to eliminate subsequent thapsigargin-induced proteolysis of ATF6{alpha} and ATF6{beta} completely (Fig. 2, lanes 1–6). It should be noted that this concentration of AEBSF was comparable with those used to inhibit Myc-mediated apoptosis in Rat-1 fibroblasts (200 µM) (28) or to inhibit the phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced differentiation of human HL-60 promyelocytic leukemia cells (190 µM) (29). In marked contrast, other serine protease inhibitors such as p-amidinophenylmethylsulfonyl fluoride and chymostatin showed no effect even at a concentration of 1 mM (Fig. 2, lanes 7–18). Time course experiments indicated that pretreatment with 300 µM AEBSF did not delay the processing but abolished the production of both pATF6{alpha}(N) and pATF6{beta}(N) in response to thapsigargin treatment (Fig. 3A), suggesting that the protease(s) responsible for cleaving ATF6{alpha} and ATF6{beta} in ER-stressed cells are sensitive to AEBSF.



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  		FIG. 1.
Effects of various protease inhibitors on the processing of ATF6{alpha} and ATF6{beta} induced by thapsigargin. HeLa cells cultured in 6-well plates were pretreated for 1 h with each of the indicated protease inhibitors at the following concentrations: 300 µM AEBSF (inhibits serine proteases), 10 µM amastatin (inhibits aminopeptidase A and leucine aminopeptidase), 50 µM antipain (inhibits trypsin-like serine proteases, papain, and some cysteine proteases), 100 µM p-amidinophenylmethylsulfonyl fluoride (APMSF, inhibits trypsin-like serine proteases), 2 µg/ml aprotinin (inhibits serine proteases), 2 µM N-acetyl-Leu-Leu-Nle-CHO (ALLN, inhibits neutral cysteine proteases and the proteasome), 100 µM chymostatin (inhibits chymotrypsin, papain, and most cysteine proteases), 100 µM cystatin (inhibits cysteine and/or thiol proteases), 10 µM E-64 (inhibits cysteine proteases), 2 µg/ml elastatinal (inhibits elastase-like serine proteases), 100 µM leupeptin (inhibits trypsin-like serine proteases and cysteine proteases), 1 µM pepstatin A (inhibits aspartic proteases), and 10 µM phosphoramidon (inhibits some metalloendopeptidases). Then the cells were treated with (+) or without (–) 300 nM thapsigargin (Tg) without removal of the protease inhibitor being tested. Two hours later, whole cell extracts were prepared and analyzed by immunoblotting using anti-ATF6{alpha} antibody (upper panel) or anti-ATF6{beta} antibody (lower panel). The positions of pATF6{alpha}(P), pATF6{alpha}(N), pATF6{beta}(P), and pATF6{beta}(N) are marked. The positions of prestained SDS-PAGE molecular weight standards (Bio-Rad) are also indicated on the left.

 


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  		FIG. 2.
Dose dependence of the effects of various serine protease inhibitors on the processing of ATF6{alpha} and ATF6{beta} induced by thapsigargin. HeLa cells in 60-mm dishes were pretreated for 1 h with one of the four serine protease inhibitors at the indicated concentrations and then treated with (+) or without (–) 300 nM thapsigargin (Tg) without removal of inhibitors. Two hours later, whole cell extracts were prepared and analyzed as described in the legend to Fig. 1. The positions of pATF6{alpha}(P), pATF6{alpha}(N), pATF6{beta}(P), and pATF6{beta}(N) are marked.

 


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  		FIG. 3.
Time course of thapsigargin-induced processing of ATF6{alpha}/{beta} and induction of BiP and Asn-S in cells pretreated with or without AEBSF. A, HeLa cells were pretreated with (+) or without (–) 300 µM AEBSF for 1 h and then treated with 300 nM thapsigargin (Tg) without removal of AEBSF for the indicated periods. Whole cell extracts were prepared and analyzed by immunoblotting using anti-ATF6{alpha} or anti-ATF6{beta} antibodies as well as anti-KDEL antibody, which recognizes BiP. The positions of pATF6{alpha}(P), pATF6{alpha}(N), pATF6{beta}(P), and pATF6{beta}(N) as well as BiP are marked. The migration positions of full-range rainbow molecular weight markers (Amersham Biosciences) are indicated on the left. B, HeLa cells were treated as in A (lanes 1–8) or incubated with 300 µM AEBSF alone for the indicated periods (lanes 9–11). Total RNA was extracted and analyzed by Northern blot hybridization using a cDNA probe specific to BiP, Asn-S, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

 

Effects of AEBSF Pretreatment on the UPR—We next determined the effects of AEBSF pretreatment on the UPR by measuring the level of BiP/GRP78, a major target of the UPR, which was strongly induced by thapsigargin treatment at both the protein (Fig. 3A, lanes 1–6) and mRNA levels (Fig. 3B, lanes 1–4). When cells were pretreated with AEBSF prior to the addition of thapsigargin, induction of BiP was not observed at either the protein (Fig. 3A, lanes 7–12) or mRNA levels (Fig. 3B, lanes 5–8), indicating that AEBSF functions as an inhibitor of the UPR. We also examined the effects of AEBSF pretreatment on ER stress inducers other than thapsigargin; namely, tunicamycin and 2-deoxyglucose, inhibitors of protein N-glycosylation (2), and dithiothreitol, which causes malfolding of proteins by disrupting disulfide bonds (2). As expected from our previous results (13, 14), the proteolysis of both ATF6{alpha} (Fig. 4A) and ATF6{beta} (data not shown) was triggered by dithiothreitol (Fig. 4A, lane 5), tunicamycin (lane 7), and 2-deoxyglucose (lane 9), as well as thapsigargin (lane 3). Importantly, pretreatment of cells with 300 µM AEBSF efficiently blocked the proteolysis of ATF6{alpha} in response to subsequent treatment with thapsigargin (Fig. 4A, lane 4), tunicamycin (lane 8), or 2-deoxyglucose (lane 10). Although the inhibitory effect of AEBSF on the processing of ATF6{alpha} induced by 1 mM dithiothreitol was not complete (lane 6), we found that AEBSF pretreatment completely inhibited the cleavage of ATF6{alpha} and ATF6{beta} and the subsequent induction of BiP by 0.5 mM dithiothreitol at both the protein and mRNA levels (Fig. 5, A and B, respectively). These results suggested that the proteolytic processing of ATF6{alpha} and ATF6{beta} mediated by a protease(s) sensitive to AEBSF is a key regulatory step in the UPR.



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  		FIG. 4.
Effects of AEBSF pretreatment on the processing of ATF6{alpha}, activation of PERK, and induction of ATF4 in cells subsequently treated with various ER stress inducers. A, HeLa cells were pretreated with (+) or without (–) 300 µM AEBSF for 1 h and then untreated (–) or treated with one of the four ER stress inducers without removal of AEBSF for the specified period prior to preparation of whole cell extracts or cell lysates; 300 nM thapsigargin (Tg) for 1 h, 1 mM dithiothreitol (DTT) for 1 h, 2 µg/ml tunicamycin (Tm) for 5 h, and 10 mM 2-deoxyglucose (2-DG) for 5 h. Whole cell extracts were analyzed by immunoblotting using anti-ATF6{alpha} antibody. PERK was immunoprecipitated from cell lysates using anti-PERK antibody and detected by immunoblotting using the same antibody. The migration positions of pATF6{alpha}(P), pATF6{alpha}(P*) (non-glycosylated form of pATF6{alpha}(P)), pATF6{alpha}(N), non-phosphorylated PERK (PERK), and phosphorylated PERK (P-PERK) are indicated. B and C, HeLa cells were pretreated with (+) or without (–) 300 µM AEBSF for 1 h and then treated with 300 nM thapsigargin (Tg, panel B) or 0.5 mM dithiothreitol (DTT, panel C) for the indicated periods. Whole cell extracts were prepared and analyzed by immunoblotting using anti-ATF4 antibody.

 


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  		FIG. 5.
Time course of dithiothreitol (DTT)-induced processing of ATF6{alpha}/{beta} and induction of BiP and Asn-S in cells pretreated with or without AEBSF. HeLa cells were pretreated with (+) or without (–) 300 µM AEBSF for 1 h and then treated with 0.5 mM dithiothreitol without removal of AEBSF for the indicated periods. Whole cell extracts and total RNA were prepared and analyzed as described in the legend to Fig. 3, A and B, respectively.

 

Nonetheless, it remains possible that AEBSF pretreatment inhibited the proteolysis of ATF6{alpha} and ATF6{beta} and subsequent induction of BiP mRNA and BiP protein in response to ER stress by somehow preventing accumulation of unfolded proteins in the ER. We therefore examined the effects of AEBSF pretreatment on ER stress-induced activation of PERK, a key mediator of translational attenuation that occurs immediately in response to ER stress (30, 31). Because PERK is a transmembrane protein kinase activated by oligomerization and autophosphorylation, its activation status can be monitored by its mobility on SDS gels (25); phosphorylated (i.e. activated) PERK migrates more slowly than inactive PERK as shown Fig. 4A (compare lane 1 with lane 3 for example). AEBSF pretreatment did not inhibit the activation of PERK induced by thapsigargin (compare lane 4 with lane 3) or dithiothreitol (compare lane 6 with lane 5), indicating that unfolded proteins were accumulated under these conditions even in the presence of AEBSF. In contrast, AEBSF pretreatment did block the activation of PERK induced by tunicamycin (compare lane 8 with lane 7) or 2-deoxyglucose (compare lane 10 with lane 9). Consistent with these observations, the deglycosylated form of pATF6{alpha}(P), pATF6{alpha}(P*), produced in cells treated with tunicamycin (lane 7) or 2-deoxyglucose (lane 9) was not detected in the presence of AEBSF (lanes 8 and 10, respectively). Thus, AEBSF pretreatment antagonized the effects of ER stress inducers that promote accumulation of unfolded proteins in the ER by inhibiting protein N-glycosylation; the reason for these unexpected results is currently not known.

ER stress-induced activation of PERK results in phosphorylation of the {alpha} subunit of eukaryotic initiation factor 2{alpha}, leading to general translational attenuation (30, 31). Paradoxically, this attenuation induces translation of the basic leucine zipper transcription factor ATF4, causing transcriptional induction of ATF4-target genes such as the basic leucine zipper transcription factor CHOP in response to ER stress (32). We recently reported evidence suggesting that the asparagine synthetase (Asn-S) gene is also a target of the PERK-eukaryotic initiation factor 2{alpha}-ATF4 pathway (24). Indeed, ATF4 was rapidly induced in cells treated with thapsigargin (Fig. 4B, lanes 1–6) or dithiothreitol (Fig. 4C, lanes 1–6), and Asn-S mRNA was induced in cells treated with thapsigargin (Fig. 3B, lanes 1–4) or dithiothreitol (Fig. 5B, lanes 1–4) accordingly. Unexpectedly, however, we found that the induction of ATF4 by thapsigargin or dithiothreitol was attenuated or delayed, respectively, in cells pretreated with AEBSF (Fig. 4, B, lanes 7–12, C, lanes 7–12, respectively) despite the fact that PERK was activated normally in these cells (Fig. 4A, lanes 4 and 6, respectively) and that activation of the PERK pathway does not require any proteolytic steps. This led to a marked reduction or significant delay of the induction of Asn-S mRNA in cells treated with thapsigargin (Fig. 3B, lanes 5–8) or dithiothreitol (Fig. 5B, lanes 5–8). Thus, AEBSF pretreatment exerts deleterious effects on not only the ATF6 pathway but also the PERK pathway. AEBSF must be used with caution because of its pleiotropic effects on mediators of the UPR.

Effects of AEBSF Treatment on the Processing of SREBP-2 and Forced Cleavage of ATF6 by S1P—As it has been reported that ATF6{alpha} is cleaved by the same enzymes that process SREBPs (18), we next examined whether AEBSF can inhibit the processing of SREBP-2 in response to sterol depletion. As shown in Fig. 6, the level of the mature form of SREBP-2, pSREBP-2(N), was markedly reduced in nuclear extracts of HeLa cells when the cells were treated with AEBSF (lane 2) as compared with no AEBSF treatment (lane 1). N-acetyl-Leu-Leu-Nle-CHO (ALLN, an inhibitor of neutral cysteine proteases and the proteasome) was employed as a positive control, and treatment with this agent caused a marked increase in the level of pSREBP-2(N) in nuclear extracts, as reported previously (7).



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  		FIG. 6.
Effects of AEBSF treatment on the processing of SREBP-2. HeLa cells were cultured for 2 days under conditions in which intracellular sterol was depleted, as described under "Experimental Procedures." During the last 3 h of the 2-day incubation period, the cells were treated (+) or not treated (–) with 300 µM AEBSF or 100 µM N-acetyl-Leu-Leu-norleucinal (ALLN). Nuclear extracts were then prepared and analyzed by immunoblotting using anti-SREBP-2 antibody.

 

Of the two enzymes that cleave SREBP and ATF6, S1P but not S2P is a serine protease (9). We therefore examined whether AEBSF inhibits S1P directly. It is known that S1P is synthesized as a prepro-protein (23, 33, 34). Cleavage of the signal sequence of S1P produces a proprotein (A form) that is still inactive enzymatically. Subsequent autocatalytic cleavage of the proprotein at the two internal sites removes the propeptide from the A form and thereby produces the B and C forms, which are active proteases. Somehow, only the C form is localized in the Golgi apparatus as carbohydrate moieties of the C but not B form are resistant to digestion with endoglycosidase H (33) or are sulfated (23). Interestingly, when S1P was forced to be expressed predominantly in the ER by replacing the C-terminal transmembrane domain of S1P with the ER retention sequence KDEL (S1P({Delta}TMD)-KDEL), soluble and active S1P (C form) was produced by autocatalytic processing, resulting in cleavage of SREBP in a stimulus-independent manner (8). We therefore determined whether such ectopic expression of soluble and active S1P in the ER causes cleavage of ATF6{alpha} and ATF6{beta} in the absence of ER stress and if so, whether AEBSF treatment can block such forced cleavage of ATF6{alpha} and ATF6{beta}. An enzymatically inactive point mutant of S1P, namely the H249A mutant (23), served as a negative control. The wild-type and mutant S1P used for this experiment were constructed as described under "Experimental Procedures" and designated S1P({Delta}TMD)-KDEL(WT) and S1P({Delta}TMD)-KDEL-(H249A), respectively.

As shown in Fig. 7A, transient introduction of S1P({Delta}TMD)-KDEL(WT) (lanes 2 and 5) but not S1P({Delta}TMD)-KDEL(H249A) (lanes 3 and 6) into HeLa cells resulted in detection of cleaved products of both ATF6{alpha} and ATF6{beta}, which migrated as a doublet in both cases (lanes 2 and 5) 24 h after transfection. As the migration position of the faster migrating band was identical to that of pATF6{alpha}(N) or pATF6{beta}(N) produced in response to thapsigargin treatment (lanes 1 and 4), we assigned the slower migrating band as the intermediate fragment, pATF6{alpha}(I) or pATF6{beta}(I), which represented ATF6{alpha} or ATF6{beta} cleaved by S1P but not cleaved by S2P yet, thus still containing the transmembrane domain. These results indicated that forced colocalization of S1P and ATF6 resulted in constitutive cleavage as in the case of that of S1P and SREBP.



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  		FIG. 7.
Effects of AEBSF treatment on cleavage of ATF6{alpha} and ATF6{beta} by soluble S1P forced to be expressed in the ER. A, HeLa cells were transfected with S1P({Delta}TMD)-KDEL(WT) or S1P({Delta}-TMD)KDEL(H249A). Twenty-four h after transfection, whole cell extracts were prepared and analyzed by immunoblotting using anti-ATF6{alpha} or anti-ATF6{beta} antibodies together with whole cell extracts prepared from HeLa cells treated with 300 nM thapsigargin (Tg) for 1 h. The positions of pATF6{alpha}(P), pATF6{alpha}(I), pATF6{alpha}(N), pATF6{beta}(P) pATF6{beta}(I), and pATF6{beta}(N) are marked. B, HeLa cells were transfected with S1P({Delta}TMD)-KDEL(WT) or S1P({Delta}TMD)-KDEL(H249A). Twenty h after transfection, aliquots of cells were harvested to prepare whole cell extracts. Alternatively, aliquots of transfected cells were further incubated in the presence (+) or absence (–) of 300 µM AEBSF for the indicated times before whole cell extracts were prepared. Whole cell extracts were analyzed by immunoblotting using anti-ATF6{alpha} or anti-ATF6{beta} antibodies as well as anti-c-Myc epitope antibody, which recognizes transfected S1P. The positions of pATF6{alpha}(P), pATF6{alpha}(I), pATF6{alpha}(N), pATF6{beta}(P), pATF6{beta}(I), and pATF6{beta}(N) as well as A, B, and C forms of S1P are marked.

 

We then examined the effects of AEBSF treatment on such forced cleavage of ATF6. As shown in Fig. 7B, 20 h after transfection, the C-form of S1P was produced in cells transfected with S1P({Delta}TMD)-KDEL(WT) (lane 1) but not in cells transfected with S1P({Delta}TMD)-KDEL(H249A) (lane 6) as expected, resulting in detection of doublet bands of cleaved ATF6{alpha} and ATF6{beta} only in cells transfected with S1P({Delta}TMD)-KDEL(WT) (compare lane 1 with lane 6), consistent with the results shown in Fig. 7A. Further incubation of transfected cells increased the expression level of the C form of S1P, leading to increased levels of the doublet bands of cleaved ATF6{alpha} and ATF6{beta} in cells transfected with S1P({Delta}TMD)-KDEL(WT) (compare lanes 2 and 3 with lane 1). Importantly, however, such incubation time (expression level)-dependent increase in cleavage was abolished by the presence of AEBSF in the culture medium (lanes 4 and 5). These results strongly suggest that the target of AEBSF is S1P, which is a serine protease involved in the cleavage of both SREBP and ATF6.

Effects of AEBSF Pretreatment on ER Stress-induced Relocation of ATF6 —We finally examined whether AEBSF pretreatment affected the intracellular transport of ATF6 in response to ER stress; both SREBP and ATF6 translocate from the ER to the nucleus via the Golgi apparatus, where sequential cleavage by S1P and S2P occurs (8, 21). As shown in Fig. 8, at time 0 in control cells, ATF6{alpha} was localized in perinuclear structures that were distinct from the punctate structure stained with anti-GM130 antibody (GM130 is a specific marker for Golgi apparatus (35)), as expected. After 15 min of treatment with dithiothreitol (0.5 mM), ATF6 had become concentrated in Golgi-like structures. After 30 min of treatment with dithiothreitol, the nucleus became stained with anti-ATF6{alpha} antibody gradually. In cells pretreated with AEBSF for 1 h, the behavior of ATF6{alpha} was indistinguishable from that in control cells from time 0 until 15 min. Importantly, however, ATF6{alpha} remained associated with the Golgi-like structures even after 30 min of treatment with dithiothreitol and the nucleus was hardly stained with anti-ATF6{alpha} antibody even at 60 min in cells pretreated with AEBSF. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF



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  		FIG. 8.
Effects of AEBSF pretreatment on dithiothreitol-induced translocation of ATF6{alpha} from the ER to the nucleus. HeLa cells on glass slides were pretreated with or without (control) 300 µM AEBSF for 1 h and then treated with 0.5 mM dithiothreitol (DTT) without removal of AEBSF for the indicated periods. Cells were fixed, double stained with anti-ATF6{alpha} and anti-GM130 antibodies, and analyzed by confocal laser scanning fluorescence microscopy. Bar, 50 µm.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
RIP is a novel mechanism regulating gene expression (9). In some cases, an active transcription factor or a specific regulator of a certain transcription factor is synthesized as a cytoplasmic part of a transmembrane protein, and excised from a larger precursor when cells receive appropriate stimuli. The cytoplasmic fragment thereby liberated from the membrane by proteolysis enters the nucleus and activates or modifies transcription of a specific set of genes. RIP is characteristic in the sense that the excision of an active form is almost always achieved by two sequential cleavages of a precursor protein. As the proteolysis is tightly regulated and the second cleavage occurs within the transmembrane domain, this process is called regulated intramembrane proteolysis. Because of sequestration of a transmembrane protein from the chromosomal DNA, this mechanism allows the cell to regulate expression of a specific set of genes only when such transcriptional control is required.

Interestingly and importantly, RIP is conserved from bacteria to humans (9). In Escherichia coli, RIP is utilized to activate the extracytoplasmic stress response, a response quite similar to the UPR in eukaryotes (36). When unfolded proteins are accumulated in the envelope of E. coli cells, the transcription factor {sigma}E is activated to induce transcription of genes encoding molecular chaperones, folding enzymes, and proteases localized in the envelope to maintain homeostasis of the periplasm. The inner membrane protein RseA is a key regulator of the extracytoplasmic stress response as its cytosolic domain inhibits the transcriptional activity of {sigma}E via direct binding (37, 38). It was recently demonstrated (39, 40) that RseA is cleaved in two steps in response to stress in the periplasm. First cleavage occurs in the periplasmic domain of RseA, which is mediated by DegS. DegS is a serine protease that passes through the inner membrane once (41, 42), thus its enzyme type and topology are quite similar to those of S1P. Second cleavage occurs within the transmembrane domain, which is mediated by YaeL, a metalloprotease spanning the inner membrane multiple times (43–45). YaeL is considered to be an E. coli homolog of S2P (9). Once the cytoplasmic region of RseA is liberated from the inner membrane by proteolysis, it looses inhibitory activity toward {sigma}E probably because it is degraded, leading to activation of {sigma}E (39, 40). YaeL can cleave RseA only after cleavage of RseA by DegS occurs. As both the substrate (periplasmic domain of RseA) and the catalytic site of DegS are colocalized in the periplasm, the proteolytic activity of DegS toward RseA must be tightly regulated so that {sigma}E is activated only when transcriptional enhancement of {sigma}E-target genes is required by the cell. Indeed, it was recently shown that a C-terminal peptide present in unfolded envelope proteins binds to the PDZ domain of DegS and prevents inhibitory interaction between the protease and PDZ domains of DegS, leading to activation of DegS (46).

In mammalian cells, RIP is coupled to intracellular transport as a substrate, such as SREBP and ATF6, and the proteases, especially S1P, are localized in the ER and Golgi apparatus, respectively (8, 18, 21). Thus, substrate proteins must relocate from the ER to the Golgi apparatus to be cleaved in response to stimuli. Because of this difference in subcellular distribution, S1P, an enzyme carrying out first cleavage, can be constitutively active even under normal conditions in contrast to bacterial DegS. In fact, when expression of S1P was forced in the ER, SREBP was cleaved in the absence of stimuli (8). We showed here that ATF6 was similarly cleaved in the absence of ER stress when expression of S1P was forced in the ER (Fig. 7). Therefore, the exit of a substrate protein from the ER appears to be the most important step in eukaryotic RIP in contrast to bacterial RIP.

We showed in this report that ER stress-induced proteolysis of ATF6{alpha} and ATF6{beta} was prevented by pretreatment of HeLa cells with AEBSF, a serine protease inhibitor (Figs. 1 and 2). AEBSF was effective in cell lines other than HeLa cells, such as Chinese hamster ovary cells (data not shown). As no intermediate fragments of ATF6{alpha} and ATF6{beta} were detected in cells pretreated with AEBSF (Figs. 1 and 2) and as sterol depletion-induced processing of SREBP-2 was also prevented by AEBSF (Fig. 6), it is very likely that AEBSF inhibits the first step in the processing and that the primary target of AEBSF is S1P. This notion was further supported by the fact that AEBSF blocked cleavage of ATF6{alpha} and ATF6{beta} induced by ectopic expression of the active form of S1P in the ER (Fig. 7). Although AEBSF pretreatment exhibited pleiotropic effects on various ER stress transducers other than ATF6 (Figs. 3, 4, 5), we showed that ER stress-induced transport of ATF6 from the ER to the Golgi apparatus occurred normally even when cellular S1P activity was inhibited by pretreatment with AEBSF (Fig. 8). Thus, AEBSF would become a useful tool to analyze the mechanism of activation of ATF6 by ER stress as described below. It should be noted that our results are consistent with previous in vitro assays to determine proteolytic activity of S1P toward peptide substrates, in which AEBSF (also known as Pefabloc) was shown to inhibit S1P purified as a secreted form potently as compared with other protease inhibitors tested (23, 47).

Recent studies indicated that the lumenal domain of ATF6 is bound by the ER chaperone BiP/GRP78 under normal conditions and that dissociation of BiP from ATF6 triggers the exit of ATF6 from the ER under ER stress conditions as BiP becomes bound to unfolded proteins accumulated in the ER (21). Perhaps, a key to fully understand the mechanism of ATF6 activation is to determine the conformational changes that occur in the lumenal domain of ATF6 in response to ER stress. Biochemical analysis to look for such changes can be conducted using microsomes isolated from cells treated or untreated with various ER stress inducers. Inclusion of AEBSF during ER stress treatment would allow us to directly compare the conformation of the lumenal domain of the ER form with that of the Golgi form, because ATF6 remains intact even after reaching the Golgi apparatus; conformational changes resulting from the cleavage event would not interfere with the results. Such analysis is now in progress.


   FOOTNOTES
 
* This work was supported in part by Ministry of Education, Culture, Sports, Science and Technology of Japan Grants 14037233 and 15GS0310 (to K. M.) and a grant from the Sumitomo Foundation (to K. M). T. O. is a recipient of Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists (05375). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§§ To whom correspondence should be addressed: Graduate School of Biostudies, Kyoto University, 46-29 Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8304, Japan. Tel.: 81-75-753-4067; Fax: 81-75-753-3718; E-mail: kazu.mori{at}bio.mbox.media.kyoto-u.ac.jp.

1 The abbreviations used are: ER, endoplasmic reticulum; AEBSF, 4-(2-aminoethyl)benzenesulfonylfluoride; Asn-S, asparagine synthetase; ERSE, endoplasmic reticulum stress response element; RIP, regulated intramembrane proteolysis; SREBP, sterol regulatory element-binding protein; S1P, Site-1 protease; S2P, Site-2 protease; UPR, unfolded protein response; WT, wild-type; BiP, immunoglobulin heavy chain-binding protein; ATF, activating transcription factor. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. David Ron for providing anti-PERK antibody. We thank Maki Shibata and Kaoru Miyagawa for technical and secretarial assistance.



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