Human Fas-associated factor 1 interacts with heat shock protein 70 and negatively regulates chaperone activity

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Human Fas-associated Factor 1 Interacts with Heat Shock Protein

70 and Negatively Regulates Chaperone Activity*

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Received for publication, June 7, 2004, and in revised form, November 27, 2004 Published, JBC Papers in Press, December 13, 2004, DOI 10.1074/jbc.M406297200

Hee-Jung Kim‡§, Eun Joo Song‡§, Yun-Suk Lee¶, Eunhee Kim¶, and Kong-Joo Lee‡储

From the ‡Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750 and¶Division of Life Science, Chungnam National University, Taejon 305-764, Korea

We examined the cell death-inducing property of hu-man Fas-associated factor 1 (hFAF1) in the heat shock signaling pathway. By employing co-immunoprecipita-tion and peptide mass fingerprinting using matrix-as-sisted laser desorption ionization time-of-flight mass spectrometry, we found that hFAF1 binds to the 70-kDa heat shock protein family (Hsc70/Hsp70). Interaction mapping indicated that the 82–180 sequence of hFAF1 directly binds to the N-terminal region containing se-quence 1–120 of Hsc70/Hsp70. This binding is very tight regardless of ATP and heat shock treatment. Hsc70/ Hsp70 and hFAF1 co-localized in the cytosol and nucleus and concentrated to the perinuclear region by heat shock treatment. We examined how hFAF1 regulates Hsp70 function, and found that hFAF1 inhibited the Hsp70 chaperone activity of refolding denatured pro-tein substrates, accelerated heat shock-induced SAPK/ JNK activation, and raised heat shock-induced cell death in a binding dependent manner. These results suggest that hFAF1 prevents cells from recovery after stress by binding to and inhibiting the chaperone activ-ity of Hsp70.

Fas-associated factor 1 (FAF1)1 _{was first identified as a} binding protein to the Fas cytoplasmic region in the yeast two-hybrid screen in mouse. Transient overexpression of mouse FAF1 (mFAF1) enhances Fas-induced apoptosis in L cells (1). Unlike mFAF1, human FAF1 (hFAF1) initiates apoptosis in BOSC23 cells only by transient overexpression. hFAF1 binds to Fas through amino acid sequence 1–201 (2). Other than Fas, casein kinase 2 subunit (CK2) is the FAF1-binding molecule reported (3). hFAF1 is phosphorylated by CK2 on 289 and 291

serine residues, and the hFAF1-CK2 complex formation in-creases when apoptosis occurs (4, 5). Recently, hFAF1 was reported as a member of the Fas death-inducing signaling complex by interacting with Fas-associated via death domain and caspase-8 (6) and as a suppressor of NF-␬B activity by cytoplasmic retention of NF-␬B p65 via physical interaction (7). Heat shock protein 70 (Hsp70) participates in the folding of newly synthesized proteins, translocation of intracellular pro-teins, assembly and disassembly of oligomeric protein struc-tures, proteolytic degradation of denatured proteins, and in controlling the activity of regulatory proteins (8 –10). As Hsp70 exerts its various roles through binding to various chaperone cofactors or co-chaperones, the fate of substrate proteins is determined by the nature of the co-chaperones. In Escherichia

coli, the Hsp70 homologue, DnaK, is shown to be assisted by

two co-chaperones, DnaJ, which yields the high substrate af-finity form for substrate binding, and GrpE, which accelerates release of substrates (11, 12). In mammalian systems, where several co-chaperones have been identified, a homologue of DnaJ, Hsp40/Hdj-1, and Hsc70-interacting protein (Hip/p48) was shown to increase the affinity for substrate protein, thus preventing aggregation of denatured proteins (13, 14). The Hsc70-Hsp90-organizing protein (Hop/p60/Sti1) interacts with the C-terminal domain of Hsc70 and serves as an adaptor molecule that forms an Hsc70-Hop-Hsp90 complex, without affecting the chaperone activity of Hsc70 (15–17). BAG-1, which is known to bind to Bcl-2 and thus exerts anti-apoptotic activity, binds to the ATPase domain of Hsp70 and attenuates Hsc70 chaperone activity (9, 18, 19). The C terminus of Hsc70-interacting protein (CHIP) inhibits its ability to refold non-native proteins (20). Many co-chaperones were identified con-taining the Hsp70 binding domain and ubiquitin-like or U box domain, but even the functions of each domain were not well understood (21).

We found that hFAF1 is a new Hsc70/Hsp70-binding protein employing immunoprecipitation and identification of the bound proteins using MALDI-TOF MS. We examined whether hFAF1 regulates the chaperone activity through binding to chaperones in the heat shock-mediated signaling pathway because hFAF1 contains two ubiquitin-like domains and one ubiquitin-like module (UBX). Transient overexpression of hFAF1 inhibits chaperone activity of Hsp70, suggesting that hFAF1 is possibly a novel co-chaperone of Hsp70. We postulate that hFAF1 plays a role in the regulation of stress-induced cell death using Hsp70 as a binding partner.

EXPERIMENTAL PROCEDURES

Plasmids—Full-length human hsp70 was subcloned into the pGEX-4T-1 vector or 3⫻ FLAG-CMV-7.1 vector pGEX-4T-1/Hsp70⌬ABD-(1–119, 429 – 640). The construct pGEX-4T-1/Hsp70N-(1–119) was prepared by PCR using the sense primer 5 ⬘-GAAGAATTCATGGCCA-AAGCCGCGGCAG-3⬘ and antisense primer 5⬘-GCATCTCGAGCGAG-ATCTCCTCGGGGTA-3⬘ and was subcloned into the pGEX-4T-1 vector. * This work was supported in part by Korea Science and Engineering

Foundation through the Center for Cell Signaling Research at Ewha Womans University, by 21C Frontier Functional Proteomics Center Project MOST FPR03B3-04-110 and FPR02A7-32-110, and by the Na-tional R & D Program for Cancer Control Grant 0420190-1. 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.

□S The on-line version of this article (available at http://www.jbc.org) contains a figure.

§ Supported by the Brain Korea 21 Project. Both authors contributed equally to this work.

储To whom correspondence should be addressed: Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul 120-750, Korea. Tel.: 82-2-3277-3038; Fax: 82-2-3277-3760; E-mail: kjl@mm.ewha.ac.kr.

1_{The abbreviations used are: FAF1, Fas-associated factor 1; hFAF1,} human Fas-associated factor 1; mFAF1, mouse FAF1; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS, mass spectrometry; GST, glutathione S-transferase; SAPK, stress-activated protein kinase; JNK, c-Jun N-terminal kinase; HBSS, Hanks’ buffered saline solution; FITC, fluorescein isothiocyanate; Hsp70, heat shock protein 70; ABD, ATP-binding domain.

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pGEX-4T-1/Hsp70⌬N-(120–640) was prepared by PCR using sense primer 5⬘-CGAGGAATTCATGTCCATGGTGCTGACC-3 and antisense primer 5⬘-GGCCTCGAGCTAATCTACCTCCTCAATG-3⬘ and was sub-cloned into the pGEX-4T-1 vector. pGEX-4T-1/Hsp70⌬PBD-(1–435, 619 – 640) was prepared as described previously (22). The constructs 2/hFAF1, 2/hFAF1-(1–201), pFLAG-CMV-2/hFAF1-(1–345), pFLAG-CMV-2/hFAF1-(82– 650), pFLAG-CMV-2/hF-AF1-(366 – 650) (hFAF1⌬N), pFLAG-CMV-2/hFAF1-(181–381), and pCDNA/hFAF1 were prepared as described previously (2). pCytluc (pRSVLL/V) encoding cytoplasmic luciferase was provided by S. Subr-amani (University of California, San Diego).

Cells and Reagents—Human embryonic kidney epithelial

(HEK293T) cells were grown and maintained in high glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. Monoclonal anti-FLAG antibody M2 was purchased from Sigma. Monoclonal anti-Hsc70/Hsp70 antibody was obtained from StressGen. Polyclonal anti-hFAF1 antibody was generated and charac-terized in our group.

Transient Transfection—HEK293T cells were transfected with ex-pression plasmid using the calcium phosphate precipitation method. Cells were seeded in 10-cm plates a day before transfection at the density of 1.5⫻ 106_{cells and transiently transfected with 6 –12}_{␮g of} expression plasmid by the calcium phosphate method. The fresh me-dium was changed at 6 h after transfection and was cultured for an additional 18 h.

Metabolic Labeling and Immunoprecipitation—Cells were metaboli-cally labeled with 2 ␮Ci/ml [35_{S]methionine in methionine half-free} medium for 18 h. Cells were disrupted with a buffer containing protease inhibitors (50 mMTris, pH 8.0, 150 mMNaCl, 2 mMEDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 mMdithiothreitol, 5 ␮g/ml aprotinin, 1 ␮g/ml leupeptin, 5 mMNa3VO4, 5 mMNaF) for 30 min on ice. The lysates were centrifuged at 12,000 rpm for 1 h, and the supernatant was incubated for 3 h at 4 °C with monoclonal anti-FLAG M2-affinity cross-linking agarose beads or incubated for 2 h with anti-FLAG antibody and for an additional 1 h with protein A beads at 4 °C. The beads were washed more than three times with 1 ml of lysis buffer. Two-dimensional Gel Electrophoresis—The protein samples were mixed with a buffer containing 9.5Murea, 2% Triton X-100, 5% ␤-mer-captoethanol, 1 mMphenylmethylsulfonyl fluoride, 5␮g/ml aprotinin, 10␮g/ml pepstatin A, 10 ␮g/ml leupeptin, 1 mMEDTA, 10 mMNa3VO4, 10 mMNaF for 30 min at room temperature and electrofocused in 7-cm Immobiline DryStrips (pH 4 –7) with IPGphor (Amersham Biosciences). The following focusing protocol was used: 50␮A per strip at 20 °C; 1) rehydration for 16 h; 2) 500 V for 1 h (step and hold); 3) 1000 V for 1 h (step and hold); 4) 8000 V for 3 h (step and hold). After electrofocusing, the strips were shaken for 15 min with equilibration buffer (1.5 M Tris-Cl, pH 8.8, 6Murea, 30% glycerol, 2% SDS, 10 mg/ml dithiothre-itol) and loaded onto mini two-dimensional SDS-polyacrylamide gel (Bio-Rad).

In Vitro Binding Assay—GST-Hsp70 deletion mutant expressed E. coli cytosol was incubated with glutathione-Sepharose 4B beads in phosphate-buffered saline containing 0.1% Triton X-100 for 3 h at 4 °C. The beads were washed three times with phosphate-buffered saline containing 0.1% Triton X-100. Binding assays were performed with Hsp70 deletion mutants bound to glutathione-Sepharose 4B beads and hFAF1 cleaved by thrombin from GST-hFAF1 in binding buffer (50 mMNaCl with 1 mg of bovine serum albumin per ml) with rocking for 3 h at 4 °C. The beads were washed five times with phosphate-buffered saline containing 0.5% Nonidet P-40, resus-pended in 2⫻ gel sample buffer, resolved with SDS-PAGE, and Coo-massie Blue-stained. After obtaining the gel image, the proteins in the gel were transferred to polyvinylidene difluoride membrane and immunostained with polyclonal anti-hFAF1 antibody.

Confocal Microscopy—For morphological studies, cells were grown on coated coverslips, transiently transfected, and treated with heat shock. The cells were gently rinsed in HBSS and fixed with 4% paraformaldehyde in HBSS for 10 min at room temperature. After washing with HBSS, the cells were permeabilized by incubating with 0.1% Triton X-100 in HBSS for 10 min at room temperature before immunostaining. Nonspecific protein absorption was inhibited by incu-bation of the cells for 1 h in HBSS containing 3% bovine serum albumin, 0.2% Tween 20, and 0.2% gelatin. For Hsc70/Hsp70 staining, the cells were incubated with mouse monoclonal Hsc70/Hsp70 antibody (Stress-Gen) diluted at 1:150 in HBSS containing 1% sucrose and 1% bovine serum albumin for 2 h at 37 °C. After washing three times with HBSS, the cells were incubated for 1 h with Texas Red-conjugated rabbit anti-mouse (Molecular Probes) diluted at 1:200 and subsequently

washed with HBSS. FLAG-hFAF1 was stained with FITC-labeled monoclonal M2 anti-FLAG antibody diluted at 1:200 for 1 h at room temperature. Confocal microscopy was performed with a Multiphoton Radiance 2000 (Bio-Rad) connected to a microscope (Nikon Eclipse TE 300).

In-gel Digestion and Mass Spectrometric Analysis—The cellular pro-teins were separated on two-dimensional gel electrophoresis and stained with Coomassie Blue or Silver. The gel spots were excised with a scalpel, crushed, and destained by washing with 25 mMNH4HCO3, 50% acetonitrile. The silver-stained gel was destained by washing with 15 mMK4FeCN6, 50 mMsodium thiosulfate. After crushing, the gels were dehydrated by addition of acetonitrile, rehydrated by adding 10 –20␮l of 25 mMammonium bicarbonate with 10 ng/␮l of sequencing grade trypsin (Promega), and incubated at 37 °C for 12–15 h. Peptides were extracted by adding 30␮l of solution containing 60% acetonitrile/ 0.1% trifluoroacetic acid. The extraction was repeated three times and completed by adding 20␮l of acetonitrile. The extracted solutions were pooled and evaporated to dryness in a SpeedVac vacuum centrifuge. Samples were reconstituted in 10␮l of 0.1% trifluoroacetic acid and treated with ZipTips containing C18 resin (Millipore) according to the manufacturer’s instructions. The washed peptides were eluted with saturated matrix solution (␣-cyano-4-hydroxycinnamic acid in 60% ace-tonitrile, 0.1% trifluoroacetic acid). Peptide mixtures were analyzed with MALDI-TOF MS using a delayed ion extraction and ion mirror reflector mass spectrometer (Voyager-DE STR; Applied Biosystems, Inc.). External calibration was carried out using sEQUAZYME peptide mass standard kit (PerSeptive Biosystems) and internal calibration, by using the autolytic peaks of trypsin. This procedure typically results in mass accuracies of 50 ppm. For interpretation of the mass spectra, we used the Ms-Fit program available on the World Wide Web site of the University of California at San Francisco (prospector.ucsf.edu/). This result were confirmed by peptide sequencing using liquid chromatog-raphy-electrospray ionization-Q-TOF tandem MS.

Luciferase Reactivation Assay—Cells were transiently transfected with pCytluc and FLAG-tagged Hsp70 or hFAF1. Twenty-four hours after transfection, the cells were transferred into tissue culture dishes in medium with 20␮g/ml cycloheximide for 30 min at 37 °C. Luciferase was inactivated by heating the cells at 45 °C for 15 min and recovered at 37 °C for various times. Luciferase activities of the harvested cells were measured using luciferase assay kit (Promega).

SAPK/JNK Assay—SAPK/JNK was assayed using GST-c-Jun-(1– 89)-peptide (provided by Dr. J. R. Woodgett) as a substrate after im-munoprecipitation with anti-JNK antibody (Santa Cruz Biotechnology). Cell pellets were homogenized with buffer A containing 20 mMHEPES, pH 7.4, 50 mM␤-glycerophosphate, 2 mMEGTA, 1 mMdithiothreitol, 1 mMNa3VO4, 1% Triton X-100, 10% glycerol, 2␮Mleupeptin, 400␮M phenylmethylsulfonyl fluoride, and 10 units/ml aprotinin. The super-natant containing 1 mg of protein was incubated with antibodies against mammalian SAPK/JNK and then with protein A-Sepharose 4B suspension. The immunocomplexes were washed three times and then incubated with a kinase buffer (50 mMTris-Cl, pH 7.4, 10 mMMgCl2, 2 mMEGTA, 1 mMdithiothreitol, 0.1% Triton X-100) supplemented with GST-c-Jun and [␥-32_{P]ATP for 20 min at 37 °C. To stop the reaction, 2}_⫻ gel sample buffer was added. Samples were subjected to 12% SDS-PAGE, and the gels were stained, destained, and dried. Phosphoryla-tion of c-Jun was measured by BAS2500 (Fuji).

RESULTS

Identification of Hsc70 and Hsp70 as hFAF1-interacting Pro-tein in Vivo—To identify the proPro-teins interacting with hFAF1,

we performed co-immunoprecipitation experiments. FLAG-tagged hFAF1 was transiently overexpressed in HEK293T cells, and cellular proteins were metabolically labeled with 2 ␮Ci/ml [35_{S]methionine. hFAF1-interacting proteins were} di-vided into two fractions and analyzed by two-dimensional gel electrophoresis. One fraction was transferred to NC membrane for autoradiogram and Western blot analysis, and the other fraction was silver-stained for visualization (Fig. 1). Protein spots detected in the autoradiogram were cut out from the corresponding silver-stained gel and subjected to in-gel diges-tion with trypsin and mass analyses using MALDI-TOF MS and electrospray ionization-Q-TOF tandem MS. We identified spot numbers 1 and 2 as heat shock protein 70 cognate (Hsc70) and heat shock protein 70 inducible (Hsp70), respectively (Ta-ble I). Binding to Hsc70 and Hsp70 was reconfirmed by

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ern blot analysis of the NC membrane with monoclonal anti-Hsc70/Hsp70 antibody (Fig. 1C). To confirm further the binding, cell lysate from the FLAG-hFAF1-overexpressed cell line was separated on gel filtration column chromatography, and the binding complex of hFAF1 and Hsc70/Hsp70 was co-eluted at the 250 –300-kDa fraction (data not shown).

To see if endogenous hFAF1 interacts with endogenous Hsc70/Hsp70 in vivo, we performed immunoprecipitation stud-ies with polyclonal anti-hFAF1 antibody in HEK293T cells. Fig. 2 shows that endogenous Hsc70/Hsp70 exists in the hFAF1 protein complex in vivo, and a small fraction of endogenous Hsc70/Hsp70 could bind hFAF1. In addition, the recombinant hFAF1 expressed in E. coli can bind to bacterial Hsp70, DnaK (data not shown), suggesting that hFAF1 has intrinsic binding property to Hsp70.

Hsc70/Hsp70 Interacts with the Amino Acids 82–180 of hFAF1 in Vivo—To characterize the hFAF1 domains necessary

for interaction with Hsp70, we examined binding between en-dogenous Hsc70/Hsp70 and a series of transiently transfected FLAG-hFAF1 deletion mutants (Fig. 3A). HEK293T cells were transfected with various FLAG-tagged hFAF1 deletion mu-tants and immunoprecipitated by anti-FLAG M2-agarose cross-linking affinity gel. The precipitates were analyzed by Western blot analysis using anti-FLAG antibody (Fig. 3B,

up-per panel) and anti-Hsc70/Hsp70 antibody (Fig. 3B, lower panel). Hsc70/Hsp70 did not bind to anti-FLAG M2-agarose

cross-linking affinity gel of vector-transfected cells, bound only negligibly to amino acid sequences of 366 – 650 and 181–381 of FLAG-tagged hFAF1, but bound well to N-terminal hFAF1 fragments containing amino acid sequences, 1–201, 1–345, and 82– 650. These data showed that the amino acid sequence 82– 180 of hFAF1 is necessary for binding with Hsc70/Hsp70. This was confirmed in Fig. 3C. The amino acids 82–180 of hFAF1 bound to endogenous Hsc70/Hsp70. This region includes the Fas death domain-binding domain (amino acid residues 1–201) (23).

hFAF1 Binds Directly to the N-terminal Domain of Hsp70 in Vitro—To examine whether hFAF1 might be associated

di-rectly or indidi-rectly with Hsp70, in an accessory protein-medi-ated process, we carried out in vitro pull down assay. We prepared various truncated forms of GST-Hsp70 (Fig. 4A) as

follows: full Hsp70, 1– 640; 1–120; 120 – 640; 1–119; 428 – 640; 1– 435; and 616 – 640. Various recombinant GST-Hsp70s were immobilized to glutathione-Sepharose beads and incubated with purified hFAF1 in the presence of 10 mMATP. We found

direct interaction between hFAF1 and GST-Hsp70-(1–120) by immunoblotting with anti-hFAF1 antibody (Fig. 4B). In the presence of various concentrations of ATP, the bindings be-tween Hsp70 and hFAF1 were not affected (Fig. 4C). These results suggest that hFAF1 directly interacts with the N ter-minus of Hsp70 and that the binding was not affected by ATP-induced conformational changes of Hsp70.

Heat Shock Does Not Affect the Interaction between Hsp70 and hFAF1—We examined whether the interaction between

hFAF1 and Hsc70/Hsp70 in vivo could be regulated by heat shock. HEK293T cells transiently transfected with pFLAG-CMV-2 vector or FLAG-tagged hFAF1 were heat-shocked at 45 °C for 45 min, recovered for various times at 37 °C, and immunoprecipitated with anti-FLAG M2-agarose cross-linking affinity gel. Immunoprecipitates were analyzed by Western blot analysis using anti-Hsc70/Hsp70 (Fig. 5, upper panel) and anti-FLAG M2 antibody (Fig. 5, lower panel). When we quan-titatively analyzed the ratio of Hsc70/Hsp70 to FLAG-hFAF1 in a linear range using LAS1000 (Fuji), Hsc70 interactions did not change during recovery after heat shock, and even the amount of Hsp70 was slightly increased in cell lysates. This suggests that the interaction between hFAF1 and Hsc70/Hsp70 is homeostatic and is not affected by heat shock treatment.

Subcellular Localization of hFAF1 and Hsc70/Hsp70 Was Changed by Heat Shock—Because the interaction between

hFAF1 and Hsc70/Hsp70 was unaffected by ATP and stress, the question arises whether the localization of the binding FIG. 1. FLAG-hFAF1 interacts with

Hsc70 and Hsp70. HEK293T cells were

transfected with pFLAG-CMV-2 vector (A) and FLAG-tagged hFAF1 (B) and la-beled with 2 ␮Ci/ml [35_{S]methionine.} Cells were lysed and immunoprecipitated with anti-FLAG M2-agarose cross-linking affinity gel. Precipitates were analyzed by two-dimensional gel electrophoresis and autoradiographed using BAS2500. Pro-tein spots detected on the autoradiograph were cut out from the corresponding sil-ver-stained gel and subjected to in-gel di-gestion with trypsin, and mass peptide fingerprint analyses were conducted. En-larged figure of the boxed section is shown at the right bottom of each gel. C, Hsc70/ Hsp70 were confirmed by Western blot analysis.

TABLE I

FLAG-hFAF1-binding proteins identified by two-dimensional gel electrophoresis and MALDI-TOF MS

Spot no. Identified_protein NCBI GI no. Mass pI _coveragePeptide

Da

1 Hsc70 13273304 70,899 5.4 29

2 Hsp70 386785 69,869 5.4 30

FIG. 2. Endogenous hFAF1 interacts with endogenous Hsc70/

Hsp70. HEK293T cells were lysed and immunoprecipitated (IP) with

mouse IgG as control (1st lane) and anti-hFAF1 polyclonal antibody (2nd lane). The precipitates were analyzed by SDS-PAGE and Western blot analysis by using anti-hFAF1 polyclonal antibody (upper panel) and anti-Hsc70/Hsp70 monoclonal antibody (lower panel). Cell lysates after immunoprecipitation were analyzed by Western blot analysis using anti-hFAF1 and anti-Hsc70/Hsp70 antibodies.

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complex is specific and is changed by heat shock. FLAG-hFAF1 transiently overexpressed in HeLa cells was treated with heat shock at 45 °C for 30 min and recovered at 37 °C after 24 h. Hsp70/Hsc70 proteins were stained with monoclonal anti-Hsc70/Hsp70 antibody, and secondary antibody was labeled with Texas Red and FLAG-hFAF1 with FITC-labeled anti-FLAG M2 monoclonal antibody. hFAF1 and Hsp70 normally appeared in the cytosol and nucleus (Fig. 6, A and B). When two images were merged, the cytosolic and nuclear regions showed bright yellow color, suggesting co-localization of two proteins (Fig. 6C). When the cells were treated with heat shock at 45 °C for 30 min, immediately after heat shock, hFAF1 and Hsp70 co-localized in perinuclear region, and speckles were bright yellow in the cytosol (Fig. 6F). After 24 h of recovery at 37 °C, hFAF1 relocalized mainly to the nucleus and cytosol, and Hsp70 showed enrichment in the nucleus when compared with the control cells (Fig. 6, G and H). Again the two proteins co-localized in the perinuclear region (Fig. 6I). These results show that the sites of co-localization of hFAF1 and Hsp70 vary between the cytosolic and nuclear region and the perinuclear

region, depending on heat shock and recovery conditions.

hFAF1 Inhibits Hsp70-mediated Reactivation of Heat-dena-tured Firefly Luciferase in Vivo—Previous reports (24, 25)

showed the activity of transiently expressed luciferase in cells can serve as a marker of chaperone activity of Hsp70 in vivo. To examine whether FLAG-tagged Hsp70 accelerates the reacti-vation of heat-denatured luciferase in vivo, we transiently transfected FLAG-tagged Hsp70 or Hsp70 ATP binding domain

deletion mutant (Hsp70_{⌬ABD-(1–119 and 429–640)) in}

HEK293T cells. We co-expressed firefly luciferase, whose en-zymatic activity during recovery from thermal inactivation is highly dependent on chaperone activity. The overexpression of each protein was confirmed by Western blot analysis using anti-Hsp70 antibody (Fig. 7A), because we had problems de-tecting FLAG-Hsp70 with anti-FLAG antibody. The cells were heat-shocked at 45 °C for 15 min and recovered for the indi-cated times at 37 °C, harvested, and lysed, and luciferase assay was performed (Fig. 7B). Overexpression of Hsp70 alone is sufficient to enhance reactivation of luciferase activity during recovery at 37 °C. However, in Hsp70⌬ABD-transfected cells, the reactivation of luciferase was slower than in Hsp70-ex-pressed cells but faster than in control cells, suggesting that heat shock-inactivated luciferase was reactivated by the chap-erone activity of Hsp70 and that ATP binding domain is not essential for chaperone activity in vivo.

To examine the effect of hFAF1 on the chaperone activity of endogenous Hsp70, HEK293T cells were transfected with var-ious amounts of FLAG-tagged hFAF1. The overexpression of FLAG-tagged hFAF1 was confirmed by Western blot analysis using anti-FLAG antibody (Fig. 7C). The reactivation of lucif-erase activity was monitored during recovery at 37 °C after heat shock. Transfection of FLAG-CMV-2 vector plasmid alone caused basal level reactivation of heat-denatured cytoplasmic luciferase after 4 h at 37 °C, whereas in the presence of FLAG-hFAF1, luciferase activity was significantly reduced. Overex-pression of hFAF1 significantly reduced the reactivation rate of luciferase activity in a dose-dependent manner, which indi-cates the inhibitory effect of hFAF1 on the chaperone activity of endogenous Hsp70 (Fig. 7D).

To confirm that the inhibitory effect of hFAF1 is through binding to endogenous Hsp70, HEK293T cells were transfected with FLAG-tagged hFAF1 or a deletion mutant, amino acid

sequence 366 – 650 of FLAG-hFAF1 (FLAG-hFAF1_{⌬N), lacking}

the N-terminal domain required for Hsp70 interaction without Hsp70 overexpression. Expression of hFAF1 and FLAG-hFAF1_{⌬N was monitored by Western blot analysis (Fig. 7E). In} the presence of FLAG-hFAF1, luciferase activity was

signifi-cantly reduced; however, expression of FLAG-hFAF1⌬N did

not inhibit Hsp70 chaperone activity (Fig. 7F). To confirm the dominant negative-like effect of the overexpression of FLAG-hFAF1⌬N, hFAF1 lacking the N-terminal domain required for Hsp70 interaction in Fig. 7F, we transfected 1 and 2 ␮g of

FLAG-hFAF1⌬N vector DNA in HEK293T cells. Transfection

of FLAG-hFAF1⌬N increased the renaturation activity of

Hsp70 in a dose-dependent manner (data not shown). To ex-amine this effect on overexpressed Hsp70, we co-transfected a

small amount of Hsp70 (0.3␮g DNA) with 1.0 ␮g of DNA of

FLAG control, FLAG-hFAF1⌬N, or hFAF1 (Fig. 7G).

Overex-pression of Hsp70 alone increased the renaturation activity compared with FLAG control. Co-transfection of FLAG-hFAF1 with Hsp70 significantly reduced the higher basal luciferase activity; however, expression of FLAG-hFAF1⌬N did not in-hibit Hsp70 chaperone activity (Fig. 7H). These studies estab-lish that hFAF1 acts as an inhibitor of the chaperone activity of Hsp70 and that the N-terminal residues of hFAF1 play a role in this inhibition of Hsp70 chaperone activity.

FIG. 3. Hsc70 and Hsp70 interact with amino acids 82–180 of

hFAF1 in vivo. A, diagram of FLAG-tagged hFAF1 fragments.

HEK293T cells were transfected with CMV-2 vector or pFLAG-CMV/hFAF1 fragments. Cells were lysed and immunoprecipitated with anti-FLAG M2-affinity cross-linking agarose beads (B and C) or with monoclonal anti-FLAG antibody (Ab) and with protein A beads. Precip-itates were analyzed by Western blot analysis using anti-FLAG M2 monoclonal antibody (upper panels of B and C) and anti-Hsc70/Hsp70 antibody (lower panels of B and C).

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Overexpression of hFAF1 Increases Cell Sensitivity to Heat Shock Stress—Activation of SAPK/JNK is required for

stress-induced apoptosis, and Hsp70 overexpression prevents

apo-ptosis by suppressing the activation of SAPK/JNK (26 –28). We have shown that cells being tolerant to stress by expressing the elevated Hsp70 were less sensitive to stress, activated the FIG. 4. hFAF1 directly interacts with

the N-terminal domain of Hsp70 in

vitro. A, diagram of GST-tagged Hsp70

fragments. B, purified recombinant hFAF1 was incubated with GST-Hsp70 deletion mutants immobilized on glutathione-Sepha-rose in the presence or absence of 10 mM ATP. Precipitates were detected by Coomas-sie Blue staining (B, upper panel) and immu-noblotting using anti-hFAF1 polyclonal an-tibody (B, lower two panels). C, HEK293T cells were transiently transfected with FLAG-tagged hFAF1. Cells were lysed and incubated with monoclonal anti-FLAG M2 antibody and protein A-Sepharose 4B beads. Various concentrations of ATP were added to the immune complex and incubated for 1 h. Immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis using Hsc70/Hsp70 (upper panel) and anti-FLAG M2 (lower panel) monoclonal antibodies.

FIG. 5. Heat shock has no effect on

the interaction between Hsc70/Hsp70 and hFAF1. HEK293T cells were

tran-siently transfected with pFLAG-CMV-2 vector or FLAG-tagged hFAF1. Cells were heat-shocked at 45 °C for 45 min or un-treated (C) and recovered for the indi-cated times. At each time point, cells were lysed and immunoprecipitated (IP) with monoclonal anti-FLAG M2 antibody. Pre-cipitates were analyzed by Western blot analysis using anti-Hsc70/Hsp70 (upper panel) and anti-FLAG M2 (lower panel) monoclonal antibodies.

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SAPK/JNK in response to a higher dose of stress, and had faster deactivation rate of SAPK/JNK in the same dose of stress. It would thus appear that kinetics of SAPK/JNK acti-vation and deactiacti-vation can serve as a marker of cellular sen-sitivity to various stresses (29).

We then examined the effect of overexpression of hFAF1 on heat shock-induced cellular response, using SAPK/JNK activa-tion as a marker. We transfected HEK293T cells with

FLAG-CMV-2 vector, FLAG-tagged hFAF1, or FLAG-tagged

hFAF1⌬N. Transfected HEK293T cells were treated with heat shock at 45 °C for 45 min, and SAPK/JNK activities were measured (Fig. 8). The kinetics of SAPK/JNK activation in both

control and FLAG-hFAF1_{⌬N-expressing cells were similar;}

there was a 13-fold activation immediately after heat shock and deactivation to the basal level after a 7-h recovery. On the other hand, SAPK/JNK activity in FLAG-hFAF1-expressing cells increased 16-fold activation immediately after heat shock and lasted up to 2 h; deactivation was significantly delayed up to 7 h. This indicated that hFAF1-expressing cells were more sensitive to heat shock and were less able to recover. Also the hyper-activation of SAPK/JNK observed in heat-shocked FLAG-hFAF1-expressing cells was absent in heat-shocked FLAG-hFAF1⌬N-expressing cells. This suggests that overex-pression of hFAF1 renders cells more sensitive to heat shock and more prone to apoptosis.

hFAF1 Overexpression Increases Heat Shock-induced Cell Death—To confirm the effect of hFAF1 on stress-induced cell

death, we examined cell viability after heat shock. HEK293T cells transfected with FLAG-CMV-2 vector, FLAG-tagged

hFAF1, FLAG-tagged hFAF1⌬N, or co-expression with Hsp70

were heat shocked at 45 °C for 45 min. After recovery at 37 °C for 24 h, the numbers of viable cells were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. FLAG-hFAF1-overexpressed HEK293T cells displayed reduced survival, compared with vector transfectants, in contrast to

FLAG-hFAF1_{⌬N-transfected cells that showed enhanced}

sur-vival (Fig. 9). This confirms that complexing with hFAF1 de-creased the chaperone activity of Hsp70 and rendered the cells

more sensitive to stress. FLAG-hFAF1⌬N-expressing cells,

which lack of Hsp70 binding affinity, showed tolerance to heat shock. The same experiments were performed in the cells over-expressed with both Hsp70 and each hFAF1 in order to confirm the phenomena. Overexpression of Hsp70 slightly raised the resistance to stress, but the co-expression with hFAF1 in-creased the sensitivity to stress. FLAG-hFAF1⌬N co-express-ing cells abolished the hFAF1 effect. This may be due to in-creased chaperone activity of Hsp70 or some unknown role of the C-terminal hFAF1. The results confirms that overexpres-sion of hFAF1 renders cells more sensitive to heat shock and more prone to apoptosis by interacting with Hsp70.

FAF1 Sequences Are Well Conserved in Vertebrates—To

de-termine whether FAF1 is well conserved through evolution, we aligned long and short forms of hFAF1s (23) with rat, mouse, quail, Danio rerio, Caenorhabditis elegans, and fly FAF1 as shown in the supplemental figure; the entire sequence of FAF1 is well conserved as well as the 82–180 amino acid region to which Hsp70 binds. It is 78 –95% identical in vertebrates and 18 –21% identical between vertebrates and invertebrates. This suggests that FAF1 plays an essential role throughout the vertebrates.

FIG. 6. Immunofluorescence analysis of FLAG-hFAF1 and Hsp70 localization. HeLa cells were transiently transfected with pFLAG-CMV-2 vector or pFLAG-pFLAG-CMV-2/hFAF1. Cells were immunostained without heat shock (A–C), after heat shock at 45 °C for 30 min (D–F), or after a recovery period of 24 h (G–I). All cells were stained with a monoclonal antibody to the Hsc70/Hsp70 followed by incubation with Texas Red-labeled secondary antibody (red). The cells were then stained with anti-FLAG M2 monoclonal antibody labeled with FITC (green). Yellow in the merged figures (C, F, and I) means co-localization of red and green fluorescence. The images were made by confocal microscopy.

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DISCUSSION

These studies demonstrate interaction between hFAF1 and Hsc70/Hsp70 and the functional significance of this interaction in heat shock-induced cell signaling. We previously identified the 1–201-amino acid region of hFAF1 as a Fas binding domain by using the yeast two-hybrid assay (23). The cellular function of hFAF1, a novel protein known to be an adaptor molecule of death receptor Fas, was identified as a member of the Fas

death-inducing signaling complex (6) and as a suppressor of NF-␬B activity by cytoplasmic retention of NF-␬B p65 via phys-ical interaction at the 181–381-amino acid region (7). In this study, by using two-dimensional gel electrophoresis and MALDI-TOF MS, gel filtration gel chromatography, and in

vitro by pull down assay, we have shown that the

82–180-amino acid region of hFAF1 directly interacts with the N ter-minus (amino acids 1–120) of Hsp70 in vivo. Hsp homologues FIG. 7. Overexpression of hFAF1 inhibits Hsp70-mediated reactivation of heat-denatured firefly luciferase. HEK293T cells were transiently transfected with pCytluc (encoding cytosolic luciferase) together with 2 vector, 2/Hsp70, or pFLAG-CMV-2/Hsp70⌬ABD, pFLAG-CMV-2/hFAF1 (A, C, and E), or pFLAG-CMV-2/hFAF1⌬N 3FLAG-CMV-7.1/Hsp70 as indicated (G), and were detected with Western blot analysis by using anti-Hsp70 antibody for FLAG-tagged Hsc70/Hsp70, Hsp70⌬ABD, and anti-FLAG or anti-FAF1 antibody for hFAF1, and hFAF1⌬N expression (A, C, E, and G) and anti-FLAG antibody for 3FLAG-Hsp70 (G). After pretreatment with cycloheximide (20 ␮g/ml) for 30 min, luciferase was inactivated by heating the cells at 45 °C for 15 min. During a subsequent recovery period at 37 °C, luciferase activity (B, D, F, and H) at each time point was measured.

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such as DnaK and DnaJ were co-purified during the purifica-tion of recombinant hFAF1 from the E. coli lysate.

Hsp70 plays an important protective role in stress-induced cell death. hFAF1 binding can affect this role of Hsp70 in two possible ways. One possibility is that hFAF1 may serve as a substrate and become refolded, degraded, or translocated by Hsp70. The second is that hFAF1 may act as modulator of

Hsp70 function. Our studies revealed that hFAF1 did not bind to the peptide binding domain, substrate-binding sites of Hsp70, but rather bound to N terminus of the ATPase domain of Hsp70, as co-chaperones Bag-1 and Hip bind to the ATPase domain. Also the expression level of hFAF1 was not varied in various stresses, indicating that hFAF1 is not a substrate of Hsp70. We therefore examined whether hFAF1 is involved in the regulation of Hsp70 activity. Transient overexpression of hFAF1 inhibited the chaperone activity of Hsp70, whereas transient expression of the N-terminally deleted hFAF1 mu-tant showed the opposite effect, indicating that hFAF1 may act as a co-chaperone and inhibit the chaperone activity of Hsp70. Recently, co-chaperones of Hsp70 such as Hsp40, Hip, Hop, Bag-1, CHIP, Chap1/PLIC-2, and Chap2/Bat3/Scythe have been identified. Hip and Hop facilitates refolding of unfolded proteins and Bag-1, CHIP, and Chap2/Bat3/Scythe attenuated the chaperone activity of Hsp70 (9, 14, 16, 17–20, 30). Because the latter proteins contain ubiquitin-like do-mains, which bind to proteasomes, they are thought to be involved in the degradation of Hsp70 substrates in protea-somes. Bag-1 contains a ubiquitin-like domain which binds to proteasome; CHIP has U box domain that has ubiquitin li-gase activity, and Chap2/Bat3/Scythe also contains a ubiq-uitin-like domain. The whole sequence of FAF1 is 78 –95% identical in vertebrates and 18 –21% identical between ver-tebrates and inverver-tebrates. It contains two ubiquitin-related domains, UAS and UBX (see the supplemental figure). This suggests that hFAF1 might play a role in protein degradation in combination with Hsp70. Our other data2_{also suggest the} involvement of hFAF1 in proteasome degradation.

To understand the biological function of hFAF1 reflected by its negative regulation of Hsp70 chaperone activity, we exam-ined the effect of hFAF1 in heat shock responses. Hsp70 seems to play an important role in preventing programmed cell death in response to heat shock by suppressing the activation of SAPK/JNK (26, 28, 31). By using the kinetics of SAPK/JNK activation and deactivation as a marker of cellular sensitivity to stress, we found that the activation of SAPK/JNK in ther-motolerant cells having elevated Hsp70 expression occurred at a higher dose of stress, and the deactivation in thermotolerant cells was much faster than in control (29). Recently we also reported that heat shock induced transient tyrosine phospho-rylations of various proteins (32). As shown in Fig. 8, heat shock-induced SAPK/JNK activation was accelerated in cells overexpressing hFAF1, and the deactivation during recovery was significantly delayed compared with vector-transfected control cells. This effect was completely abolished by overex-pression of the N-terminal truncated hFAF1, lacking affinity for Hsp70 binding. This indicates that overexpression of hFAF1 renders cells more sensitive to heat shock, probably due to inhibition of Hsp70 chaperone activity. It is possible that Hsp70 chaperone activity is related to the inhibition of SAPK/ JNK activity induced by various stresses and to its anti-apo-ptotic effects. Volloch et al. (27) showed that ABD of Hsp70 is not necessary for the inhibition of SAPK/JNK. It seems that the chaperone activity of Hsp70, based on ATP hydrolysis as energy source, does not affect SAPK/JNK activity. However, the relative roles of ABD and chaperone function of Hsp70 are not clear (33). In this study, the ABD deletion mutant of Hsp70 showed elevated chaperone activity, although lower than that of the full form of Hsp70, suggesting that ABD, having a smaller domain (amino acids 120 – 428) in this study than the conventional ABD-(1– 428), is not indispensable for the

chap-2_{E. J. Song, S. H. Yim, E. Kim, N. S. Kim, and K.-J. Lee, unpublished} data.

FIG. 8. Overexpression of FLAG-hFAF1 accelerates heat

shock-induced SAPK/JNK activation. HEK293T cells were

tran-siently transfected with pFLAG-CMV-2 vector, pFLAG-CMV-2/hFAF1, or pFLAG-CMV-2/hFAF1⌬N and exposed to heat shock at 45 °C for 45 min. SAPK/JNK activities were measured after the indicated recovery times. C, control without heat shock treatment.

FIG. 9. Overexpression of FLAG-hFAF1 increases heat

shock-induced cell death. HEK293T cells were transiently transfected with

the pFLAG-CMV-2 vector, pFLAG-CMV-2/hFAF1, or pFLAG-CMV-2/ hFAF1⌬N, co-expressed with 3FLAG-CMV-7.1/Hsp70 and each hFAF1, and exposed to heat shock at 45 °C for 45 min. After recovery at 37 °C for 24 h, the percentage of viable cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

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erone activity. Thus, the possibility that chaperone activity of Hsp70 can affect SAPK/JNK activation should not be excluded. We then examined how the increased cellular sensitivity to heat shock in the hFAF1-overexpressed cell line is related to cell death. HEK293T cells with transient overexpression of FLAG-hFAF1 showed lower viability than vector-transfected cells following severe heat shock treatment. Also, the FLAG-hFAF1⌬N deletion mutant lacking the binding site to Hsp70 showed increased viability than vector-transfected cells, sug-gesting that hFAF1 renders cells more sensitive to heat shock by inhibiting the chaperone activity of Hsp70. The viability of FLAG-hFAF1⌬N-transfected cells increases possibly because the deletion mutant acts as a dominant negative mutant to the full form of hFAF1. It is possible that hFAF1 has multiple domains, and binding to the C terminus may regulate ubiquiti-nation pathway. This hypothesis needs to be verified in future studies.

hFAF1 was originally identified as a Fas-associated protein. Amino acid sequence 1–201 is the binding domain for the Fas death domain (23). This region overlaps with amino acid resi-dues 82–180, the Hsp70 interaction region. It is possible that hFAF1 binds to Fas or Hsp70, depending in the cellular condi-tions. In a previous finding, overexpression of Hsp70 did not protect cells from Fas-induced apoptosis (34). Further studies of the interaction between Fas and Hsp70 with hFAF1 are needed to explain this discrepancy.

The cellular function of hFAF1, as a novel protein, is still unclear. This study suggests that hFAF1 may play a role in the stress-induced signaling pathway through binding to Hsp70 and inhibition of the chaperone activity. Our study suggests that hFAF1 is a previously unrecognized new co-chaperone of Hsp70 having cell death-inducing potential. These functions of hFAF1 may possibly be regulated by post-translational modi-fications (4) and by changes in binding at multidomains of hFAF1.

Acknowledgments—We thank Dr. S. Subramani for providing cyto-plasmic luciferase and Dr. R. I. Morimoto for providing Hsp70 clone.

REFERENCES

1. Chu, K., Niu, X., and Williams, L. T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11894 –11898

2. Ryu, S. W., and Kim, E. (2001) Biochem. Biophys. Res. Commun. 286,

1027–1032

3. Kusk, M., Ahmed, R., Thomsen, B., Bendixen, C., Issinger, O. G., and Bold-yreff, B. (1999) Mol. Cell. Biochem. 191, 51–58

4. Jensen, H. H., Hjerrild, M., Guerra, B., Larsen, M. R., Hojrup, P., and Bold-yreff, B. (2001) Int. J. Biochem. Cell Biol. 33, 577–589

5. Guerra, B., Boldyreff, B., and Issinger, O. G. (2001) Int. J. Oncol. 19, 1117–1126

6. Ryu, S. W., Lee, S. J., Park, M. Y., Jun, J. I., Jung, Y. K., and Kim, E. (2003)

J. Biol. Chem. 278, 24003–24010

7. Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol.

Chem. 279, 2544 –2549

8. Hartl, F. U. (1996) Nature 381, 571–580

9. Ho¨hfeld, J., and Jentsch, S. (1997) EMBO J. 16, 6209 – 6216 10. Bukau, B., and Horwich, A. L. (1998) Cell 92, 351–366

11. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C., and Zylicz, M. (1991)

Proc. Natl. Acad. Sci. U. S. A. 88, 2874 –2878

12. McCarty, J. S., Buchberger, A., Reinstein, J., and Bukau, B. (1995) J. Mol.

Biol. 249, 126 –137

13. Minami, Y., Ho¨hfeld, J., Ohtsuka, K., and Hartl, F. U. (1996) J. Biol. Chem. 271, 19617–19624

14. Ho¨hfeld, J., Minami, Y., and Hartl, F. U. (1995) Cell 83, 589 –598 15. Demand, J., Luders, J., and Ho¨hfeld, J. (1998) Mol. Cell. Biol. 8, 2023–2028 16. Schumacher, R. J., Hurst, R., Sullivan, W. P., McMahon, N. J., Toft, D. O., and

Matts, R. L. (1994) J. Biol. Chem. 269, 9493–9499

17. Smith, D. F., Sullivan, W. P., Marion, T. N., Zaitsu, K., Madden, B., McCor-mick, D. J., and Toft, D. O. (1993) Mol. Cell. Biol. 13, 869 – 876 18. Takayama, S., Bimston, D. N., Matsuzawa, S., Freeman, B. C., Aime-Sempe,

C., Xie, Z., Morimoto, R. I., and Reed, J. C. (1997) EMBO J. 16, 4887– 4896 19. Zeiner, M., Gebauer, M., and Gehring, U. (1997) EMBO J. 16, 5483–5490 20. Ballinger, C. A., Connell, P., Wu, Y., Hu, Z., Thompson, L. J., Yin, L, Y., and

Patterson, C. (1999) Mol. Cell. Biol. 19, 4535– 4545

21. King, F. W., Wawrzynow, A., Hohfeld, J., and Zylicz, M. (2001) EMBO J. 20, 6297– 6305

22. Park, H. S., Lee, J. S., Huh, S. H., Seo, J. S., and Choi, E. J. (2001) EMBO J. 20, 446 – 456

23. Ryu, S. W., Chae, S. K., Lee, K. J., and Kim, E. (1999) Biochem. Biophys. Res.

Commun. 262, 388 –394

24. Michels, A. A., Nguyen, V. T., Konings, A. W., Kampinga, H. H., Bensaude, O. (1995) Eur. J. Biochem. 234, 382–389

25. Nollen, E. A., Brunsting, J. F., Song, J., Kampinga, H. H., and Morimoto, R. I. (2000) Mol. Cell. Biol. 20, 1083–1088

26. Gabai, V. L., Meriin, A. B., Mosser, D. D., Caron, A. W., Rits, S., Shifrin, V. I., and Sherman, M. Y. (1997) J. Biol. Chem. 272, 18033–18037

27. Volloch, V., Mosser, D. D., Massie, B., and Sherman, M. Y. (1998) Cell Stress

Chaperones 3, 265–271

28. Meriin, A. B., Yaglom, J. A., Gabai, V. L., Zon, L., Ganiatsas, S., Mosser, D. D., Zon, L., and Sherman, M. Y. (1999) Mol. Cell. Biol. 19, 2547–2555 29. Kim, H. J., and Lee, K. J. (2000) Mol. Cell. Biochem. 229, 139 –151 30. Thress, K., Song, J., Morimoto, R. I., and Kornbluth, S. (2001) EMBO J. 20,

1033–1041

31. Volloch, V., Gabai, V. L., Rits, S., and Sherman, M. Y. (1999) FEBS Lett. 461, 73–76

32. Kim, H. J., Song, E. J., and Lee, K. J. (2002) J. Biol. Chem. 277, 23193–23207 33. Stege, G. J., Li, L., Kampinga, H. H., Konings, A. W., and Li, G. C. (1994) Exp.

Cell Res. 214, 279 –284

34. Liossis, S. N., Ding, X. Z., Kiang, J. G., and Tsokos, G. C. (1997) J. Immunol. 158, 5668 –5675

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zGT

Hsp70 binding domain

Human - -M A S- -NM D R E M I L A D F QA C T G I E N I D E A I T L L E QN NWD L V A A I N GV I P QE N G I L Q S E Y 56 Human(s) - - - MI L A D F QA C T G I E N I D E A I T L L E QN NWD L V A A I N GV I P QE N G I L Q S E Y 48 Rat - -M A S- -NM D R E M I L A D F QA C T G I E N I D E A I T L L E QN NWD L V A A I N GV I P QE N G I L Q SD F 56 Mouse - -M A S- -NM DL PM I L A D F QA C T G I E N I D E A I T L L E QN NWD L V A A I N GV I P QE N G I L Q SD F 56 Quail - -M A S- -NM D R E M I L A D F QA C T G I E N I D E A I T L L E QN NWD LWA A I N GV I P QE N G I L Q S E Y 56 D.rerio - -M A SD SNM D R E M I L A D F QA C T G I D N I A E A I T L L ELN NWD L V A A I N GV I P QE N G I L Q SS F 58 C.elegans M D L T A S L E DDQR E K L R QY T E FTH QQD Y E VA IG TLA S LNWNLE QA IE A H L M QE D - - K N D D E 58 Fly - M E A D G L T N E Q T E K V L Q FQD LT G I EDM N V C R D VLI R H QWD LE VAF Q - - - 45 : : : * : : . * : * : * * : : Human G GE T I P GP A F N P A S H P- - - -A S A P T S S S S S A F R P V M P S R Q I V 94 Human(s) G GE T I P GP A F N P A S H P- - - -A S A P T S S S S S A F R P V M P S R Q I V 86 Rat G GE TMP GPTFDP A SPP- - - -APA PA PS S S-A F R P V M P S R Q I V 93 Mouse G GE TMP GPTFDP A S H P- - - -APASTPS S S-A F R P V M P S R Q I V 93 Quail G GE TL QGP AY GPTS HS - - - T TAS - -S S S S A F RHV M P STQ I V 92 D.rerio S SEA S Q T GP Y GPHSQS E A T A P T P S A P P P S A S E S A S A S S SSSPPS S S SPA FCP VQP S RHI V118 C.elegans D PEI L E T I P A A A S G R N - - - -AGAS SS SR F E P E V I N I E D D E M P 96 Fly - - - EQL N 49 . : Human E R Q P RM L D F R V E Y R D R N V D V V L E D T C T V GE I K Q I L E N E L Q I P V S KM L L K GWK T G D V E D S T 154 Human(s) E R Q P RM L D F R V E Y R D R N V D V V L E D T C T V GE I K Q I L E N E L Q I P V S KM L L K GWK T G D V E D S T 146 Rat E R Q P RM L D F R V E Y R D R N V D V V L E DSC T V GE I K Q I L E N E L Q I P VPKM L L K GWK T G D V E D S T153 Mouse E R Q P RM L D F R V E Y R D R N V D V V L E DSC T V GE I K Q I L E N E L Q I P VPKM L L K GWK T G D V E D S T153 Quail E R Q P RM L D F R V E Y R D R N V D V V L E DS ST V GDIT HI L E N E L Q I PAS KM L L K GWK T G D VDD S T152 D.rerio E R Q S RM LSF R V E Y RQRS I EV V L E DA ST V GE I KTI L ETE L QVP V S KMQL K GWKSG D VSD S T178 C.elegans A T R GRR R GR AVT P D E T T T V D N QV K R L R I D D GS S S S SNGA A T H H R GA A I P R QKRGQA T E PT 156 Fly IRE GRP TM F A A S T D VRA P AVL N D R F L QQV F S A NM P GGR T V S RVP S GP V P R S FT G- - I I GY 107 . * . : * Human V L K S L H L P K N N S L Y V L T P D L P P P S S S S H A GA L QE S L N QN F M L I I T H R E V QR E Y N L N F S GS 214 Human(s) V L K S L H L P K N N S L Y V L T P D L P P P S S S S H A GA L QE- - - 180 Rat V L K S L H L P K N N S L Y V L T P D L P P P S S S S H A GA L QE S L N QN F M L I I T H R E V QR E Y N L N F S GS 213 Mouse V L K S L H L P K N N S L Y V L T P D L P P P S S S S H A GA L QE S L N QN F M L I I T H R E V QR E Y N L N F S GS 213 Quail V L KTL H L P K N N S L Y V L T P D L P P P S S S-HFGA L QE S L N QN F M L I I T H R E V QGE Y N L N F S GS 211 D.rerio V LRS L H L P K N N S L Y V L T P DI APS ASTSQN SAQQG L SN QNY LLVISH R EAQRDYSL N FP AS 238 C.elegans P S SSGS S S A S FSS R R GTR A NPVPP P N QE P A H P E S A R QN GG ILA S RHN N H N N QQNN H H H H H216 Fly VI N F V F QY F Y S TLT S I V S A F V N L GGGN EAR L V T D PLG - - - 144 : . . Human S T I QE V K R N V Y D L T S I P V R H Q L WE GWP T S A T D D S M C L A E S G L S Y P C H R L T V GR R S S P A Q T274 Human(s) -Rat S TVQE V K R N V Y D L T S I P V R H Q L WE GWPAS A T D D S M C L A E S G L S Y P C H R L T V GR RTS PVQ T 273 Mouse S TVQE V K R N V Y D L T S I P V R H Q L WE GWPAS A T D D S M C L A E S G L S Y P C H R L T V GR RTS PVQ T 273 Quail S T I QE V K R N V Y D L T S I P V R H QQWE GWPPS A T D D S MTL AVS GV N FP C H R L T V GR R S S PVQ T 271 D.rerio KT I QE V K R NI SD L TNI P V R H QQWDGWPAS A T D D S MTL AIS GIS Y P C HHL T VCR R S S P ALT 298 C.elegans Q RIP I N PRRVD V F N - - - V D S D E DD D S MA IAY E D D D D GVHE V H H S E V C E K D R A 265 Fly -Human R E Q S E E Q I T D V HM V S D S D GD D F E D A T E F GV D D GE V F GM A S S A L R K S P MM P E N A E N E G D A L 334 Human(s) -Rat R E Q S E E QSTVV HM V S D S D GD D F E D ASE F GVVD GE V F GM A S ST MR K S P MM P E N A E N E G D A L 333 Mouse R E Q S E E QST D V HM V S D S D GD D F E D ASE F GV D D GE V F GM A S STL R K S P MM P E N A E N E G D A L 333 Quail R E Q S E E QCT D V HM V S D S D GD D F E D A T E F GV D D GEMF GVA S S A L R K S P MM P E N A E N E G D A L 331 D.rerio V DQT - D E CT D V HM V S D SEGD D F E D APE F GV DE SEIF GMGS SS CR K S P MM P E NSE N E G D A L 357 C.elegans I S A F L A P K L GN E VVA R GSG- - - P P N GR IP MI P D G F S S V SD A L 304 Fly - - - D V146

50

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UBX

Human L Q F T A E F S S R- - - -Y GD C H P V F F I GS L E A A F Q E A F Y V-K A R D R K L L A I Y L H H D- -E S V 385 Human(s) - - - -F S S R- - - -Y GD C H P V F F I GS L E A A F Q E A F Y V-K A R D R K L L A I Y L H H D- -E S V 225 Rat L Q F T A E F S S R- - - -Y GD C H P V F F I GS L E A A F Q E A F Y V-K A R D R K L L A I Y L H H D- -E S V 384 Mouse L Q F T A E F S S R- - - -YSD C H P V FYI GS L E A A F Q E A F Y V-K A R D R K L L A I Y L H H D- -E S V 384 Quail L Q F T A E F S S RR - - - - -Y GD C H P VYF I GS L E A A F Q E A F YG - K A R D R K L L A I Y L H H D- -E S V 383 D.rerio LHF T A E F SRR- - - -Y GD C H P V FYI GS L E A ASQ E A F YG - K A R D R K L L A I Y L HND- -E S V 408 C.elegans R NFVAIF SDRF C S T P Q T QA F MPPFY T E PLPA AV KE A FD H P N S E HRR PLL FY INH D- - RSI 362 Fly M KFI REY Y ER- - - Y P EH P V FY QGT Y A QAL N DAK QE - - - L R FLI VY L HKDP A K N P 194 : * * : : . * : : * : * . * : : : * . . Human L T N V F C S QM L C A E S I V S Y L S QN F I TWA WD L T K D S N R A R F L TM C N R H F G- - -S V V A Q T I R T 442 Human(s) L T N V F C S QM L C A E S I V S Y L S QN F I TWA WD L T K D S N R A R F L TM C N R H F G- - -S V V A Q T I R T 282 Rat L T N V F C S QM L C A E S I V S Y L S QN F I TWA WD L T K DAN R A R F L TM C N R H F G- - -S VIA Q T I R T441 Mouse L T N V F C S QM L C A E S I V S Y L S QN F I TWA WD L T K DTN R A R F L TM C N R H F G- - -S VIA Q T I R T441 Quail L T N V F C S QM LFA E S I V S Y LTQEF I TWGWD L T KE AN R ASF L TM CTR H F G- - - DV V A Q T I R T 440 D.rerio LSN V F C S QMMC ADS I V S Y L S QN F I TWA WDVT KE ANKA RLL TM CTR H F G- - -S V V A Q T I RS 465 C.elegans A ANIFAS QVL CSET V S T L I R H QY V L F PWDITSD SN L M LF LE Y L QA A NM GD V R T I IQR L A M422 Fly D V E SF CR N TLSARSV I DYI N T H T L LWGCDV A T P E G - - - Y R V M QS235 : * . : : : : : : . : : * : : . . : : Human Q K T D Q K P L F L I I M G K R S S N E V L N V I QGN T T V D E L MM R L M A A M E I F T A QQQE D I K D GD E R E 502 Human(s) Q K T D Q K P L F L I I M G K R S S N E V L N V I QGN T T V D E L MM R L M A A M E I F T A QQQE D I K D GD E R E 342 Rat Q K T D QFP L F L I I M G K R S S N E V L N V I QGN T T V D E L MM R L M A A M E I FSA QQQE D I K DED E R E 501 Mouse Q K T D QFP L F L I I M G K R S S N E V L N V I QGN T T V D E L MM R L M A A M E I FSA QQQE D I K DED E R E 501 Quail Q K T D QFP L F L I I M G K R S S N E V L N V I QGN T T V D E L MM R L M A A M E I FSA QQQE D I K DED E R E 500 D.rerio YK T D QFP LLL IVM G K RTS N E V L N V I QGN T T V D E L MM R L MGA M E I F T A QQQE D I K DED E R E 525 C.elegans SKI E S FP LM AIV V K ERNSY R L V D Y C RGT DTSDQVME KLL S GV S E Y S D I RM N E QS E R RE R E 482 Fly I T V R S YPT M V MIS L R A N RMM I V G R F EGD CTP EE LL RR LQS V T N A N E V WL S QA R ADR LE RN 295 . . * : : : . . : : . . * * : : : : : * . . : : * * : Human A R E N V K R E QD E A Y R L S L E A D R A K R E A R E R E M- - - -A E Q F R L E Q I R K-E QE E E R E A I R L S L 557 Human(s) A R E N V K R E QD E A Y R L S L E A D R A K R E A R E R E M- - - -A E Q F R L E Q I R K-E QE E E R E A I R L S L 397 Rat A R E N V K R E QD E A Y R L S L E A D R A K R E AHE R E M- - - -A E Q F R L E Q I R K-E QE E E R E A I R L S L 556 Mouse A R E N V K R E QD E A Y R L S L E A D R A K R E AHE R E M- - - -A E Q F R L E Q I R K-E QE E E R E A I R L S L 556 Quail A R E N V K R E QD E A Y RIS L E A D R A K R E AQE R E M- - - -A E Q F R L E Q I R KKE QE E E R E A I R L S L 556 D.rerio A R EMV K R E QD E A Y RVS L E A D RKK R E AQE R EE - - - -A E QVRQERI R K-E QE E E R E A I R L S L 580 C.elegans ER EA I R N QQE A EYK AS LAA DKARME AK QQEI - - - - EE Q-R L EE ER KL R EE E EE C V RRQ T V 537 Fly F T Q T L RRQQD E A YE QS LLA DE EKE R QRQRER D A V R QA E E A VE QARR D V E L R K EEI ARQ K I 355 : : : . : * : * . * * * * . : . : : : * : * . * : : . : . * . : Human E QA L P P E P K E E N-A E P V S K L R I R T P S GE F L E R R F L A S N K L Q I V F D F V A S K G F P WD E Y K L L 616 Human(s) E QA L P P E P K E E N-A E P V S K L R I R T P S GE F L E R R F L A S N K L Q I V F D F V A S K G F P WD E Y K L L 456 Rat E QA L P P E PEE E N-A E P V S K L R I R T P S GE F L E R R F L A S N K L Q I V F D F V A S K G F P WD EFK L L 615 Mouse E QA L P P E P K E E N-A E P V S K L R I R T P S GE F L E R R F L A S N K L Q I V F D F V A S K G F P WD EFK L L 615 Quail E QSL P P E P K E ES - TESV S K L R I R T P S GE FFE R R F L A SSK L QVV F D F V AYK G F P WEEFK L L 615 D.rerio E QA L P P E P K E EG - GE PIS K L R I R T P S GE F L E R R F LG T CK L QV LF D F V A S K GYPSD EFK L L 639 C.elegans A S T VPEE PP A S A P L A E I I N V K FRLPEGGQDMR R FR R L E S IQT L I N Y L SS K GY S PDK FKY F 597 Fly ELAT L V P S E P A A DAV GA I A V V F K LP S GT RL E R R FN Q T D S V L DVY H Y L F C H P D S PD EF E I T415 : . : : : * . * * * * . : : . : : : . : : : : Human S-T F P R R D V T-Q L D P N K S- - - -L L E V K L F P QE T L F L E A K E- - - 650 Human(s) S-T F P R R D V T-Q L D P N K S- - - -L L E V K L F P QE T L F L E A K E- - - 490 Rat S-T F P R R D V T-Q L D P N K S- - - -L L E VNL F P QE T L F LLA K E- - - 649 Mouse S-T F P R R D V T-Q L D P N K S- - - -L L E VNL F P QE T L F LQA K E- - - 649 Quail G -T F P R R D V T-Q L D P N K S- - - -LQE V K LYP QE T L F L E A K E- - - 649 D.rerio T -T F P R RN IT-Q L D PA WN - - - -LVEAK L F P QE T L F L E A KS K I D P K S L E 681 C.elegans N S DF PK K E ITR H FDL S H N - - - F A D TKWP A REQ IFVEE I - - - 632 Fly T - NF PKRV L F S K A N L D A A GE T G T A K E T L T K TLQAVGLK N RELL FV N D LEA - - - 464 * * : : : : : : . : * : * :

51

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Hee-Jung Kim, Eun Joo Song, Yun-Suk Lee, Eunhee Kim and Kong-Joo Lee

Regulates Chaperone Activity