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CHIP interacts with heat shock factor 1 during heat stress
FEBS 30135
FEBS Letters 579 (2005) 6559–6563
CHIP interacts with heat shock factor 1 during heat stress Soo-A Kima,b, Jung-Hoon Yoona,b, Do-Kyung Kima, Su-Gwan Kima, Sang-Gun Ahna,b,* b
a Oral Biology Research Institute, Chosun University College of Dentistry, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea Department of Pathology, BK 21 Project, Chosun University College of Dentistry, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea
Received 8 September 2005; revised 21 October 2005; accepted 21 October 2005 Available online 2 November 2005 Edited by Gianni Cesareni
Abstract Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1–290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Heat shock factor 1; Carboxyl terminus of the heat shock cognate protein 70-interacting protein; Heat shock cognate protein 70; Heat shock protein 70; Heat shock protein 90; Heat stress
1. Introduction Heat shock factors (HSFs) regulate the expression of heat shock proteins (Hsps) against a variety of environmental and developmental stresses [1–4]. Mammals have multiple distinct HSF genes, encoding isoforms denoted HSF1, HSF2 and HSF4. HSF1 is the predominant HSF isoform that responds to thermal and oxidative stress to activate the expression of Hsp genes [2,5]. HSF1 is composed of an amino-terminal DNA-binding domain (DBD), an adjacent coiled-coil trimerization domain (Leucine zipper 1-3, LZ1-3), a central regulatory domain (RD), a second coiled-coil domain (Leucine zipper 4, LZ4), and a carboxyl-terminal transcriptional activation domain (AD) (Fig. 1A). The transcriptional activity of HSF1 is tightly controlled. Under normal physiological conditions, HSF1 largely localizes to the cytoplasm as a monomeric form with low DNA-binding activity [6,7]. Intramolecular interactions
* Corresponding author. Fax: +82 62 223 3205. E-mail address: [email protected] (S.-G. Ahn).
Abbreviations: HSF1, heat shock factor 1; CHIP, carboxyl terminus of the Hsc70-interacting protein; Hsc70, heat shock cognate protein 70; Hsp90, heat shock protein 90; GST, glutathione S-transferase
between LZ1-3 and LZ4 restrain HSF1 in an inactive state [8–10]. Upon sensing stress, HSF1 undergoes the transition from a monomeric to a homotrimeric complex in which monomers associate through the formation of a three-stranded coiled-coil by the trimerization domain [9,11]. However, precise molecular mechanisms by which HSF1 senses thermal stress to switch from the monomer to the homotrimeric form are poorly understood. One of the most pronounced consequences of heat stress is the unfolding and the misfolding of proteins. To avoid the cellular damage, aberrant proteins must be either refolded by molecular chaperones or eliminated by the ubiquitin-proteasome protein degradation system. Carboxyl terminus of the Hsc70-interacting protein (CHIP) is a co-chaperone interacts with heat shock cognate protein 70 (Hsc70) and Hsp90 molecular chaperones via a tetratricopeptide repeat motif and inhibits chaperone-dependent protein folding [12–14]. CHIP also stimulates protein degradation by acting as an E3 ubiquitin ligase via a modified ring finger domain called a U-box [13–17]. Recently, Dai and colleagues [18] have shown that CHIP regulates the stress-chaperone response through induced trimerization and transcriptional activation of HSF1. Although they reported the functional relationship between CHIP and HSF1, the molecular mechanism of interaction remains to be elucidated. In this study, we demonstrate that HSF1 directly interacts with CHIP through its N-terminal region. Interestingly, interaction between CHIP and full-length HSF1 was induced by heat stress suggesting conformational change of HSF1 is required for their interaction. Finally, we observed structural change of HSF1 by circular dichorism (CD) spectra under heat stressed conditions. Our results demonstrate a previously unreported molecular mechanism of HSF1–CHIP interaction.
2. Materials and methods 2.1. Plasmids Bacterial expression vectors for recombinant glutathione S-transferase (GST)-tagged full-length HSF1 (FL) and C-terminal deleted HSF1 (a.a. 1–290) fusion proteins have been described [19]. The open reading frames of Hsc70 (1941 nucleotides) and CHIP (912 nucleotides) corresponding to GenBank Accession Nos. BC016179 and AF129085 were amplified by PCR using human skeletal muscle cDNA as a template (CLONTECH). Vectors for the expression of bacterial recombinant His-tagged Hsc70 and CHIP were created using the pET21a vector (Stratagene). The pET-Hsc70 vector was created by inserting a DNA fragment containing the Hsc70 open reading frame without a stop codon into the 5 0 -BamHI and 3 0 -XhoI sites of pET21a in-frame with the six-histidine tag. The pET-CHIP construct was created by inserting a DNA fragment containing the CHIP open reading frame without a
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.10.043
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S.-A Kim et al. / FEBS Letters 579 (2005) 6559–6563
Fig. 1. HSF1 interacts with CHIP through its N-terminal region. (A) Schematic diagram of the HSF1 domain organization. HSF1 is composed of an amino-terminal DBD, an adjacent coiled-coil trimerization domain (LZ1-3), a central RD, a second coiled-coil domain (LZ4), and a carboxylterminal transcriptional AD. Domain boundaries were obtained from the SMART and COIL programs. (B) Purified GST-tagged bacterial recombinant HSF1 (1–290) or HSF1 (FL) (1 lg) were incubated equimolar His-tagged CHIP. As a control, equimolar GST was incubated with Histagged CHIP. GST or GST-HSF1 was pulled down by glutathione-agarose beads. Co-precipitated proteins were resolved on SDS–PAGE and detected by Western blot analysis using anti-His antibody. (C) Purified bacterial recombinant GST-tagged HSF1 (1–290), GST-tagged HSF1 (FL) and His-tagged CHIP were monitored by Coomassie blue staining.
stop codon into the 5 0 -NdeI and 3 0 -NotI sites of pET21a in-frame with the six-histidine tag. Mammalian expression vector for HSF1 have been described previously [19]. Vectors for mammalian expression of N-terminally FLAG-tagged CHIP fusion protein was created by inserting a DNA fragment containing the complete CHIP open reading frame into the 5 0 -KpnI and 3 0 -NotI sites of pCDNA3.1-NF. 2.2. Protein expression and purification Bacterial recombinant HSF1 was expressed as an N-terminally GSTtagged fusion protein in Escherichia coli BL21 (DE3) Codon Plus cells (Stratagene) and purified by using glutathione-agarose affinity resin as described [19]. Recombinant Hsc70 and CHIP were expressed as C-terminally His-tagged fusion proteins in E. coli BL21 (DE3) Codon Plus cells and purified by using Ni2+-agarose affinity resin as described [20]. 2.3. GST pull-down assay GST-tagged HSF1 (1 lg) was added to equimolar His-tagged CHIP in reaction buffer (20 mM Tris–Cl, pH 8.0, 200 mM NaCl, 0.1% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 1% BSA). After 2 h incubation at 4 C, glutathione-agarose beads were added and incubated for another 1 h at 4 C. Beads were washed four times with TBST (20 mM Tris–Cl, pH 8.0, 200 mM NaCl, 0.1% Tween-20) and boiled in SDS–PAGE sample buffer. Proteins were resolved by SDS–PAGE and immunoblotted with anti-His antibody (Qiagen). 2.4. Cell culture and immunoprecipitation HEK 293 cells were maintained at 37 C with 5% CO2 in DMEM containing 10% FCS, 50 U/ml penicillin and 50 lg/ml streptomycin. For immunoprecipitation, HEK 293 cells were transiently transfected by using the FuGENE 6 reagent (Roche Molecular Biochemicals) according to the manufacturers protocol. Thirty hours after transfection, the cells were washed twice with PBS and lysed in RIPA buffer (PBS supplemented with 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM PMSF, 1 lg/ml aprotinin, 1 mM sodium orthovanadate). The cell lysates were harvested and incubated at 4 C for 30 min and cleared by centrifugation at 10 000 · g for 10 min. The supernatant
was incubated with anti-FLAG antibody (Sigma–Aldrich) for 3 h, after which protein G–Sepharose (Amersham Pharmacia) was added and incubated another 1 h. The immunoprecipitates were washed four times with RIPA buffer containing 0.05% SDS and boiled in SDS–PAGE sample buffer. Proteins were resolved by SDS–PAGE and immunoblotted with anti-HSF1 antibody (Abcam), anti-FLAG antibody (Sigma–Aldrich), anti-Hsp70 antibody (Santa Cruz Biotechnology), anti-Hsc70 antibody (Santa Cruz Biotechnology), or anti-Hsp90 antibody (Santa Cruz Biotechnology). 2.5. Characterization of proteins by circular dichroism For CD experiments, purified recombinant HSF1 was dialyzed in 20 mM HEPES buffer, pH 7.5. The protein was diluted with HEPES buffer to a final concentration of 0.3 mg/ml and treated heat shock at 42 C for 5 min. CD scans were performed on a Jasco J-810 spectropolarimeter in the far-UV range at 25 C as previously described [21]. A cell of 0.1 cm optical path was used to obtain spectra at a scan speed of 50 nm/min. Spectra were averaged from four individual scans and results were presented as mean molar ellipticity.
3. Results and discussion 3.1. HSF1 interacts with CHIP through its N-terminal region To investigate the molecular mechanism of interaction between HSF1 and CHIP, we examined the binding in vitro using GST pull-down assays. GST-tagged full-length HSF1 (FL) and C-terminal truncated HSF1 (a.a. 1–290) were expressed in bacteria and purified. Bacterial recombinant Histagged CHIP was also purified. Interestingly, CHIP specifically interacts with C-terminal truncated HSF1 (1–290), encompassing the N-terminal DBD and coiled-coil motif (LZ1-3) (Fig. 1B). Under non-stressed conditions, full-length HSF1
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(FL) did not show interaction with CHIP, suggesting that the C-terminus of HSF1 affects the binding of HSF1 with CHIP (Fig. 1B). GST control did not show any interaction with CHIP (Fig. 1B). Coomassie blue staining showed the equivalent level of purified proteins (Fig. 1C). These results suggest that HSF1 directly interacts with CHIP through its N-terminal region and the C-terminal region of HSF1 may affect the interaction with CHIP under normal conditions. 3.2. Interaction between HSF1 and CHIP does not affected by Hsc70 Originally, CHIP was identified as an Hsc70 interacting protein. Additionally, we and others have previously shown that Hsc70 interacts with HSF1 [22,23]. Therefore, we next examined whether Hsc70 can affect the interaction between HSF1 and CHIP using GST pull-down assays. As shown in Fig. 2 (upper panel), CHIP strongly interacts with C-terminal deleted HSF1 (1–290) in the absence of Hsc70. Furthermore, the presence of Hsc70 does not affect the interaction between HSF1 and CHIP. Full-length HSF1 does not show interaction with CHIP in the absence of Hsc70. However, full-length HSF1 showed very weak interaction with CHIP in the presence of Hsc70 (Fig. 2, upper panel). This interaction may be caused by the indirect interaction between full-length HSF1 and CHIP through the direct interaction between full-length HSF1 and Hsc70. Consistent with our previous report, Hsc70 interacts with full-length HSF1 (FL) but not with C-terminal deleted HSF1 (1–290) (Fig. 2, lower panel) [22]. Collectively, these results demonstrate that Hsc70 does not affect the direct interaction between the N-terminal region of HSF1 and CHIP.
Fig. 3. Heat shock induces the interaction between full length HSF1 and CHIP in vitro. Bacterial recombinant GST-tagged HSF1 (1–290) or HSF1 (FL) (1 lg) were incubated with equimolar His-tagged CHIP. Samples were untreated () or heat shocked at 42 C (+) for 10 min. HSF1 was pulled down by glutathione-agarose beads. Co-precipitated proteins were resolved on SDS–PAGE and detected by Western blot analysis using anti-His antibody.
To confirm the interaction between full-length HSF1 and CHIP in vivo, HEK 293 cells were transfected with expression vector for FLAG-tagged CHIP (pCDN3.1-FLAG-CHIP). Forty eight hours after transfection, cells were heat shocked at 42 C for 1 h. Cell lysates were prepared from heat-treated (+) or -untreated () cells and subjected to immunoprecipitation using anti-FLAG antibody, followed by Western blot analysis using anti-HSF1 antibody to detect endogenous HSF1. As shown in Fig. 4 (upper panel), heat shock treatment greatly induced the interaction of CHIP with endogenous HSF1. No corresponding interaction was observed in the heat untreated control samples, confirming that the interaction between HSF1 and CHIP was induced by heat stress. Previous studies have reported that CHIP interacts with molecular chaperones, such as Hsp70, Hsp90 and Hsc70.
3.3. Interaction of CHIP with full-length HSF1 is induced by heat stress To examine whether heat stress affects the interaction between full-length HSF1 and CHIP, GST-tagged HSF1 was heat shocked at 42 C for 10 min and then incubated with His-tagged CHIP at room temperature for 60 min. As expected, full-length HSF1 did not show any interaction with CHIP under non-stressed conditions (Fig. 3). However, when the full-length HSF1 was heat stressed, the interaction between full-length HSF1 and CHIP was largely induced in vitro (Fig. 3).
Fig. 2. Hsc70 does not affect the interaction between HSF1 and CHIP in vitro. Bacterial recombinant GST-tagged HSF1 (1–290) or HSF1 (FL) (1 lg) were incubated with equimolar His-tagged CHIP and/or Hsc70. HSF1 was pulled down by glutathione-agarose beads. Coprecipitated proteins were resolved on SDS–PAGE and detected by Western blot analysis using anti-His antibody.
Fig. 4. Heat shock induces the interaction between HSF1 and CHIP in vivo. HEK 293 cells were transfected with the FLAG-tagged CHIP expression vector (pCDNA3.1-FLAG-CHIP). Forty eight hours after transfection, cells were untreated () or heat shocked at 42 C (+) for 1 h. Cell extracts were subjected to immunoprecipitation using antiFLAG antibody, and endogenous HSF1, Hsp70, Hsp90 and Hsc70 were detected by Western blot analysis.
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Therefore, we next examined the interaction between CHIP and molecular chaperones under heat stressed conditions. In contrast to HSF1, Hsp70 and Hsp90 strongly interact with CHIP under non-stressed conditions (Fig. 4). Upon heat treatment, Hsp70 and Hsp90 were significantly dissociated from CHIP protein complex (Fig. 4). These results demonstrate that dissociation of molecular chaperones, such as Hsp90 and Hsp70, from CHIP has reverse correlation with formation of HSF1 and CHIP protein complex. CHIP contains the U-box motif and has U-box dependent E3 ubiquitin ligase activity, which stimulates the ubiquitination of unfolded proteins [15,16]. CHIP also interacts with Hsp90 and antagonizes the proper chaperoning activity [12,14,24]. For example, CHIP inhibits Hsp40-stimulated ATPase activity of Hsp70, suggesting that CHIP blocks the forward reaction of the Hsp70 molecular chaperone [14,24]. Additionally, Ryosuke and colleagues [25] reported that CHIP is increased during the ER stress and promotes the release of molecular chaperones from multi-protein complexes. Considering our results, it is suggested that heat stress induces the interaction between HSF1 and CHIP and leads to the activation of CHIP signaling pathway. Unlike Hsp70 and Hsp90, the interaction between CHIP and Hsc70 was not affected by heat stress (Fig. 4). This result extends our previous report showing that the interaction between HSF1 and Hsc70 was not affected by heat stress [22]. 3.4. The secondary structure of HSF1 is modulated by heat stress Our data show that the interaction between full-length HSF1 and CHIP was largely induced by heat stress. These results strongly suggest that heat stress may induce the conformational change of HSF1 which plays an important role in HSF1–CHIP interaction. To investigate this possibility, purified recombinant HSF1 was heat treated at 42 C for 5 min and the effect of heat stress was analyzed by CD spectroscopy. Interestingly, the structural change of HSF1 was observed under heat-treated conditions. As shown in Fig. 5, CD analysis of heat treated HSF1 showed different degree of secondary structure near 210 nm compared with non-stressed HSF1, demonstrating the different a-helix contents. This result confirms that the overall structure of HSF1 is altered by heat stress. Under normal physiological conditions, intramolecular interactions between LZ1-3 and LZ4 restrain HSF1 in an inac-
Fig. 5. Circular dichroism analysis of heat stressed HSF1. Bacterial recombinant HSF1 (0.3 mg/ml) was untreated or heat shocked at 42 C for 10 min. The CD spectra were recorded in the far UV region (200– 250 nm) at 20 C. The spectra were presented as mean residue ellipticity, expressed as deg Æ cm2/dmol. Each spectrum is the average of four separate scans.
S.-A Kim et al. / FEBS Letters 579 (2005) 6559–6563
Fig. 6. Model for the interaction of HSF1 and CHIP. Under normal conditions, HSF1 is found as an inactive monomer restrained by interaction between LZ1-3 and LZ4. Conversion from the monomer to the trimer occurs constitutively when the cells are heat stressed (42 C) that would be disrupted intramolecular interactions. The conformational changes of HSF1 induce the interaction with CHIP and the dissociation of molecular chaperones from multi-protein complexes.
tive monomeric form [8–10]. Upon heat stress, intramolecular interactions are disrupted and HSF1 forms homotrimeric complex (Fig. 6) [9,11]. In our results, CHIP showed weak indirect interaction with full-length HSF1 through the direct interaction with Hsc70 under non-stressed conditions (Figs. 2 and 6). Upon heat stress, conformational changes of HSF1 occur, leading to the direct interaction between HSF1 and CHIP (Fig. 6). Hsc70 may recruit and/or stabilize the association of CHIP with HSF1. It will now be interesting and important to determine the role of CHIP on the function of HSF1. Acknowledgements: We thank Dr. Dennis Thiele for helpful suggestions. This work was supported by Grant No. RT104-03-03 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE), and BK 21 Project.
References [1] Abravaya, K., Myers, M.P., Murphy, S.P. and Morimoto, R.I. (1992) The human heat shock protein hsp. Genes Dev. 6, 1153– 1164. [2] Morano, K.A. and Thiele, D.J. (1999) Heat shock factor function and regulation in response to cellular stress, growth, and differentiation signals. Gene Expr. 7, 271–282. [3] Morimoto, R.I., Kroeger, P.E. and Cotto, J.J. (1996) The transcriptional regulation of heat shock genes: a plethora of heat shock factors and regulatory conditions in: Stress-Inducible Cellular Responses (Feige, U., Morimoto, R.I., Yahara, I. and Polla, B.S., Eds.), pp. 139–163, Birkhauser, Basel. [4] Voellmy, R. (1996) Sensing stress and responding to stress in: Stress-Inducible Cellular Responses (Feige, U., Morimoto, R.I., Yahara, I. and Polla, B.S., Eds.), pp. 121–137, Birkhauser, Basel. [5] Wu, C. (1995) Heat shock transcription factors: structure and regulation. Annu. Rev. Cell Dev. Biol. 11, 441–469. [6] Morimoto, R.I., Tissieres, A. and Georgopoulos, C. (1994) The biology of the heat shock proteins and molecular chaperones Cold Spring Harbor Monograph Series, vol. 26, p. 610, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [7] Sarge, K.D., Murphy, S.P. and Morimoto, R.I. (1993) Activation of heat shock gene transcription by heat shock factor 1 involves oligomerization, acquisition of DNA-binding activity, and nuclear localization and can occur in the absence of stress. Mol. Cell. Biol. 13, 1392–1407. [8] Rabindran, S.K., Haroun, R.I., Clos, J., Wisniewski, J. and Wu, C. (1993) Regulation of heat shock factor trimer formation: role of a conserved leucine zipper. Science 259, 230–234. [9] Zuo, J., Baler, R., Dahl, G. and Voellmy, R. (1994) Activation of the DNA-binding ability of human heat shock transcription factor 1 may involve the transition from an intramolecular to an intermolecular triple-stranded coiled-coil structure. Mol. Cell. Biol. 14, 7557–7568.
S.-A Kim et al. / FEBS Letters 579 (2005) 6559–6563 [10] Farkas, T., Kutskova, Y.A. and Zimarino, V. (1998) Intramolecular repression of mouse heat shock factor 1. Mol. Cell. Biol. 18, 906–918. [11] Peteranderl, R. and Nelson, H.C. (1992) Trimerization of the heat shock transcription factor by a triple-stranded alpha-helical coiled-coil. Biochemistry 31, 12272–12276. [12] Connell, P., Ballinger, C.A., Jiang, J., Wu, Y., Thompson, L.J., Ho¨hfeld, J. and Patterson, C. (2001) The co-chaperone CHIP regulates protein triage decisions mediated by heat-shock proteins. Nat. Cell Biol. 3, 93–96. [13] Meacham, G.C., Patterson, C., Zhang, W., Younger, J.M. and Cyr, D.M. (2001) The Hsc70 co-chaperone CHIP targets immature CFTR for proteasomal degradation. Nat. Cell Biol. 3, 100– 105. [14] McDonough, H. and Patterson, C. (2003) CHIP: a link between the chaperone and proteasome systems. Cell Stress Chaperones 8, 303–308. [15] Jiang, J., Ballinger, C.A., Wu, Y., Dai, Q., Cyr, D.M., Ho¨hfeld, J. and Patterson, C. (2001) CHIP is a U-box-dependent E3 ubiquitin ligase. J. Biol. Chem. 276, 42938–42944. [16] Murata, S., Minami, Y., Minami, M., Chiba, T. and Tanaka, K. (2001) CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein. EMBO Rep. 2, 1133– 1138. [17] Murata, S., Chiba, T. and Tanaka, K. (2003) CHIP: a qualitycontrol E3 ligase collaborating with molecular chaperones. Int. J. Biochem. Cell Biol. 35, 572–578. [18] Dai, Q., Zhang, C., Wu, Y., McDonough, H., Li, H.H., Madamanchi, N., Xu, W., Neckers, L., Cyr, D. and Patterson,
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C. (2003) CHIP activates HSF1 and confers protection against apoptosis and cellular stress. EMBO J. 22, 5446–5458. Ahn, S.G. and Thiele, D.J. (2003) Redox regulation of mammalian heat shock factor 1 is essential for Hsp gene activation and protection from stress. Genes Dev. 17, 516–528. Goodson, M.L. and Sarge, K.D. (1995) Heat-inducible DNA binding of purified heat shock transcription factor 1. J. Biol. Chem. 270, 2447–2450. Ellis, H.R. and Poole, L.B. (1997) Roles for the two cysteine residues of Ahpc in catalysis of peroxide reduction by alkyl hydroperoxide reductase from Salmonella typhimurium. Biochemistry 36, 13349–13356. Ahn, S.G., Kim, S.A., Yoon, J.H. and Vacratsis, P. (2005) Heat shock cognate 70 is required for the activation of heat shock factor 1 in mammalian cells. Biochem. J. 392, 145–152. Nunes, S.L. and Calderwood, S.K. (1995) Heat shock factor-1 and the heat shock cognate 70 protein associate in high molecular weight complexes in the cytoplasm of NIH-3T3 cells. Biochem. Biophys. Res. Commun. 213, 1–6. Ballinger, C.A., Connell, P., Wu, Y., Hu, Z., Thompson, L.J., Yin, L.Y. and Patterson, C. (1999) Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. Mol. Cell. Biol. 19, 4535–4545. Imai, Y., Soda, M., Hatakeyama, S., Akagi, T., Hashikawa, T., Nakayama, K.I. and Takahashi, R. (2002) CHIP is associated with Parkin, a gene responsible for familial Parkinsons disease, and enhances its ubiquitin ligase activity. Mol. Cell 10, 55–67.
FEBS Letters 579 (2005) 6559–6563
CHIP interacts with heat shock factor 1 during heat stress Soo-A Kima,b, Jung-Hoon Yoona,b, Do-Kyung Kima, Su-Gwan Kima, Sang-Gun Ahna,b,* b
a Oral Biology Research Institute, Chosun University College of Dentistry, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea Department of Pathology, BK 21 Project, Chosun University College of Dentistry, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea
Received 8 September 2005; revised 21 October 2005; accepted 21 October 2005 Available online 2 November 2005 Edited by Gianni Cesareni
Abstract Heat shock factor 1 (HSF1) is a major transactivator of heat shock genes in response to stress and mediates cell protection against various harmful conditions. In this study, we identified the interaction of CHIP (carboxyl terminus of the heat shock cognate protein 70-interacting protein) with the N-terminus of HSF1. Using GST full-down assay, we found that CHIP directly interacts with C-terminal deleted HSF1 (a.a. 1–290) but not with full-length HSF1 under non-stressed conditions. Interestingly, interaction of CHIP with full-length HSF1 was induced by heat shock treatment. The structural change of HSF1 was observed under heat stressed conditions by CD spectra. These observations demonstrate the direct interaction between HSF1 and CHIP and this interaction requires conformational change of HSF1 by heat stress. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Heat shock factor 1; Carboxyl terminus of the heat shock cognate protein 70-interacting protein; Heat shock cognate protein 70; Heat shock protein 70; Heat shock protein 90; Heat stress
1. Introduction Heat shock factors (HSFs) regulate the expression of heat shock proteins (Hsps) against a variety of environmental and developmental stresses [1–4]. Mammals have multiple distinct HSF genes, encoding isoforms denoted HSF1, HSF2 and HSF4. HSF1 is the predominant HSF isoform that responds to thermal and oxidative stress to activate the expression of Hsp genes [2,5]. HSF1 is composed of an amino-terminal DNA-binding domain (DBD), an adjacent coiled-coil trimerization domain (Leucine zipper 1-3, LZ1-3), a central regulatory domain (RD), a second coiled-coil domain (Leucine zipper 4, LZ4), and a carboxyl-terminal transcriptional activation domain (AD) (Fig. 1A). The transcriptional activity of HSF1 is tightly controlled. Under normal physiological conditions, HSF1 largely localizes to the cytoplasm as a monomeric form with low DNA-binding activity [6,7]. Intramolecular interactions
* Corresponding author. Fax: +82 62 223 3205. E-mail address: [email protected] (S.-G. Ahn).
Abbreviations: HSF1, heat shock factor 1; CHIP, carboxyl terminus of the Hsc70-interacting protein; Hsc70, heat shock cognate protein 70; Hsp90, heat shock protein 90; GST, glutathione S-transferase
between LZ1-3 and LZ4 restrain HSF1 in an inactive state [8–10]. Upon sensing stress, HSF1 undergoes the transition from a monomeric to a homotrimeric complex in which monomers associate through the formation of a three-stranded coiled-coil by the trimerization domain [9,11]. However, precise molecular mechanisms by which HSF1 senses thermal stress to switch from the monomer to the homotrimeric form are poorly understood. One of the most pronounced consequences of heat stress is the unfolding and the misfolding of proteins. To avoid the cellular damage, aberrant proteins must be either refolded by molecular chaperones or eliminated by the ubiquitin-proteasome protein degradation system. Carboxyl terminus of the Hsc70-interacting protein (CHIP) is a co-chaperone interacts with heat shock cognate protein 70 (Hsc70) and Hsp90 molecular chaperones via a tetratricopeptide repeat motif and inhibits chaperone-dependent protein folding [12–14]. CHIP also stimulates protein degradation by acting as an E3 ubiquitin ligase via a modified ring finger domain called a U-box [13–17]. Recently, Dai and colleagues [18] have shown that CHIP regulates the stress-chaperone response through induced trimerization and transcriptional activation of HSF1. Although they reported the functional relationship between CHIP and HSF1, the molecular mechanism of interaction remains to be elucidated. In this study, we demonstrate that HSF1 directly interacts with CHIP through its N-terminal region. Interestingly, interaction between CHIP and full-length HSF1 was induced by heat stress suggesting conformational change of HSF1 is required for their interaction. Finally, we observed structural change of HSF1 by circular dichorism (CD) spectra under heat stressed conditions. Our results demonstrate a previously unreported molecular mechanism of HSF1–CHIP interaction.
2. Materials and methods 2.1. Plasmids Bacterial expression vectors for recombinant glutathione S-transferase (GST)-tagged full-length HSF1 (FL) and C-terminal deleted HSF1 (a.a. 1–290) fusion proteins have been described [19]. The open reading frames of Hsc70 (1941 nucleotides) and CHIP (912 nucleotides) corresponding to GenBank Accession Nos. BC016179 and AF129085 were amplified by PCR using human skeletal muscle cDNA as a template (CLONTECH). Vectors for the expression of bacterial recombinant His-tagged Hsc70 and CHIP were created using the pET21a vector (Stratagene). The pET-Hsc70 vector was created by inserting a DNA fragment containing the Hsc70 open reading frame without a stop codon into the 5 0 -BamHI and 3 0 -XhoI sites of pET21a in-frame with the six-histidine tag. The pET-CHIP construct was created by inserting a DNA fragment containing the CHIP open reading frame without a
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.10.043
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Fig. 1. HSF1 interacts with CHIP through its N-terminal region. (A) Schematic diagram of the HSF1 domain organization. HSF1 is composed of an amino-terminal DBD, an adjacent coiled-coil trimerization domain (LZ1-3), a central RD, a second coiled-coil domain (LZ4), and a carboxylterminal transcriptional AD. Domain boundaries were obtained from the SMART and COIL programs. (B) Purified GST-tagged bacterial recombinant HSF1 (1–290) or HSF1 (FL) (1 lg) were incubated equimolar His-tagged CHIP. As a control, equimolar GST was incubated with Histagged CHIP. GST or GST-HSF1 was pulled down by glutathione-agarose beads. Co-precipitated proteins were resolved on SDS–PAGE and detected by Western blot analysis using anti-His antibody. (C) Purified bacterial recombinant GST-tagged HSF1 (1–290), GST-tagged HSF1 (FL) and His-tagged CHIP were monitored by Coomassie blue staining.
stop codon into the 5 0 -NdeI and 3 0 -NotI sites of pET21a in-frame with the six-histidine tag. Mammalian expression vector for HSF1 have been described previously [19]. Vectors for mammalian expression of N-terminally FLAG-tagged CHIP fusion protein was created by inserting a DNA fragment containing the complete CHIP open reading frame into the 5 0 -KpnI and 3 0 -NotI sites of pCDNA3.1-NF. 2.2. Protein expression and purification Bacterial recombinant HSF1 was expressed as an N-terminally GSTtagged fusion protein in Escherichia coli BL21 (DE3) Codon Plus cells (Stratagene) and purified by using glutathione-agarose affinity resin as described [19]. Recombinant Hsc70 and CHIP were expressed as C-terminally His-tagged fusion proteins in E. coli BL21 (DE3) Codon Plus cells and purified by using Ni2+-agarose affinity resin as described [20]. 2.3. GST pull-down assay GST-tagged HSF1 (1 lg) was added to equimolar His-tagged CHIP in reaction buffer (20 mM Tris–Cl, pH 8.0, 200 mM NaCl, 0.1% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 1% BSA). After 2 h incubation at 4 C, glutathione-agarose beads were added and incubated for another 1 h at 4 C. Beads were washed four times with TBST (20 mM Tris–Cl, pH 8.0, 200 mM NaCl, 0.1% Tween-20) and boiled in SDS–PAGE sample buffer. Proteins were resolved by SDS–PAGE and immunoblotted with anti-His antibody (Qiagen). 2.4. Cell culture and immunoprecipitation HEK 293 cells were maintained at 37 C with 5% CO2 in DMEM containing 10% FCS, 50 U/ml penicillin and 50 lg/ml streptomycin. For immunoprecipitation, HEK 293 cells were transiently transfected by using the FuGENE 6 reagent (Roche Molecular Biochemicals) according to the manufacturers protocol. Thirty hours after transfection, the cells were washed twice with PBS and lysed in RIPA buffer (PBS supplemented with 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM PMSF, 1 lg/ml aprotinin, 1 mM sodium orthovanadate). The cell lysates were harvested and incubated at 4 C for 30 min and cleared by centrifugation at 10 000 · g for 10 min. The supernatant
was incubated with anti-FLAG antibody (Sigma–Aldrich) for 3 h, after which protein G–Sepharose (Amersham Pharmacia) was added and incubated another 1 h. The immunoprecipitates were washed four times with RIPA buffer containing 0.05% SDS and boiled in SDS–PAGE sample buffer. Proteins were resolved by SDS–PAGE and immunoblotted with anti-HSF1 antibody (Abcam), anti-FLAG antibody (Sigma–Aldrich), anti-Hsp70 antibody (Santa Cruz Biotechnology), anti-Hsc70 antibody (Santa Cruz Biotechnology), or anti-Hsp90 antibody (Santa Cruz Biotechnology). 2.5. Characterization of proteins by circular dichroism For CD experiments, purified recombinant HSF1 was dialyzed in 20 mM HEPES buffer, pH 7.5. The protein was diluted with HEPES buffer to a final concentration of 0.3 mg/ml and treated heat shock at 42 C for 5 min. CD scans were performed on a Jasco J-810 spectropolarimeter in the far-UV range at 25 C as previously described [21]. A cell of 0.1 cm optical path was used to obtain spectra at a scan speed of 50 nm/min. Spectra were averaged from four individual scans and results were presented as mean molar ellipticity.
3. Results and discussion 3.1. HSF1 interacts with CHIP through its N-terminal region To investigate the molecular mechanism of interaction between HSF1 and CHIP, we examined the binding in vitro using GST pull-down assays. GST-tagged full-length HSF1 (FL) and C-terminal truncated HSF1 (a.a. 1–290) were expressed in bacteria and purified. Bacterial recombinant Histagged CHIP was also purified. Interestingly, CHIP specifically interacts with C-terminal truncated HSF1 (1–290), encompassing the N-terminal DBD and coiled-coil motif (LZ1-3) (Fig. 1B). Under non-stressed conditions, full-length HSF1
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(FL) did not show interaction with CHIP, suggesting that the C-terminus of HSF1 affects the binding of HSF1 with CHIP (Fig. 1B). GST control did not show any interaction with CHIP (Fig. 1B). Coomassie blue staining showed the equivalent level of purified proteins (Fig. 1C). These results suggest that HSF1 directly interacts with CHIP through its N-terminal region and the C-terminal region of HSF1 may affect the interaction with CHIP under normal conditions. 3.2. Interaction between HSF1 and CHIP does not affected by Hsc70 Originally, CHIP was identified as an Hsc70 interacting protein. Additionally, we and others have previously shown that Hsc70 interacts with HSF1 [22,23]. Therefore, we next examined whether Hsc70 can affect the interaction between HSF1 and CHIP using GST pull-down assays. As shown in Fig. 2 (upper panel), CHIP strongly interacts with C-terminal deleted HSF1 (1–290) in the absence of Hsc70. Furthermore, the presence of Hsc70 does not affect the interaction between HSF1 and CHIP. Full-length HSF1 does not show interaction with CHIP in the absence of Hsc70. However, full-length HSF1 showed very weak interaction with CHIP in the presence of Hsc70 (Fig. 2, upper panel). This interaction may be caused by the indirect interaction between full-length HSF1 and CHIP through the direct interaction between full-length HSF1 and Hsc70. Consistent with our previous report, Hsc70 interacts with full-length HSF1 (FL) but not with C-terminal deleted HSF1 (1–290) (Fig. 2, lower panel) [22]. Collectively, these results demonstrate that Hsc70 does not affect the direct interaction between the N-terminal region of HSF1 and CHIP.
Fig. 3. Heat shock induces the interaction between full length HSF1 and CHIP in vitro. Bacterial recombinant GST-tagged HSF1 (1–290) or HSF1 (FL) (1 lg) were incubated with equimolar His-tagged CHIP. Samples were untreated () or heat shocked at 42 C (+) for 10 min. HSF1 was pulled down by glutathione-agarose beads. Co-precipitated proteins were resolved on SDS–PAGE and detected by Western blot analysis using anti-His antibody.
To confirm the interaction between full-length HSF1 and CHIP in vivo, HEK 293 cells were transfected with expression vector for FLAG-tagged CHIP (pCDN3.1-FLAG-CHIP). Forty eight hours after transfection, cells were heat shocked at 42 C for 1 h. Cell lysates were prepared from heat-treated (+) or -untreated () cells and subjected to immunoprecipitation using anti-FLAG antibody, followed by Western blot analysis using anti-HSF1 antibody to detect endogenous HSF1. As shown in Fig. 4 (upper panel), heat shock treatment greatly induced the interaction of CHIP with endogenous HSF1. No corresponding interaction was observed in the heat untreated control samples, confirming that the interaction between HSF1 and CHIP was induced by heat stress. Previous studies have reported that CHIP interacts with molecular chaperones, such as Hsp70, Hsp90 and Hsc70.
3.3. Interaction of CHIP with full-length HSF1 is induced by heat stress To examine whether heat stress affects the interaction between full-length HSF1 and CHIP, GST-tagged HSF1 was heat shocked at 42 C for 10 min and then incubated with His-tagged CHIP at room temperature for 60 min. As expected, full-length HSF1 did not show any interaction with CHIP under non-stressed conditions (Fig. 3). However, when the full-length HSF1 was heat stressed, the interaction between full-length HSF1 and CHIP was largely induced in vitro (Fig. 3).
Fig. 2. Hsc70 does not affect the interaction between HSF1 and CHIP in vitro. Bacterial recombinant GST-tagged HSF1 (1–290) or HSF1 (FL) (1 lg) were incubated with equimolar His-tagged CHIP and/or Hsc70. HSF1 was pulled down by glutathione-agarose beads. Coprecipitated proteins were resolved on SDS–PAGE and detected by Western blot analysis using anti-His antibody.
Fig. 4. Heat shock induces the interaction between HSF1 and CHIP in vivo. HEK 293 cells were transfected with the FLAG-tagged CHIP expression vector (pCDNA3.1-FLAG-CHIP). Forty eight hours after transfection, cells were untreated () or heat shocked at 42 C (+) for 1 h. Cell extracts were subjected to immunoprecipitation using antiFLAG antibody, and endogenous HSF1, Hsp70, Hsp90 and Hsc70 were detected by Western blot analysis.
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Therefore, we next examined the interaction between CHIP and molecular chaperones under heat stressed conditions. In contrast to HSF1, Hsp70 and Hsp90 strongly interact with CHIP under non-stressed conditions (Fig. 4). Upon heat treatment, Hsp70 and Hsp90 were significantly dissociated from CHIP protein complex (Fig. 4). These results demonstrate that dissociation of molecular chaperones, such as Hsp90 and Hsp70, from CHIP has reverse correlation with formation of HSF1 and CHIP protein complex. CHIP contains the U-box motif and has U-box dependent E3 ubiquitin ligase activity, which stimulates the ubiquitination of unfolded proteins [15,16]. CHIP also interacts with Hsp90 and antagonizes the proper chaperoning activity [12,14,24]. For example, CHIP inhibits Hsp40-stimulated ATPase activity of Hsp70, suggesting that CHIP blocks the forward reaction of the Hsp70 molecular chaperone [14,24]. Additionally, Ryosuke and colleagues [25] reported that CHIP is increased during the ER stress and promotes the release of molecular chaperones from multi-protein complexes. Considering our results, it is suggested that heat stress induces the interaction between HSF1 and CHIP and leads to the activation of CHIP signaling pathway. Unlike Hsp70 and Hsp90, the interaction between CHIP and Hsc70 was not affected by heat stress (Fig. 4). This result extends our previous report showing that the interaction between HSF1 and Hsc70 was not affected by heat stress [22]. 3.4. The secondary structure of HSF1 is modulated by heat stress Our data show that the interaction between full-length HSF1 and CHIP was largely induced by heat stress. These results strongly suggest that heat stress may induce the conformational change of HSF1 which plays an important role in HSF1–CHIP interaction. To investigate this possibility, purified recombinant HSF1 was heat treated at 42 C for 5 min and the effect of heat stress was analyzed by CD spectroscopy. Interestingly, the structural change of HSF1 was observed under heat-treated conditions. As shown in Fig. 5, CD analysis of heat treated HSF1 showed different degree of secondary structure near 210 nm compared with non-stressed HSF1, demonstrating the different a-helix contents. This result confirms that the overall structure of HSF1 is altered by heat stress. Under normal physiological conditions, intramolecular interactions between LZ1-3 and LZ4 restrain HSF1 in an inac-
Fig. 5. Circular dichroism analysis of heat stressed HSF1. Bacterial recombinant HSF1 (0.3 mg/ml) was untreated or heat shocked at 42 C for 10 min. The CD spectra were recorded in the far UV region (200– 250 nm) at 20 C. The spectra were presented as mean residue ellipticity, expressed as deg Æ cm2/dmol. Each spectrum is the average of four separate scans.
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Fig. 6. Model for the interaction of HSF1 and CHIP. Under normal conditions, HSF1 is found as an inactive monomer restrained by interaction between LZ1-3 and LZ4. Conversion from the monomer to the trimer occurs constitutively when the cells are heat stressed (42 C) that would be disrupted intramolecular interactions. The conformational changes of HSF1 induce the interaction with CHIP and the dissociation of molecular chaperones from multi-protein complexes.
tive monomeric form [8–10]. Upon heat stress, intramolecular interactions are disrupted and HSF1 forms homotrimeric complex (Fig. 6) [9,11]. In our results, CHIP showed weak indirect interaction with full-length HSF1 through the direct interaction with Hsc70 under non-stressed conditions (Figs. 2 and 6). Upon heat stress, conformational changes of HSF1 occur, leading to the direct interaction between HSF1 and CHIP (Fig. 6). Hsc70 may recruit and/or stabilize the association of CHIP with HSF1. It will now be interesting and important to determine the role of CHIP on the function of HSF1. Acknowledgements: We thank Dr. Dennis Thiele for helpful suggestions. This work was supported by Grant No. RT104-03-03 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE), and BK 21 Project.
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