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• W2048325346 abstract "The major stress protein transcription factor, heat shock factor (HSF1), is tightly regulated through a multilayered activation-deactivation process involving oligomerization, post-translational modification, and interaction with the heat shock protein (Hsp90)-containing multichaperone complex. Conditions of proteotoxic stress, such as heat shock, trigger reversible assembly of latent HSF1 monomers into DNA-binding homotrimers that bind with high affinity to cognate heat shock elements. Transactivation is a second and independently regulated function of HSF1 that is accompanied by hyperphosphorylation and appears to involve a number of signaling events. Association of HSF1 with Hsp90 chaperone complexes provides additional regulatory complexity, however, not all the co-chaperones have been identified, and the specific molecular interactions throughout the activation/deactivation pathway remain to be determined. Here we demonstrate that protein phosphatase 5 (PP5), a tetratricopeptide domain-containing component of Hsp90-steroid receptor complexes, functions as a negative modulator of HSF1 activity. Physical interactions between PP5 and HSF1-Hsp90 complexes were observed in co-immunoprecipitation and gel mobility supershift experiments. Overexpression of PP5 or activation of endogenous phosphatase activity resulted in diminished HSF1 DNA binding and transcriptional activities, and accelerated recovery. Conversely, microinjection of PP5 antibodies, or inhibition of its phosphatase activity in vivo, significantly delayed trimer disassembly after heat shock. Inhibition of PP5 activity did not activate HSF1 in unstressed cells. These results indicate that PP5 is a negative modulator of HSF1 activity. The major stress protein transcription factor, heat shock factor (HSF1), is tightly regulated through a multilayered activation-deactivation process involving oligomerization, post-translational modification, and interaction with the heat shock protein (Hsp90)-containing multichaperone complex. Conditions of proteotoxic stress, such as heat shock, trigger reversible assembly of latent HSF1 monomers into DNA-binding homotrimers that bind with high affinity to cognate heat shock elements. Transactivation is a second and independently regulated function of HSF1 that is accompanied by hyperphosphorylation and appears to involve a number of signaling events. Association of HSF1 with Hsp90 chaperone complexes provides additional regulatory complexity, however, not all the co-chaperones have been identified, and the specific molecular interactions throughout the activation/deactivation pathway remain to be determined. Here we demonstrate that protein phosphatase 5 (PP5), a tetratricopeptide domain-containing component of Hsp90-steroid receptor complexes, functions as a negative modulator of HSF1 activity. Physical interactions between PP5 and HSF1-Hsp90 complexes were observed in co-immunoprecipitation and gel mobility supershift experiments. Overexpression of PP5 or activation of endogenous phosphatase activity resulted in diminished HSF1 DNA binding and transcriptional activities, and accelerated recovery. Conversely, microinjection of PP5 antibodies, or inhibition of its phosphatase activity in vivo, significantly delayed trimer disassembly after heat shock. Inhibition of PP5 activity did not activate HSF1 in unstressed cells. These results indicate that PP5 is a negative modulator of HSF1 activity. The cellular stress response protects against environmental or physiological perturbations that result in aberrant folding and aggregation of proteins. Stress induces rapid synthesis of a set of molecular chaperones known as heat shock proteins (Hsps) 1The abbreviations used are: Hsp, heat shock protein; Ab, antibody; HSF1, heat shock factor 1; PP5, protein phosphatase 5; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation; GR, glucocorticoid receptor; TPR, tetratricopeptide repeat; CMV, cytomegalovirus; HSE, heat shock element. 1The abbreviations used are: Hsp, heat shock protein; Ab, antibody; HSF1, heat shock factor 1; PP5, protein phosphatase 5; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation; GR, glucocorticoid receptor; TPR, tetratricopeptide repeat; CMV, cytomegalovirus; HSE, heat shock element. (1Morimoto R. Tissiers A. Georgopoulos C. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar) that aid the folding, transport, regulation, and degradation of cellular proteins. The highly conserved transcription factor HSF1 is the key regulatory protein responsible for the up-regulation of Hsp expression in higher eukaryotes (1Morimoto R. Tissiers A. Georgopoulos C. Stress Proteins in Biology and Medicine. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar, 2Christians E.S. Yan L.J. Benjamin I.J. Crit. Care Med. 2002; 30: S43-S50Crossref Scopus (187) Google Scholar, 3Voellmy R. Cell Stress Chaperones. 2004; 9: 122-133Crossref PubMed Scopus (223) Google Scholar). The mechanism of HSF1 regulation is highly complex, involving multifactorial control by phosphorylation, cellular compartmentalization, and protein-protein interaction with chaperone complexes. It is not yet known how these multiple layers of control are integrated to result in cellular regulation of HSF1 and the stress response. HSF1 exists under normal conditions as non-DNA-binding monomers, poised for rapid conversion to homotrimers with high affinity DNA binding activity to the heat shock elements in Hsp gene promoters (4Baler R. Dahl G. Voellmy R. Mol. Cell. Biol. 1993; 13: 2486-2496Crossref PubMed Scopus (390) Google Scholar, 5Sarge K.D. Murphy S.P. Morimoto R.I. Mol. Cell. Biol. 1993; 13: 1392-1407Crossref PubMed Scopus (740) Google Scholar). This oligomeric switching involves rearrangement of hydrophobic interactions between 3 hydrophobic heptad repeats distributed along the HSF1 molecule (4Baler R. Dahl G. Voellmy R. Mol. Cell. Biol. 1993; 13: 2486-2496Crossref PubMed Scopus (390) Google Scholar, 6Westwood J.T. Clos J. Wu C. Nature. 1991; 353: 822-827Crossref PubMed Scopus (307) Google Scholar, 7Westwood J.T. Wu C. Mol. Cell. Biol. 1993; 13: 3481-3486Crossref PubMed Scopus (185) Google Scholar, 8Zuo J. Baler R. Dahl G. Voellmy R. Mol. Cell. Biol. 1994; 14: 7557-7568Crossref PubMed Scopus (164) Google Scholar), and it is likely that there is dynamic recycling between monomeric and trimeric complexes during induction and recovery phases of the HSF1 activation/deactivation pathway. Acquisition of transcriptional competence is a second independent step in the stress-activation process that is regulated through a central regulatory region (9Zuo J. Rungger D. Voellmy R. Mol. Cell. Biol. 1995; 15: 4319-4330Crossref PubMed Scopus (203) Google Scholar) and multiple phosphorylation events (3Voellmy R. Cell Stress Chaperones. 2004; 9: 122-133Crossref PubMed Scopus (223) Google Scholar, 10Xia W. Voellmy R. J. Biol. Chem. 1997; 272: 4094-4102Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Although hyperphosphorylation is generally associated with increased transcriptional activity, elucidating the effect of specific phosphorylation on HSF1 activity is proving to be a complicated task. Several kinases have been reported to phosphorylate HSF1 on specific residues and either enhance or repress its activities, including calcium/calmodulin-dependent protein kinase II (11Holmberg C.I. Hietakangas V. Mikhailov A. Rantanen J.O. Kallio M. Meinander A. Hellman J. Morrice N. MacKintosh C. Morimoto R.I. Eriksson J.E. Sistonen L. EMBO J. 2001; 20: 3800-3810Crossref PubMed Scopus (244) Google Scholar), casein kinase 2 (12Soncin F. Zhang X. Chu B. Wang X. Asea A. Ann Stevenson M. Sacks D.B. Calderwood S.K. Biochem. Biophys. Res. Commun. 2003; 303: 700-706Crossref PubMed Scopus (57) Google Scholar), protein kinase Cα (13Yang R.C. Jao H.C. Huang L.J. Wang S.J. Hsu C. Exp. Cell Res. 2004; 296: 276-284Crossref PubMed Scopus (12) Google Scholar), glycogen synthase kinase 3 (14Xavier I.J. Mercier P.A. McLoughlin C.M. Ali A. Woodgett J.R. Ovsenek N. J. Biol. Chem. 2000; 275: 29147-29152Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 15Chu B. Zhong R. Soncin F. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 1998; 273: 18640-18646Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), and extracellular signal-regulated kinase 1 (16Chu B. Soncin F. Price B.D. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 1996; 271: 30847-30857Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 17Wang X. Grammatikakis N. Siganou A. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2004; 279: 49460-49469Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In addition, there is evidence that the activities of HSF1 are influenced by Hsp90 multichaperone complexes (18Ali A. Bharadwaj S. O'Carroll R. Ovsenek N. Mol. Cell. Biol. 1998; 18: 4949-4960Crossref PubMed Scopus (236) Google Scholar, 19Zou J. Guo Y. Guettouche T. Smith D.F. Voellmy R. Cell. 1998; 94: 471-480Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar, 21Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 22Nair S.C. Toran E.J. Rimerman R.A. Hjermstad S. Smithgall T.E. Smith D.F. Cell Stress Chaperones. 1996; 1: 237-250Crossref PubMed Scopus (196) Google Scholar). The role of Hsp90 chaperone complexes on transcription factor regulation has been most extensively characterized in the experimental model of steroid receptor maturation (23Riggs D. Cox M. Cheung-Flynn J. Prapapanich V. Carrigan P. Smith D. Crit. Rev. Biochem. Mol. Biol. 2004; 39: 279-295Crossref PubMed Scopus (106) Google Scholar, 24Pratt W.B. Toft D.O. Bull. Exp. Biol. Med. 2003; 228: 111-133Crossref Scopus (1239) Google Scholar). Hsp90 is the key molecular chaperone in mature receptor complexes together with p23 and one of the immunophilins (Cyp40, FKBP51, or FKBP52). Assembly of steroid receptor complexes is a highly dynamic process involving Hsp70 and accessory chaperones Hsp40, Hip, and Hop (24Pratt W.B. Toft D.O. Bull. Exp. Biol. Med. 2003; 228: 111-133Crossref Scopus (1239) Google Scholar). Using human HeLa cells extracts and rabbit reticulocyte lysates, Voellmy and colleagues (3Voellmy R. Cell Stress Chaperones. 2004; 9: 122-133Crossref PubMed Scopus (223) Google Scholar, 19Zou J. Guo Y. Guettouche T. Smith D.F. Voellmy R. Cell. 1998; 94: 471-480Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, 21Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) have shown Hsp90 association with HSF1 monomers, as well as HSF1 trimer association with an Hsp90-immunophilin-p23 complex, and have proposed a model for transcriptional repression of HSF1 activity by Hsp90. Our laboratory has examined HSF1 regulation by Hsp90 in the Xenopus oocyte model system, and reported HSF1 interaction with multiple components of the Hsp90 foldosome (18Ali A. Bharadwaj S. O'Carroll R. Ovsenek N. Mol. Cell. Biol. 1998; 18: 4949-4960Crossref PubMed Scopus (236) Google Scholar, 20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar). Hsp90, P23, and FKBP52 remain associated with the activated HSE binding HSF1 complex (20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar). Conversion to monomers after heat shock was accelerated by overexpression of Hsp90, Hsp70, Hip, and Hop and delayed by cyclophilins (Cyp-40, FKBP51, and FKBP52). HSF1 was activated in the absence of heat shock by microinjection of Abs against Hsp90 and p23, and microinjected Abs against several of the co-chaperones also affected the DNA-binding properties of HSF1. Further evidence supporting Hsp90-mediated control of HSF has been reported in yeast (25Duina A.A. Kalton H.M. Gaber R.F. J. Biol. Chem. 1998; 273: 18974-18978Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The precise molecular interactions and specific roles of individual components of the Hsp90 foldosome in regulating trimerization, DNA binding, and transcriptional activity, at each stage of the HSF1 activation/deactivation pathway, has not yet been determined. However, the initial studies with HeLa cells and Xenopus oocytes suggest that the multichaperone interactions that may regulate HSF1 share common features with the steroid receptor maturation pathway (22Nair S.C. Toran E.J. Rimerman R.A. Hjermstad S. Smithgall T.E. Smith D.F. Cell Stress Chaperones. 1996; 1: 237-250Crossref PubMed Scopus (196) Google Scholar). PP5 (protein phosphatase 5) is a major component of steroid receptor-Hsp90 heterocomplexes, and is involved in the regulation of glucocorticoid receptor (GR) and estrogen receptor function (Refs. 26Chen M.S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 27Silverstein A.M. Galigniana M.D. Chen M.S. Owens-Grillo J.K. Chinkers M. Pratt W.B. J. Biol. Chem. 1997; 272: 16224-16230Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 28Zuo Z. Urban G. Scammell J.G. Dean N.M. McLean T.K. Aragon I. Honkanen R.E. Biochemistry. 1999; 38: 8849-8857Crossref PubMed Scopus (110) Google Scholar, 29Ramsey A.J. Russell L.C. Whitt S.R. Chinkers M. J. Biol. Chem. 2000; 275: 17857-17862Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 30Ikeda K. Ogawa S. Tsukui T. Horie-Inoue K. Ouchi Y. Kato S. Muramatsu M. Inoue S. Mol. Endocrinol. 2004; 18: 1131-1143Crossref PubMed Scopus (37) Google Scholar, for review, see Refs. 31Chinkers M. Trends Endocrinol. Metab. 2001; 12: 28-32Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar and 32Chinkers M. Top. Curr. Genet. 2004; 5: 107-130Crossref Google Scholar). PP5 is a 58-kDa member of the PPP family of serine/threonine protein phosphatases that contains a tetratricopeptide repeat (TPR) domain not found in other members of this family. The N-terminal TPR domain of PP5 functions both to inhibit the C-terminal phosphatase domain and to mediate interactions with the MEEVD domain of Hsp90 (33Yang J. Roe S.M. Cliff M.J. Williams M.A. Ladbury J.E. Cohen P.T. Barford D. EMBO J. 2005; 24: 1-10Crossref PubMed Scopus (153) Google Scholar). Hsp90 activates PP5 by disrupting TPR-phosphatase domain interactions, permitting substrate access to the active phosphatase domain (33Yang J. Roe S.M. Cliff M.J. Williams M.A. Ladbury J.E. Cohen P.T. Barford D. EMBO J. 2005; 24: 1-10Crossref PubMed Scopus (153) Google Scholar, 34Kang H. Sayner S.L. Gross K.L. Russell L.C. Chinkers M. Biochemistry. 2001; 40: 10485-10490Crossref PubMed Scopus (73) Google Scholar). Here we investigate the potential interaction of PP5 with HSF1-Hsp90 complexes, and subsequent regulation of HSF1 activities. Oocyte Manipulations—Xenopus laevis oocytes were obtained, fractionated, and microinjected as described previously (14Xavier I.J. Mercier P.A. McLoughlin C.M. Ali A. Woodgett J.R. Ovsenek N. J. Biol. Chem. 2000; 275: 29147-29152Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar). Chemical stresses were performed for 2 h at 18 °C with the specified concentration of chemical stressors. Oocytes were treated for 2 h with 4 μm fostriecin (Calbiochem), or 100 nm okadaic acid (Sigma) at 18 °C prior to further experimentation as indicated. In all experiments, a minimum of 25 oocytes were used for each sample, and each experiment was performed using oocytes from the same batch. Plasmid DNAs for microinjection were as follows: pCMV-PP5 (43Chinkers M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11075-11079Crossref PubMed Scopus (135) Google Scholar), pCMV-PP5Flag, pET30-K97A, and pET-R101A (26Chen M.S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In some experiments, oocytes were microinjected into the nucleus with 7.5 milliunits of PP1 or PP2A (Calbiochem catalog numbers 539527 and 539508) and then incubated for 1 h at 18 °C prior to further experimentation. Protein Extracts and Gel Mobility Shift Assays—DNA mobility shift assays were performed by using radiolabeled oligonucleotide probes as described previously (44Ovsenek N. Williams G.T. Morimoto R.I. Heikkila J.J. Dev. Genet. 1990; 11: 97-109Crossref PubMed Scopus (21) Google Scholar). For gel mobility supershift analysis, Abs were added into DNA binding reactions at a final dilution of 1:100 (v/v). DNA binding activity was quantified using NIH Image (version 1.6.1) software, and results were expressed in arbitrary densitometry units as a percentage of the signal obtained in DNA binding reactions from uninjected untreated, heat-shocked controls. Antibodies—Abs used for gel mobility shift analysis and Western blotting were as follows: anti-Hsp90 monoclonal Ab (Stressgen, Victoria, British Columbia, Canada, catalog number SPA-830), anti-PP5 polyclonal Ab (M. Chinkers, University of South Alabama), anti-p23 monoclonal Ab (clone JJ3; gift from D. Toft, Mayo Graduate School), anti-FKBP52 monoclonal Ab (clone Hi52c gift from D. Smith, University of Nebraska), anti-Hip monoclonal Ab (clone 2G6 Hi52c gift from D. Smith, University of Nebraska), anti-Hop monoclonal Ab (clone F5; gift from D. Smith, University of Nebraska), anti-PP1 polyclonal Ab (Cal-biochem catalog number 539517), PP2A (Upstate Biotechnology catalog number 05-421), anti-YY1 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number sc-372G), anti-proliferating cell nuclear antigen monoclonal Ab (Santa Cruz Biotechnology, catalog number sc-56), anti-IκB (Santa Cruz Biotechnology, catalog number sc-1643), and anti-FLAG (Sigma, catalog number F4042). Immunoblotting—Protein extracts were fractionated as described (14Xavier I.J. Mercier P.A. McLoughlin C.M. Ali A. Woodgett J.R. Ovsenek N. J. Biol. Chem. 2000; 275: 29147-29152Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar). Secondary Abs were goat anti-rabbit (Bio-Rad catalog number 70–5045) or anti-mouse (Bio-Rad catalog number 170–5044), diluted 1:10,000 in TBST and 2.5% (w/v) milk. Blots were then washed in TBST and developed for peroxidase activity using chemiluminescence (Renaissance system, PerkinElmer Life Sciences) and autoradiography. Immunoprecipitation (IP)—IPs were performed using the modified method of Firestone and Winguth (45Firestone G.L. Winguth S.D. Methods Enzymol. 1990; 182: 688-700Crossref PubMed Scopus (59) Google Scholar) as described (20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar). Oocyte cell extracts for IP reactions were prepared using TETN-250 buffer (25 mm Tris-HCl (pH 7.5), 5 mm EDTA (pH 8.0), 250 mm NaCl, 1% (v/v) Triton X-100, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). IP reactions contained 4 oocyte equivalents in TETN-250 in a total volume of 200 μl, with a 30-μl bed volume of protein A/G-Sepharose resin (Amersham Bio-sciences, catalog number 17-0469-01). IPs were carried out for 30 min at 23 °C with 2 μl of pre-absorbed IP Ab. Following reactions, resin was washed with TETN-250 (6 × 0.5 ml), bound proteins were eluted with SDS-PAGE sample buffer, and aliquots were analyzed by Western blotting. Transcription Assays—Chloramphenicol acetyltransferase assays were performed as previously described (18Ali A. Bharadwaj S. O'Carroll R. Ovsenek N. Mol. Cell. Biol. 1998; 18: 4949-4960Crossref PubMed Scopus (236) Google Scholar, 20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar). Overexpression of PP5 Reduces HSF1 DNA Binding upon Heat Shock—To determine the potential role of PP5 in HSF1 regulation, oocytes were microinjected with pCMV6-PP5 (26Chen M.S. Silverstein A.M. Pratt W.B. Chinkers M. J. Biol. Chem. 1996; 271: 32315-32320Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), which codes for rat PP5 under control of the constitutively active CMV promoter. Western blot analysis (Fig. 1A) showed steadily increasing quantities of plasmid-derived PP5 over the time course of incubation, with a substantial increase relative to endogenous PP5 seen after 12 h of expression. Heat shock did not affect steady state levels of either endogenous or recombinant PP5, nor did overexpression of PP5 have any effect on the levels of HSF1, before or after heat shock. The heat-inducible activation of HSF1 oligomerization/DNA binding was compared in uninjected controls and PP5 expressing (12 h) oocytes by EMSA (Fig. 1B). The amount of HSF1-HSE complex formation was significantly reduced in PP5 expressing oocytes relative to controls. DNA binding activity of other transcription factors such as CAAT box transcription factor (Fig. 1B, lower panel) or SP1 (data not shown) were not affected by expression of exogenous PP5, ruling out a general suppressive effect on DNA binding activities in the oocyte. HSF1 activation was compared in oocytes expressing PP5 for 3, 6, or 12 h prior to heat shock (Fig. 1C). The reduction of HSF1 activation was proportional to the amount of exogenous PP5, suggesting a direct link between increased PP5 concentrations and HSF1 regulation. The relative reduction in HSF1 DNA binding activity was more prominent at the early phases of heat shock. After 60 min of heat shock, HSF1-HSE complex formation relative to controls was reduced by 20% in all samples, suggesting that lengthy heat shock diminished the impact of PP5 on activation. We next tested the effect of PP5 overexpression on the induction of HSF1 by chemical stresses. HSF1 activation was reduced in PP5 expressing oocytes in response to salicylate, arsenite, cadmium, and ethanol treatments (Fig. 1D). Subcellular Localization of PP5—We next compared the subcellular localization of endogenous and plasmid-derived PP5 in control and microinjected oocytes (Fig. 2). Endogenous PP5 was predominantly localized in the cytoplasm with low but detectable amounts in a single oocyte nucleus. Heat shock resulted in modest reproducible translocation of PP5 into the nucleus (lanes 5 and 6). Exogenous PP5 expressed from microinjected pCMV-FLAG-PP5 behaved similarly to endogenous PP5, both in relative distribution and nuclear translocation induced by heat shock. The total mass of the nucleus is ∼5% of the whole cell, and longer film exposure revealed the presence of FLAG-tagged PP5 in the nucleus prior to heat shock (data not shown). Controls using the cytoplasmic marker IκB and the nuclear marker proliferating cell nuclear antigen demonstrate the purity of subcellular fractions. PP5 Interaction with HSF1—HSF1 is an exclusively nuclear protein in oocytes (35Mercier P.A. Foksa J. Ovsenek N. Westwood J.T. J. Biol. Chem. 1997; 272: 14147-14151Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), and detection of nuclear PP5 made plausible a physical interaction between these proteins. Co-IP experiments were performed to test for interaction between endogenous PP5 and HSF1. Co-IP of PP5 using an Ab against HSF1 (Fig. 3A, lanes 1 and 2), and reciprocal pull-down of HSF1 using an Ab against PP5 (lanes 10 and 11) was observed, confirming a physical interaction between PP5 and HSF1 in oocytes. These interactions were detected in unshocked and heat-shocked samples with no consistent or reproducible difference observed in the co-IP signal after heat shock. This suggests that PP5 is a component of Hsp90 heterocomplexes with both inactive monomeric and activated trimeric HSF1. The protein-protein association here is presumably through interaction between the TPR domain of PP5 and the C terminus of Hsp90, although it remains possible that PP5 could interact directly with HSF1. Control IP reactions with unrelated Abs failed to bring down PP5 or HSF1 in both non-shocked or heat-shocked oocytes (Fig. 3A, lanes 4–9 and 12–18). We examined whether exogenous plasmid-derived PP5 in microinjection experiments associates with HSF1 in a similar manner as endogenous PP5. Co-IP of FLAG-tagged PP5 with HSF1 was observed in both non-shocked and heat-shocked samples (Fig. 3B, lanes 3 and 4), showing a direct interaction between exogenous PP5 and HSF1 complexes. These data provide evidence of a biochemical link between PP5 overexpression and changes in HSF1 activity and functional properties observed in PP5 expressing oocytes (see Figs. 1 and 4, 5, 6).Fig. 5Effect of PP5 on HSF1 deactivation during recovery. A, the decay of HSF1 binding activity was compared between uninjected controls (Control) and plasmid-microinjected oocytes expressing PP5. Oocytes were either not heat shocked (N) or heat shocked at 33 °C for 1 h (H) prior to recovery (times indicated) at 18 °C and analyzed by EMSA with HSE. B, EMSA of oocytes microinjected with 7.5 milliunits of PP1 or PP2A and subjected to heat shock and recovery as above. C, EMSA of oocytes microinjected with anti-PP5 Ab or anti-YY1 Ab, then subjected to heat shock and recovery (left panel, lanes 1–15), and Western blot of co-immunoprecipitation with HSF1 Ab (right panel). Oocyte samples were uninjected (Control, lanes 16 and 17), anti-PP5 Ab-injected (lanes 18 and 19), and anti-YY1 Ab-injected (lanes 20 and 21). D, EMSA of oocytes treated with 100 nm okadaic acid for 2 h prior to heat shock and recovery. E, EMSA of oocytes expressing PP5, C-90, or treated with 4 μm fostriecin. In each panel, comparisons were made between oocytes obtained from a single ovary, each experiment was repeated at least 3 times, and representative data are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Effect of PP5 on HSF1-mediated transcription. Oocytes were microinjected with 100 pg of Hsp70-chloramphenicol acetyltransferase (left panels), or 100 pg of CMV-chloramphenicol acetyltransferase reporter plasmids (right panels), then incubated for 12 h at 18 °C prior to a 1-h heat shock at 33 °C (H). N, no heat shock. Following heat shock, oocytes were further incubated for 12 h at 18 °C for expression, then subjected to chloramphenicol acetyltransferase assays. Treated samples (left panel, third and fourth lanes; right panels, third and fourth lanes) were manipulated as follows: A, co-microinjection of pCMV-PP5 for overexpression of PP5. B, microinjection of 7.5 milliunits of PP1 enzyme prior to heat shock. C, microinjection of 7.5 milliunits of PP2A enzyme prior to heat shock. D, treatment with 4 μm fostriecin for 2 h prior to heat shock. E, treatment with 100 nm okadaic acid for 2 h prior to heat shock. F, control treatment with Me2SO (DMSO; 1:500 v/v) for 2 h prior to heat shock. Each panel contains an individual experiment using a batch of oocytes obtained from a single ovary to minimize variation, and comparisons were made only within each panel set. Experiments were repeated at least 5 times, and representative results are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further confirm the presence of PP5 with activated HSF1 trimers, mobility supershift analyses were performed using Abs against PP5 and other components of HSF1-Hsp90 heterocomplexes. We have previously observed supershifts of HSF1-HSE complexes with anti-Hsp90, p23, and FKBP52 Abs (20Bharadwaj S. Ali A. Ovsenek N. Mol. Cell. Biol. 1999; 19: 8033-8041Crossref PubMed Scopus (144) Google Scholar), results consistent with findings of pull-down experiments of others using HeLa cell extracts (21Guo Y. Guettouche T. Fenna M. Boellmann F. Pratt W.B. Toft D.O. Smith D.F. Voellmy R. J. Biol. Chem. 2001; 276: 45791-45799Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Distinct supershifted complexes with clearly reduced mobility were observed with Abs against PP5, HSF1, Hsp90, p23, Hip, and FKBP52 (Fig. 3C). This suggests that PP5 is a component of active trimeric HSF1 complexes along with Hsp90, p23, FKBP52, and Hip. HSF1-HSE complexes were not affected by Hop Abs (lanes 13 and 14), or control Abs against YY1 transcription factor or proliferating cell nuclear antigen (lanes 17–20). PP1 and PP2A Abs were also tested in these assays to test for possible interactions with the HSF1 heterocomplex, however, no supershift was observed (lanes 21–26). Effect of PP5 Phosphatase Activity on the DNA Binding Activity of HSF1—Co-IP and mobility supershift experiments (Fig. 3) establish PP5 as a component of the HSF1-Hsp90 heterocomplex, and we observed significantly reduced activation of HSF1 DNA binding after expression of exogenous PP5 (Fig. 1). The next series of experiments were aimed at determining whether the inhibitory effect on HSF1 was because of the specific PP5 phosphatase activity. Oocytes did not survive treatments with arachidonic acid, a well established activator of PP5 (36Chen M.X. Cohen P.T. FEBS Lett. 1997; 400: 136-140Crossref PubMed Scopus (116) Google Scholar, 37Skinner J. Sincl" @default.
• W2048325346 created "2016-06-24" @default.
• W2048325346 creator A5010238664 @default.
• W2048325346 creator A5013210249 @default.
• W2048325346 creator A5064725400 @default.
• W2048325346 creator A5072252990 @default.
• W2048325346 creator A5072811641 @default.
• W2048325346 date "2005-08-01" @default.
• W2048325346 modified "2023-10-17" @default.
• W2048325346 title "Protein Phosphatase 5 Is a Negative Modulator of Heat Shock Factor 1" @default.
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