Phosphorylation of HSF1 by MAPKAP Kinase 2 on Serine 121, Inhibits Transcriptional Activity and Promotes HSP90 Binding

2,4XiaoZhe Wang, 1,4Md Abdul Khaleque, 1Mei Juan Zhao, 1Rong Zhong, 3Matthias Gaestel

and 1,5Stuart K. Calderwood 1Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 2 Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115 3 Institute of Biochemistry, Medical School Hanover, 30625, Hanover, Germany 4Equal contributors

Running title: Regulation of HSF1 by MK2 phosphorylation* This work was supported by National Institutes of Health grants CA47407, CA31303, and CA50642. 5Communicating author: Dr Stuart K. Calderwood, Beth Israel Deaconess Medical Center, 21-27 Burlington Avenue, Room 553B, Boston, MA 02215, phone 617-632-0628; Email: scalderw@bidmc.harvard.edu Heat shock transcription factor 1 (HSF1) monitors the structural integrity of intracellular proteins and its regulation is essential for the health and longevity of eukaryotic organisms. HSF1 also plays a role in the acute inflammatory response in the negative regulation of cytokine gene transcription. Here we show, for the first time that HSF1 is regulated by the pro-inflammatory protein kinase MAPKAP kinase 2 (MK2). We have shown that MK2 directly phosphorylates HSF1 and inhibits activity by decreasing its ability to bind the heat shock elements (HSE) found in the promoters of target genes encoding the HSP molecular chaperones and cytokine genes. We show that activation of HSF1 to bind HSE in hsp promoters is inhibited through the phosphorylation of a specific residue, serine 121 by MK2. A potential mechanism for MK2-induced HSF1 inactivation is suggested by the findings that phosphorylation of serine 121 enhances HSF1 binding to HSP90, a major repressor of HSF1. Dephosphorylation of serine 121 in cells exposed to non-steroidal anti-inflammatory drugs leads to HSP90 dissociation from HSF1 which then forms active DNA binding trimers. These experiments indicate a novel mechanism for the regulation of HSF1 by pro-inflammatory signaling and may permit HSF1 to respond rapidly to

extracellular events, permitting optimal physiological regulation.

Heat shock factor 1 (HSF1) is the transcriptional activator of HSP molecular chaperone genes during stress (1, 2). The hsf1 gene plays an essential role in protection of cells from heat shock by regulating the induction of cytoprotective HSP as well as in protection against the effects of endotoxins through its ability to repress the transcription of proinflammatory cytokines through inhibition of factors involved in cytokine transcription such as NF-IL6 and NFκB (3, 4). Aging is associated with the degeneration of HSP expression with time and the loss of resistance to cellular oxidants: elevated HSF1 leads to significant increase in lifespan in C. elegans (5-7). In cancer, the converse situation applies and malignant transformation is associated with aberrantly high levels of HSP (8, 9). These clinical phenomena reflect the role of HSP molecular chaperones in cellular regulation, as either up- or down-regulation of HSP expression can profoundly modulate multiple key proteins within the cell (10). It is therefore clear that elucidating the molecular mechanisms that control HSP expression in mammalian cells is essential.

Under normal conditions, most HSF1 is inactive and maintained in a compacted monomeric form (11-13). Inactive HSF1 lacks the

1

http://www.jbc.org/cgi/doi/10.1074/jbc.M505822200The latest version is at JBC Papers in Press. Published on November 8, 2005 as Manuscript M505822200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

ability to bind to the heat shock elements (HSE) in hsp promoters, is unable to trans-activate hsp genes and fails to repress the promoters of proinflammatory cytokines (13-16). Activation of HSF1 is a complex process involving monomer to trimer transition and DNA binding; hyperphosphorylation and capacity to activate target promoters (12, 17-19). Trimerization of HSF1 is governed by leucine zipper domains in the amino terminus and is subject to intramolecular negative regulation by a fourth leucine zipper in the carboxy-terminus (15). This form of regulation is sufficient to regulate monomer to trimer transition in purified HSF1 in vitro (1). However, additional levels of regulation exist in the cell. Activation is controlled by HSF1 binding to the molecular chaperone HSP90, and HSP90 binding maintains the monomeric state (13, 20). This form of regulation is also found in other transcription factor families including the nuclear receptors (21). Activation of HSF1 by heat shock causes release of HSF1 from HSP90 in a process thought to involve the sequestration of HSP90 by denatured proteins (13). The monomer to trimer transition can also be induced by high concentrations of the non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen and sodium salicylate (NaSal) (22, 23). These compounds derive the majority of their anti-inflammatory activity through inhibition of the cyclooxygenase (COX) enzymes (24, 25). However, the induction of trimeric HSF1 involves NSAID concentrations 1000 fold greater than are required for inhibition of COX activity (22, 23). At these concentrations, the NSAIDs are effective inhibitors of protein kinases such as RSK2, ERK, and IKKα (26, 27). This led us to propose the hypothesis that HSF1 activation is negatively regulated by a protein kinase that is inhibited by NSAIDs. HSF1 is subject to reciprocal regulation by proinflammatory and anti-inflammatory signals in myeloid cells; while proinflammatory lipopolysaccharides (28) repress HSF1, the NSAIDs activate HSF1 and reverse the effects of LPS (29). Our preliminary studies show that inhibitors of LPS activation of mitogen activated protein kinase activate protein kinase 2 (MAPKAP kinase 2 or MK2) activate the binding of HSF1 to HSE. Such compounds include tyrosine kinase inhibitors AG126 and herbimycin A, p38 kinase inhibitor SB203580, and NSAIDs sodium

salicylate, aspirin and ibuprofen (S. K. Calderwood, unpublished). MK2 is an inducible kinase activated by p38MAPK and through p38 is regulated by cell stress, LPS and the cytokines IL1β and TNFα (30-32). MK2 plays an important physiological role in the acute inflammatory response (33), enhancing the expression of proinflammatory cytokines through mRNA stabilization and increasing expression of inflammatory enzymes including lipoxygenase and phospholipase A (28, 34-37). Inhibition of HSF1 by MK2, as we suggest here, would be consonant with a role in inflammation, as HSF1 inhibition would relieve the repression on cytokine promoters (3, 4, 38). In the current study, we have examined the effects of MK2 on the regulation of HSF1. We show that activated MK2 is a potent inhibitor of HSF1 and inhibits HSF1 binding to HSE and trans activation of the HSP70B promoter. Our studies indicate that MK2 directly phosphorylates HSF1 on a specific serine residue 121 (S121) that this event may mediate some of the intracellular effects of MK2 on HSF1. Mutation of the S121 residue in HSF1 to alanine prevented the inhibitory effects of MK2. Activation of MK2 led to an increase in HSF1-HSP90 binding while S121 mutation to alanine prevented the effects of MK2 on HSF1-HSP90 binding, and permitted HSF1 association with HSE. As HSP90 binding to HSF1 is known to be involved in the inactivation of the factor, our data suggest a potential mechanism for HSF1 inhibition by MK2. Our experiments therefore indicate that the proinflammatory protein kinase MK2 is an inhibitor of HSF1 and may exert its effects on the protein by direct phosphorylation.

MATERIALS AND METHODS Plasmid Construction and Mutagenesis The human HSF1 cDNA was amplified and subcloned into the pHM6 (Roche) vector to generate pHM6HSF1 wt (pHA-HSF1 wt) which was used as a template for site-directed mutagenesis using the QuikChangeTM site-directed mutagenesis kit (Stratagene) as described previously (39). Specific single, double and triple mutations (pHA- S121A, pHA- S121D, pHA-T120A, pHA-T120AS121A, pHA-S123A, pHA-

2

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

T124A, pHA-S127A, pHA-T527A, pHA-S529A, pHA-T120A/S121A, pHA-S121A/S123A, pHA-T527A/S529A, pHA-T120A /S121A / S123A, pHA-T120A/S121A/T527A, pHA-T120A/S121A/S529A) were introduced. All constructs and mutants were confirmed by DNA sequencing. pcDNA3-Myc-MK2, MK2 dominant negative mutant pcDNA3-MK2 K59R, HSP70B promoter luciferase reporter construct (pGLHSP70B), pGEX2T-HSF1, and pcDNA3 FLAG-HSF1 were described previously (40, 41). In Vitro Kinase Assays Purified MK2 (0.1 U, Upstate) was incubated for 20 min at 300C with 0.25 mM synthetic peptide substrate KKPLNRTLSVASLPG (95% pure) and 0.5 µCi [γ-32P] ATP (PerkinElmer) in 25 µl of GNM buffer (60 mM ß-glycerophosphate, 30 mM p-nitrophenylphosphate, 25 mM MOPS, 15 mM MgCl2, 150 mM ATP, 0.1 mM sodium orthovanadate, 5 mM EGTA, and 1 mM DTT, pH 7.0). Phosphopeptides were isolated on p81 filters (Pierce), washed in ice-cold 75 mM phosphoric acid, and assayed by Cerenkov counting. In order to detect kinetics of phosphorylation of GST-HSF1 by MK2, fusion protein GST-HSF1 was purified from bacterial lysates as described (4), in vitro kinase assay were performed with 5µg GST-HSF1 in a 30µl volume of reaction buffer with 0.2 Unit of MK2 as described above at 300C for 0, 1, 2, 4, 6 hr, and stopped by addition of SDS sample buffer, then analyzed by 10% SDS-PAGE and X-ray film autoradiography. Mass Spectrometric (MALDI-TOF) analysis of Tryptic Peptides In vitro phosphorylation of GST-HSF1 by MK2 was carried out for 4hr as described above with radioisotope, then analyzed by 10% SDS-PAGE. The silver-stained GST-HSF1 bands were cut out and in-gel-digested with sequencing grade modified trypsin (Promega), followed by reverse phase HPLC purification and concentration step as described previously (42). The 32P activity of each fraction was detected using scintillation counting. Molecular masses of 32P-labled HPLC fractions from tryptic peptides were analyzed by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Voyager DESTR).

Cell Culture and Transfection HeLa cells were maintained in HAM’s F-12 (Mediatech) with 10% heat inactivated fetal bovine serum (FBS). Mouse embryonic fibroblasts (MEFs; mk2+/+) (28) were cultured in DMEM and 10% heat inactivated FBS, 100µg of streptomycin/ml (complete medium). HeLa cells (2.5 X 105 cells/well) in 6-well plates were transfected with the plasmids indicated in the Figure legends in triplicate using FuGENE6 (Roche) as described (43). pSV-β-galactosidase plasmid was co-transfected as an internal control for transfection efficiency. pHM6 empty vector was used as a blank plasmid to balance the amount of DNA transfected in transient transfection. Luciferase and β-galactosidase activity assays were performed after 24hr of transfection according to the Promega protocol. Luciferase activity was normalized to β-galactosidase activity. Results were expressed as relative luciferase activity of the appropriate control. Western Analysis Cells were lysed, protein concentration was quantified in the cell lysates by DC protein assay (Bio-Rad) and samples were subjected to SDS-PAGE. Western analysis was performed as described (44). The following antibodies were used: anti-HSF1 monoclonal antibody (Stressgen), anti-HSP90 monoclonal antibody (Stressgen), anti-MK2 polyclonal antibody (Upstate), anti-phospho-MK2 (Thr334) polyclonal antibody (Cell signaling), anti-HA.11 monoclonal antibody (Covance). Electrophoretic Mobility Shift Assay (EMSA) Nuclear extracts were incubated with a double-stranded, 32P-labeled consensus HSE from human HSP70B promoter probe, and analyzed by EMSA as described (39). Gel filtration Whole cell extracts (2 mg) were applied to a 24-ml Superdex 200 HR 10/30 column (Pharmacia)

equilibrated with 20 mM Tris-HCl at pH 7.5, 0.5 M NaCl, 10% glycerol, and 1 mM DTT. Fractions (1-ml column) were collected and analyzed by SDS-PAGE followed by Western blot analysis. The calibration was performed using thyroglobulin (669 kDa), ferritin (440 kDa),

3

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

catalase (232kDa), adolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen

A (25 kDa) as protein standards. Co-Immunoprecipitation HeLa cells (1.2 x 106 cells) were transfected with the plasmids indicated in figure legends using FuGENE6 (Roche) as described (39). After 24 hr of transfection, cells were either untreated or treated with 20 mM sodium salicylate (NaSal) in HEPES buffer, pH 6.8 for 30 min at 370C or heat shocked at 430C for 30 min. Cells were then harvested and lysed using immunoprecipitation buffer [(20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonylfluoride, 1 mM sodium orthovanadate and10 mM NaF)]. Immunoprecipitation was performed as described (39) using anti-HA (Y-11) polyclonal antibody (Santa Cruz), followed by analysis with 10%SDS-PAGE and immunoblot analysis. Generation of Antibodies Specific for HSF1-phosphoserine-121 Antibodies were prepared against HSF1-phosphoserine-121 using the phosphopeptide AcRKVT (pS) VSTC-amide (R7C) that spans the S121 site. Phosphopeptide was coupled to bovine serum albumen and injected into rabbits with 3 subsequent boosting injections. Peptides and antiserum were prepared commercially by Biosource International. Anti-HSF1-phosphoserine 121 antibody (pS121 Ab) was then cleared by passage through an affinity column coupled to unphosphorylated peptide R7C and purified by affinity chromatography on phospho-R7C –agarose.

RESULTS

(1) MK2 inhibits the ability of HSF1 to activate the HSP70B promoter and reduces HSF1 binding to DNA.

We first examined the effects of MK2 activation on the ability of intracellular HA-HSF1 to activate the HSP70B promoter. Overexpression

of HA-HSF1 led to the activation of heat shock promoter (HSP70B) activity and such HSF1 activation of the HSP70B promoter activity was inhibited by 50-60 % when cells were co-transfected with pMyc-MK2 (Fig. 1A). We next examined potential mechanisms underlying the regulation of HSF1 activity by MK2. Our hypothesis predicts that MK2 may be an inhibitor of the first step in HSF1 activation which involves the release of HSF1 from inhibitory complexes and the formation of HSE-binding HSF1 trimers (11, 12). We tested this hypothesis by examining the effects of Myc-MK2 overexpression on the binding of HSF1 in nuclear extracts to heat shock elements (HSE) using EMSA (Fig. 1B). HSF1-HSE binding was assayed in cells transfected with HA-HSF1 as this permitted us to distinguish the transfected HSF1 from endogenous HSF1. Overexpression of HA-HSF1 increased binding to HSE (Fig. 1B, lane 4) and HSF1-HSE complexes could be identified by supershift assay with anti-HA antibody (Fig.1B, lane 5). Previous studies have shown that expression of HSF1 from exogenous promoters can lead to the trimerization and DNA binding of HSF1 in the absence of stress (45). This may be due to the titration of endogenous inhibitors of trimerization or to the increase in HSF1 concentration facilitating the kinetically rare event of trimer formation. The electrophoretic mobility of the HA-HSF1-HSE complexes derived from the transfected cells was similar to the mobility of such complexes extracted from heat shocked cells (used here as a control) (Fig. 1B, lanes 1, 2). The rate of migration of the HA-HSF1-HSE complexes in the gels was further retarded by anti-HA antibody binding (Fig. 1B, lane 5), migrating at a similar rate to HSF1-HSE complexes from heat shocked cells bound to anti-HSF1 antibody (Fig. 1B, lane 2). Overexpression of Myc-MK2 strongly inhibited HA-HSF1 binding to HSE whether incubated without or with anti-HA antibody (Fig. 1B, lanes 6, 7). We also examined the physical association of overexpressed Myc-MK2 with HA-HSF1. Myc-MK2 overexpression led to its activation as indicated by phosphorylation on threonine 334 (data not shown). Such active, overexpressed Myc-MK2 became bound to HA-HSF1 and was recovered in anti-HA immunoprecipitates and detected by immunoblot assay with antibodies specific for the Myc tag (total Myc-MK2) or

4

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

phospho-MK2 (pThr334; Thr334-phosphorylated Myc-MK2) (Fig. 1C). Sodium salicylate (NaSal) appeared to reduce pThr334 levels. The reason for this is not clear although in similar studies carried out on RSK2, NaSal reduced RSK2 autophosphorylation by unknown mechanisms (27). In control cells in which HA-HSF1 was not transfected, we did not observe phospho-MK2 in the HA immunoprecipitates (Fig 1C). (2) MK2 directly phosphorylates HSF1

We next examined the hypothesis that HSF1 is a direct substrate for phosphorylation by MK2 and that this phosphorylation may mediate the inhibitory effects of MK2 (Fig. 2A). When a purified GST-HSF1 fusion protein was incubated with MK2 in vitro, we observed 32P-incorporation into GST-HSF1 in a time-dependent manner that reached a maximum by 4 hr. We estimate incorporation at this point at around 1.3 moles of orthophosphate per mole of GST-HSF1 (Fig 2A). However as the peptide mapping analysis shown later indicates phosphorylation of GST-HSF1 is at at least 6 sites, phosphorylation of individual serines / threonines is likely to be sub-stoichiometric. We next investigated whether this interaction could be inhibited by NaSal. We incubated GST-HSF1 with purified MK2 and determined the effect of incubation with 20mM NaSal on 32P-incorporation. This concentration of NaSal inhibited 32P-incorporation by approximately 50% (Fig. 2B, compare lanes 2 and 3). We next examined in more detail whether MK2 activity is inhibited by the doses of NaSal that activate the binding of HSF1 to the hsp promoters in vivo (22,23) by using a well-characterized substrate peptide derived from the hsp27 sequence (32). The experiments indicate that MK2 activity is inhibited in a dose-dependent manner, with approximately 50% inhibition at a concentration of 20 mM, the concentration that inhibits HSF1 phosphorylation (Fig. 2B, C) and activates the binding of HSF1 to HSE in vivo (22,23). We next investigated the identity of the amino acid residues within HSF1 phosphorylated by MK2. GST-HSF1 was phosphorylated by MK2 as in Fig. 2A in the presence of [γ-32P] ATP

for 4hr at 300C in vitro, then analyzed by SDS-PAGE and visualized by silver staining as in Fig. 2D. (GST, unconjugated to HSF1 was not

phosphorylated in these circumstances Fig. 2D). The non-phospho-HSF1 (control, Fig. 2D, upper panel, lane 3) and phospho-HSF1 (Fig. 2D, upper panel, lane 4) bands were excised from the gel and in-gel-digested with sequencing grade-modified trypsin, followed by reverse phase HPLC purification and concentration. Radioactivity in each HPLC fraction was determined by liquid scintillation counting, 32P-containing fractions eluted by HPLC were then analyzed by MALDI-TOF mass spectrometry (Fig. 2D, E). Two major phosphopeptide species were resolved by this analysis, including one (118-127) with the sequence (R)KVTSVSTLK(S) containing four potential phosphorylation sites and a second peptide 523-529 (K)AKDPTVS(-) from the extreme C-terminus of HSF1 containing T527 and S529 (Fig. 2E, F). Only one of these potential phosphate acceptor sites, serine 121 resembles a consensus site for MK2 phosphorylation (H-X-R/K-X-X-S/T) (Fig. 2F). This motif resembles a MK2 site in possessing an upstream lysine residue in -3 position and hydrophobic residue at –6 relative to serine 121. The sequence deviates from the consensus derived in studies using synthetic peptides and purified MK2 carried out by Stokoe et al (1993) in that I-115 is at the –6 position relative to serine 121 rather than – 5 as would be predicted from the consensus (Fig. 2F). Serine 121 is highly conserved across mammalian and avian species, consistent with an important role in HSF1 regulation (Fig. 2G). This sequence was not conserved in yeast or Drosophila HSF1 suggesting that this form of regulation may be confined to vertebrates. Our MS analysis carried out in duplicate therefore indicates MK2 phosphorylation of HSF1 in vitro at these sites only. Previous analysis of HSF1 phosphorylation by MK2 by two dimensional peptide mapping also indicated phosphorylation in peptides corresponding in chromatographic behavior to the peptides indicated in Fig 2F (B. Chu & S.K. Calderrwood, unpublished data). (3) Role of serine 121 in repression of HSF1 by MK2

We next examined the potential functional roles of all the serine and threonine residues in HSF1 that we identified as MK2 regulated phosphoacceptor sites. We examined their potential role in regulation by point mutations of

5

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

residues corresponding to these phospho Ser / Thr residues characterized by MALDI-TOF (Fig. 2; Table 1A, B). We also constructed HSF1 mutants with amino acid substitutions at multiple sites, as the MALDI-TOF analysis suggested that some of the phosphopeptides isolated after MK2 treatment are multiply phosphorylated (Table 1A). The HSF1 mutant proteins were initially tested for their ability to activate the transcription of a hsp70B-based promoter reporter construct after co-transfection into cells in vivo, as described in Fig. 1A. The HSP70B promoter was strongly inhibited when cells were co-transfected with wild-type HSF1 and Myc-MK2, consistent with the result in Fig. 1A, while overexpressed dominant negative MK2 (DN) failed to inhibit HSF1 (Fig. 3A). Transactivation of HSF1 was enhanced by mutation of serine 121 to alanine and the activity of the S121A construct was not inhibited by MK2 overexpression, suggesting that Ser121 mediates MK2 inhibition of HSF1 activity (Table 1A, Fig. 3A). Mutation of Ser 121 to aspartate (S121D), in an effort to mimic phospho-Ser121-HSF1, led to an HSF1 molecule with diminished capacity for HSF1 activation (Fig. 3A), consistent with the hypothesis that negative charge at Ser121 may be inhibitory for HSF1 activity. Mutations at other potential MK2 phosphorylation sites within peptide VTSVSTLK to yield T120A, S123A, T124A were not effective and the mutants behaved essentially as wild-type HSF1 (Table 1A). Likewise the T527A and S529A mutants behaved like wild-type HSF1 (Table 1A). When multiple mutations containing the S121A mutant were prepared, we found that all behaved essentially as the S121A single mutant, and these included T120A/S121A, S121A/S123A, T120A/S121A/S123A, T120A/S121A/T527A, T120A/S121A/S529A (Table 1A). In addition, the T527A/S529A double mutant behaved like wild-type HSF1 (Table 1A). Thus, although multiply phosphorylated HSF1 peptides can be isolated from HSF1 after in vitro phosphorylation by MK2, these changes appear to have little significance for HSF1 regulation in vivo. Only Ser121 appears to play a significant role in the response of HSF1 to MK2 in vivo (Table 1A). We also screened the mutant forms of HSF1 for ability to bind to HSE in the in vitro EMSA conditions described in Fig. 1B. EMSA was carried out on nuclear extracts from cells after overexpression of wild type HSF1

and mutants in vivo (Table 1B). The results of these experiments paralleled the findings in the trans-activation experiments (Table 1A), indicating that all constructs with the exception of S121A mutants behave in an essentially similar manner to wild type HSF1 (Table 1A, B). The effects of S121 mutation on HSF1-HSE binding are shown in Fig. 3B. Wild-type HA-HSF1 is induced to bind HSE after overexpression in cells in vivo and its identity validated by anti-HA supershift (Fig. 3B, lanes 4, 5). Binding of HSF1 to HSE is enhanced by the mutation HA-S121A and the intensity of the supershifted band is strongly enhanced (Fig. 3B, lanes 6, 7). Binding of HSF1 to HSE was partially inhibited by HA-S121D mutation and the intensity of the supershifted band markedly reduced (Fig. 3B, lanes 8, 9). These effects are not as apparent in the non-supershifted complex and this may be because this complex contains both endogenous wild-type HSF1 as well as HA-S121D, whereas the anti-HA antibody supershifted complex only contains HA-S121D free of endogenous HSF1 (Fig. 3B, lanes 8, 9). Our data indicate a role for serine 121 in the repression of HSF1 binding to HSE and the HSP70 promoter activity by MK2. It is significant that this site has recently been shown to be phosphorylated in HSF1 in human cells in vivo (46). Although those studies do not explore the potential regulatory role of serine 121 in HSF1 activity in vivo, they do concur with the present study that this site is one of the serine residues in HSF1 that can be phosphorylated under physiological conditions. (4) Phosphorylation of HSF1 on serine 121 promotes HSP90 binding to HSF1. Ability of HSF1 to bind DNA is regulated by reversible binding to HSP90 as well as a number of co-chaperones, and HSP90 complexes are thought to maintain HSF1 in an inactive, non-DNA binding form (13,20). We have therefore examined the hypothesis that phosphorylation of HSF1 by MK2 may influence HSP90 binding and thus repress HSF1. When we examined the distribution of HSF1, HSP90 and MK2 by gel filtration, we found some overlap in their retention by the column. The hydrodynamic behavior of HSF1 from whole cell extracts indicates its association in complexes of median Mr 440-232 kDa (Fig. 4A), consistent with a previous report

6

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

(47). Although HSP90 fractions tended toward higher Mr fractions (median 440 kDa), and MK2 fractions tended to a smaller complex (<158 kDa) (Fig. 4A), two areas of potential HSP90 and MK2 overlap with HSF1 were observed (Fig. 4A, at fraction 45, 83, lanes 4, 15), that could correspond to an HSF1-HSP90-MK2 complex.

We next carried out co-immunoprecipitation experiments from cells overexpressing FLAG-HSF1 with or without Myc-MK2 or Myc-MK2 (DN) to analyze the potential interaction of FLAG-HSF1 and endogenous HSP90 (Fig. 4B). Previous studies have indicated that co-immunoprecipitation of HSF1 and HSP90 is difficult to demonstrate evidently due to the transient nature of the interaction (48). However we were able to detect FLAG-HSF1 co-immunoprecipitation with HSP90 under basal conditions, presumably due to the increased levels of overexpressed FLAG-HSF1 (Fig. 4B, lanes 2). Overexpression of Myc-MK2 led to increased FLAG-HSF1 / HSP90 binding above the endogenous level (Fig. 4B, compare lanes 2 and 3, anti-HSP90 immunoblot) but no changes were seen when Myc-MK2 (DN) were overexpressed (Fig. 4B, lane 4). No FLAG-HSF1 / Myc-MK2 co-association was seen if the immunoprecipitation was carried out under denaturing conditions (data not shown) performed as described (39), indicating true co-immunoprecipitation of the molecules under native conditions (Fig. 4B, lane 2). As NaSal is an MK2 inhibitor (Fig. 2), we next examined its role in HSF1-HSP90 binding (Fig. 4C). We used here HA-HSF1 rather than FLAG-HSF1 to ensure that the results were not influenced by the type of affinity tag used in the experiments. Incubation of cells with 20mM NaSal led to an approximate 50 % decrease in HA-HSF1 binding to endogenous HSP90, consistent with a role for MK2 in enhancing the association of HSF1 with HSP90 (Fig. 4C, D, compare lanes 3 and 4, anti-HSP90 immunoblot). To test a role for serine 121 in these effects, we then examined the effect of S121A mutation on HSP90 binding. HSP90 binding to HSF1 was markedly reduced with S121A mutation (Fig. 4C, D, lane 7, anti-HSP90 immunoblot). However the S121D mutant form of HSF1 bound effectively to HSP90, indicating a role for negative charge in the 121 position in HSP90 binding (Fig. 4C, D, lane 8, anti-HSP90 immunoblot). Overexpression of Myc-

MK2 increases the binding of HSF1 to HSP90 (Fig. 4C, D, compare lanes 3 and 5, anti-HSP90 immunoblot), consistent with the result in Fig. 4B. We also examined the association of Myc-MK2 with HA-HSF1. We found strong binding between HSF1 and MK2 after overexpression of Myc-tagged MK2 (Fig. 4C, D, lane 5, anti-Myc-MK2 immunoblot). In contrast to HSP90, S121A mutation did not inhibit binding of Myc- MK2 to HSF1 and the association was, if anything enhanced (Fig. 4C, D, lane 7, anti-Myc-MK2 immunoblot). S121D mutation by contrast inhibited Myc-MK2 binding to HSF1 (Fig. 4C, D, lane 8, anti- Myc-MK2 immunoblot). These experiments suggest that HSP90 and MK2 bind independently to HSF1 and may under certain circumstances compete for binding, and that MK2 may bind preferably to HSF1 when serine 121 is unphosphorylated or that HSP90 binding may hinder HSF1 binding to MK2.

In order to examine more directly the role of serine 121 in HSF1 activity, we prepared an antibody that specifically reacts with the phospho-serine 121-HSF1. As can be seen, the immunoblot experiments using anti-phospho-S121 antibodies indicate that immunoprecipitated HA-HSF1 is constitutively phosphorylated on serine 121 and this activity is inhibited by NaSal and stimulated by approximately 20 % through Myc-MK2 overexpression (Fig. 4 E, F, lanes 3-6). In cells transfected with HA-S121A, or HA-S121D, little evidence of immunoreactivity is seen with or without addition of NaSal, indicating the specificity of the antibody for phospho-S121 (Fig. 4E, F, lanes 7-10). The degree of phosphorylation of HSF1 on serine 121 thus correlates well with its ability to bind to HSP90 (Fig. 4). This small increase in HSF1 phosphorylation appears to be sufficient to inhibit the DNA binding and trans-activation of transfected HA-HSF1 (Fig 1A, B). It is known that only a small fraction of transfected HSF1 is able to bind HSE and activate hsp transcription and this fraction appears to be the fraction that is not phosphorylated at serine 121 or bound to hsp90 (45, 49). Forced expression of MK2 appears to lead to phosphorylation of free HSF1 on serine 121 and this is associated with hsp90 binding and inhibition of HSE binding (Fig 4B, 4E).

7

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

(5) Heat shock can override the effects of MK2 on HSP90 binding to HSF1

When cells are exposed to heat shock, HSP90 rapidly dissociates from HSF1 permitting HSF1 to migrate to the nucleus and activate HSP promoters and repress cytokine genes despite the fact that MK2 is strongly activated by the same heat shock conditions (30). We therefore examined whether heat shock can overturn the ability of MK2 to stabilize HSF1-HSP90 interactions. Indeed, we observed rapid dissociation of HSF1 from HSP90 in heat shocked cells even in the presence of overexpressed Myc-tagged MK2 (Fig. 5A). Exposure to heat shock decreased the association of the Myc-MK2 with HSF1 and led to dephosphorylation of S121 (Fig. 5A). Dephosphorylation of S121 may thus participate in the rapid escape of HSF1 from HSP90 mediated repression observed during heat shock (Fig. 5A).

As many of these experiments have been carried out on HSF1 overexpressed in cells in order to permit the examination of serine121 mutants, we also carried out studies on the role of S121 phosphorylation in endogenous HSF1 (Fig 5B). HeLa cells were either exposed to sodium salicylate or heat shock and then analyzed by immunoprecipitation with anti-phospho-S121 antibodies. In control cells endogenous HSF1 was immunoprecipitated by the antibody while NaSal or heat treatment markedly reduced the degree of immunoprecipitation indicating S121 phosphorylation under resting conditions and significant loss of S121 phosphorylation under these circumstances (Fig 5B).

DISCUSSION

Our experiments suggest a mechanism whereby inflammatory signals can repress HSF1 through downstream convergence on MK2 and inhibition of endogenous hsp expression in monocytes and macrophages. We show that MK2 directly inhibits HSF1 activity HSF1 through a mechanism involving phosphorylation on serine 121 and that anti-inflammatory drugs can antagonize this effect and activate HSF1 by direct inhibition of MK2 (Fig. 1, 2). Our experiments suggest that MK2 exerts its effects at least partially by phosphorylation of HSF1 on S121 and by inhibiting association of the phosphorylated

HSF1 with HSE in hsp gene promoters (Fig. 1, 3). MK2 can thus, by inhibiting HSF1 effectively repress endogenous hsp expression. We have also identified a potential mechanism whereby HSF1 phosphorylation on serine 121 by MK2 regulates HSF1. Previous studies have shown that HSF1 is maintained in an inactive monomeric form by its association with inhibitory protein complexes containing HSP90 (13). Our current studies suggest that HSF1 regulation by serine 121 phosphorylation involves this mode of control as HSF1 binding to HSP90 is enhanced by serine 121 phosphorylation or faux-phosphorylation through serine 121 to aspartate mutation and decreased when HSF1-HSP90 binding is reduced by NaSal treatment and completely inhibited by serine121-alanine mutation (Fig. 4). Constitutive repression of HSF1 under resting non-stress conditions has been previously ascribed to the cooperative engagement of two types of regulation, including: (1) intramolecular masking of the trimerization domains and (2) binding of the inhibitory HSP90 containing molecular chaperone complex (12, 14, 15). HSF1 activation by stress was suggested to involve the sequestration of HSP90 in protein aggregates generated by protein damage (13). The addition of a third component, involving serine 121, to these regulatory interactions may offer sharper, more flexible transcriptional control of HSF1 activity and greater responsiveness to physiological circumstances such as activation of the acute phase response. Previous studies show that the linker region enclosing Ser121 controls the monomer to trimer transition (50). In addition, serine 121 is immediately adjacent to one of the bipartite nuclear localization signals in HSF1 (116-118) and HSP90 binding adjacent to serine 121 could act to mask this domain (51). In the acute phase response, cytokine expression is ultimately followed by repression in the resolution of the response (52, 53). This may involve the febrile activation of HSF1 which can repress cytokines directly by binding to their promoters or indirectly by the induction of hsp70, an inhibitor of the proinflammatory transcription factor NFκB (54). Under these circumstances, MK2 can be overridden by two potential mechanisms. Firstly, accumulation of denatured / aggregated proteins that sequester hsp90 and activate HSF1 independently of S121 phosphorylation (55, 56). Secondly, our data show that S121 becomes at

8

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

least partially dephosphorylated by stress even when MK2 is active (Fig 5B). However, the exact role of S121 phosphorylation in HSF1 activation by stress is not yet clear, and more information is required to determine the quantitative levels of S121 phosphorylation after stress and whether S121 dephosphorylation (as in Fig 5) plays a causal role. It is also possible that the effects of MK2 on HSF1 may also involve other cellular effects of MK2, which is a pleiotropic factor involved in a range of cellular processes (57, 58). Inhibition of HSF1 by inflammatory signals could also involve additional phosphorylation sites in HSF1. LPS can lead to the activation of the ERK cascade, and ERK activation has been shown to lead to repression of HSF1 through inhibitory phosphorylation site serine 307 which can lead to the nuclear export of HSF1 (59-62).

HSP90 has been shown to interact with three classes of polypeptides in the cell, including: (a) co-chaperones such as p23 and FKBP52, (b) client proteins that bind stably to HSP90, most notably protein kinases and steroid hormone receptors and (c) unfolded proteins and peptides

that are chaperoned by HSP90 (63,64). HSF1 is similar to other HSP90 client proteins in that it forms a super-complex with HSP90, immunophilins and p23 that is functionally inactive (13, 20). Its interaction however differs in quality from many other clients in that dissociation from HSP90 during heat shock or treatment with HSP90 inhibitors does not destabilize HSF1 and does not target it for destruction as with a multitude of other substrates (65). It therefore seems that the HSP90 complex functions as a reversible HSF1 repressor and is not needed for HSF1 stabilization. The physicochemical nature of the interaction may thus be different from other HSP90 clients such as steroid hormone receptors in which the chaperone domains of HSP90 bind a disordered / unstable region of the receptor (65, 66). However, it is also possible that other HSP90 binding proteins may be regulated in a phosphorylation dependent manner like HSF1, as observed recently for the asialoglycoprotein receptor (67). Serine 121 is located in the linker domain of HSF1, between the DNA binding domain and the first coiled-coil region (50).

REFERENCES

1. Wu, C. (1995) Annu.Rev.Cell Dev.Biol. 11, 441-469 2. McMillan, D. R., Xiao,X., Shao,L., Graves,K., and Bejamin,I.J. (1998) J.Biol.Chem. 273, 7523-

7528 3. Cahill, C. M., Waterman, W. R., Auron, P. E., and Calderwood, S. K. (1996) J Biol Chem 271,

24874-24879 4. Xie, Y., Zhong, R., Chen, C., and Calderwood, S. K. (2003) J Biol Chem. 278, 4687-4698 5. Gutsmann-Conrad, A., Heydari, A. R., You, S., and Richardson, A. (1998) Exp Cell Res 241,

404-413 6. Heydari, A. R., You, S., Takahashi, R., Gutsmann-Conrad, A., Sarge, K. D., and Richardson, A.

(2000) Exp Cell Res 256, 83-93 7. Hsu, A. L., Murphy, C. T., and Kenyon, C. (2003) Science 300, 1142-1145 8. Ciocca, D. R., Clark, G. M., Tandon, A. K., Fuqua, S. A., Welch, W. J., and McGuire, W. L.

(1993) J Natl Cancer Inst 85, 570-574 9. Cornford, P. A., Dodson, A. R., Parsons, K. F., Desmond, A. D., Woolfenden, A., Fordham, M.,

Neoptolemos, J. P., Ke, Y., and Foster, C. S. (2000) Cancer Res 60, 7099-7105 10. Nollen, E. A., and Morimoto, R. I. (2002) J Cell Sci 115, 2809-2816. 11. Rabindran, S. K., Gioorgi, G., Clos, J., and Wu, C. (1991) Proc Natl Acad Sci U S A 88, 6906-

6910 12. Zuo, J., Rungger, D., and Voellmy, R. (1995) Mol. Cell. Biol. 15, 4319-4330 13. Zuo, J., Guo, Y., Guettouche,T., Smith, D., and Voellmy, R. (1998) Cell 94, 471-480 14. Westwood, J. T., and Wu, C. (1993) Mol. Cell. Biol. 13, 3481-3486 15. Rabindran, S. K., Haroun, R.I., Clos, J., Wisniewski, J., and Wu, C. (1993) Science 259, 230-234 16. Xie, Y., Chen, C., Stevenson, M. A., Auron, P. E., and Calderwood, S. K. (2002) J Biol Chem

277, 11802-11810

9

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

17. Sarge, K. D., Murphy, S.P., and Morimoto, R.I. (1993) Mol. Cell. Biol. 13, 1392-1407 18. Hensold, J. O., Hunt, C. R., Calderwood, S. K., Houseman, D. E., and Kingston, R. E. (1990) Mol

Cell Biol 10, 1600-1608 19. Price, B. D., and Calderwood, S. K. (1991) Mol Cell BIol 11, 3365-3368 20. Bharadwaj, S., Ali, A., and Ovsenek, N. (1999) Mol Cell Biol 19, 8033-8041 21. Pratt, W. B., Galigniana, M. D., Harrell, J. M., and DeFranco, D. B. (2004) Cell Signal 16, 857-

872 22. Jurvivich, D. A., Sistonen, L., Kroes, R. A., and Morimoto, R. I. (1992) Science 255, 1243-1245 23. Housby, J. N., Cahill, C. M., Chu, B., Prevelege, R., Bickford, K., Stevenson, M. A., and

Calderwood, S. K. (1999) Cytokine 11, 347-358 24. Brooks, P. M., and Day, R. O. (1991) N Engl J Med 324, 1716-1725 25. Borne, R. F. (1995), "Nonsteroidal Anti-inflammatory Drugs" in "Principles of medicinal

Chemistry", 4th edition, W.O. Foye, Ed., Williams & Wilkins, Baltimore, pp. 535-580 26. Palayoor, S. T., Youmell, M. Y., Calderwood, S. K., Coleman, C. N., and Price, B. D. (1999)

Oncogene 18, 7389-7394 27. Stevenson, M. A., Zhao, M. J., Asea, A., Coleman, C. N., and Calderwood, S. K. (1999) J

Immunol 163, 5608-5616 28. Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H. D., and Gaestel,

M. (1999) Nat Cell Biol 1, 94-97 29. Soncin, F., and Calderwood, S. K. (1996) Biochem Biophys Res Commun 229, 479-484 30. Rouse, J., Cohen, P., Trigon, S., Morange, M., Llamazares, A. A., Zamarillo, D., Hunt, T., and

Nebreda, A. (1994) Cell 78, 1027-1037 31. Stokoe, D., Campbell., D. G., Nakielny, S., Hidaka, H., Leevers, S. L., Marshall, C., and Cohen,

P. (1992) EMBO J . 11, 3985-3994 32. Stokoe, D., Caudwell, B., Cohen, P. T. W., and Cohen, P. (1993) Biochem J 296, 843-849 33. Dalal, S. N., Schweitzer, C.M., Gan, J., and Decaprio, J. (1999) Mol.Cell. Biol. 19, 4465-4479 34. Beyaert, R., Cuenda, A., Vanden Berghe, W., Plaisance, S., Lee, J. C., Haegeman, G., Cohen, P.,

and Fiers, W. (1986) EMBO J 15, 1914-1923 35. Degousee, N., Stefanski, E., Lindsay, T. F., Ford, D. A., Shahani, R., Andrews, C. A., Thuerauf,

D. J., Glembotski, C. C., Nevalainen, T. J., Tischfield, J., and Rubin, B. B. (2001) J Biol Chem 276, 43842-43849

36. Neininger, A., Kontoyiannis, D., Kotlyarov, A., Winzen, R., Eckert, R., Volk, H. D., Holtmann, H., Kollias, G., and Gaestel, M. (2002) J Biol Chem 277, 3065-3068

37. Werz, O., Szellas, D., Steinhilber, D., and Radmark, O. (2002) J Biol Chem 277, 14793-14800 38. Singh, I. S., He, J. R., Calderwood, S., and Hasday, J. D. (2002) J Biol Chem 277, 4981-4988 39. Wang, X., Grammatikakis, N., Siganou, A., and Calderwood, S. K. (2003) Mol Cell Biol 23,

6013-6026 40. Chen, C., Xie, Y., Stevenson, M. A., Auron, P. E., and Calderwood, S. K. (1997) J Biol Chem

272, 26803-26806 41. Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C.Y., Shyu, A.B., Muller, M., Gaestel, M.,

Resch, K., Holtmann, H. (1999) EMBO J. 18 42. Shevchenko, A., Wilm,M., Vorm, O., and Mann, M. (1996) Anal. Chem. 68, 850-858 43. Wang, X. Z., Asea, A., Xie,Y., Kabingu,E., Stevenson, M.A., and Calderwood, S.K. (2000) Cell

Stress & Chaperones 5, 432-437 44. Stevenson, M. A., and Calderwood, S. K. (1990) Mol Cell Biol 10, 1234-1238 45. Zuo, J., Rungger, D., and Voellmy, R. (1995) Mol Cell Biol 15, 4319-4330 46. Guettouche, T., Boellmann, F., Lane, W. S., and Voellmy, R. (2005) BMC Biochem 6, 4 47. Nakai, A., Kawazoe, Y., Tanabe, M., Nagata, K., Morimoto, R.I. (1995) Mol Cell Biol., 5268-

5278 48. Guo, Y., Guettouche, T., Fenna, M., Boellmann, F., Pratt, W. B., Toft, D. O., Smith, D. F., and

Voellmy, R. (2001) J. Biol. Chem. 276, 45791-45799

10

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

49. Zuo, J., Baler, R., Dahl, G., and Voellmy, R. (1994) Mol Cell Biol 14, 7557-7568 50. Liu, P. C., and Thiele, D. J. (1999) J Biol Chem 274, 17219-17225 51. Sheldon, L. A., and Kingston, R. E. (1993) Genes & Development 7, 1549-1558 52. Gewert, K., Svensson, U., Andersson, K., Holst, E., and Sundler, R. (1999) Cell Signal 11, 665-

670 53. Knudsen, P. J., Dinarello, C. A., and Strom, T. B. (1987) J Immunol 139, 4129-4134 54. Calderwood, S. (2005) Methods 23, 139-148 55. Bharadwaj, S., Ali, A., and Ovsenek, N. (1999) Mol. Cell. Biol. 19, 8033-8041 56. Zou, J., Guo, Y., Guettouche, T., Smith, D. F., and Voellmy, R. (1998) Cell 94, 471-480 57. Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H. D., and Gaestel,

M. (1999) Nat Cell Biol 1, 94-97 58. Neininger, A., Kontoyiannis, D., Kotlyarov, A., Winzen, R., Eckert, R., Volk, H. D., Holtmann,

H., Kollias, G., and Gaestel, M. (2002) J Biol Chem 277, 3065-3068 59. Chu, B., Zhong, R., Soncin, F., Stevenson, M. A., and Calderwood, S. K. (1998) J. Biol. Chem.

273, 18640-18646 60. Wang, X., Grammatikakis, N., Siganou, A., Stevenson, M. A., and Calderwood, S. K. (2004) J

Biol Chem 61. Wang, X., Grammatikakis, N., Siganou, A., and Calderwood, S. K. (2003) Mol Cell Biol 23,

6013-6026 62. Calderwood, S. K. (2005) Methods 35, 139-148 63. Scheibel, T., Weikl, T., and Buchner, J. (1998) Proc Natl Acad Sci U S A 95, 1495-1499 64. Buchner, J. (1999) TIBS 24, 136-142 65. Neckers, L., and Ivy, S. P. (2003) Curr Opin Oncol 15, 419-424 66. Citri, A., Kochupurakkal, B. S., and Yarden, Y. (2004) Cell Cycle 3, 51-60 67. Huang, T., Deng, H., Wolkoff, A.W., Stockert, R.J. (2002) J Biol Chem. 277, 37798-37803 68. Wang, X., Grammatikakis, N., Siganou, A., Stevenson, M. A., and Calderwood, S. K. (2004) J

Biol Chem. 279, 32651-32659

ACKNOWLEDGMENTS

We thank Drs Jacques Landry, Fabrice Soncin and Boyang Chu for help and advice in the early stages of this study and Jim Lee for performing the HPLC and mass spectrometry. We thank Dr Richard Voellmy for the kind gift of materials. Competing interests statement: The authors declare that they have no competing financial interests.

FIGURE LEGENDS FIG. 1. Effects of MK2 overexpression on the transcriptional activation of the HSP70B promoter and HSF1-HSE binding activity. A, HeLa cells were co-transfected in triplicate with the HSP70B promoter luciferase reporter plasmid pGLHSP70B and expression plasmids pHA-HSF1wt, with or without pMyc-MK2; pCMV-lacZ plasmid was co-transfected into each culture as an internal control for transfection efficiency. Cells were harvested after 24hr of transfection and proteins were extracted and assayed for corrected luciferase activity as described in MATERIALS AND METHODS. Relative luciferase activity was then expressed as a percentage of the activity in cells co-transfected with pGLHSP70B and HSF1wt. Relative luciferase values shown in the histogram are the mean ± SD from three independent experiments. B, HeLa cells were transfected with empty vector pHM6 (lane 3), with pHA-HSF1 alone (lanes 4 and 5), or with pHA-HSF1 and Myc-MK2 (lanes 6 and 7). Nuclear extracts were then prepared after 24hr of transfection, and analyzed by EMSA. Gel supershift assay with anti-

11

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

HSF1 and anti-HA antibodies was carried out as described in (39) (lanes 2, 5, 7). Nuclear extracts from heat shocked (HS) HeLa cells (lanes 1 and 2) were used as positive controls to demonstrate the migration of HSF1-HSE complexes. Equal loading of nuclear extracts in lanes 3-7 in the incubations was verified by Western blot analysis with anti-HA antibody. C, HeLa cells were transfected with the indicated plasmids for 24 hr then exposed to 20 mM sodium salicylate (NaSal) for 30 min (lanes 2, 4, 6 and 8). Immunoprecipitation was carried out with anti-HA (Y-11) polyclonal antibody as described in MATERIALS AND METHODS. Immunoprecipitated materials were then immunoblotted with anti-phospho-MK2 (Thr334) polyclonal antibody, anti-Myc monoclonal antibody or anti-HA monoclonal antibody. Heavy chain IgG was used as a control loading. FIG. 2. MK 2 directly phosphorylates HSF1 in vitro. A, GST-HSF1 fusion protein was incubated with purified MK2 at the different time course (0, 1hr, 2hr, 4hr, 6hr) with [γ-32P] ATP labeling as described in MATERIALS AND METHODS, then the reaction was stopped by addition of SDS sample buffer before analysis by 10% SDS-PAGE and X-ray film autoradiography. Quantitation of relative density (moles phosphate per mole GST-HSF1) from γ-32P-labled GST-HSF1 bands is shown below the panels. B, Phosphorylation of GST-HSF1 fusion protein by purified MK2 with and without 20mM Sodium Salicylate (NaSal). GST-HSF1 was incubated with MK2 as in MATERIALS AND METHODS, and then analyzed by 10% SDS-PAGE and X-ray film autoradiography. C, Dose-response curve for the inhibition of purified MK2 by NaSal. Purified MK2 was incubated for 20 min with specific peptide substrate and 0.5 µCi [γ-32P] ATP as described in MATERIALS AND METHODS. MK2 is inhibited by 50% at the concentration of 20 mM NaSal that activate HSF1 in vivo. Assays were conducted in triplicate and were repeated three times with consistent findings. D, GST-HSF1 fusion protein was incubated without or with purified MK2 in the presence of [γ-32P] ATP for 4hr at 300C, and then analyzed by 10% SDS-PAGE and followed by silver staining. The γ-32P-phosphopeptides digested with sequencing grade- modified trypsin from silver-stained SDS-PAGE were fractionated by reverse phase HPLC as described in MATERIALS AND METHODS. Radioactivity in each fraction was determined by liquid scintillation counting. Arrows indicate 32P-containing fractions eluted by HPLC. E, 32P- phosphopeptides fractions shown by the arrows in (A) are then analyzed by MALDI-TOF. The labeled peaks at m/z 1282.5849 and 877.1530 (indicated by arrows) correspond to two peptides 118-127: (R)KVTSVSTLK(S); 523-529: (K)AKDPTVS(-). F, Summary of the two phosphorylated HSF1 32P- tryptic peptides observed by MALDI-TOF. Only one of the 32P- phosphopeptides which is 118-127 (R)KVTSVSTLK (S) contains phosphorylation consensus sequence of MK2, and serine 121 resembles a consensus site for MK2 phosphorylation. G, Alignment of the sequences surrounding the H-X-R/K-X-X-S/T consensus sequence in human HSF1 with HSF1 sequences in other organisms. FIG. 3. Effects of MK2 overexpression on the transcriptional activation of the HSP70B promoter and HSF1-HSE binding activity by wild-type HSF1 or serine mutants. A, HeLa cells were co-transfected in triplicate with pGLHSP70B and expression plasmids pHA-HSF1 wt, pHA-S121A, pHA-S121D with or without Myc-MK2; Myc-MK2 dominant negative (DN) mutant as indicated. In addition, pCMV-lacZ plasmid was co-transfected into each culture as an internal control for transfection efficiency. Experiments were carried out four times with reproducible findings as described in Figure legend 1A. B, mk2+/+ cells were transfected with empty vector pHM6 (lane 3), with pHA-HSF1 wt alone (lanes 4 and 5), or with pHA-S121A (lanes 6 and 7), pHA-S121D (lanes 8 and 9). Nuclear extracts were then prepared after 24hr of transfection, EMSA and gel supershift assay were carried out as described in Figure legend 1B. Nuclear extracts from heat shocked (HS) HeLa cells (lanes 1 and 2) were used as positive controls to demonstrate the migration of HSF1-HSE complexes. Equal loading of nuclear extracts in lanes 3-9 in the incubations was verified by Western blot analysis of the same nuclear extracts with anti-HA antibody. C, Quantitation of relative HSF1 –HSE binding from (B) is shown using densitometric analysis of the shifted or supershifted bands on the autoradiographs.

12

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

FIG. 4. Role of Serine 121 in HSF1-HSP90 binding. A, Gel filtration profile of HSF1, HSP90 and MK2. Whole cell extracts were analyzed by gel filtration. Fractions (1-ml column) were collected and analyzed by SDS-PAGE followed by Western blot with anti-HSF1, anti-HSP90 and anti-MK2 antibodies. The peaks corresponding to proteins with known Mr are denoted on the top. B, Co-immunoprecipitation of endogenous HSP90 with FLAG-HSF1 in HeLa cells transfected with empty expression vector (lane 1) or pFLAG-HSF1 (lanes 2-4) with pMyc-MK2 (lane 3) or pMyc-MK2 dominant negative (DN) mutant (lane 4). Immunoprecipitates were analyzed by immunoblot with anti-HSP90 and anti-FLAG antibodies. The HSP90 bands are quantitated by densitometry in the lower panel. The whole experiment was repeated twice with consistent findings. C, HeLa cells were transfected with the indicated plasmids for 24 hr and then treated with 20 mM NaSal for 30 min (lanes 2, 4 and 6). Whole cell extracts were subjected to immunoprecipitation with anti-HA (Y-11) polyclonal antibody and then immunoblotted with anti- HSP90, anti-Myc and anti-HA antibodies. The heavy chain IgG band was used as a loading control. The experiment was repeated twice with consistent findings. D, Relative levels of HSP90 and Myc-MK2 in the HA-HSF1 immunoprecipitate were quantitated by densitometry. E, HeLa cells were transfected with the indicated plasmids. After 24hr, transfected cells were treated with 20 mM NaSal for 30 min. Immunoprecipitation was carried out using anti-HA (Y-11) polyclonal antibody. Immunoprecipitated materials were then analyzed by Western blot using anti-phospho-S121 and anti-HA antibodies. Experiments were carried out in duplicate with similar findings. F, Quantitation of relative density of phospho-HSF1 (pS121) from (A) is shown. FIG. 5. Effects of heat shock on serine 121 phosphorylation. A. HeLa cells were transfected with the indicated plasmids for 24 hr, transfected cells were either untreated or treated with heat shock at 430C, 30 min. Whole cell extracts were immunoprecipitated with anti-HA (Y-11) antibody as indicated. Immune complexes were analyzed by SDS–PAGE, and immunoblotted with anti-HSP90, anti-Myc, anti-phospho-S121 and anti-HA antibodies. The heavy chain IgG band was used as a loading control. Experiments were repeated reproducibly three times. B. Phosphorylation of endogenous intracellular HSF1 on serine 121. HeLa cells were grown in HAM’s F-12 (Mediatech) media with 10% heat inactivated fetal bovine serum (FBS) and heat shocked at 430C for 30 min or exposed to 20 mM sodium salicylate (NaSal) for 30 min. Immunoprecipitation was carried out with anti-phospho HSF1 (serine 121). HeLa lysate (lanes 1-3) and immunoprecipitated materials (lanes 4-6) were then analyzed by Western blot using anti-HSF1 antibody. Experiments were carried out in duplicate with similar findings. The histogram shows relative levels of HSF1 in the anti-phospho HSF1 (serine 121) immunoprecipitate were quantitated by densitometry.

13

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

Table 1. (A). Effects of MK2 overexpression on the transcriptional activation of the HSP70B promoter by wild-type HSF1 and a series of the mutants

Constructs Transcriptional activity on HSP70B promoter

pHA-HSF1wt + +pHA-HSF1wt + Myc-MK2 +pHA-T120A + +pHA-T120A + Myc-MK2 +pHA-S121A + + + + pHA-S121A + Myc-MK2 + + + + pHA-S121D +pHA-S121D + Myc-MK2 +pHA-S123A + +pHA-S123A+ Myc-MK2 + + pHA-T124A + +pHA-T124A + Myc-MK2 + + pHA-T527A + +pHA-T527A+ Myc-MK2 +pHA-S529A + +pHA-S529A+ Myc-MK2 +pHA-T120A/S121A + + + + pHA-T120A/S121A + Myc-MK2 + + + + pHA- S121A/S123A + + + + pHA-S121A/S123A + Myc-MK2 + + + + pHA-T527A/S529A + +pHA-T527A/S529A + Myc-MK2 +pHA-T120A/S121A/S123A + + + + pHA-T120A/S121A/S123A + Myc-MK2 + + + + pHA-T120A/S121A/T527A + + + + pHA-T120A/S121A/T527A + Myc-MK2 + + + + pHA-T120A/S121A/S529A + + + + pHA-T120A/S121A/S529A + Myc-MK2 + + + +

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

(B). Effects of MK2 overexpression on HSF1-HSE binding activity by wild-type HSF1 and a series of the mutants

Constructs HSF1-HSE DNA-binding activitypHA-HSF1wt + +pHA-T120A + +pHA-S121A + + + + pHA-S121D +pHA-S123A + +pHA-T124A + +pHA-T527A + +pHA-S529A + +

pHA-T120A/S121A + + + + pHA- S121A/S123A + + + + pHA-T527A/S529A ND

pHA-T120A/S121A/S123A NDpHA-T120A/S121A/T527A NDpHA-T120A/S121A/S529A ND

* ND: Not determined

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from

Stuart K. CalderwoodXiaoZhe Wang, Md Abdul Khaleque, Mei Juan Zhao, Rong Zhong, Matthias Gaestel and

activity and promotes HSP90 bindingPhosphorylation of HSF1 By MAPKAP kinase 2 on serine 121, inhibits transcriptional

published online November 8, 2005J. Biol. Chem. 

  10.1074/jbc.M505822200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on July 31, 2020http://w

ww

.jbc.org/D

ownloaded from