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A Central Role for the Hsp90·Cdc37 Molecular Chaperone Module in Interleukin-1 Receptor-associated-kinase-dependent Signaling by Toll-like Receptors

2005; Elsevier BV; Volume: 280; Issue: 11 Linguagem: Inglês

10.1074/jbc.m409745200

ISSN

1083-351X

Autores

Dominic De Nardo, Paul Masendycz, Sokwei Ho, Maddalena Cross, Andrew J. Fleetwood, Eric C. Reynolds, John A. Hamilton, Glen M. Scholz,

Tópico(s)

Veterinary medicine and infectious diseases

Resumo

Toll-like receptors (TLRs) serve crucial roles in innate immunity by mediating the activation of macrophages by microbial pathogens. The protein kinase interleukin-1 receptor associated kinase (IRAK-1) is a key component of TLR signaling pathways via its interaction with TRAF6, which subsequently leads to the activation of MAP kinases and various transcription factors. IRAK-1 is degraded following TLR activation, and this has been proposed to contribute to tolerance in macrophages by limiting further TLR-mediated signaling. Using a mass spectrometric-based approach, we have identified a cohort of chaperones and co-chaperones including Hsp90 and Cdc37, which bind to IRAK-1 but not IRAK-4 in 293T cells. Pharmacologic inhibition of Hsp90 led to a rapid decline in the expression level of IRAK-1, whereas overexpression of Cdc37 enhanced the activation and oligomerization of IRAK-1 in 293T cells. Significantly, the inhibition of Hsp90 in macrophages resulted in the destabilization and degradation of IRAK-1 but not IRAK-4. Concomitant with the loss of IRAK-1 expression was a reduction in the activation of p38 MAP kinase and Erk1/2 following stimulation with the bacterially derived TLR ligands, lipopolysaccharide and CpG DNA. Moreover, TLR ligand-induced expression of proinflammatory cytokines was also reduced. Thus we conclude that the level of on-going support provided to IRAK-1 by the Hsp90-Cdc37 chaperone module directly influences the magnitude of TLR-mediated macrophage activation. In addition, because further TLR signaling depends on the synthesis of new IRAK-1, the Hsp90-Cdc37 chaperone module could also contribute to tolerance in macrophages by controlling the rate at which nascent IRAK-1 is folded into a functional conformation. Toll-like receptors (TLRs) serve crucial roles in innate immunity by mediating the activation of macrophages by microbial pathogens. The protein kinase interleukin-1 receptor associated kinase (IRAK-1) is a key component of TLR signaling pathways via its interaction with TRAF6, which subsequently leads to the activation of MAP kinases and various transcription factors. IRAK-1 is degraded following TLR activation, and this has been proposed to contribute to tolerance in macrophages by limiting further TLR-mediated signaling. Using a mass spectrometric-based approach, we have identified a cohort of chaperones and co-chaperones including Hsp90 and Cdc37, which bind to IRAK-1 but not IRAK-4 in 293T cells. Pharmacologic inhibition of Hsp90 led to a rapid decline in the expression level of IRAK-1, whereas overexpression of Cdc37 enhanced the activation and oligomerization of IRAK-1 in 293T cells. Significantly, the inhibition of Hsp90 in macrophages resulted in the destabilization and degradation of IRAK-1 but not IRAK-4. Concomitant with the loss of IRAK-1 expression was a reduction in the activation of p38 MAP kinase and Erk1/2 following stimulation with the bacterially derived TLR ligands, lipopolysaccharide and CpG DNA. Moreover, TLR ligand-induced expression of proinflammatory cytokines was also reduced. Thus we conclude that the level of on-going support provided to IRAK-1 by the Hsp90-Cdc37 chaperone module directly influences the magnitude of TLR-mediated macrophage activation. In addition, because further TLR signaling depends on the synthesis of new IRAK-1, the Hsp90-Cdc37 chaperone module could also contribute to tolerance in macrophages by controlling the rate at which nascent IRAK-1 is folded into a functional conformation. The innate immune system represents the first line of defense of the host against infection by microbial pathogens. Macrophages are a key component of the innate immune system as they have the capacity to secrete inflammatory cytokines (e.g. TNFα 1The abbreviations used are: TNFα, tumor necrosis factor α; IL, interleukin; MAP, mitogen-activated protein; BMMs, bone marrow-derived macrophages; GA, geldanamycin; IRAK, interleukin-1 receptor associated kinase; LPS, lipopolysaccharide; RAD, radicicol; TLR, Toll-like receptor; KD, kinase-dead; HA, hemagglutinin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; Hsc, heat shock cognate protein; IFN-γ, interferon-γ. and IL-1), as well as phagocytose, and degrade microbial pathogens. Subsequent presentation of pathogen-derived peptides to T-helper cells by macrophages is important for the adaptive immune response (1Medzhitov R. Janeway Jr., C.A. Semin. Immunol. 1998; 10: 351-353Crossref PubMed Scopus (303) Google Scholar). 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Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (234) Google Scholar). Notably, IRAK-1-deficient mice are less susceptible to the lethal effects of LPS than their wild-type counterparts (14Swantek J.L. Tsen M.F. Cobb M.H. Thomas J.A. J. Immunol. 2000; 164: 4301-4306Crossref PubMed Scopus (234) Google Scholar). IRAK-1 also participates in signaling by TLR2, TLR5, TLR7, and TLR9 (6Chuang T.H. Lee J. Kline L. Mathison J.C. Ulevitch R.J. J. Leukocyte Biol. 2002; 71: 538-544PubMed Google Scholar, 15Sato S. Takeuchi O. Fujita T. Tomizawa H. Takeda K. Akira S. Int. Immunol. 2002; 14: 783-791Crossref PubMed Scopus (155) Google Scholar, 16Moors M.A. Li L. Mizel S.B. Infect. Immun. 2001; 69: 4424-4429Crossref PubMed Scopus (78) Google Scholar). IRAK-1 is rapidly phosphorylated, ubiquitinated, and then degraded via the proteasome following its activation by TLR ligands or IL-1 (17Hu J. Jacinto R. McCall C. Li L. J. Immunol. 2002; 168: 3910-3914Crossref PubMed Scopus (75) Google Scholar, 18Li L. Cousart S. Hu J. McCall C.E. J. Biol. Chem. 2000; 275: 23340-23345Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 19Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Such degradation of IRAK-1 may represent a negative feedback mechanism to prevent prolonged activation of cells in response to TLR ligands or IL-1. IRAK-1 is also subject to negative regulation via its interaction with Tollip and IRAK-M (20Zhang G. Ghosh S. J. Biol. Chem. 2002; 277: 7059-7065Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar, 21Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar). Tollip forms a complex with IRAK-1 in resting cells and prevents its activation in the absence of an appropriate stimulus (e.g. TLR ligand) (20Zhang G. Ghosh S. J. Biol. Chem. 2002; 277: 7059-7065Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). IRAK-M is a catalytically inactive kinase that suppresses IRAK-1 function by inhibiting phosphorylation of IRAK-1 or preventing its release from the receptor complex (21Kobayashi K. Hernandez L.D. Galan J.E. Janeway Jr., C.A. Medzhitov R. Flavell R.A. Cell. 2002; 110: 191-202Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar). Here we report that the stable expression of functional IRAK-1 in cells was dependent on its interaction with Hsp90. Hsp90 is a molecular chaperone that acts in concert with heat shock cognate protein (Hsc)/Hsp70 and various co-chaperones (e.g. Hop and Cdc37) to fold client proteins (e.g. steroid receptors and protein kinases) into functional conformations (22Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar). Hop serves to coordinate interactions between Hsp90 and Hsc/Hsp70 during client protein folding and is typically associated with "early/intermediate" folding complexes consisting of the client protein, Hsp70 and Hsp90 (22Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Crossref PubMed Scopus (1543) Google Scholar, 23Johnson B.D. Schumacher R.J. Ross E.D. Toft D.O. J. Biol. Chem. 1998; 273: 3679-3686Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Cdc37 specifically stabilizes the interaction of Hsp90 with client protein kinases, thereby promoting the formation of "mature" complexes containing the client protein kinase, Hsp90, and Cdc37 (24Grammatikakis N. Lin J.H. Grammatikakis A. Tsichlis P.N. Cochran B.H. Mol. Cell. Biol. 1999; 19: 1661-1672Crossref PubMed Scopus (229) Google Scholar, 25Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (181) Google Scholar, 26Roe S.M. Ali M.M. Meyer P. Vaughan C.K. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. Cell. 2004; 116: 87-98Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 27Siligardi G. Panaretou B. Meyer P. Singh S. Woolfson D.N. Piper P.W. Pearl L.H. Prodromou C. J. Biol. Chem. 2002; 277: 20151-20159Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 28Stepanova L. Leng X. Parker S.B. Harper J.W. Genes Dev. 1996; 10: 1491-1502Crossref PubMed Scopus (448) Google Scholar). The client protein kinase is subsequently released from the chaperone complex following its folding into a functional conformation. Inhibition of Hsp90 in macrophages resulted in the rapid loss of IRAK-1 expression and culminated in the impaired ability of TLR4 and TLR9 ligands to activate p38 MAP kinase and Erk1/2 and to stimulate the expression of inflammatory cytokines. Thus Hsp90 may play an important role in controlling the inflammatory response of macrophages by directly modulating the stability and hence signal transducing capacity of IRAK-1. Reagents—Geldanamycin, LPS (Escherichia coli 0111:B4) and anti-FLAG, horseradish peroxidase-conjugated anti-FLAG, and agarose-coupled anti-FLAG antibodies were obtained from Sigma. Radicicol was obtained from Calbiochem, and the murine CpG oligonucleotide ODN1860 was from InvivoGen. The anti-Hsp90 antibody was from Affinity BioReagents, Inc. The anti-IRAK-1 antibody was obtained from Santa Cruz Biotechnology, Inc., and the anti-IRAK-4 antibody was from UBI, Inc. Anti-phospho-p38 MAP kinase, anti-p38 MAP kinase, anti-phospho-Erk1/2, and anti-Erk1 antibodies were from Cell Signaling Technology. The rabbit polyclonal anti-Cdc37 antibody (used for immunoprecipitation) was generated in this laboratory, whereas the mouse polyclonal anti-Cdc37 antibody (used for Western blotting) was a gift from Dr. Steven Hartson (Oklahoma State University). The anti-Hsp70 and anti-Hop antibodies were gifts from Dr. David Toft (Mayo Clinic, Rochester, MN). Plasmids—Expression vectors encoding FLAG-tagged versions of wild-type IRAK-1 (pIC-FLAG-IRAK-1) and kinase-dead IRAK-1 (pIC-FLAG-IRAK-1 KD) were generous gifts of Dr. Sankar Ghosh (Yale University) (20Zhang G. Ghosh S. J. Biol. Chem. 2002; 277: 7059-7065Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). An expression vector encoding FLAG-tagged IRAK-4 (i.e. FLAG-IRAK-4) was constructed by PCR using Pfu DNA polymerase and the plasmid pTriplEx-mIRAK-4 (from Dr. Wen-Chen Yeh, University of Toronto) as template. The resulting 1.4-kb PCR product was digested with MluI and subcloned into the corresponding site in pEF-BOS-FLAG (29Scholz G. Hartson S.D. Cartledge K. Hall N. Shao J. Dunn A.R. Matts R.L. Mol. Cell. Biol. 2000; 20: 6984-6995Crossref PubMed Scopus (35) Google Scholar). An expression vector encoding HA-tagged Cdc37 (i.e. HA-Cdc37) was created by excising the cDNA for Cdc37 from pEF-FLAG-Cdc37 (29Scholz G. Hartson S.D. Cartledge K. Hall N. Shao J. Dunn A.R. Matts R.L. Mol. Cell. Biol. 2000; 20: 6984-6995Crossref PubMed Scopus (35) Google Scholar) with MluI and subcloning it into the corresponding site in pEF-BOS-HA. Cell Culture and Transient Transfections—Human 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and transiently transfected using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions (29Scholz G. Hartson S.D. Cartledge K. Hall N. Shao J. Dunn A.R. Matts R.L. Mol. Cell. Biol. 2000; 20: 6984-6995Crossref PubMed Scopus (35) Google Scholar). Bone marrow-derived macrophages were prepared from 8-week-old female C57BL/6 mice as described previously (30Jaworowski A. Wilson N.J. Christy E. Byrne R. Hamilton J.A. J. Biol. Chem. 1999; 274: 15127-15133Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Cell Lysis, Western Blotting, and Immunoprecipitation—Cells were lysed directly in tissue culture dishes with Nonidet P-40 lysis buffer (20 mm Hepes, pH 7.4, 100 mm NaCl, 2 mm EGTA, 1 mm dithiothreitol, 1.0% Nonidet P-40, 10% glycerol, 1 mm sodium orthovanadate, 0.1 mm sodium molybdate, and Complete™ protease inhibitors) for 60 min on ice. Lysates were clarified by centrifugation at 13,000 × g for 10 min at 4 °C, and then protein concentrations were measured with a Bio-Rad protein assay kit. Western blotting and immunoprecipitation of cell lysates were performed by standard techniques (29Scholz G. Hartson S.D. Cartledge K. Hall N. Shao J. Dunn A.R. Matts R.L. Mol. Cell. Biol. 2000; 20: 6984-6995Crossref PubMed Scopus (35) Google Scholar, 31Scholz G.M. Hartson S.D. Cartledge K. Volk L. Matts R.L. Dunn A.R. Cell Growth & Differ. 2001; 12: 409-417PubMed Google Scholar). Mass Spectrometry—Protein bands were excised from gels stained with colloidal Coomassie Blue G-250 and digested in situ with trypsin. The peptides then were extracted, and peptide mass fingerprinting was performed using a Voyager DE™ MALDI-TOF mass spectrometer (Applied Biosystems) in linear mode or an Ultraflex™ MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) in reflectron mode. The mass spectra were analyzed by searching the Mascot search engine (www.matrixscience.com) based on the NCBI and SwissProt protein databases. In Vitro Kinase Assays—Anti-FLAG immunoprecipitates were incubated at 37 °C for 20 min in 20 μl of kinase buffer (20 mm Hepes, pH 7.4, 25 mm MgCl2, 10 mm β-glycerophosphate, 1 mm sodium orthovanadate, and 20 μm ATP) containing 10 μCi of [γ-32P]ATP. The ability of immunoprecipitated IRAK-1 to phosphorylate an exogenous substrate was tested by supplementing the kinase reactions with 2.5 μg of myelin basic protein. Reactions were terminated with SDS-PAGE sample buffer and heated for 5 min at 95 °C. The reactions then were subjected to SDS-PAGE followed by transfer to a nitrocellulose filter and exposure to x-ray film. High Pressure Liquid Chromatography Size-Exclusion Chromatography—Transfected 293T cells were lysed by Dounce homogenization in lysis buffer containing 0.1% Nonidet P-40. The lysates (500 μl) then were applied to a Superose-6 column (HR 10/30, Amersham Biosciences) equilibrated with column buffer (20 mm Hepes, pH 7.4, 100 mm NaCl, 10 mm NaF, 2 mm EGTA, and 10% glycerol), and elution was performed at a flow rate of 0.4 ml/min with fractions collected at each minute. The column was calibrated with thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), and equine myoglobin (17 kDa). Real-Time PCR Analysis of Gene Expression—Total RNA was isolated from bone marrow-derived macrophages (BMMs) with an RNeasy Mini kit (Qiagen) and then reverse-transcribed using SuperScript III reverse transcriptase (Invitrogen). Quantitative PCR was performed using an ABI PRISM 7900HT sequence detection system and pre-developed TaqMan probe/primer combinations for TNFα, IL-1β, and 18 S rRNA (ABI). Threshold cycle numbers were transformed using the ΔΔCt and relative value method as described by the manufacturer and expressed relative to 18 S rRNA. IRAK-1 Forms a Complex with a Cohort of Molecular Chaperones and Co-chaperones in 293T Cells—To identify novel regulators or effectors of IRAK-1, FLAG-tagged versions of wild-type and kinase-dead IRAK-1 (i.e. FLAG-IRAK-1 and FLAG-IRAK-1 KD, respectively) were transiently expressed in human 293T cells and then affinity-purified using anti-FLAG antibodies coupled to agarose beads. IRAK-1-binding proteins were detected by subjecting the immunoprecipitates to SDS-PAGE followed by staining of the gel with colloidal Coomassie Blue G-250. As shown in Fig. 1A, five distinct proteins (designated "1", "2", "3" "4," and "5") were detected in anti-FLAG immunoprecipitates derived from 293T cells expressing FLAG-IRAK-1 (lane 2) or FLAG-IRAK-1 KD (lane 3) but not from empty vector control-transfected cells (lane 1). These five protein bands were excised from the gel and digested in situ with trypsin and the resulting peptides subjected to mass spectrometry (MALDI-TOF or MALDI-TOF/TOF). The identities of the proteins (in order 1–5) were established as Hsp90, IRAK-1, Hsc70, Hsp70, and Hsp90-organizing protein (Hop). To confirm the mass spectrometric-based identification of the IRAK-1-binding proteins, anti-FLAG immunoprecipitates of FLAG-IRAK-1 were also subjected to Western blotting. As shown in Fig. 1B, Hsp90, Hsp70, and Hop were detected in anti-FLAG immunoprecipitates derived from 293T cells expressing either wild-type or kinase-dead FLAG-IRAK-1 (lanes 2 and 3, respectively) but not in immunoprecipitates from cells transfected with empty control vector (lane 1). Cdc37 is a 50-kDa protein that recruits a subset of protein kinases to Hsp90 to facilitate their folding into active conformations (24Grammatikakis N. Lin J.H. Grammatikakis A. Tsichlis P.N. Cochran B.H. Mol. Cell. Biol. 1999; 19: 1661-1672Crossref PubMed Scopus (229) Google Scholar, 25Kimura Y. Rutherford S.L. Miyata Y. Yahara I. Freeman B.C. Yue L. Morimoto R.I. Lindquist S. Genes Dev. 1997; 11: 1775-1785Crossref PubMed Scopus (181) Google Scholar, 28Stepanova L. Leng X. Parker S.B. Harper J.W. Genes Dev. 1996; 10: 1491-1502Crossref PubMed Scopus (448) Google Scholar, 29Scholz G. Hartson S.D. Cartledge K. Hall N. Shao J. Dunn A.R. Matts R.L. Mol. Cell. Biol. 2000; 20: 6984-6995Crossref PubMed Scopus (35) Google Scholar). Although a 50-kDa protein was not detected in the anti-FLAG immunoprecipitates, the heavy chain of the anti-FLAG antibody could have obscured the presence of Cdc37 (Fig. 1A). Indeed, Western blotting of the immunoprecipitates with an anti-Cdc37 antibody revealed co-immunoprecipitation of Cdc37 with both wild-type and kinase-dead FLAG-IRAK-1 (Fig. 1B, lanes 2 and 3). Reciprocal immunoprecipitation experiments with an anti-Cdc37 antibody demonstrated co-immunoprecipitation of FLAG-IRAK-1 (kinase-active and kinase-dead) and Hsp90 with Cdc37 (Fig. 1C, lanes 2 and 3). However, neither Hsp70 nor Hop was found to co-immunoprecipitate with Cdc37 (Fig. 1C, lanes 1–4). These findings suggest that FLAG-IRAK-1 is present in at least two distinct heterocomplexes, one consisting of FLAG-IRAK-1, Hsp90, Hsp70, and Hop and another consisting of FLAG-IRAK-1, Hsp90, and Cdc37. To examine the specificity of the interaction of IRAK-1 with Hsp90, Hsp70, Hop, and Cdc37, the ability of IRAK-4 to form a complex with these proteins was also determined. IRAK-4 is structurally related to IRAK-1 and has been proposed to mediate the activation of IRAK-1 in response to engagement of Toll-like receptors by their ligands (32Li S. Strelow A. Fontana E.J. Wesche H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5567-5572Crossref PubMed Scopus (549) Google Scholar). No interaction of FLAG-tagged IRAK-4 with Hsp90, Hsp70, Hop, or Cdc37 was observed (Fig. 1, B and C, lane 4). Thus the interaction of IRAK-1 with Hsp90, Hsp70, Hop, and Cdc37 appears to be specific and not the consequence of its overexpression in 293T cells. Stable Expression of IRAK-1 in 293T Cells Is Dependent on Hsp90 Activity—The importance of Hsp90 for IRAK-1 function was directly addressed by treating 293T cells expressing FLAG-IRAK-1 with two structurally dissimilar Hsp90 inhibitors, geldanamycin (GA) and radicicol (RAD) (33Schulte T.W. Akinaga S. Soga S. Sullivan W. Stensgard B. Toft D. Neckers L.M. Cell Stress Chaperones. 1998; 3: 100-108Crossref PubMed Scopus (365) Google Scholar, 34Shiotsu Y. Neckers L.M. Wortman I. An W.G. Schulte T.W. Soga S. Murakata C. Tamaoki T. Akinaga S. Blood. 2000; 96: 2284-2291Crossref PubMed Google Scholar, 35Roe S.M. Prodromou C. O'Brien R. Ladbury J.E. Piper P.W. Pearl L.H. J. Med. Chem. 1999; 42: 260-266Crossref PubMed Scopus (896) Google Scholar, 36Stebbins C.E. Russo A.A. Schneider C. Rosen N. Hartl F.U. Pavletich N.P. Cell. 1997; 89: 239-250Abstract Full Text Full Text PDF PubMed Scopus (1257) Google Scholar, 37Whitesell L. Mimnaugh E.G. De Costa B. Myers C.E. Neckers L.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8324-8328Crossref PubMed Scopus (1337) Google Scholar). Consistent with an earlier report by Li et al. (38Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar), several electrophoretically distinct forms of FLAG-IRAK-1 were observed upon its overexpression in 293T cells (Fig. 2A). Yamin and Miller (19Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) have previously shown that the more slowly migrating forms represent phosphorylated IRAK-1. Thus overexpression of FLAG-IRAK-1 in 293T cells results in ligand-independent autoactivation of the protein kinase (38Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). Treatment of the cells with either GA or RAD resulted in a dose-dependent decrease in the expression level of phosphorylated (i.e. activated) FLAG-IRAK-1 (Fig. 2A). Time course experiments with GA revealed a decrease in the level of phosphorylated IRAK-1 within 2 h of treating the cells with the Hsp90 inhibitor, which diminished significantly further by 8 h (Fig. 2B, lanes 3–6). To directly assess the effect of GA on the enzymatic activity of FLAG-IRAK-1, the capacity of immunoprecipitated FLAG-IRAK-1 to autophosphorylate as well as phosphorylate an exogenous substrate (i.e. myelin basic protein) was measured. These in vitro kinase assays confirmed a time-dependent loss of catalytically active FLAG-IRAK-1 in 293T cells treated with GA (Fig. 2C). To investigate the effect of GA on the composition of IRAK-1 heterocomplexes, FLAG-tagged IRAK-1 was immunoprecipitated from lysates of transfected 293T cells and subjected to Western blotting. The data presented in Fig. 2D clearly revealed that treatment of the transfected cells with GA reduced but did not abolish the association of Hsp90 with FLAG-IRAK-1. By contrast, the levels of Hsp70 and Hop associated with FLAG-IRAK-1 increased in response to GA treatment, whereas the association of Cdc37 with FLAG-IRAK-1 was abolished within 60 min of treating the cells with GA (Fig. 2D, lane 2 versus lane 3). These findings clearly indicate that Hsp90 activity is required for the stable expression of catalytically active IRAK-1 in 293T cells. Furthermore, they suggest that the interaction of Cdc37 with IRAK-1 is necessary for the stabilization of IRAK-1 by Hsp90. Hsp90 Is Required for the Stable Expression of IRAK-1 in Macrophages—To establish whether a functional relationship also exists between endogenous IRAK-1 and Hsp90 in macrophages, primary mouse BMMs were treated with different concentrations of GA or RAD for 8 h and the expression levels of IRAK-1 were evaluated by Western blotting. As shown in Fig. 3A, the treatment of BMMs with either GA or RAD induced a dose-dependent decrease in the expression level of endogenous IRAK-1. Neither GA nor RAD had any significant effect on the expression levels of endogenous IRAK-4 or p38 MAP kinase (Fig. 3A). The expression levels of Hsp90 and Cdc37 were essentially unaltered in GA- or RAD-treated BMMs (data not shown). GA promoted the relatively rapid degradation of IRAK-1 in BMMs with the half-life of the protein kinase estimated to be ∼3 h in GA-treated macrophages (Fig. 3B). To establish whether the loss of IRAK-1 in GA-treated BMMs was the result of degradation of nascent and/or mature (i.e. folded) IRAK-1, the half-life of IRAK-1 in macrophages containing functional Hsp90 was established. BMMs were treated with the protein synthesis inhibitor cycloheximide, and IRAK-1 levels were monitored by Western blotting. These experiments revealed that the half-life of mature IRAK-1 in macrophages is greater than 8 h (Fig. 3C). Likewise, the half-lives of IRAK-4, p38 MAP kinase, and Erk1/2 in BMMs were also greater than 8 h (data not shown). Thus our finding that the half-life of IRAK-1 is reduced in GA-treated BMMs (3 h versus >8 h) suggests that, even following its folding into a mature and functional conformation, IRAK-1 is still dependent on Hsp90 activity for its continued stable expression in macrophages. The functional dependence of IRAK-1 on Hsp90 and Cdc37 in macrophages was further illustrated by demonstrating co-immunoprecipitation of Hsp90 and Cdc37 with endogenous IRAK-1 (Fig. 3D). Consistent with the data presented in Fig. 2D, treatment of BMMs with GA for 1 h led to a reduction in the association of Hsp90 and Cdc37 with endogenous IRAK-1 (Fig. 3D). Cdc37 Enhances Autoactivation of IRAK-1 in 293T Cells— The finding that GA-induced degradation of IRAK-1 correlated with the loss of Cdc37 from IRAK-1 heterocomplexes suggested that Cdc37 played an important role in the stabilization of IRAK-1 by Hsp90. To address this issue, the effect of Cdc37 overexpression on IRAK-1 expression and activity was investigated. The data presented in Fig. 4A revealed that the proportion of total FLAG-IRAK-1 that was hyperphosphorylated (and thus activated) was higher in 293T cells expressing HA-Cdc37 compared with cells expressing FLAG-IRAK-1 alone (lane 4 versus lane 2). Notably, the enhanced activation of FLAG-IRAK-1 in 293T cells co-expressing HA-Cdc37 was still dependent on Hsp90 activity (Fig. 4A, lane 5 versus lane 4). Examination of the composition of FLAG-IRAK-1 heterocomplexes revealed that HA-Cdc37 competed with endogenous Cdc37 for binding to FLAG-IRAK-1 (Fig. 4B, lane 4 versus lane 2). Expression of HA-Cdc37 did not appear to affect the level of Hsp90 associated with FLAG-IRAK-1 (Fig. 4B, lane 4 versus lane 2). However, the association of Hsp70 and Hop with FLAG-IRAK-1 was markedly reduced upon co-expression of the kinase with HA-Cdc37 (lane 4 versus lane 2), although treatment of identically transfected cells wi

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