A Proteomic Investigation of Ligand-dependent HSP90 Complexes Reveals CHORDC1 as a Novel ADP-dependent HSP90-interacting Protein
2009; Elsevier BV; Volume: 9; Issue: 2 Linguagem: Inglês
10.1074/mcp.m900261-mcp200
ISSN1535-9484
Autores Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoStructural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes. Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes. The dimeric HSP90 ATPase is a highly abundant molecular chaperone that functions in later stages of protein folding pathways to assist the folding and activation of a specific set of proteins known as "clients." The essential functions of HSP90 are underscored by the growing list of client proteins functioning in diverse biological processes such as cell division, metabolism, signal transduction, transcription, and immunity. Pharmacological inhibition of HSP90 ATPase activity is generally believed to cause proteolytic degradation of the client protein, leading to alteration of the associated biological processes. HSP90 has become an important target for anticancer therapies because of its role in stabilizing proteins directly involved in oncogenic pathways (1Powers M.V. Workman P. Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors.Endocr.-Relat. Cancer. 2006; 13: S125-S135Crossref PubMed Scopus (255) Google Scholar). Consequently, intensive efforts have been put forth to develop HSP90 inhibitors.HSP90 complexes contain several co-chaperones and accessory proteins that modulate ATPase activity and client protein interactions (2Pearl L.H. Prodromou C. Structure and mechanism of the Hsp90 molecular chaperone machinery.Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (884) Google Scholar). It is believed that these co-chaperone interactions may direct HSP90 activity to particular complexes and structures within the cell, thereby introducing a degree of selectivity for particular subsets of client proteins (3Lee Y.T. Jacob J. Michowski W. Nowotny M. Kuznicki J. Chazin W.J. Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain.J. Biol. Chem. 2004; 279: 16511-16517Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It has also been proposed that the binding of co-chaperones can physically alter the client protein binding surfaces on HSP90, thus restricting chaperone activity to a discrete subset of client protein substrates (4Riggs D.L. Cox M.B. Cheung-Flynn J. Prapapanich V. Carrigan P.E. Smith D.F. Functional specificity of co-chaperone interactions with Hsp90 client proteins.Crit. Rev. Biochem. Mol. Biol. 2004; 39: 279-295Crossref PubMed Scopus (108) Google Scholar, 5Pearl L.H. Hsp90 and Cdc37—a chaperone cancer conspiracy.Curr. Opin. Genet. Dev. 2005; 15: 55-61Crossref PubMed Scopus (200) Google Scholar). For example, the co-chaperone CDC37 is found almost exclusively in HSP90 complexes containing kinase client proteins and may therefore serve as a kinase-specific adaptor (6Roe S.M. Ali M.M. Meyer P. Vaughan C.K. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. The mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37).Cell. 2004; 116: 87-98Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar).There is increasing evidence that constituents of HSP90 complexes are influenced by the ligand-bound state of HSP90 (7Siligardi G. Hu B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle.J. Biol. Chem. 2004; 279: 51989-51998Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8Colombo G. Morra G. Meli M. Verkhivker G. Understanding ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7976-7981Crossref PubMed Scopus (61) Google Scholar). The co-chaperone AHSA1 binds exclusively to the ATP-bound HSP90 and stimulates ATP hydrolysis, and this interaction is thought to influence client protein activation (9Meyer P. Prodromou C. Liao C. Hu B. Mark Roe S. Vaughan C.K. Vlasic I. Panaretou B. Piper P.W. Pearl L.H. Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery.EMBO J. 2004; 23: 511-519Crossref PubMed Scopus (191) Google Scholar, 10Lotz G.P. Lin H. Harst A. Obermann W.M. Aha1 binds to the middle domain of Hsp90, contributes to client protein activation, and stimulates the ATPase activity of the molecular chaperone.J. Biol. Chem. 2003; 278: 17228-17235Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. Cramer R. Mollapour M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1.Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 12Harst A. Lin H. Obermann W.M. Aha1 competes with Hop, p50 and p23 for binding to the molecular chaperone Hsp90 and contributes to kinase and hormone receptor activation.Biochem. J. 2005; 387: 789-796Crossref PubMed Scopus (102) Google Scholar). In addition, several studies have shown the co-chaperone p23 to preferentially interact with the ATP-bound HSP90, inhibiting ATP hydrolysis (7Siligardi G. Hu B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle.J. Biol. Chem. 2004; 279: 51989-51998Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13McLaughlin S.H. Sobott F. Yao Z.P. Zhang W. Nielsen P.R. Grossmann J.G. Laue E.D. Robinson C.V. Jackson S.E. The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins.J. Mol. Biol. 2006; 356: 746-758Crossref PubMed Scopus (155) Google Scholar, 14Sullivan W.P. Owen B.A. Toft D.O. The influence of ATP and p23 on the conformation of hsp90.J. Biol. Chem. 2002; 277: 45942-45948Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (485) Google Scholar). Inhibition of ATP binding and hydrolysis by the pharmacological inhibitor geldanamycin is generally observed to disrupt HSP90 interactions with co-chaperones and client proteins; however, some exceptions exist where geldanamycin has no effect or stabilizes interactions (16Whitesell L. Cook P. Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells.Mol. Endocrinol. 1996; 10: 705-712Crossref PubMed Scopus (253) Google Scholar, 17Young J.C. Hartl F.U. Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23.EMBO J. 2000; 19: 5930-5940Crossref PubMed Scopus (197) Google Scholar). Less is understood about how ADP binding affects HSP90 interactions. Crystallographic evidence from the Escherichia coli HSP90 homolog HtpG suggests that the ADP-bound HSP90 adopts a conformation distinct from the ATP-bound form, and it was proposed that this conformation may alter the constituents of HSP90 complexes (18Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements.Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 19Krukenberg K.A. Förster F. Rice L.M. Sali A. Agard D.A. Multiple conformations of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90.Structure. 2008; 16: 755-765Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 20Morra G. Verkhivker G. Colombo G. Modeling signal propagation mechanisms and ligand-based conformational dynamics of the Hsp90 molecular chaperone full-length dimer.PLoS Comput. Biol. 2009; 5: e1000323Crossref PubMed Scopus (121) Google Scholar).Several groups have recently reported proteomics studies of the HSP90 interactome; however, these studies were focused on documenting HSP90 interactions under single experimental conditions with isolated proteins or were not designed to study how the dynamic interactions of this chaperone complex are influenced by HSP90 ligands (21Zhao R. Davey M. Hsu Y.C. Kaplanek P. Tong A. Parsons A.B. Krogan N. Cagney G. Mai D. Greenblatt J. Boone C. Emili A. Houry W.A. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone.Cell. 2005; 120: 715-727Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 22Falsone S.F. Gesslbauer B. Tirk F. Piccinini A.M. Kungl A.J. A proteomic snapshot of the human heat shock protein 90 interactome.FEBS Lett. 2005; 579: 6350-6354Crossref PubMed Scopus (77) Google Scholar, 23Te J. Jia L. Rogers J. Miller A. Hartson S.D. Novel subunits of the mammalian Hsp90 signal transduction chaperone.J. Proteome Res. 2007; 6: 1963-1973Crossref PubMed Scopus (50) Google Scholar, 24Falsone S.F. Gesslbauer B. Rek A. Kungl A.J. A proteomic approach towards the Hsp90-dependent ubiquitinylated proteome.Proteomics. 2007; 7: 2375-2383Crossref PubMed Scopus (27) Google Scholar, 25Schumacher J.A. Crockett D.K. Elenitoba-Johnson K.S. Lim M.S. Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells.Proteomics. 2007; 7: 2603-2616Crossref PubMed Scopus (30) Google Scholar, 26Maloney A. Clarke P.A. Naaby-Hansen S. Stein R. Koopman J.O. Akpan A. Yang A. Zvelebil M. Cramer R. Stimson L. Aherne W. Banerji U. Judson I. Sharp S. Powers M. deBilly E. Salmons J. Walton M. Burlingame A. Waterfield M. Workman P. Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin.Cancer Res. 2007; 67: 3239-3253Crossref PubMed Scopus (114) Google Scholar, 27Tsaytler P.A. Krijgsveld J. Goerdayal S.S. Rüdiger S. Egmond M.R. Novel Hsp90 partners discovered using complementary proteomic approaches.Cell Stress Chaperones. 2009; 14: 629-638Crossref PubMed Scopus (37) Google Scholar). To gain proteome-wide resolution on the effects of ligands on the HSP90 interactome, we affinity-purified human HSP90 complexes from HEK293T cells in the presence of excess ATP, ADP, or the HSP90 inhibitor geldanamycin and used LC-MS/MS and spectral counting to quantify the effects of these ligands on the constituents of the HSP90 interactome. The dimeric HSP90 ATPase is a highly abundant molecular chaperone that functions in later stages of protein folding pathways to assist the folding and activation of a specific set of proteins known as "clients." The essential functions of HSP90 are underscored by the growing list of client proteins functioning in diverse biological processes such as cell division, metabolism, signal transduction, transcription, and immunity. Pharmacological inhibition of HSP90 ATPase activity is generally believed to cause proteolytic degradation of the client protein, leading to alteration of the associated biological processes. HSP90 has become an important target for anticancer therapies because of its role in stabilizing proteins directly involved in oncogenic pathways (1Powers M.V. Workman P. Targeting of multiple signalling pathways by heat shock protein 90 molecular chaperone inhibitors.Endocr.-Relat. Cancer. 2006; 13: S125-S135Crossref PubMed Scopus (255) Google Scholar). Consequently, intensive efforts have been put forth to develop HSP90 inhibitors. HSP90 complexes contain several co-chaperones and accessory proteins that modulate ATPase activity and client protein interactions (2Pearl L.H. Prodromou C. Structure and mechanism of the Hsp90 molecular chaperone machinery.Annu. Rev. Biochem. 2006; 75: 271-294Crossref PubMed Scopus (884) Google Scholar). It is believed that these co-chaperone interactions may direct HSP90 activity to particular complexes and structures within the cell, thereby introducing a degree of selectivity for particular subsets of client proteins (3Lee Y.T. Jacob J. Michowski W. Nowotny M. Kuznicki J. Chazin W.J. Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain.J. Biol. Chem. 2004; 279: 16511-16517Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It has also been proposed that the binding of co-chaperones can physically alter the client protein binding surfaces on HSP90, thus restricting chaperone activity to a discrete subset of client protein substrates (4Riggs D.L. Cox M.B. Cheung-Flynn J. Prapapanich V. Carrigan P.E. Smith D.F. Functional specificity of co-chaperone interactions with Hsp90 client proteins.Crit. Rev. Biochem. Mol. Biol. 2004; 39: 279-295Crossref PubMed Scopus (108) Google Scholar, 5Pearl L.H. Hsp90 and Cdc37—a chaperone cancer conspiracy.Curr. Opin. Genet. Dev. 2005; 15: 55-61Crossref PubMed Scopus (200) Google Scholar). For example, the co-chaperone CDC37 is found almost exclusively in HSP90 complexes containing kinase client proteins and may therefore serve as a kinase-specific adaptor (6Roe S.M. Ali M.M. Meyer P. Vaughan C.K. Panaretou B. Piper P.W. Prodromou C. Pearl L.H. The mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37).Cell. 2004; 116: 87-98Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). There is increasing evidence that constituents of HSP90 complexes are influenced by the ligand-bound state of HSP90 (7Siligardi G. Hu B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle.J. Biol. Chem. 2004; 279: 51989-51998Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 8Colombo G. Morra G. Meli M. Verkhivker G. Understanding ligand-based modulation of the Hsp90 molecular chaperone dynamics at atomic resolution.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 7976-7981Crossref PubMed Scopus (61) Google Scholar). The co-chaperone AHSA1 binds exclusively to the ATP-bound HSP90 and stimulates ATP hydrolysis, and this interaction is thought to influence client protein activation (9Meyer P. Prodromou C. Liao C. Hu B. Mark Roe S. Vaughan C.K. Vlasic I. Panaretou B. Piper P.W. Pearl L.H. Structural basis for recruitment of the ATPase activator Aha1 to the Hsp90 chaperone machinery.EMBO J. 2004; 23: 511-519Crossref PubMed Scopus (191) Google Scholar, 10Lotz G.P. Lin H. Harst A. Obermann W.M. Aha1 binds to the middle domain of Hsp90, contributes to client protein activation, and stimulates the ATPase activity of the molecular chaperone.J. Biol. Chem. 2003; 278: 17228-17235Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 11Panaretou B. Siligardi G. Meyer P. Maloney A. Sullivan J.K. Singh S. Millson S.H. Clarke P.A. Naaby-Hansen S. Stein R. Cramer R. Mollapour M. Workman P. Piper P.W. Pearl L.H. Prodromou C. Activation of the ATPase activity of hsp90 by the stress-regulated cochaperone aha1.Mol. Cell. 2002; 10: 1307-1318Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar, 12Harst A. Lin H. Obermann W.M. Aha1 competes with Hop, p50 and p23 for binding to the molecular chaperone Hsp90 and contributes to kinase and hormone receptor activation.Biochem. J. 2005; 387: 789-796Crossref PubMed Scopus (102) Google Scholar). In addition, several studies have shown the co-chaperone p23 to preferentially interact with the ATP-bound HSP90, inhibiting ATP hydrolysis (7Siligardi G. Hu B. Panaretou B. Piper P.W. Pearl L.H. Prodromou C. Co-chaperone regulation of conformational switching in the Hsp90 ATPase cycle.J. Biol. Chem. 2004; 279: 51989-51998Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 13McLaughlin S.H. Sobott F. Yao Z.P. Zhang W. Nielsen P.R. Grossmann J.G. Laue E.D. Robinson C.V. Jackson S.E. The co-chaperone p23 arrests the Hsp90 ATPase cycle to trap client proteins.J. Mol. Biol. 2006; 356: 746-758Crossref PubMed Scopus (155) Google Scholar, 14Sullivan W.P. Owen B.A. Toft D.O. The influence of ATP and p23 on the conformation of hsp90.J. Biol. Chem. 2002; 277: 45942-45948Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Obermann W.M. Sondermann H. Russo A.A. Pavletich N.P. Hartl F.U. In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.J. Cell Biol. 1998; 143: 901-910Crossref PubMed Scopus (485) Google Scholar). Inhibition of ATP binding and hydrolysis by the pharmacological inhibitor geldanamycin is generally observed to disrupt HSP90 interactions with co-chaperones and client proteins; however, some exceptions exist where geldanamycin has no effect or stabilizes interactions (16Whitesell L. Cook P. Stable and specific binding of heat shock protein 90 by geldanamycin disrupts glucocorticoid receptor function in intact cells.Mol. Endocrinol. 1996; 10: 705-712Crossref PubMed Scopus (253) Google Scholar, 17Young J.C. Hartl F.U. Polypeptide release by Hsp90 involves ATP hydrolysis and is enhanced by the co-chaperone p23.EMBO J. 2000; 19: 5930-5940Crossref PubMed Scopus (197) Google Scholar). Less is understood about how ADP binding affects HSP90 interactions. Crystallographic evidence from the Escherichia coli HSP90 homolog HtpG suggests that the ADP-bound HSP90 adopts a conformation distinct from the ATP-bound form, and it was proposed that this conformation may alter the constituents of HSP90 complexes (18Shiau A.K. Harris S.F. Southworth D.R. Agard D.A. Structural Analysis of E. coli hsp90 reveals dramatic nucleotide-dependent conformational rearrangements.Cell. 2006; 127: 329-340Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 19Krukenberg K.A. Förster F. Rice L.M. Sali A. Agard D.A. Multiple conformations of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90.Structure. 2008; 16: 755-765Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 20Morra G. Verkhivker G. Colombo G. Modeling signal propagation mechanisms and ligand-based conformational dynamics of the Hsp90 molecular chaperone full-length dimer.PLoS Comput. Biol. 2009; 5: e1000323Crossref PubMed Scopus (121) Google Scholar). Several groups have recently reported proteomics studies of the HSP90 interactome; however, these studies were focused on documenting HSP90 interactions under single experimental conditions with isolated proteins or were not designed to study how the dynamic interactions of this chaperone complex are influenced by HSP90 ligands (21Zhao R. Davey M. Hsu Y.C. Kaplanek P. Tong A. Parsons A.B. Krogan N. Cagney G. Mai D. Greenblatt J. Boone C. Emili A. Houry W.A. Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone.Cell. 2005; 120: 715-727Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar, 22Falsone S.F. Gesslbauer B. Tirk F. Piccinini A.M. Kungl A.J. A proteomic snapshot of the human heat shock protein 90 interactome.FEBS Lett. 2005; 579: 6350-6354Crossref PubMed Scopus (77) Google Scholar, 23Te J. Jia L. Rogers J. Miller A. Hartson S.D. Novel subunits of the mammalian Hsp90 signal transduction chaperone.J. Proteome Res. 2007; 6: 1963-1973Crossref PubMed Scopus (50) Google Scholar, 24Falsone S.F. Gesslbauer B. Rek A. Kungl A.J. A proteomic approach towards the Hsp90-dependent ubiquitinylated proteome.Proteomics. 2007; 7: 2375-2383Crossref PubMed Scopus (27) Google Scholar, 25Schumacher J.A. Crockett D.K. Elenitoba-Johnson K.S. Lim M.S. Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells.Proteomics. 2007; 7: 2603-2616Crossref PubMed Scopus (30) Google Scholar, 26Maloney A. Clarke P.A. Naaby-Hansen S. Stein R. Koopman J.O. Akpan A. Yang A. Zvelebil M. Cramer R. Stimson L. Aherne W. Banerji U. Judson I. Sharp S. Powers M. deBilly E. Salmons J. Walton M. Burlingame A. Waterfield M. Workman P. Gene and protein expression profiling of human ovarian cancer cells treated with the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin.Cancer Res. 2007; 67: 3239-3253Crossref PubMed Scopus (114) Google Scholar, 27Tsaytler P.A. Krijgsveld J. Goerdayal S.S. Rüdiger S. Egmond M.R. Novel Hsp90 partners discovered using complementary proteomic approaches.Cell Stress Chaperones. 2009; 14: 629-638Crossref PubMed Scopus (37) Google Scholar). To gain proteome-wide resolution on the effects of ligands on the HSP90 interactome, we affinity-purified human HSP90 complexes from HEK293T cells in the presence of excess ATP, ADP, or the HSP90 inhibitor geldanamycin and used LC-MS/MS and spectral counting to quantify the effects of these ligands on the constituents of the HSP90 interactome. We thank the Clinical Division at the Fred Hutchinson Cancer Research Center for supporting this research. We also thank Eric Foss, Phil Gafken, Jason Hogan, and Tomoko Hamma at the Fred Hutchinson Cancer Research Center for critical input and technical support. Supplementary Material Download .zip (17.0 MB) Help with zip files Download .zip (17.0 MB) Help with zip files
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