Regulation of the Dynamics of hsp90 Action on the Glucocorticoid Receptor by Acetylation/Deacetylation of the Chaperone

Abstract

It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 “client” protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601–607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR·hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR·hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR·hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR·hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR·hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a ∼100-fold shift to the right in the steroid dose response for gene activation. It is known that inhibition of histone deacetylases (HDACs) leads to acetylation of the abundant protein chaperone hsp90. In a recent study, we have shown that knockdown of HDAC6 by a specific small interfering RNA leads to hyperacetylation of hsp90 and that the glucocorticoid receptor (GR), an established hsp90 “client” protein, is defective in ligand binding, nuclear translocation, and gene activation in HDAC6-deficient cells (Kovacs, J. J., Murphy, P. J. M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C. V., Yoshida, M., Toft, D. O., Pratt, W. B., and Yao, T-P. (2005) Mol. Cell 18, 601–607). Using human embryonic kidney wild-type and HDAC6 (small interfering RNA) knockdown cells transiently expressing the mouse GR, we show here that the intrinsic properties of the receptor protein itself are not affected by HDAC6 knockdown, but the knockdown cytosol has a markedly decreased ability to assemble stable GR·hsp90 heterocomplexes and generate stable steroid binding activity under cell-free conditions. HDAC6 knockdown cytosol has the same ability to carry out dynamic GR·hsp90 heterocomplex assembly as wild-type cytosol. Addition of purified hsp90 to HDAC6 knockdown cytosol restores stable GR·hsp90 heterocomplex assembly to the level of wild-type cytosol. hsp90 from HDAC6 knockdown cytosol has decreased ATP-binding affinity, and it does not assemble stable GR·hsp90 heterocomplexes when it is a component of a purified five-protein assembly system. Incubation of knockdown cell hsp90 with purified HDAC6 converts the hsp90 to wild-type behavior. Thus, acetylation of hsp90 results in dynamic GR·hsp90 heterocomplex assembly/disassembly, and this is manifest in the cell as a ∼100-fold shift to the right in the steroid dose response for gene activation. The hsp90/hsp70-based chaperone machinery regulates a wide variety of proteins involved in cellular signaling through the assembly of client protein·hsp90 heterocomplexes (reviewed in Ref. 1Pratt W.B. Toft D.O. Exp. Biol. Med. 2003; 228: 111-133Crossref PubMed Scopus (1239) Google Scholar). The chaperones hsp90 and hsp70 play a key role in the balance between maintenance of protein integrity and degradation by the ubiquitin-proteasomal pathway, and inhibition of hsp90 function leads to degradation of hsp90 client proteins (2Schneider C. Sepp-Lorenzino L. Nimmesgern E. Ouerfelli O. Danishefsky S. Rosen N. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14536-14541Crossref PubMed Scopus (367) Google Scholar, 3Isaacs J.S. Xu W. Neckers L. Cancer Cell. 2003; 3: 213-217Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). Yu et al. (4Yu X. Guo Z.S. Marcu M.G. Neckers L. Nguyen D.M. Chen G.A. Schrump D.S. J. Natl. Cancer Inst. 2002; 94: 504-513Crossref PubMed Scopus (358) Google Scholar) have reported that treatment of cells with an inhibitor of histone deacetylases (see Refs. 5Yoshida M. Furumai R. Nishiyama M. Komatsu Y. Nishino N. Horinouchi S. Cancer Chemother. Pharmacol. 2001; 48: S20-S26Crossref PubMed Scopus (219) Google Scholar and 6Marks P.A. Richon V.M. Breslow R. Rifkind R.A. Curr. Opin. Oncol. 2001; 13: 477-483Crossref PubMed Scopus (505) Google Scholar for a review of deacetylase inhibitors) leads to acetylation of hsp90 and depletion of several hsp90 client proteins, including Raf-1, ErbB2, and mutant p53. This suggests that hsp90 function is affected by acetylation/deacetylation. Histone deacetylase (HDAC) 3The abbreviations used are: HDAC, histone deacetylase; GR, glucocorticoid receptor; hsp, heat shock protein; CFTR, cystic fibrosis transmembrane conducting regulator; KD, knockdown; CAT, chloramphenicol acetyltransferase; MMTV, mouse mammary tumor virus; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.3The abbreviations used are: HDAC, histone deacetylase; GR, glucocorticoid receptor; hsp, heat shock protein; CFTR, cystic fibrosis transmembrane conducting regulator; KD, knockdown; CAT, chloramphenicol acetyltransferase; MMTV, mouse mammary tumor virus; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. 6 is a cytoplasmic HDAC that is associated with microtubules (7Hubbert C. Guardiola A. Shao R. Kawaguchi Y. Ito A. Nixon A. Yoshida M. Wang X.F. Yao T-P. Nature. 2002; 417: 455-458Crossref PubMed Scopus (1721) Google Scholar) and has been shown to regulate aggresome formation in response to misfolded protein stress (8Kawaguchi Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T-P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). The cystic fibrosis transmembrane conducting regulator (CFTR) is a client protein of hsp90 (9Loo M.A. Jensen T.J. Cui L. Hou Y. Chang X.B. Riordan J.R. EMBO J. 1998; 17: 6879-6887Crossref PubMed Scopus (294) Google Scholar), and a mutant form, CFTR-Δ508, is prone to misfolding and aggresome formation (reviewed in Ref. 10Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1572) Google Scholar). Misfolded CFTR-Δ508 moves in a dynein-dependent fashion along microtubules to form the perinuclear aggresome (10Kopito R.R. Trends Cell Biol. 2000; 10: 524-530Abstract Full Text Full Text PDF PubMed Scopus (1572) Google Scholar). HDAC6 is a component of these aggresomes and cells deficient in HDAC6 cannot form aggresomes properly, apparently because of a failure to load polyubiquitinated misfolded protein onto the dynein motor for transport to aggresomes (8Kawaguchi Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T-P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). Because ubiquitylated CFTR-Δ508 is coimmunoadsorbed from cytosol with HDAC6 and HDAC6 is coadsorbed with dynein, it was proposed that HDAC6 directly links ubiquitylated CFTR-Δ508 to the motor protein (8Kawaguchi Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T-P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). Other hsp90 client proteins, such as the glucocorticoid receptor (GR) (11Harrell J.M. Murphy P.J.M. Morishima Y. Chen H. Mansfield J.F. Galigniana M.D. Pratt W.B. J. Biol. Chem. 2004; 279: 54647-54654Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and the tumor suppressor protein p53 (12Giannakakou P. Sackett D.L. Ward Y. Webster K.R. Blagosklonny M.V. Fojo T. Nat. Cell Biol. 2000; 2: 709-717Crossref PubMed Scopus (301) Google Scholar, 13Galigniana M.D. Harrell J.M. O'Hagen H.M. Ljungman M. Pratt W.B. J. Biol. Chem. 2004; 279: 22483-22489Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), have been shown to utilize an hsp90-dependent mechanism for dynein-dependent retrograde movement along microtubules. In this movement system, client protein·hsp90 heterocomplexes formed by the hsp90/hsp70-based chaperone machinery are linked via hsp90-binding immunophilins to the dynein/dynactin motor protein complex (Refs. 11Harrell J.M. Murphy P.J.M. Morishima Y. Chen H. Mansfield J.F. Galigniana M.D. Pratt W.B. J. Biol. Chem. 2004; 279: 54647-54654Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar and 13Galigniana M.D. Harrell J.M. O'Hagen H.M. Ljungman M. Pratt W.B. J. Biol. Chem. 2004; 279: 22483-22489Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar and reviewed in Ref. 14Pratt W.B. Galigniana M.D. Harrell J.M. DeFranco D.B. Cell. Signal. 2004; 16: 857-872Crossref PubMed Scopus (235) Google Scholar). Because HDAC6 could be required for proper hsp90 function in forming these movement complexes rather than acting itself to directly link the client protein to the dynein motor, we have examined the effect of HDAC6 on hsp90 acetylation and function (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). that HDAC6 hsp90 in and that purified HDAC6 hsp90 in (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). of histone deacetylases by knockdown of HDAC6 by specific small interfering RNA leads to hyperacetylation of and hsp90 in cytosol of HDAC6 knockdown cells has of the hsp90 to it than the hsp90 in wild-type cytosol (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The in HDAC6-deficient cells is defective in ligand binding, nuclear translocation, and gene that HDAC6 is required for hsp90 function in (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). we proposed that HDAC6 activity is for it was not that rather than the component of the hsp90/hsp70-based chaperone was the of HDAC6 In this we have examined the role of HDAC6 on GR·hsp90 heterocomplex assembly and steroid binding activity in show that cytosol from HDAC6 knockdown cells has a decreased ability to assemble stable GR·hsp90 heterocomplexes and that the assembly activity be to the level of wild-type cytosol by of purified hsp90. The stable heterocomplex assembly activity of hsp90 purified from HDAC6 knockdown cytosol be by it with purified in stable heterocomplex the hsp90 of HDAC6 knockdown cells is to carry out dynamic assembly of GR·hsp90 The hsp90 has a for than the wild-type and the dynamic GR·hsp90 heterocomplex that in HDAC6 knockdown cells is manifest as a ∼100-fold shift to the right wild-type cells in the dose response of for activation. with (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), that hsp90 function is through acetylation/deacetylation. was from and and from and and from was from The used to the mouse and the used to human from The used to hsp90 was from The used to was a from Toft The used to the mouse was by and the used to hsp90 was by The mouse and the mouse mammary tumor by of human embryonic kidney cells expressing a small interfering RNA HDAC6 small interfering RNA (8Kawaguchi Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T-P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). The HDAC6 and used to HDAC6 and in the Yao and have been (7Hubbert C. Guardiola A. Shao R. Kawaguchi Y. Ito A. Nixon A. Yoshida M. Wang X.F. Yao T-P. Nature. 2002; 417: 455-458Crossref PubMed Scopus (1721) Google Scholar, Y. Kovacs J.J. McLaurin A. Vance J.M. Ito A. Yao T-P. Cell. 2003; 115: 727-738Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar). Cell and cells in with by and Cell in in of and with and of inhibitor and by The was for and the to as was and was in and cytosol was as Y. Murphy P.J.M. Pratt W.B. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of and cells as in to and with of of and of of with was and the for of wild-type and knockdown cells as in to and for with of of and of The was with and the cells for the cells for with of of from of steroid of cell cytosol by for with of protein to of in of TES, was of associated hsp90 by the for an with of in The with of by a with of GR·hsp90 of chaperones with of cell cytosol and of an and heterocomplex with purified proteins, with of knockdown wild-type of purified of purified human 6 of purified human of purified to with and of the system. The for with of the by the the of the the with of with and for steroid binding and for hsp90. In the shown in the five-protein purified knockdown hsp90 was for with an from of cells to of the to for heterocomplex for of to be for steroid binding, a of cytosol was of a of with and by The steroid binding is as of of cytosol. to be for steroid binding to stable GR·hsp90 heterocomplexes in of with of and by of steroid binding under dynamic GR·hsp90 assembly was the assembly and and In the steroid binding is as of from of cell cytosol. and on and to The with for GR, for for The a with the to the of and purified as by and Pratt Pratt W.B. 2003; PubMed Scopus Google Scholar). hsp90 was purified from HDAC6 knockdown cells by Pratt W.B. 2003; PubMed Scopus Google Scholar), it was and to purified wild-type hsp90 in stable GR·hsp90 heterocomplex assembly in the five-protein assembly system. Because hsp90 to when the of the is and be with a we used a of to hsp90 and to the ATP-binding properties of hsp90 from knockdown and wild-type This hsp90 from activity and hsp90 from knockdown cytosol that does not stable GR·hsp90 heterocomplex assembly of cytosol in was to a of the was with of and the was with a of in hsp90 was with the and the was of hsp70 and ATP-binding proteins by with The by and to by It is to the in small and to gene was by activity in wild-type and knockdown cell a of the in et al. J.R. Nature. PubMed Scopus Google Scholar). with and for with of in and by the cell to Cell for and the protein of the was by of the of protein for in The to a purified and for of and was and the The was for and of the was by hsp90 to and in HDAC6 an inhibitor of histone has been reported to cells of several hsp90 client proteins ErbB2, (4Yu X. Guo Z.S. Marcu M.G. Neckers L. Nguyen D.M. Chen G.A. Schrump D.S. J. Natl. Cancer Inst. 2002; 94: 504-513Crossref PubMed Scopus (358) Google Scholar). knockdown of HDAC6 in cells results in decreased glucocorticoid binding activity in the level of human the effect of HDAC6 knockdown, we to the and directly activity in formation of GR·hsp90 Because we to an the human GR, we transiently the mouse in we decreased steroid binding activity in HDAC6 knockdown cells in the level of was from wild-type and HDAC6 knockdown and the to coadsorbed hsp90 and shown in is hsp90 in from knockdown cells with wild-type The in HDAC6 to with GR·hsp90 in cell-free assembly P. Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and in Y. Murphy P.J.M. Y. Pratt W.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it is that acetylation of of the itself could for decreased steroid binding activity and decreased of GR·hsp90 heterocomplexes from HDAC6 knockdown we not acetylation of the from knockdown cells under acetylation of knockdown hsp90 could be the from HDAC6 knockdown cells could form stable GR·hsp90 the was with shown in the from knockdown cells the same ability to form hsp90 heterocomplexes with the same steroid binding activity as from wild-type Thus, the from HDAC6 knockdown cells to be and to a receptor in the of wild-type hsp90 chaperone HDAC6 GR·hsp90 HDAC6 knockdown cells deficient GR·hsp90 heterocomplex was from of and with from wild-type and knockdown It be in that cytosol from HDAC6 knockdown cells has a ability to form GR·hsp90 heterocomplexes and generate steroid binding In client proteins form heterocomplexes with hsp90 that are stable to and inhibition of hsp90 function by leads to degradation via the J.S. Xu W. Neckers L. Cancer Cell. 2003; 3: 213-217Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). This is the for the L. P. Mol. 1996; 10: PubMed Scopus Google Scholar), and it is that the level of in HDAC6 knockdown cells is the same as that in wild-type cells the ability of knockdown cells to form GR·hsp90 in hsp90 client proteins in a dynamic of heterocomplex assembly/disassembly, with that client protein·hsp90 heterocomplexes are with This is the with for with hsp90 in a dynamic that is of a of hsp90 Morishima Y. Murphy P.J.M. Pratt W.B. Y. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). such a dynamic of heterocomplex to degradation S. Pratt W.B. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The a dynamic of hsp90 heterocomplex in when is from the purified assembly P. Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). a dynamic assembly be by the assembly Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The to the receptor as GR·hsp90 complexes are steroid binding that the chaperone machinery has out hsp90-dependent of the steroid binding In with from wild-type and HDAC6 knockdown cells in the of and and with to stable heterocomplex assembly with in the of to dynamic heterocomplex assembly the HDAC6 knockdown cytosol is deficient stable GR·hsp90 heterocomplex it has the same activity as wild-type cytosol dynamic heterocomplex hsp90 of HDAC6 to the of the decreased stable heterocomplex assembly activity of HDAC6 knockdown cytosol was because of function of purified hsp90 was to knockdown cytosol and GR·hsp90 heterocomplex assembly activity and steroid binding activity shown in of purified hsp90 stable heterocomplex assembly activity and steroid binding activity to the of wild-type cytosol. This suggests that of the assembly machinery than hsp90 are not affected by HDAC6 have reported that hsp90 from knockdown cells has to it (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it is that acetylation of hsp90 and that the of could this have shown that the of to hsp90 in is and that is the component of the hsp90/hsp70-based chaperone in Y. Murphy P.J.M. Y. Pratt W.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). purified is to to with is an in stable GR·hsp90 heterocomplex and steroid binding activity Y. Murphy P.J.M. Y. Pratt W.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in stable assembly is when purified is to wild-type cytosol Addition of purified to HDAC6 knockdown cytosol the same in stable but it does not the in assembly with to wild-type cytosol Thus, it that acetylation of hsp90 for and it is that acetylation hsp90 to to hsp90 from HDAC6 ATP-binding from HDAC6 knockdown cells are deficient stable GR·hsp90 heterocomplex but have of the stable assembly activity of from wild-type cells The ability to form stable heterocomplexes suggests that is a of and hsp90 in knockdown cytosol. purified hsp90 from knockdown cytosol on and Pratt W.B. 2003; PubMed Scopus Google Scholar). The purified hsp90 was for GR·hsp90 heterocomplex assembly activity in a five-protein purified purified human purified human and purified the of Pratt W.B. 2003; PubMed Scopus Google Scholar). the purified HDAC6 knockdown cell hsp90 the same activity stable GR·hsp90 heterocomplex assembly as hsp90 purified from wild-type cells not This that the knockdown cell hsp90 was and when knockdown cytosol was GR·hsp90 heterocomplex the of we a of hsp90 from the of cytosol with of hsp90 is that the chaperone is by under but in the it is to the in Pratt W.B. 2003; PubMed Scopus Google Scholar). Thus, we HDAC6 knockdown wild-type cytosol in to a of and with a of The by and for stable and dynamic GR·hsp90 assembly in the purified five-protein system. shown in the HDAC6 knockdown cell hsp90 from a and hsp90 from the wild-type cell This suggests that the hsp90 in knockdown cells has a ATP-binding than the hsp90 in wild-type The hsp90 purified from HDAC6 knockdown cells has stable GR·hsp90 heterocomplex assembly activity in the purified five-protein but it dynamic assembly activity Incubation with HDAC6 GR·hsp90 to have shown that hsp90 a HDAC6 mutant does not (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the in the five-protein assembly purified HDAC6 knockdown cell hsp90 was with and stable GR·hsp90 heterocomplex assembly was It be that the stable heterocomplex assembly activity of purified knockdown cell hsp90 is to the level of purified wild-type hsp90 by with Incubation of wild-type hsp90 with does not ability to generate steroid binding not The to the in HDAC6 a the of the ligand binding that is required for hsp90 binding and steroid binding activity M. Pratt W.B. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of in this of the to a mutant that in dynamic GR·hsp90 heterocomplex in S. Murphy P.J.M. Chen J. L. Pratt W.B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The for gene is to the right in cells expressing the mutant with the wild-type S. Murphy P.J.M. Chen J. L. Pratt W.B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Because GR·hsp90 heterocomplex is dynamic in HDAC6 knockdown we the same with to the dose response for HDAC6 knockdown cells transiently with an for with of to of shown in the dose response for activity is ∼100-fold to the right in HDAC6 knockdown wild-type In to the right shift in the dose is a in the of the wild-type and HDAC6 knockdown cells have the same of protein, and we not the activity is The an of HDAC6 on proteins than hsp90 involved in the we have shown that specific depletion of HDAC6 glucocorticoid in cells deficient in steroid binding activity and and in stable heterocomplex assembly the level of the intrinsic ability to be stable GR·hsp90 heterocomplexes with steroid binding activity are affected by HDAC6 knockdown from knockdown cells is deficient in ability to assemble stable GR·hsp90 heterocomplexes but the assembly activity is to the level of wild-type cytosol by of purified hsp90 This suggests that hsp90 is the component of the assembly machinery that is affected by depletion of with hsp90 purified from HDAC6 knockdown cells is deficient stable GR·hsp90 heterocomplex assembly when it is the hsp90 component of a purified five-protein assembly The in stable assembly by hsp90 from knockdown cytosol is by with HDAC6 between HDAC6 knockdown and wild-type acetylation hsp90 The hsp90 has decreased ATP-binding it has binding (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and it is of but not GR·hsp90 heterocomplex assembly decreased of hsp90 for was when cells with the HDAC inhibitor (4Yu X. Guo Z.S. Marcu M.G. Neckers L. Nguyen D.M. Chen G.A. Schrump D.S. J. Natl. Cancer Inst. 2002; 94: 504-513Crossref PubMed Scopus (358) Google Scholar). The (reviewed in Ref. Toft D.O. Cell 2003; PubMed Scopus Google Scholar) to the of hsp90 W. N. Toft D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Toft D.O. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), and to have an steroid binding the hsp90 Toft D.O. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The hsp90 in HDAC6 knockdown cells as a component of the chaperone machinery to the steroid binding of the GR, as shown in but the is because does not to the GR·hsp90 itself in GR·hsp90 complexes in the steroid binding form P. Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is that the hsp90 a binding but it is that binding does not acetylation an binding on hsp90 such that and the chaperone a binding hsp90 a binding the that is the binding for the hsp90 inhibitor P. J. E. H. E. Neckers Toft D.O. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, C. Rosen N. Hartl F.U. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, C. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), and it binding M.G. A. M. Neckers J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, C. D. P. J. J.M. C. M.G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus (125) Google Scholar), but it is not known acetylation of these to decreased binding for of the mechanism for the decreased binding, the acetylation of hsp90 the chaperone of stable GR·hsp90 The dynamic assembly with hsp90 with hsp90 when is not in a purified assembly P. Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Pratt W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the cell with HDAC6 of the hsp90 is and in the ATP-binding In the HDAC6 knockdown the of hsp90 is but from knockdown cells have hsp90 with ATP-binding that is of stable GR·hsp90 heterocomplex assembly The HDAC6 knockdown cells HDAC6 protein (15Kovacs J.J. Murphy P.J.M. Gaillard S. Zhao X. Wu J-T. Nicchitta C.V. Yoshida M. Toft D.O. Pratt W.B. Yao T.P. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), and it is that activity of for the of hsp90 activity in cytosol from knockdown it is that HDAC6 activity is for response to The wild-type in HDAC6 knockdown cells the mutant S. Murphy P.J.M. Chen J. L. Pratt W.B. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) in that the dose response for is of to the right with to wild-type cells expressing a level of This is the the in is a of the a of the in the cell have an steroid binding and be to Because of of the the of that the steroid binding is in the cell is and a of steroid be to hsp90 and the ligand binding Thus, the in HDAC6-deficient cells is of ligand binding, nuclear translocation, and gene and the HDAC6 with hsp90 acetylation a in of steroid are required to the same and Toft for and used in this