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Len Neckers - One of the best experts on this subject based on the ideXlab platform.
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Endoplasmic reticulum vacuolization and valosin-containing protein relocalization result from simultaneous hsp90 inhibition by Geldanamycin and proteasome inhibition by velcade.
Molecular cancer research : MCR, 2006Co-Authors: Edward G. Mimnaugh, Xitong Yuan, Michele D. Vos, Len NeckersAbstract:Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER--associated protein degradation, a sequential process requiring valosin-containing protein (VCP)-dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with Geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from Geldanamycin and Velcade exposure overwhelms the ability of the VCP--centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the "small VCP--interacting protein" exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP--enhanced yellow fluorescent protein fluorescent construct, Geldanamycin plus Velcade treatment redistributed VCP--enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous Geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination.
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endoplasmic reticulum vacuolization and valosin containing protein relocalization result from simultaneous hsp90 inhibition by Geldanamycin and proteasome inhibition by velcade
Molecular Cancer Research, 2006Co-Authors: Edward G. Mimnaugh, Wanping Xu, Xitong Yuan, Len NeckersAbstract:Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER-associated protein degradation, a sequential process requiring valosin-containing protein (VCP)–dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with Geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from Geldanamycin and Velcade exposure overwhelms the ability of the VCP-centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the “small VCP-interacting protein” exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP-enhanced yellow fluorescent protein fluorescent construct, Geldanamycin plus Velcade treatment redistributed VCP-enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous Geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination. (Mol Cancer Res 2006;4(9):667–81)
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simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination causes endoplasmic reticulum derived cytosolic vacuolization and enhances antitumor activity
Molecular Cancer Therapeutics, 2004Co-Authors: Edward G. Mimnaugh, Xitong Yuan, Michele D. Vos, Jennifer S Isaacs, Kheem S Bisht, David Gius, Len NeckersAbstract:The ansamycin antibiotic, Geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the Geldanamycin analogue, 17-N-allylamino-17-demethoxy Geldanamycin, are in separate clinical trials as new anticancer drugs. We hypothesized that destabilization of hsp 90 client proteins with Geldanamycin, while blocking their degradation with bortezomib, would promote the accumulation of aggregated, ubiquitinated, and potentially cytotoxic proteins. Indeed, Geldanamycin plus bortezomib inhibited MCF-7 tumor cell proliferation significantly more than either drug alone. Importantly, while control cells were unaffected, human papillomavirus E6 and E7 transformed fibroblasts were selectively sensitive to Geldanamycin plus bortezomib. Geldanamycin alone slightly increased protein ubiquitination, but when Geldanamycin was combined with bortezomib, protein ubiquitination was massively increased, beyond the amount stabilized by bortezomib alone. In Geldanamycin plus bortezomib-treated cells, ubiquitinated proteins were mostly detergent insoluble, indicating that they were aggregated. Individually, both Geldanamycin and bortezomib induced hsp 90, hsp 70, and GRP78 stress proteins, but the drug combination superinduced these chaperones and caused them to become detergent insoluble. Geldanamycin plus bortezomib also induced the formation of abundant, perinuclear vacuoles, which were neither lysosomes nor autophagosomes and did not contain engulfed cytosolic ubiquitin or hsp 70. Fluorescence marker experiments indicated that these vacuoles were endoplasmic reticulum derived and that their formation was prevented by cycloheximide, suggesting a role for protein synthesis in their genesis. These observations support a mechanism whereby the Geldanamycin plus bortezomib combination simultaneously disrupts hsp 90 and proteasome function, promotes the accumulation of aggregated, ubiquitinated proteins, and results in enhanced antitumor activity.
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heat shock protein 90 modulates the unfolded protein response by stabilizing ire1α
Molecular and Cellular Biology, 2002Co-Authors: Monica G Marcu, Linda M Hendershot, Melissa Doyle, Anne Bertolotti, David Ron, Len NeckersAbstract:The molecular chaperone HSP90 regulates stability and function of multiple protein kinases. The HSP90-binding drug Geldanamycin interferes with this activity and promotes proteasome-dependent degradation of most HSP90 client proteins. Geldanamycin also binds to GRP94, the HSP90 paralog located in the endoplasmic reticulum (ER). Because two of three ER stress sensors are transmembrane kinases, namely IRE1α and PERK, we investigated whether HSP90 is necessary for the stability and function of these proteins. We found that HSP90 associates with the cytoplasmic domains of both kinases. Both Geldanamycin and the HSP90-specific inhibitor, 514, led to the dissociation of HSP90 from the kinases and a concomitant turnover of newly synthesized and existing pools of these proteins, demonstrating that the continued association of HSP90 with the kinases was required to maintain their stability. Further, the previously reported ability of Geldanamycin to stimulate ER stress-dependent transcription apparently depends on its interaction with GRP94, not HSP90, since Geldanamycin but not 514 led to up-regulation of BiP. However, this effect is eventually superseded by HSP90-dependent destabilization of unfolded protein response signaling. These data establish a role for HSP90 in the cellular transcriptional response to ER stress and demonstrate that chaperone systems on both sides of the ER membrane serve to integrate this signal transduction cascade.
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Geldanamycin stimulated destabilization of mutated p53 is mediated by the proteasome in vivo
Oncogene, 1997Co-Authors: Luke Whitesell, Theodor W. Schulte, Patrick D Sutphin, Mikhail V Blagosklonny, Len NeckersAbstract:Mutation of the tumor suppressor gene p53 is the most common genetic abnormality detected in human cancers. Wild type p53 is a short-lived protein with very low basal intracellular levels. Most mutated forms of the protein, however, display markedly increased intracellular levels as an essential feature of their positive transforming activity. In this report, we have used selective inhibitors of the 20S proteasome to demonstrate that processing of p53 by ubiquitination and proteasome-mediated degradation is impaired by commonly occuring mutations of the protein. We found that this impairment of p53 turnover can be reversed by treatment of tumor cells with the benzoquinone ansamycin, Geldanamycin, leading to a marked reduction in intracellular p53 levels. Finally, using cells which over-express a mutant p53 protein, we were able to demonstrate that restoration of proteasome-mediated degradation by Geldanamycin is accompanied by p53 polyubiquitination. Although much remains to be learned about the mechanisms involved, our data demonstrate that selective de-stabilization of mutant transforming proteins such as p53 can be achieved pharmacologically with agents such as Geldanamycin which modify the function of molecular chaperone proteins within tumor cells.
Leonard M Neckers - One of the best experts on this subject based on the ideXlab platform.
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Geldanamycin abrogates ErbB2 association with proteasome-resistant beta-catenin in melanoma cells, increases beta-catenin-E-cadherin association, and decreases beta-catenin-sensitive transcription.
Cancer Research, 2001Co-Authors: Paolo Bonvini, Antonio García De Herreros, Mireia Duñach, Won G. An, Angelo Rosolen, Phongmai Nguyen, Jane B Trepel, Leonard M NeckersAbstract:β-catenin undergoes both serine and tyrosine phosphorylation. Serine phosphorylation in the amino terminus targets β-catenin for proteasome degradation, whereas tyrosine phosphorylation in the COOH terminus influences interaction with E-cadherin. We examined the tyrosine phosphorylation status of β-catenin in melanoma cells expressing proteasome-resistant β-catenin, as well as the effects that perturbation of β-catenin tyrosine phosphorylation had on its association with E-cadherin and on its transcriptional activity.β -catenin is tyrosine phosphorylated in three melanoma cell lines and associates with both the ErbB2 receptor tyrosine kinase and the LAR receptor tyrosine phosphatase. Geldanamycin, a drug which destabilizes ErbB2, caused rapid cellular depletion of the kinase and loss of its association with β-catenin without perturbing either LAR orβ -catenin levels or LAR/β-catenin association. Geldanamycin also stimulated tyrosine dephosphorylation of β-catenin and increasedβ -catenin/E-cadherin association, resulting in substantially decreased cell motility. Geldanamycin also decreased the nuclearβ -catenin level and inhibited β-catenin-driven transcription, as assessed using two different β-catenin-sensitive reporters and the endogenous cyclin D1 gene. These findings were confirmed by transient transfection of two β-catenin point mutants, Tyr-654Phe and Tyr-654Glu, which, respectively, mimic the dephosphorylated and phosphorylated states of Tyr-654, a tyrosine residue contained within the β-catenin-ErbB2-binding domain. These data demonstrate that the functional activity of proteasome-resistant β-catenin is regulated further by Geldanamycin-sensitive tyrosine phosphorylation in melanoma cells.
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novobiocin and related coumarins and depletion of heat shock protein 90 dependent signaling proteins
Journal of the National Cancer Institute, 2000Co-Authors: Monica G Marcu, Theodore W Schulte, Leonard M NeckersAbstract:BACKGROUND: Heat shock protein 90 (Hsp90) interacts with and stabilizes several oncogenic protein kinases (e.g., p185(erbB2), p60(v-src), and Raf-1) and is required for the stability and dominant-negative function of mutated p53 protein. Two unrelated antibiotics, Geldanamycin and radicicol, bind specifically to an atypical nucleotide-binding pocket of Hsp90, a site that shares homology with the adenosine triphosphate (ATP)-binding domain of bacterial DNA gyrase B. This interaction leads to destabilization of proteins that interact with Hsp90. Since the nucleotide-binding site of gyrase B is targeted by coumarin antibiotics (e.g., novobiocin), we investigated whether these drugs can also interact with Hsp90 and affect its activity. METHODS: We used immobilized novobiocin, Geldanamycin, or radicicol to isolate either endogenous Hsp90 from cell lysates or Hsp90 deletion fragments translated in vitro. Effects of the coumarin antibiotics novobiocin, chlorobiocin, and coumermycin A1 on several proteins interacting with Hsp90 were assessed in vitro and in vivo. RESULTS: Hsp90 binding to immobilized novobiocin was competed by soluble coumarins and ATP but not by Geldanamycin or radicicol. A carboxy-terminal Hsp90 fragment bound immobilized novobiocin but not immobilized Geldanamycin, while a Geldanamycin-binding amino-terminal fragment did not bind novobiocin. All three coumarins markedly reduced cellular levels of p185(erbB2), p60(v-src), Raf-1, and mutated p53. Furthermore, novobiocin reduced Raf-1 levels in the spleens of mice treated with the drug. CONCLUSIONS: These coumarin antibiotics, particularly novobiocin, represent a first-generation alternative to other Hsp90-targeting drugs that are not as well tolerated. Novobiocin's unique interaction with Hsp90 identifies an additional site on this protein amenable to pharmacologic interference with small molecules.
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Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with Geldanamycin.
Cell stress & chaperones, 1998Co-Authors: Theodor W. Schulte, David O. Toft, William J. Sullivan, Bridget Stensgard, Shiro Akinaga, Shiro Soga, Leonard M NeckersAbstract:The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind Geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to Geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with Geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the Geldanamycin binding site. Radicicol, as does Geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185erbB2, Raf-1 and mutant p53, similar to Geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.
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Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Luke Whitesell, Charles E Myers, B. Costa, Edward G. Mimnaugh, Leonard M NeckersAbstract:Abstract The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, Geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized Geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized Geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble Geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.
Edward G. Mimnaugh - One of the best experts on this subject based on the ideXlab platform.
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Endoplasmic reticulum vacuolization and valosin-containing protein relocalization result from simultaneous hsp90 inhibition by Geldanamycin and proteasome inhibition by velcade.
Molecular cancer research : MCR, 2006Co-Authors: Edward G. Mimnaugh, Xitong Yuan, Michele D. Vos, Len NeckersAbstract:Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER--associated protein degradation, a sequential process requiring valosin-containing protein (VCP)-dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with Geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from Geldanamycin and Velcade exposure overwhelms the ability of the VCP--centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the "small VCP--interacting protein" exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP--enhanced yellow fluorescent protein fluorescent construct, Geldanamycin plus Velcade treatment redistributed VCP--enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous Geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination.
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endoplasmic reticulum vacuolization and valosin containing protein relocalization result from simultaneous hsp90 inhibition by Geldanamycin and proteasome inhibition by velcade
Molecular Cancer Research, 2006Co-Authors: Edward G. Mimnaugh, Wanping Xu, Xitong Yuan, Len NeckersAbstract:Geldanamycin and Velcade, new anticancer drugs with novel mechanisms of action, are currently undergoing extensive clinical trials. Geldanamycin interrupts Hsp90 chaperone activity and causes down-regulation of its many client proteins by the ubiquitin-proteasome pathway; Velcade is a specific proteasome inhibitor. Misfolded Hsp90 clients within the endoplasmic reticulum (ER) lumen are cleared by ER-associated protein degradation, a sequential process requiring valosin-containing protein (VCP)–dependent retrotranslocation followed by ubiquitination and proteasomal proteolysis. Cotreatment of cells with Geldanamycin and Velcade prevents destruction of destabilized, ubiquitinated Hsp90 client proteins, causing them to accumulate. Here, we report that misfolded protein accumulation within the ER resulting from Geldanamycin and Velcade exposure overwhelms the ability of the VCP-centered machine to maintain the ER secretory pathway, causing the ER to distend into conspicuous vacuoles. Overexpression of dominant-negative VCP or the “small VCP-interacting protein” exactly recapitulated the vacuolated phenotype provoked by the drugs, associating loss of VCP function with ER vacuolization. In cells transfected with a VCP-enhanced yellow fluorescent protein fluorescent construct, Geldanamycin plus Velcade treatment redistributed VCP-enhanced yellow fluorescent protein from the cytoplasm and ER into perinuclear aggresomes. In further support of the view that compromise of VCP function is responsible for ER vacuolization, small interfering RNA interference of VCP expression induced ER vacuolization that was markedly increased by Velcade. VCP knockdown by small interfering RNA eventually deconstructed both the ER and Golgi and interdicted protein trafficking through the secretory pathway to the plasma membrane. Thus, simultaneous Geldanamycin and Velcade treatment has far-reaching secondary cytotoxic consequences that likely contribute to the cytotoxic activity of this anticancer drug combination. (Mol Cancer Res 2006;4(9):667–81)
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simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination causes endoplasmic reticulum derived cytosolic vacuolization and enhances antitumor activity
Molecular Cancer Therapeutics, 2004Co-Authors: Edward G. Mimnaugh, Xitong Yuan, Michele D. Vos, Jennifer S Isaacs, Kheem S Bisht, David Gius, Len NeckersAbstract:The ansamycin antibiotic, Geldanamycin, targets the hsp 90 protein chaperone and promotes ubiquitin-dependent proteasomal degradation of its numerous client proteins. Bortezomib is a specific and potent proteasome inhibitor. Both bortezomib and the Geldanamycin analogue, 17-N-allylamino-17-demethoxy Geldanamycin, are in separate clinical trials as new anticancer drugs. We hypothesized that destabilization of hsp 90 client proteins with Geldanamycin, while blocking their degradation with bortezomib, would promote the accumulation of aggregated, ubiquitinated, and potentially cytotoxic proteins. Indeed, Geldanamycin plus bortezomib inhibited MCF-7 tumor cell proliferation significantly more than either drug alone. Importantly, while control cells were unaffected, human papillomavirus E6 and E7 transformed fibroblasts were selectively sensitive to Geldanamycin plus bortezomib. Geldanamycin alone slightly increased protein ubiquitination, but when Geldanamycin was combined with bortezomib, protein ubiquitination was massively increased, beyond the amount stabilized by bortezomib alone. In Geldanamycin plus bortezomib-treated cells, ubiquitinated proteins were mostly detergent insoluble, indicating that they were aggregated. Individually, both Geldanamycin and bortezomib induced hsp 90, hsp 70, and GRP78 stress proteins, but the drug combination superinduced these chaperones and caused them to become detergent insoluble. Geldanamycin plus bortezomib also induced the formation of abundant, perinuclear vacuoles, which were neither lysosomes nor autophagosomes and did not contain engulfed cytosolic ubiquitin or hsp 70. Fluorescence marker experiments indicated that these vacuoles were endoplasmic reticulum derived and that their formation was prevented by cycloheximide, suggesting a role for protein synthesis in their genesis. These observations support a mechanism whereby the Geldanamycin plus bortezomib combination simultaneously disrupts hsp 90 and proteasome function, promotes the accumulation of aggregated, ubiquitinated proteins, and results in enhanced antitumor activity.
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polyubiquitination and proteasomal degradation of the p185c erbb 2 receptor protein tyrosine kinase induced by Geldanamycin
Journal of Biological Chemistry, 1996Co-Authors: Edward G. Mimnaugh, Christine Chavany, Len NeckersAbstract:Treatment of SKBr3 human breast carcinoma cells with the benzoquinoid ansamycin, Geldanamycin, rapidly depletes p185c-erbB-2 protein-tyrosine kinase. Loss of p185c-erbB-2 is initiated by disruption of a heteromeric complex between p185c-erbB-2 and the 94-kDa glucose-regulated protein, GRP94, to which Geldanamycin binds avidly. Here we report that within minutes of exposure to Geldanamycin, mature p185c-erbB-2 becomes polyubiquitinated. Treatment of cells with the specific proteasome proteolytic inhibitor, lactacystin, blocked Geldanamycin-induced degradation of p185c-erbB-2 and enhanced the accumulation of polyubiquitinated p185c-erbB-2. Following Geldanamycin and lactacystin treatment, a higher molecular weight form of p185c-erbB-2, which likely represents ubiquitin-p185c-erbB-2 conjugates, was detected by anti-p185c-erbB-2 immunoblotting. Nascent p185c-erbB-2 synthesized in the presence of Geldanamycin is incompletely glycosylated and remains sequestered in the endoplasmic reticulum. While this immature form of the protein is not ubiquitinated in the presence of Geldanamycin, its marked, drug-induced instability is nonetheless antagonized by lactacystin. Thus, the rapid depletion of mature p185c-erbB-2 caused by Geldanamycin and the marked, drug-stimulated decrease in half-life of the newly synthesized protein are both mediated by the proteasome, although only the former phenomenon involves polyubiquitination.
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Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation
Proceedings of the National Academy of Sciences of the United States of America, 1994Co-Authors: Luke Whitesell, Charles E Myers, B. Costa, Edward G. Mimnaugh, Leonard M NeckersAbstract:Abstract The molecular mechanisms by which oncogenic tyrosine kinases induce cellular transformation are unclear. Herbimycin A, Geldanamycin, and certain other benzoquinone ansamycins display an unusual capacity to revert tyrosine kinase-induced oncogenic transformation. As an approach to the study of v-src-mediated transformation, we examined ansamycin action in transformed cells and found that drug-induced reversion could be achieved without direct inhibition of src phosphorylating activity. To identify mechanisms other than kinase inhibition for drug-mediated reversion, we prepared a solid phase-immobilized Geldanamycin derivative and affinity precipitated the molecular targets with which the drug interacted. In a range of cell lines, immobilized Geldanamycin bound elements of a major class of heat shock protein (HSP90) in a stable and pharmacologically specific manner. Consistent with these binding data, we found that soluble Geldanamycin and herbimycin A inhibited specifically the formation of a previously described src-HSP90 heteroprotein complex. A related benzoquinone ansamycin that failed to revert transformed cells did not inhibit the formation of this complex. These results demonstrate that HSP participation in multimolecular complex formation is required for src-mediated transformation and can provide a target for drug modulation.
William B Pratt - One of the best experts on this subject based on the ideXlab platform.
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retrograde transport of the glucocorticoid receptor in neurites requires dynamic assembly of complexes with the protein chaperone hsp90 and is linked to the chip component of the machinery for proteasomal degradation
Molecular Brain Research, 2004Co-Authors: Mario D Galigniana, Jennifer M Harrell, Paul R Housley, Cam Patterson, Stephen K Fisher, William B PrattAbstract:Here, we have used a chimera of green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to study retrograde movement of a model soluble (i.e., non-vesicle-associated) protein in axons and dendrites of cultured NT2-N neurons. It is known that in non-neuronal cells, the GFP-GR moves from cytoplasm to the nucleus in a steroid-dependent manner by a rapid, hsp90-dependent mechanism. When rapid movement is inhibited by Geldanamycin (GA), a specific inhibitor of the protein chaperone hsp90, the GFP-GR translocates slowly to the nucleus by diffusion. Here we show that GFP-GR expressed in hormone-free neurons is localized in both cytoplasm and neurites, and upon treatment with dexamethasone (DEX), it moves to the nucleus. In neurites, movement by diffusion is not possible, and we show that movement of the GFP-GR from neurites is blocked by Geldanamycin, suggesting that the hsp90-dependent movement machinery is required for retrograde movement. In cells treated with both dexamethasone and Geldanamycin, the GFP-GR becomes concentrated in fluorescent globules located periodically along the neurites. Carboxyl terminus of Hsc70-interacting protein (CHIP), the E3 ubiquitin ligase for the GR, also concentrates in the same loci in a steroid-dependent and Geldanamycin-dependent manner. If Geldanamycin is removed, the GFP-GR exits the globules and continues its retrograde movement. However, in the continued presence of Geldanamycin, the GFP-GR in the globules undergoes proteasomal degradation, suggesting that the globules function as degradasomes. This is the first evidence for a linkage between receptor trafficking along neurites and receptor degradation by the proteasome.
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hsp90 is required for heme binding and activation of apo neuronal nitric oxide synthase Geldanamycin mediated oxidant generation is unrelated to any action of hsp90
Journal of Biological Chemistry, 2002Co-Authors: Scott S Billecke, Andrew T Bender, Kimon C Kanelakis, Patrick J Murphy, Ezra R Lowe, Yasuhiko Kamada, William B Pratt, Yoichi OsawaAbstract:It is established that neuronal NO synthase (nNOS) is associated with the chaperone hsp90, although the functional role for this interaction has not been defined. We have discovered that inhibition of hsp90 by radicicol or Geldanamycin nearly prevents the heme-mediated activation and assembly of heme-deficient apo-nNOS in insect cells. This effect is concentration-dependent with over 75% inhibition achieved at 20 microm radicicol. The ferrous carbonyl complex of nNOS is not formed when hsp90 is inhibited, indicating that functional heme insertion is prevented. We propose that the hsp90-based chaperone machinery facilitates functional heme entry into apo-nNOS by the opening of the hydrophobic heme-binding cleft in the protein. Previously, it has been reported that the hsp90 inhibitor Geldanamycin uncouples endothelial NOS activity and increases endothelial NOS-dependent O(2)() production. Geldanamycin is an ansamycin benzoquinone, and we show here that it causes oxidant production from nNOS in insect cells as well as with the purified protein. At a concentration of 20 microm, Geldanamycin causes a 3-fold increase in NADPH oxidation and hydrogen peroxide formation from purified nNOS, whereas the non-quinone hsp90 inhibitor radicicol had no effect. Thus, consistent with the known propensity of other quinones, Geldanamycin directly redox cycles with nNOS by a process independent of any action on hsp90, cautioning against the use of Geldanamycin as a specific inhibitor of hsp90 in redox-active systems.
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heat shock protein 90 dependent Geldanamycin inhibited movement of the glucocorticoid receptor through the cytoplasm to the nucleus requires intact cytoskeleton
Molecular Endocrinology, 1998Co-Authors: Mario D Galigniana, Paul R Housley, Jennifer L Scruggs, James Herrington, Michael J Welsh, Christin Cartersu, William B PrattAbstract:We use here a chimera of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) to test the notion that the protein chaperone heat shock protein-90 (hsp90) is required for steroid-dependent translocation of the receptor through the cytoplasm along cytoskeletal tracks. The GFP-GR fusion protein undergoes steroid-mediated translocation from the cytoplasm to the nucleus, where it is transcriptionally active. Treatment of 3T3 cells containing steroid-bound GFP-GR with Geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, inhibits dexamethasone-dependent translocation from the cytoplasm to the nucleus. The t1/2 for translocation in the absence of Geldanamycin is approximately 5 min, and the t1/2 in the presence of Geldanamycin is approximately 45 min. In cells treated for 1 h with the cytoskeletal disrupting agents colcemid, cytochalasin D, and beta,beta'-iminodipropionitrile to completely disrupt the microtubule, microfilament, and intermediate filament networks, respectively, the GFP-GR still translocates rapidly to the nucleus in a strictly dexamethasone-dependent manner but translocation is no longer affected by Geldanamycin. After withdrawal of the cytoskeletal disrupting agents for 3 h, normal cytoskeletal architecture is restored, and Geldanamycin inhibition of dexamethasone-dependent GFP-GR translocation is restored. We suggest that in cells without an intact cytoskeletal system, the GFP-GR moves through the cytoplasm by diffusion. However, under physiological conditions in which the cytoskeleton is intact, diffusion is limited, and the GFP-GR utilizes a movement machinery that is dependent upon hsp90 chaperone activity. In contrast to the GR, GFP-STAT5B, a signaling protein that is not complexed with hsp90, undergoes GH-dependent translocation to the nucleus in a manner that is not dependent upon hsp90 chaperone activity.
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Geldanamycin a heat shock protein 90 binding benzoquinone ansamycin inhibits steroid dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus
Biochemistry, 1997Co-Authors: Michael J Czar, Mario D Galigniana, And Adam M Silverstein, William B PrattAbstract:When they are translated, steroid receptors are assembled into a multiprotein complex containing hsp90, p23, an immunophilin, and often some hsp70. Some of the receptors, such as that for progesterone, have nuclear localization signals that are functional in the absence of hormone, and they move into the nucleus where they exist in the same multiprotein heterocomplex with hsp90. Other receptors, such as the glucocorticoid receptor, are localized predominantly in the cytoplasm in the absence of hormone and move into the nucleus in a hormone-dependent fashion. We have previously proposed that hsp90 and the immunophilin play a role in receptor trafficking [Pratt, W. B. (1993) J. Biol. Chem. 268, 21455-21458]. In this work, we show that treatment of L cells with Geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, impedes dexamethasone-dependent trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Because Geldanamycin treatment of hormone-free cells causes a rapid loss of steroid binding activity, receptors were prebound with dexamethasone by incubating cells with hormone at 0 degrees C prior to shifting the temperature to 37 degrees C for 20 min to permit receptor transformation and translocation in the presence or absence of Geldanamycin. Geldanamycin does not cause steroid to dissociate from prebound receptors, and it does not inhibit hormone-mediated receptor transformation assayed by conversion to the DNA-binding state. However, as reported previously for the progesterone receptor, Geldanamycin blocks assembly of the glucocorticoid receptor-hsp90 heterocomplex at an intermediate state of assembly where the receptor is bound to hsp70 and p60, both of which are required components in the assembly mechanism. Our observations support the proposal that dynamic association of receptors with hsp90 is required for receptor translocation from the cytoplasm to the nucleus.
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the hsp90 binding antibiotic Geldanamycin decreases raf levels and epidermal growth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of raf kinase
Journal of Biological Chemistry, 1997Co-Authors: Louis F Stancato, Adam M Silverstein, Janet K Owensgrillo, Yuhua Chow, Richard Jove, William B PrattAbstract:We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras·Raf, Ras·Src, Raf·Mek, and Raf·Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with Geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study Geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf·hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with Geldanamycin treatment of 3Y1 cells. To determine if Geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that Geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by Geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.
Laurence H Pearl - One of the best experts on this subject based on the ideXlab platform.
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regulation of hsp90 atpase activity by the co chaperone cdc37p p50cdc37
Journal of Biological Chemistry, 2002Co-Authors: Giuliano Siligardi, Peter W Piper, Laurence H Pearl, Barry Panaretou, Philippe Meyer, Shradha Singh, Derek N Woolfson, Chrisostomos ProdromouAbstract:In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace Geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with Geldanamycin-bound Hsp90 and may be sequestered in Geldanamycin-inhibited Hsp90 complexes in vivo.
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structural basis for inhibition of the hsp90 molecular chaperone by the antitumor antibiotics radicicol and Geldanamycin
Journal of Medicinal Chemistry, 1999Co-Authors: Chrisostomos Prodromou, Ronan Obrien, John E Ladbury, Peter W Piper, Laurence H PearlAbstract:The cellular activity of several regulatory and signal transduction proteins, which depend on the Hsp90 molecular chaperone for folding, is markedly decreased by Geldanamycin and by radicicol (monorden). We now show that these unrelated compounds both bind to the N-terminal ATP/ADP-binding domain of Hsp90, with radicicol displaying nanomolar affinity, and both inhibit the inherent ATPase activity of Hsp90 which is essential for its function in vivo. Crystal structure determinations of Hsp90 N-terminal domain complexes with Geldanamycin and radicicol identify key aspects of their nucleotide mimicry and suggest a rational basis for the design of novel antichaperone drugs.
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identification and structural characterization of the atp adp binding site in the hsp90 molecular chaperone
Cell, 1997Co-Authors: Chrisostomos Prodromou, Ronan Obrien, John E Ladbury, Peter W Piper, Laurence H PearlAbstract:Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B. This site is the same as that identified for the antitumor agent Geldanamycin, suggesting that Geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested. These results finally resolve the question of the direct involvement of ATP in Hsp90 function.