Steroid hormone receptors contain a conserved sequence of amino acids within the steroid binding ... more Steroid hormone receptors contain a conserved sequence of amino acids within the steroid binding domain, and we have previously speculated that this conserved region is the site of interaction of the glucocorticoid receptor with hsp90 (Danielsen, M., Northrop, J. P., and Ringold, G. M. (1986) EMBO J. 5, 2513-2522; Pratt, W. B., Jolly, D. J., Pratt, D. V., Hollenberg, S. M., Giguere, V., Cadepond, F. M., Schweizer-Groyer, G., Catelli, M.-G., Evans, R. M., and Baulieu, E.-E. (1988) J. Biol. Chem. 263, 267-273). In this work, we transfect COS-7 cells with three mutants of the mouse glucocorticoid receptor deleted for all or part of this conserved region. The mutant receptor missing the entire conserved region is very unstable and is found predominantly as cleavage products. Approximately one-third of the cleavage products have lost most or all of the steroid binding domain. This mutant receptor has a constitutive activity that is about one-third that of the steroid-bound wild type receptor in stimulating transcription from a reporter gene. We propose that the partial constitutive activity results from proteolytic cleavage of the steroid binding domain from the rest of the receptor, thus removing the functional repression determined by this domain. This mutant receptor is associated with hsp90 in cytosols prepared in the presence of molybdate but, when molybdate is not present, the receptor is unstable and there is very little receptor-associated hsp90. This observation is consistent with the proposal that binding of hsp90 helps to stabilize the glucocorticoid receptor against proteolysis, and it demonstrates that the site of molybdate interaction with the receptor lies outside of the conserved sequence. Our data are interpreted according to a two-site model in which hsp90 interacts with the steroid binding domain at two sites. One site is in the conserved sequence, and the other is at a transition metal oxyanion binding site, located between the conserved sequence and the COOH terminus.
A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the w... more A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.
To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorti... more To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stressactivated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE 2 E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE 2 E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the existence of HSF1-regulated genes that serve to elevate the response of steroid receptors under stress conditions. (Molecular Endocrinology 18:
Typescript. "In partial fulfillment of the requirements for the degree of Doctor of Philosop... more Typescript. "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin R. Sanchez. Thesis (Ph. D.)--Medical College of Ohio, 2002. Includes bibliographical references (leaves 126-145).
glutárica tipo 1: presentación de un caso y revisión de la literatura. Iatreia. 2015 Abr-Jun;28(2... more glutárica tipo 1: presentación de un caso y revisión de la literatura. Iatreia. 2015 Abr-Jun;28(2):193-197.
414 P Pa al la ab br ra as s c cl la av ve es s Recubrimiento por plasma. Nanomateriales. Cermet.... more 414 P Pa al la ab br ra as s c cl la av ve es s Recubrimiento por plasma. Nanomateriales. Cermet. Emisión acústica. Fractura.
Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, sug... more Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, suggesting cross-talk between the heat shock and GR signal pathways. An unresolved question concerning this cross-talk is whether heat shock factor (HSF1) activity is required for this response. We addressed this issue by modulating HSF1 activity with compounds acting by distinct mechanisms: sodium vanadate (SV), an inhibitor of protein phosphatases; and wortmannin, an inhibitor of DNA-dependent protein kinase. Using HSF1- and GR-responsive CAT reporters, we demonstrate that SV inhibits both HSF1 activity and the stress potentiation of GR, while having no effect on the hormone-free GR or HSF1. Paradoxically, SV increased hormone-induced GR activity in the absence of stress. In contrast, wortmannin increased HSF1 activity in stressed cells and had no effect on HSF1 in the absence of stress. Using the pMMTV-CAT reporter containing the negative regulatory element 1 site for DNA-dependent prote...
The Journal of steroid biochemistry and molecular biology, 1993
The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules ... more The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules of the 90-kDa heat shock protein (hsp90) and one molecule of the 56-kDa heat shock protein (hsp56). In the absence of hormone, this complex is found in the cytosolic fraction of cells, and upon hormone-binding the complex dissociates and the GR is recovered in the nuclear pellet fraction. Given the association of heat shock proteins with the cytosolic form of the GR, we have examined the effects of heat shock on GR subcellular localization and transcription enhancement activity in a series of Chinese hamster ovary (CHO) cells which express either low levels of endogenous GR (CHOd cells), high levels of the mouse GR (WCL2 cells), or high levels of mutated mouse GR unable to bind DNA (NB cells). It was found that heat shock treatment of WCL2 cells results in wild-type mouse GR that is recovered almost entirely within the nuclear pellet fraction, a response similar to that seen in hormone-t...
Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocortic... more Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocorticoid-binding capacity. The steroid-binding capacity of extracted cytosol can be restored by adding dithiothreitol or by incubating with boiled liver cytosol at 20 degrees C in the presence of 10 mM sodium molybdate. Two components of boiled cytosol are required for receptor activation: NADPH and an endogenous heat-stable protein with an apparent Mr of 12,300 by Sephadex G-50 chromatography. This endogenous receptor-activating protein coelutes on Sephadex G-50 chromatography with endogenous thioredoxin activity, and it can be replaced in the activating system by purified Escherichia coli thioredoxin. These observations suggest that glucocorticoid receptors in cytosol preparations are maintained in a reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system.
We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells c... more We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclon...
Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound recept... more Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor c...
The recently-described p59 protein has been shown to be associated with untransformed steroid rec... more The recently-described p59 protein has been shown to be associated with untransformed steroid receptors present in rabbit uterus and rat liver cytosols (Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275; Renoir, J.-M., Radanyi, C., Faber, L. E., and Baulieu, E.-E. (1990) J. Biol. Chem. 265, 10740-10745), while a smaller version of this protein (p56) interacts with glucocorticoid receptors in human IM-9 cell cytosols (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). In addition to interacting with glucocorticoid receptors, the p56 protein of IM-9 cell cytosol is also found as part of a large heteromeric complex that contains both the 70-kDa and 90-kDa heat shock proteins (hsp70 and hsp90, respectively). Given this association of p56 with the two major stress proteins, I have speculated that p56 may itself be a heat shock protein. In this paper, the effect of heat stress o...
Background: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes... more Background: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.
This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabi... more This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabilized cytosol prepared from j'P-labeled L-cells. In our early work, we showed that cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90 and a 98-100 kdalton protein) that elute from an affinity resin of deoxycorticosterone agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor. Both phosphoproteins are immunoadsorbed onto protein-A-Sepharose from molybdate-stabilized cytosol incubated with a monoclonal antibody against the receptor. The 98-100 kdalton phosphoprotein binds steroid and the 90 kdalton phosphoprotein is a structurally different, nonsteroid-binding protein that is bound to the untransformed, molybdate-stabilized glucocorticoid receptor. The 90 kdalton protein reacts on Western blots with a monoclonal antibody raised against a 90 kdalton protein from the water mold Achlya ambisexualis.
Treatment of rat liver cytosol with hydrogen peroxide (H,O,) or sodium molybdate (Moo:-) inhibits... more Treatment of rat liver cytosol with hydrogen peroxide (H,O,) or sodium molybdate (Moo:-) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25°C. Dithiothreitol (DTT) prevents the stabilization of receptors by H,O1. Heating (25°C) of immune pellets formed by immunoadsorption of L-cell murine giucocorticoid receptor complexes to protein-Adepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heal shock protein (hsp90) from the steroid binding protein. Such the~al-induced dissociation of hsp90 is inhibited by H102. Pre~~ment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H,O, to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.
Glucocorticoids control a host of bodily responses, ranging from carbohydrate metabolism in the l... more Glucocorticoids control a host of bodily responses, ranging from carbohydrate metabolism in the liver to immunity and inflammation in the lymph system. In response to stress, glucocorticoid levels are known to rise-a response thought to provide a protective function against the stress event. It is now understood that the major function of glucocorticoids under stress is to protect not against the stress event itself but against overstimulation by host defenses (e.g., inflammation). Control of these responses is achieved by the glucocorticoid receptor, a member of the steroid receptor transcription factor family. The oldest, most conserved, and most ubiquitous of the stress responses is induced expression of heat shock proteins that act as chaperones against stress-induced denaturation of protein. Expression of heat shock protein genes is controlled by heat shock transcription factor 1. In this work, we review our observations and those of other laboratories demonstrating a relationship between the glucocorticoid and heat shock responses. We show that complex but reciprocal mechanisms of regulation occur between glucocorticoid receptor and heat shock transcription factor 1 and present a model of coordinated action that likely serves to fully reestablish homeostasis following stress.
The International Journal of Biochemistry & Cell Biology, 2005
The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug ... more The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug FK506. FKBP52 exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity, which is inhibited by the binding of FK506--properties that it shares with the smaller but better-studied immunophilin, FKBP12. Unlike FKBP12, however, FKBP52 does not mediate the immunosuppressive actions of FK506 and, due to its larger size, contains additional numerous functional domains. One such structure is a series of tetratricopeptide repeat (TPR) domains, which serve as binding sites for the ubiquitous and abundant molecular chaperone, Hsp90. It is this property as a TPR protein that best characterizes the known cellular roles of FKBP52. Here, we review the structural features of FKBP52 and relate them to the evolving and diverse functions of this protein. Although the most recognized role of FKBP52 is in regulation of steroid receptor signaling, other less well-known functions are also discussed.
Steroid hormone receptors contain a conserved sequence of amino acids within the steroid binding ... more Steroid hormone receptors contain a conserved sequence of amino acids within the steroid binding domain, and we have previously speculated that this conserved region is the site of interaction of the glucocorticoid receptor with hsp90 (Danielsen, M., Northrop, J. P., and Ringold, G. M. (1986) EMBO J. 5, 2513-2522; Pratt, W. B., Jolly, D. J., Pratt, D. V., Hollenberg, S. M., Giguere, V., Cadepond, F. M., Schweizer-Groyer, G., Catelli, M.-G., Evans, R. M., and Baulieu, E.-E. (1988) J. Biol. Chem. 263, 267-273). In this work, we transfect COS-7 cells with three mutants of the mouse glucocorticoid receptor deleted for all or part of this conserved region. The mutant receptor missing the entire conserved region is very unstable and is found predominantly as cleavage products. Approximately one-third of the cleavage products have lost most or all of the steroid binding domain. This mutant receptor has a constitutive activity that is about one-third that of the steroid-bound wild type receptor in stimulating transcription from a reporter gene. We propose that the partial constitutive activity results from proteolytic cleavage of the steroid binding domain from the rest of the receptor, thus removing the functional repression determined by this domain. This mutant receptor is associated with hsp90 in cytosols prepared in the presence of molybdate but, when molybdate is not present, the receptor is unstable and there is very little receptor-associated hsp90. This observation is consistent with the proposal that binding of hsp90 helps to stabilize the glucocorticoid receptor against proteolysis, and it demonstrates that the site of molybdate interaction with the receptor lies outside of the conserved sequence. Our data are interpreted according to a two-site model in which hsp90 interacts with the steroid binding domain at two sites. One site is in the conserved sequence, and the other is at a transition metal oxyanion binding site, located between the conserved sequence and the COOH terminus.
A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the w... more A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.
To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorti... more To further define the role of heat shock factor 1 (HSF1) in the stress potentiation of glucocorticoid receptor (GR) activity, we placed a constitutively active mutant of human HSF1 (hHSF1-E189) under the control of a doxycycline (DOX)-inducible vector. In mouse L929 cells, DOX-induced expression of hHSF1-E189 correlated with in vivo occupancy of the human heat shock protein 70 (hHsp70) promoter (chromatin-immunoprecipitation assay) and with increased activity under nonstress conditions at the hHsp70 promoter controlling expression of chloramphenicol acetyl transferase (CAT) (p2500-CAT). Comparison of hHSF1-E189 against stressactivated, endogenous HSF1 for DNA-binding, p2500-CAT, and Hsp70 protein expression activities showed the mutant factor to have lower, but clearly detectable, activities as compared with wild-type factor. Thus, the hHSF1-E189 mutant is capable of replicating these key functions of endogenous HSF1, albeit at reduced levels. To assess the involvement of hHSF1-E189 in GR activity, DOX-induced expression of hHSF1-E189 was performed in L929 cells expressing the minimal pGRE 2 E1B-CAT reporter. hHSF1-E189 protein expression in these cells was maximal at 24 h of DOX and remained constant up to 72 h. hHSF1-E189 expressed under these conditions was found both in the cytosolic and nuclear compartments, in a state capable of binding DNA. More importantly, GR activity at the pGRE 2 E1B-CAT promoter was found to increase after DOX-induced expression of hHSF1-E189. The potentiation of GR by hHSF1-E189 occurred at saturating concentrations of hormone and was dependent on at least 48 h of hHSF1-E189 up-regulation, suggesting that time was needed for an HSF1-induced factor to accumulate to a threshold level. Initial efforts to characterize how hHSF1-E189 controls GR signaling showed that it does not occur through alterations of GR protein levels or changes in GR hormone binding capacity. In summary, our observations provide the first molecular evidence for the existence of HSF1-regulated genes that serve to elevate the response of steroid receptors under stress conditions. (Molecular Endocrinology 18:
Typescript. "In partial fulfillment of the requirements for the degree of Doctor of Philosop... more Typescript. "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Medical Sciences." Major advisor: Edwin R. Sanchez. Thesis (Ph. D.)--Medical College of Ohio, 2002. Includes bibliographical references (leaves 126-145).
glutárica tipo 1: presentación de un caso y revisión de la literatura. Iatreia. 2015 Abr-Jun;28(2... more glutárica tipo 1: presentación de un caso y revisión de la literatura. Iatreia. 2015 Abr-Jun;28(2):193-197.
414 P Pa al la ab br ra as s c cl la av ve es s Recubrimiento por plasma. Nanomateriales. Cermet.... more 414 P Pa al la ab br ra as s c cl la av ve es s Recubrimiento por plasma. Nanomateriales. Cermet. Emisión acústica. Fractura.
Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, sug... more Heat shock and other forms of stress increase glucocorticoid receptor (GR) activity in cells, suggesting cross-talk between the heat shock and GR signal pathways. An unresolved question concerning this cross-talk is whether heat shock factor (HSF1) activity is required for this response. We addressed this issue by modulating HSF1 activity with compounds acting by distinct mechanisms: sodium vanadate (SV), an inhibitor of protein phosphatases; and wortmannin, an inhibitor of DNA-dependent protein kinase. Using HSF1- and GR-responsive CAT reporters, we demonstrate that SV inhibits both HSF1 activity and the stress potentiation of GR, while having no effect on the hormone-free GR or HSF1. Paradoxically, SV increased hormone-induced GR activity in the absence of stress. In contrast, wortmannin increased HSF1 activity in stressed cells and had no effect on HSF1 in the absence of stress. Using the pMMTV-CAT reporter containing the negative regulatory element 1 site for DNA-dependent prote...
The Journal of steroid biochemistry and molecular biology, 1993
The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules ... more The untransformed glucocorticoid receptor (GR) is a heteromeric complex containing two molecules of the 90-kDa heat shock protein (hsp90) and one molecule of the 56-kDa heat shock protein (hsp56). In the absence of hormone, this complex is found in the cytosolic fraction of cells, and upon hormone-binding the complex dissociates and the GR is recovered in the nuclear pellet fraction. Given the association of heat shock proteins with the cytosolic form of the GR, we have examined the effects of heat shock on GR subcellular localization and transcription enhancement activity in a series of Chinese hamster ovary (CHO) cells which express either low levels of endogenous GR (CHOd cells), high levels of the mouse GR (WCL2 cells), or high levels of mutated mouse GR unable to bind DNA (NB cells). It was found that heat shock treatment of WCL2 cells results in wild-type mouse GR that is recovered almost entirely within the nuclear pellet fraction, a response similar to that seen in hormone-t...
Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocortic... more Extraction of rat liver cytosol with 10% charcoal at 4 degrees C inactivates specific glucocorticoid-binding capacity. The steroid-binding capacity of extracted cytosol can be restored by adding dithiothreitol or by incubating with boiled liver cytosol at 20 degrees C in the presence of 10 mM sodium molybdate. Two components of boiled cytosol are required for receptor activation: NADPH and an endogenous heat-stable protein with an apparent Mr of 12,300 by Sephadex G-50 chromatography. This endogenous receptor-activating protein coelutes on Sephadex G-50 chromatography with endogenous thioredoxin activity, and it can be replaced in the activating system by purified Escherichia coli thioredoxin. These observations suggest that glucocorticoid receptors in cytosol preparations are maintained in a reduced, steroid-binding state by a NADPH-dependent, thioredoxin-mediated reducing system.
We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells c... more We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclon...
Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound recept... more Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor c...
The recently-described p59 protein has been shown to be associated with untransformed steroid rec... more The recently-described p59 protein has been shown to be associated with untransformed steroid receptors present in rabbit uterus and rat liver cytosols (Tai, P. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275; Renoir, J.-M., Radanyi, C., Faber, L. E., and Baulieu, E.-E. (1990) J. Biol. Chem. 265, 10740-10745), while a smaller version of this protein (p56) interacts with glucocorticoid receptors in human IM-9 cell cytosols (Sanchez, E. R., Faber, L. E., Henzel, W. J., and Pratt, W. B. (1990) Biochemistry 29, 5145-5152). In addition to interacting with glucocorticoid receptors, the p56 protein of IM-9 cell cytosol is also found as part of a large heteromeric complex that contains both the 70-kDa and 90-kDa heat shock proteins (hsp70 and hsp90, respectively). Given this association of p56 with the two major stress proteins, I have speculated that p56 may itself be a heat shock protein. In this paper, the effect of heat stress o...
Background: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes... more Background: Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.
This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabi... more This paper summarizes our work performed with glucocorticoid-binding complexes in molybdate-stabilized cytosol prepared from j'P-labeled L-cells. In our early work, we showed that cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90 and a 98-100 kdalton protein) that elute from an affinity resin of deoxycorticosterone agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor. Both phosphoproteins are immunoadsorbed onto protein-A-Sepharose from molybdate-stabilized cytosol incubated with a monoclonal antibody against the receptor. The 98-100 kdalton phosphoprotein binds steroid and the 90 kdalton phosphoprotein is a structurally different, nonsteroid-binding protein that is bound to the untransformed, molybdate-stabilized glucocorticoid receptor. The 90 kdalton protein reacts on Western blots with a monoclonal antibody raised against a 90 kdalton protein from the water mold Achlya ambisexualis.
Treatment of rat liver cytosol with hydrogen peroxide (H,O,) or sodium molybdate (Moo:-) inhibits... more Treatment of rat liver cytosol with hydrogen peroxide (H,O,) or sodium molybdate (Moo:-) inhibits thermal inactivation of glucocorticoid receptor steroid-binding capacity at 25°C. Dithiothreitol (DTT) prevents the stabilization of receptors by H,O1. Heating (25°C) of immune pellets formed by immunoadsorption of L-cell murine giucocorticoid receptor complexes to protein-Adepharose with an anti-receptor monoclonal antibody (BuGR2) results in dissociation of the M 90,000 heal shock protein (hsp90) from the steroid binding protein. Such the~al-induced dissociation of hsp90 is inhibited by H102. Pre~~ment of immunoadsorbed receptor complexes with the thiol derivatizing agent, methyl methanethiosulfonate (MMTS) prevents the ability of H,O, to stabilize the hsp90-receptor interaction. These data suggest a role for hsp90 in maintaining an active steroid-binding conformation of the glucocorticoid receptor.
Glucocorticoids control a host of bodily responses, ranging from carbohydrate metabolism in the l... more Glucocorticoids control a host of bodily responses, ranging from carbohydrate metabolism in the liver to immunity and inflammation in the lymph system. In response to stress, glucocorticoid levels are known to rise-a response thought to provide a protective function against the stress event. It is now understood that the major function of glucocorticoids under stress is to protect not against the stress event itself but against overstimulation by host defenses (e.g., inflammation). Control of these responses is achieved by the glucocorticoid receptor, a member of the steroid receptor transcription factor family. The oldest, most conserved, and most ubiquitous of the stress responses is induced expression of heat shock proteins that act as chaperones against stress-induced denaturation of protein. Expression of heat shock protein genes is controlled by heat shock transcription factor 1. In this work, we review our observations and those of other laboratories demonstrating a relationship between the glucocorticoid and heat shock responses. We show that complex but reciprocal mechanisms of regulation occur between glucocorticoid receptor and heat shock transcription factor 1 and present a model of coordinated action that likely serves to fully reestablish homeostasis following stress.
The International Journal of Biochemistry & Cell Biology, 2005
The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug ... more The large molecular-weight immunophilin, FKBP52, is a known target of the immunosuppressive drug FK506. FKBP52 exhibits peptidyl-prolyl cis-trans isomerase (PPIase) activity, which is inhibited by the binding of FK506--properties that it shares with the smaller but better-studied immunophilin, FKBP12. Unlike FKBP12, however, FKBP52 does not mediate the immunosuppressive actions of FK506 and, due to its larger size, contains additional numerous functional domains. One such structure is a series of tetratricopeptide repeat (TPR) domains, which serve as binding sites for the ubiquitous and abundant molecular chaperone, Hsp90. It is this property as a TPR protein that best characterizes the known cellular roles of FKBP52. Here, we review the structural features of FKBP52 and relate them to the evolving and diverse functions of this protein. Although the most recognized role of FKBP52 is in regulation of steroid receptor signaling, other less well-known functions are also discussed.
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Papers by Edwin Sanchez