Reactive cysteines of the 90-kDa heat shock protein, Hsp90

The 90-kDa heat shock protein (Hsp90) is the most abundant molecular chaperone of the eukaryotic cytoplasm. Its cysteine groups participate in the interactions of Hsp90 with the heme-regulated eIF-2alpha kinase and molybdate, a stabilizer of Hsp90-protein complexes. In our present studies we investigated the reactivity of the sulfhydryl groups of Hsp90. Our data indicate that Hsp90 as well as two Hsp90 peptides containing Cys-521 and Cys-589/590 are able to reduce cytochrome c. The effect of Hsp90 can be blocked by sulfhydryl reagents including arsenite and cadmium, which indicates the involvement of the vicinal cysteines Cys589/590 in the reduction of cytochrome c. Hsp90 neither reduces the disulfide bonds of insulin nor possesses a NADPH:quinone oxidoreductase activity. Oxidizing conditions impair the chaperone activity of Hsp90 toward citrate synthase. The high and specific reactivity of Hsp90 cysteine groups toward cytochrome c may indicate a role of this chaperone in modulation of the redox status of the cytosol in resting and apoptotic cells.     

Evidence that the 90-kDa phosphoprotein associated with the untransformed L-cell glucocorticoid receptor is a murine heat shock protein

Two phosphoproteins are adsorbed to protein-A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98-100-kDa phosphoprotein that contains the steroid-binding site and the other is a 90-kDa nonsteroid-binding phosphoprotein that is associated with the untransformed, molybdate-stabilized receptor (Housley, P. R., Sanchez, E. R., Westphal, H.M., Beato, M., and Pratt, W.B. (1985) J. Biol. Chem. 260, in press). In this paper we show that the 90-kDa receptor-associated phosphoprotein is an abundant cytosolic protein that reacts with a monoclonal antibody that recognizes the 90-kDa phosphoprotein that binds steroid receptors in the chicken oviduct. The 90-kDa protein immunoadsorbed from L-cell cytosol with this antibody reacts on Western blots with rabbit antiserum prepared against the 89-kDa chicken heat shock protein. Immunoadsorption of molybdate-stabilized cytosol by antibodies against the glucocorticoid receptor results in the presence of a 90-kDa protein that interacts on Western blots with the antiserum against the chicken heat shock protein. The association between the 90-kDa protein and the receptor is only seen by this technique when molybdate is present to stabilize the complex; and when steroid-bound receptors are incubated at 25 degrees C to transform them to the DNA-binding state, the 90-kDa protein dissociates. These observations are consistent with the proposal that the untransformed glucocorticoid receptor in L-cells exists in a complex with the murine 90-kDa heat shock protein.     

Association of the Ah receptor with the 90-kDa heat shock protein

Partially purified Ah receptor preparations were used to produce a monoclonal antibody, designated as 8D3, that is capable of immunoprecipitating the Ah receptor. Hepa 1c1c7 cytosol was photoaffinity-labeled with [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin followed by immunoprecipitation, and the resulting precipitate was applied to a sodium dodecyl sulfate-polyacrylamide electrophoretic gel. These gels were stained with Coomassie Blue and revealed the presence of a major immunoprecipitated 90-kDa protein, and after autoradiography a radiolabeled 95-kDa protein (Ah receptor) was detected. The 90-kDa protein was determined to be the 90-kDa heat shock protein (HSP90) by western blot analysis using an antibody (AC88) previously shown to be specific for HSP90. An increase in the sedimentation of the Ah receptor on sucrose density gradients was seen upon addition of monoclonal antibody 8D3 to Hepa 1c1c7 cytosol. Monoclonal antibody 8D3 immunoprecipitates the Ah receptor from Hepa 1 cells (murine), HeLa cells (human), and rat liver cytosolic extracts, indicating that the Ah receptor is complexed with HSP90 in several mammalian species tested. These results illustrate another physicochemical property that the supergene family of soluble steroid receptors and the Ah receptor have in common.     

Calmodulin-regulated binding of the 90-kDa heat shock protein to actin filaments

We have found that the 90-kDa heat shock protein (HSP90) prepared from a mouse lymphoma exists in homodimeric form under physiological conditions and has the ability to bind to F-actin (Koyasu, S., Nishida, E., Kadowaki, T., Matsuzaki, F., Iida, K., Harada, F., Kasuga, M., Sakai, H., and Yahara, I. (1986) Proc. Natl. Acad. Sci. U.S.A., in press). Here we show that calmodulin regulates the binding of HSP90 to F-actin in a Ca2+-dependent manner. The binding of HSP90 to F-actin occurred optimally under physiological solution conditions, i.e. in 2 mM MgCl2 + 100 mM KCl. The binding was saturable in a molar ratio of about 1 HSP90 (dimer) to 10 actins. HSP90 was dissociated from F-actin by the binding of tropomyosin to F-actin. Calmodulin was found to inhibit the binding of HSP90 to F-actin in a Ca2+-dependent manner. Moreover, the equilibrium gel filtration demonstrated that calmodulin binds to HSP90 in the presence of Ca2+, but not in the absence of Ca2+. These data indicate that HSP90 complexed with Ca2+-calmodulin is unable to bind to F-actin. Ca2+-dependent interaction of HSP90 with calmodulin as well as calmodulin-regulated binding of HSP90 to F-actin revealed here may provide new insight into the function of HSP90 and the regulation of actin structure in cells.     

Regulation of cytosolic prostaglandin E2 synthase by 90-kDa heat shock protein

Cytosolic prostaglandin (PG) E(2) synthase (cPGES) is constitutively expressed in a wide variety of cells and converts cyclooxygenase (COX)-1-derived PGH(2) to PGE(2). Given the fact that cPGES is identical to p23, a heat shock protein 90 (Hsp90)-binding protein, we herein examined the effect of Hsp90 on PGE(2) generation by cPGES. Incubation of cPGES with Hsp90 resulted in a significant increase in PGES activity in vitro. Association of cPGES with Hsp90 was increased in cells stimulated with A23187 or bradykinin, accompanied by concomitant increases in cPGES activity and PGE(2) production. Moreover, treatment of cells with Hsp90 inhibitors, which destabilized the cPGES/Hsp90 complex, reduced cPGES activity and PGE(2) production to basal levels. These results suggest that the regulation of cPGES activity in cells depends on its association with Hsp90 and provide the first line of evidence that eicosanoid biosynthesis is under the control of the molecular chaperone.     

Characterization of the 90 kDa heat shock protein (HSP90)-associated ATP/GTPase

The 90 kDa heat shock protein (HSP90) is an ATP-binding molecular chaperone with an associated ATPase activity having nucleoplasmin and HSP70-binding homology domains and containing Ca-binding EF-hands and a nuclear localization signal. Here we characterize the HSP90-associated ATPase and show that it is (i) a P-type ATPase inhibited by molybdate and vanadate, (ii) able to hydrolyze methylfluorescein phosphate with a 5–6-fold higher affinity, (iii) a 3-times better GTPase than ATPase in the presence of calcium and (iv) HSP27 and F-actin, but not HSP10 can “convert” the HSP90-associated ATPase activity to HSP90 autokinase activity. The HSP90-associated ATP/GTPase may participate in the regulation of complex formation of HSP90 with other proteins, such as F-actin, tubulin and heat shock proteins.     

Possible involvement of the 90-kDa heat shock protein in the regulation of protein synthesis

The heme-sensitive eukaryotic initiation factor (eIF)-2 alpha kinase regulates translational activity in reticulocytes by phosphorylation of the smallest subunit of eukaryotic peptide initiation factor 2, eIF-2. Highly purified preparations of the kinase contain an abundant 90-kDa polypeptide which appears to modulate the activity of the enzyme. The physical properties and structural characteristics of the reticulocyte 90-kDa peptide are similar to those of the 90-kDa heat shock protein (hsp 90) from HeLa and other mammalian cells. The reticulocyte and HeLa cell proteins are shown to be immunologically cross-reactive. A direct comparison of the two proteins by one-dimensional peptide mapping of large peptides generated by limited proteolysis and by reversed-phase high performance liquid chromatography analysis of tryptic peptides indicates that they represent the same protein species. Like the 90-kDa reticulocyte protein, HeLa cell hsp 90 causes increased eIF-2 alpha phosphorylation by the heme-sensitive kinase and is a potent inhibitor of protein synthesis in the reticulocyte lysate system. A potential mechanism for the latter inhibition is inferred. These results implicate hsp 90 in the regulation of protein synthesis via its interaction with and perhaps regulation of the heme-sensitive kinase and phosphorylation of eIF-2 alpha.     

Three-step purification method and characterization of the bovine brain 90-kDa heat shock protein

A protein that cross-reacted with antibody against the 90-kDa heat shock protein (HSP90) of a mouse lymphoma cell line was purified from bovine brain by three steps. Fifty milligrams of the 90-kDa protein was recovered from 350 g of the brain cortex. The sedimentation coefficient and Stokes radius of the purified protein were 6.0 s and 6.7 nm, respectively. The molecular weight was calculated to be 170,000. The molecule was composed of two identical 90-kDa subunits. A partial amino acid sequence (23 residues) of this protein was homologous (96%) to human HSP90 (the sequence of 174-196). These facts led to the identification of the 90-kDa brain protein with HSP90. In bovine tissues, the brain contained this protein at a remarkably high concentration. The brain HSP90 was separable from glucocorticoid receptor by heparin-agarose and DNA-cellulose columns. It is concluded that HSP90 is present in brain cytosol and mostly as free molecules. Immunohistochemical studies showed that the protein was localized in nerve excitable cells. It was not found in nuclei but in cytosol.     

Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90

The 90-kDa heat shock protein, HSP90, of the mouse has two isoforms, alpha and beta, which are electrophoretically separable. We have investigated the native forms of HSP90 molecules under physiological conditions and determined their isoform compositions. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that HSP90 purified from mouse lymphoma L5178Y cells consists of approximately 40% alpha and 60% beta isoforms. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the purified HSP90 exists predominantly as a dimer, but a considerable amount of monomer was also detected. Western blotting using polyclonal anti-mouse HSP90 antibodies revealed that the native forms of HSP90 in the crude L5178Y cell lysates are also dimer and monomer. The nondenaturing polyacrylamide gel electrophoresis resolved the dimeric forms into two separate bands that were identified as alpha/alpha and beta/beta homodimers by two methods: sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. In addition, the results showed that the monomeric form consists mainly of the beta isoform. Both the alpha and beta isoforms were shown to bind equally to actin filaments.     

Function of 90-kDa heat shock protein in cellular differentiation of human embryonal carcinoma cells

Summary Heat shock proteins (HSPs) have been recognized as molecules that maintain cellular homeostasis during changes in the environment. Here we report that HSP90 functions not only in stress responses but also in certain aspects of cellular differentiation. We found that HSP90 slowed remarkably high expression in undifferentiated human embryonal carcinoma (EC) cells, which were subsequently dramatically down-regulated during in vitro cellular differentiation, following retinoic acid (RA) treatment, at the protein level. Surprisingly, heat shock treatment also triggered the down-regulation of HSP90 within 48 h at the protein level. Furthermore, the heat treatment induced cellular differentiation into neural cells. This down-regulation of HSP90 by heat treatment was shifted to an up-regulation attern after cellular differentiation in response to RA treatment. In order to clarify the functions of HSP90 in cellular differentiation, we conducted various experiments, including overexpression of HSP90 via gene transfer. We showed that the RA-induced differentiation of EC cells into a neural cell lineage was inhibited by overexpression of the HSP90α or-β isoform via the gene transfer method. On the other hand, the overexpression of HSP90β alone impaired cellular differentiation into trophoectoderm. These results show that down-regulation of HSP90 is a physiological critical event in the differentiation of human EC cells and that specific HSP90 isoforms may be involved in differentiation into specific cell lineages.     

The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity

The 90-kDa heat shock protein (hsp-90) is an abundant cytosolic protein believed to play a role in maintenance of protein trafficking and closely associated with several steroid hormone receptors. Incubation of highly purified hsp-90 with [gamma-32P]ATP results in its autophosphorylation on serine residues. There are several lines of evidence which suggest that this activity is due to a kinase intrinsic to hsp-90 rather than some closely associated protein kinases: 1) the phosphorylation persists after the removal of casein kinase II by heparin chromatography and after immunoprecipitation of hsp-90 with anti-hsp-90 antibodies. 2) The approximate kM for ATP of the reaction is 0.16 mM, higher than that of many other protein kinases. 3) Phosphorylation is not affected by a number of activators and inhibitors of other known kinases which might associate with hsp-90. 4) The phosphorylation displays a unique cation dependence being most active in the presence of Ca2+ and practically inactive with Mg2+, although the autophosphorylation in the presence of Mg2+ is activated by histones and polyamines. 5) The activity is remarkably heat-stable; incubation of hsp-90 for 20 min at 95 degrees C results in only a 60% decrease in autophosphorylation. 6) Finally, and most importantly, purified hsp-90 can be labeled with azido-ATP and it is able to bind to ATP-agarose. The binding shows similar cation dependence to the autophosphorylation. These data are in agreement with the presence of a consensus sequence for ATP binding sites in the primary structure of the protein similar to that observed in the 70-kDa heat-shock proteins. Our data suggest the 90-kDa heat shock protein possesses an enzymatic activity analogous in many respects to the similar activity of the 70-kDa heat shock proteins. This may represent an important, previously unrecognized function of hsp-90.     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc     

Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases

We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [-³²P]ATP/Mg²⁺ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [-³²P]ATP·Mg²⁺ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 , and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 .     

Association of the 90-kDa heat shock protein does not affect the ligand-binding ability of androgen receptor

An N-terminal truncated androgen receptor with putative DNA- and ligand- binding domains (AR438) and that with a ligand-binding domain (AR612) were expressed under control of the T7 promoter in E. coli or translated in vitro with rabbit reticulocyte lysate, and their ligand-binding properties and the interaction with HSP90 were investigated. Bacterially expressed AR438 and AR612 bound a synthetic androgen, [³H]R1881, with apparent dissociation constant of 2.6 ± 0.2 and 3.1 ± 0.7 nM, respectively, values which are comparable to those of androgen receptor in target tissues. The recombinant androgen receptors sedimented at the 4–5 S region irrespective of the presence of 10 mM tungstate, indicating that the receptor exists free from HtpG, which is the bacterial homolog of eukaryotic HSP90. The apparent dissociation constant of truncated androgen receptors translated in vitro was 0.1 nM for AR438 and 0.2 nM for AR612. Sedimentation coefficients of in vitro translated molecules were converted from 7–8 S in the presence of tungstate to 3 S in the absence of tungstate. Both AR438 and AR612 translated in vitro were retained by anti-rat HSP90 antibody-protein A Sepharose. Exposure to 0.3 M NaCl in the presence of ligand caused dissociation of AR438 and AR612 from HSP90, and concomitantly, the DNA-cellulose binding ability of AR438 was enhanced. Thus, we conclude that the androgen receptor associates with HSP90 through the ligand-binding domain and that this association prevents the interaction of the androgen receptor with DNA. However, HSP90 seems to have little effect on the ligand-binding characteristics of the androgen receptor.     

The 90-kDa heat shock protein protects mammalian cells from thermal stress but not from viral infection

Using an anti-sense RNA approach, a mouse cell line containing dramatically reduced levels of the 90-kDa heat shock protein (hsp90) has been isolated. This line shows reduced growth at mildly elevated temperatures and reduced survival at highly elevated temperatures but is no more sensitive to herpes simplex virus infection than the parental cells. This is the first direct evidence of a role for an individual mammalian hsp in thermotolerance and suggests that individual hsps may be of different value in the response to various types of stress.     

Cytoplasmic 8 S glucocorticoid receptor binds to actin filaments through the 90-kDa heat shock protein moiety

The glucocorticoid receptor exists in the cytoplasm of hormone-untreated cells as a complex with the 90-kDa heat shock protein (HSP90). Glucocorticoids induce dissociation of the glucocorticoid binding protein from HSP90 and translocation of the receptor to the nucleus. HSP90 binds to actin filaments, and calmodulin or tropomyosin inhibits the binding. We present here evidence that the HSP90-containing glucocorticoid receptor complexes (8 S receptor) bind to filamentous actin in vitro while the HSP90-free form of the receptor does not. The binding was detectable for both the crude cytosolic fractions and the partially purified 8 S glucocorticoid receptor. Purified HSP90 or tropomyosin completely abolished the binding. Calmodulin also inhibited the binding in a Ca(2+)-dependent manner. From these results, we conclude that the glucocorticoid receptor complex is able to bind actin filaments via the HSP90 moiety. The binding may provide an anchoring mechanism for the glucocorticoid receptor in the cytoplasm.