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.     

Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts

A polyclonal antibody, R2, was raised against a fusion protein consisting of a portion of plant hsp90 fused to the trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2 antiserum, hsp90 and two additional proteins with approximate molecular masses of 49 and 45 kDa were detected in the immunoprecipitates. These results are consistent with the idea that hsp90:protein heterocomplexes exist in plant cells.     

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 .     

The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity.

We found that a preparation of the 90-kDa heat shock protein, HSP90, purified to apparent homogeneity, contains a serine/threonine kinase which phosphorylates HSP90. The protein kinase was identified as casein kinase II (CKII) according to its properties. The protein kinase was separable from HSP90 by adsorption to heparin-Sepharose or phosphocellulose. CKII was coimmunoprecipitated with HSP90 by anti-HSP90 antibodies from cell extracts. Sucrose density gradient centrifugation analysis revealed that an addition of anti-HSP90 antibodies to cell extracts induces a shift of the sedimentation peak of CKII toward the bottom of a centrifuge tube. These results suggest that CKII is associated with HSP90 in cell lysates at low salt conditions. Furthermore, the CKII.HSP90 complex was reconstituted from purified HSP90-free CKII and CKII-free HSP90. In a buffer at low ionic strength, CKII forms large aggregates, but HSP90 dissociates the aggregates. Finally, we found that HSP90 activates CKII; an addition of HSP90 to CKII dramatically increased phosphorylation of exogenous substrates as well as the CKII beta subunit. Taken altogether, these observations suggest that CKII is structurally and functionally active when it forms a complex with HSP90.     

Identification of heat shock protein 90-associated 84-kDa phosphoprotein

In eukaryotic cells, HSP90 is associated with several protein kinases and regulates their activities. HSP90 was also reported to possess an autophosphorylase activity. In this study, we examined in vitro autophosphorylation of HSP90, which was purified from chick muscle. We show that HSP90 was not phosphorylated in vitro, but an 84-kDa protein (p84) was highly phosphorylated. P84 was neither HSP90 nor its degradative product, as it was not detected by an antibody (BF4) specific to HSP90 in denaturing immunoprecipitation and Western blot analysis. Phosphorylation of a protein similar to p84 was also detected with purified human brain and HeLa HSP90, indicating that p84 is present in many different types of cells. P84 appeared to exist as large complexes, as determined by HPLC and native gel electrophoresis. Native immunoprecipitation using anti-HSP90 (BF4)-conjugated Affi-gel revealed that this phosphoprotein is specifically associated with HSP90. The interaction of p84 and HSP90 was not affected by p84 phosphorylation. In addition, p84 phosphorylation was prevented by the presence of divalent cations such as Mg(2+) and Mn(2+). In contrast, p84 phosphorylation was significantly activated by addition of exogenous Ca(2+) between 100 and 500 microM, although it was blocked by higher concentrations (>1 mM) of Ca(2+). HSP90, but not p84, could be phosphorylated by casein kinase II. Finally, p84 phosphorylation was specifically prevented by hemin, but not by other kinase inhibitors, indicating that p84 phosphorylation may be regulated by heme-regulated protein kinase.     

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.     

Effect of the 90 kDa heat shock protein, HSP90, on glucocorticoid receptor binding to DNA-cellulose

Glucocorticoid receptors in the IM-9 human lymphoblastoid cell line were affinity labeled with [3H]dexamethasone 21-mesylate and activated to a DNA-binding form by filtration through a Bio-Gel A-1.5m column. The 90 kDa heat shock protein, HSP90, was identified by labeling IM-9 cells with 35S-methionine at both 37 degrees C and 42 degrees C and purified to near homogeneity by sequential chromatography through DE52 and hydroxyapatite. Addition of purified HSP90 to activated, affinity labeled glucocorticoid receptors in a molecular ratio of 16 to 1 inhibited the binding of the receptors to DNA-cellulose. HSP90 did not affect the binding of other proteins to DNA-cellulose, indicating that the inhibitory effect of HSP90 was specific for the glucocorticoid receptor. These results suggest that HSP90 may associate with the glucocorticoid receptor, masking its DNA-binding site and thereby inhibiting receptor interaction with DNA.     

Phosphorylation of glucocorticoid receptor-associated and free forms of the approximately 90-kDa heat shock protein before and after receptor activation

Several lines of evidence have suggested that glucocorticoid receptor function may be regulated by phosphorylation-dephosphorylation reactions, and it has been proposed that dephosphorylation accompanies activation to the DNA-binding form. The phosphate content of the approximately 100-kDa steroid-binding protein has been determined directly and was found not to change during activation in intact cells (Mendel, D.B., Bodwell, J.E., and Munck, A. (1987) J. Biol. Chem. 262, 5644-5648). We have now determined the effect of interaction with the receptor and of activation on the phosphate content of the approximately 90-kDa heat shock protein (Hsp 90), which is thought to be a non-steroid-binding subunit of nonactivated glucocorticoid receptors that dissociates on activation. Monoclonal antibodies AC88 and BuGR2 were used to purify free Hsp 90 and cytosolic nonactivated glucocorticoid-receptor complexes, respectively, from WEHI-7 cells grown in the presence of 32Pi and [35S] methionine. Cell-free activation of the nonactivated receptor-antibody complexes immobilized on protein A-Sepharose minicolumns allowed the recovery of the Hsp 90 dissociated from the complexes during activation. Proteins were separated by denaturing polyacrylamide gel electrophoresis, and the 32P/35S ratio, which was used as a measure of the phosphate content relative to protein, was determined for the free, receptor-associated, and dissociated forms of the Hsp 90, as well as for the approximately 100-kDa steroid-binding protein of non-activated and activated receptors. The three forms of the Hsp 90 had the same phosphate contents, as did the approximately 100-kDa steroid-binding protein before and after activation. Based upon these results, we conclude that no net change in the phosphorylation occurs when the Hsp 90 associates with the approximately 100-kDa steroid-binding protein to form nonactivated receptors and that neither protein component of nonactivated complexes is dephosphorylated when they dissociate during thermal activation under cell-free conditions.     

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.     

Evidence that the 90-kDa heat shock protein (HSP90) exists in cytosol in heteromeric complexes containing HSP70 and three other proteins with Mr of 63,000, 56,000, and 50,000

Monoclonal antibody (mAb) 8D3 and 3G3 are unique antibodies capable of precipitating both free 90-kDa heat shock protein (HSP90) and HSP90-protein complexes. Immunoprecipitation of [35S]methionine-labeled Hepa 1c1c7 cytosolic extracts were performed using mAb 8D3 or 3G3. The resulting immunoprecipitates can be dissociated from the mAb with a 500 mM NaCl wash. These washes were subjected to both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Five major protein spots were detected in addition to HSP90 with the following relative molecular weights: 68,000, 63,000, 56,000, 50,000, and 188,000. On Western blots mAb 3G3 was capable of specifically binding to HSP90. Each of these proteins was localized on two-dimensional gels. Using one-dimensional gel electrophoresis and immunochemical localization on Western blots, the p68 spot was identified as HSP70, and the p56 spot was found to cross-react with polyclonal antibody JP-1 raised against a 59-kDa protein. This 59-kDa protein has been found previously to be associated with several steroid hormone receptors in rabbit uterine cytosol. Immunoprecipitation of [32P]orthophosphate-labeled Hepa 1c1c7 cytosol with mAb 8D3 or 3G3 revealed two major phosphorylated proteins with relative molecular weights of 90,000 and 50,000. The identities of p63 and p188 are currently unknown. This is the first report examining the major proteins that are complexed with HSP90 in mammalian cells.     

HSP100, a 100-kDa heat shock protein, is a Ca2+-calmodulin-regulated actin-binding protein

The 100-kDa heat shock protein, HSP100, was purified from mouse lymphoma cells. Amino acid sequences of three peptide fragments which were obtained from the purified protein by lysylendopeptidase digestion were completely or nearly identical with those of a mouse endoplasmic reticulum protein, ERp99, of a hamster glucose-regulated protein, GRP94, and of a chicken heat shock protein, HSP108, all of which have been known to have strong homology with the 90-kDa heat shock protein, HSP90. HSP100 bound to actin filaments and an apparent Kd for the binding was determined to be 8 x 10(-7) M in 2 mM MgCl2 + 100 mM KCl. Calmodulin inhibited the binding in a Ca2+-dependent manner. Equilibrium gel filtration demonstrated that HSP100 has an ability to bind to calmodulin only in the presence of Ca2+. Moreover, HSP100 competed with HSP90 for binding to actin filaments. These results together with our previous findings that HSP90 and HSP100 have similar physicochemical properties (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. 83, 8054-8058) and HSP90 is a calmodulin-regulated actin-binding protein (Nishida, E., Koyasu, S., Sakai, H., and Yahara, I. (1986) J. Biol. Chem. 261, 16033-16036), strongly suggest that HSP100 is structurally and functionally related to HSP90.     

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.     

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.