The non-DNA-binding heterooligomeric form of mammalian steroid hormone receptors contains a hsp90-bound 59-kilodalton protein

Untransformed cytosol receptors for progesterone (PR), androgen (AR), estrogen (ER), and glucocorticosteroid (GR) in rabbit tissues contain a 59-kDa protein (p59) (Tai, P.K.K., Maeda, Y., Nakao, K., Wakim, N.G., Duhring, J.L., and Faber, L.E. (1986) Biochemistry 25, 5269-5275) and a 90-kDa heat shock protein (hsp90). In the present study, receptors from calf uterus (PR, AR, ER, and GR) and from human breast cancer MCF7 cells (PR and GR) were also shown to be comprised of hsp90 and p59. These heterooligomer receptor complexes were stabilized both by transition metal oxyanions (molybdate and tungstate) and chemical cross-linking with dimethylpimelimidate. In 0.4 M KCl, tungstate-stabilized (but not molybdate-stabilized) PR, AR, ER, and GR retained hsp90, but lost p59. Dimethylpimelimidate cross-linking prevented p59 dissociation from hsp90-receptor complexes. Stabilization with tungstate and/or cross-linking permitted immunoaffinity purification of untransformed rabbit as well as calf PR and ER on EC1-Affi-Gel 10 column (an anti-p59 immunoadsorbant). Combined immunoaffinity purification and cross-linking experiments indicated that p59 is bound to hsp90 in the cytosol. We propose that in the nontransformed steroid receptor, p59 interacts with hsp90 rather than with the hormone binding subunit.     

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.     

Stable association of hsp90 and p23, but Not hsp70, with active human telomerase

The ribonucleoprotein telomerase holoenzyme is minimally composed of a catalytic subunit, hTERT, and its associated template RNA component, hTR. We have previously found two additional components of the telomerase holoenzyme, the chaperones p23 and heat shock protein (hsp) 90, both of which are required for efficient telomerase assembly in vitro and in vivo. Both hsp90 and p23 bind specifically to hTERT and influence its proper assembly with the template RNA, hTR. We report here that the hsp70 chaperone also associates with hTERT in the absence of hTR and dissociates when telomerase is folded into its active state, similar to what occurs with other chaperone targets. Our data also indicate that hsp90 and p23 remain associated with functional telomerase complexes, which differs from other hsp90-folded enzymes that require only a transient hsp90.p23 binding. Our data suggest that components of the hsp90 chaperone complex, while required for telomerase assembly, remain associated with active enzyme, which may ultimately provide critical insight into the biochemical properties of telomerase assembly.     

Identification of two hsp90 genes in carp

Two hsp90 cDNA isoforms (hsp90alpha and hsp90beta) were isolated from the common carp (Cyprinus carpio). Gene-specific probes and primers were selected and used in Northern blot hybridization and RT-PCR reactions to measure the basal hsp90 mRNA levels and to follow the inducer-specific expression of the hsp90 genes in different tissues during in vivo studies. The hsp90beta gene is largely constitutively expressed at a fairly high level in all the examined tissues (brain, liver and kidney) and is slightly inducible by an elevated temperature. Hsp90alpha mRNA is present in the brain, but is hardly detectable in the kidney and liver of unstressed animals. In the brain, this gene is greatly upregulated following thermal stress, whereas in the liver and kidney heat shock has only minor effects on its expression. Hsp90alpha, but not hsp90beta, responds to an elevated level of Cd in a dose-, time- and tissue-dependent manner.     

Identification of a 60-kilodalton stress-related protein, p60, which interacts with hsp90 and hsp70.

Immunoaffinity purification of hsp90 from chick oviduct cytosol reveals two major proteins, hsp70 and a 60-kDa protein (p60), copurifying with hsp90. A similar result is obtained when hsp90 is immunoaffinity purified from chick liver and brain cytosols, avian fibroblasts, and rabbit reticulocyte lysate. This p60 is the same protein previously identified in certain assembly complexes of chick progesterone receptor generated in a cell-free reconstitution system. Tryptic and cyanogen bromide peptide fragments were generated from gel-purified p60, and partial N-terminal sequences were determined from eight peptides. The sequences show a striking similarity to the sequence of a 63-kDa human protein (IEF SSP 3521) whose abundance is increased in MRC-5 fibroblasts following simian virus 40 transformation. A monoclonal antibody was prepared against avian p60; Western immunoblot analysis showed that p60 was present in each of eight chick tissues examined and in each of the human, rat, rabbit, and Xenopus tissues tested. Immunoaffinity purifications from both chick oviduct cytosol and rabbit reticulocyte lysate using anti-p60 and anti-hsp70 monoclonal antibodies confirm that there is a relatively abundant complex in these extracts containing hsp90, hsp70, and p60. This complex appears to comprise an important functional unit in the assembly of progesterone receptor complexes. However, judging from the abundance and widespread occurrence of this multiprotein complex, hsp90, hsp70, and p60 probably function interactively in other systems as well.     

Saturation, competition, and specificity in interaction of heat shock proteins (hsp) gp96, hsp90, and hsp70 with CD11b+ cells

Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.     

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.     

The hsp90 cochaperone p23 is the limiting component of the multiprotein hsp90/hsp70-based chaperone system in vivo where it acts to stabilize the client protein: hsp90 complex

A variety of signaling proteins form heterocomplexes with and are regulated by the heat shock protein chaperone hsp90. These complexes are formed by a multiprotein machinery, including hsp90 and hsp70 as essential and abundant components and Hop, hsp40, and p23 as non-essential cochaperones that are present in much lower abundance in cells. Overexpression of signaling proteins can overwhelm the capacity of this machinery to properly assemble heterocomplexes with hsp90. Here, we show that the limiting component of this assembly machinery in vitro in reticulocyte lysate and in vivo in Sf9 cells is p23. Only a fraction of glucocorticoid receptors (GR) overexpressed in Sf9 cells are in heterocomplex with hsp90 and have steroid binding activity, with the majority of the receptors present as both insoluble and cytosolic GR aggregates. Coexpression of p23 with the GR increases the proportion of cytosolic receptors that are in stable GR.hsp90 heterocomplexes with steroid binding activity, a strictly hsp90-dependent activity for the GR. Coexpression of p23 eliminates the insoluble GR aggregates and shifts the cytosolic receptor from very large aggregates without steroid binding activity to approximately 600-kDa heterocomplexes with steroid binding activity. These data lead us to conclude that p23 acts in vivo to stabilize hsp90 binding to client protein.     

Nuclear localization and the heat shock proteins

The highly conserved heat shock proteins (HSP) belong to a subset of cellular proteins that localize to the nucleus. HSPs are atypical nuclear proteins in that they localize to the nucleus selectively, rather than invariably. Nuclear localization of HSPs is associated with cell stress and cell growth. This aspect of HSPs is highly conserved with nuclear localization occurring in response to a wide variety of cell stresses. Nuclear localization is likely important for at least some of the heat shock proteins’ protective functions; little is known about the function of the heat shock proteins in the nucleus. Nuclear localization is signalled by the presence of a basic nuclear localization sequence (NLS) within a protein. Though most is known about HSP 72’s nuclear localization, the NLS(s) has not been definitively identified for any of the heat shock proteins. Likely more is involved than presence of a NLS; since the heat shock proteins only localize to the nucleus under selective conditions, nuclear localization must be regulated. HSPs also function as chaperons of nuclear transport, facilitating the movement of other macromolecules across the nuclear membrane. The mechanisms involved in chaperoning of proteins by HSPs into the nucleus are still being identified.     

The influence of ATP and p23 on the conformation of hsp90

The chaperoning activity of the heat shock protein hsp90 is directed, in part, by the binding and hydrolysis of ATP and also by association with co-chaperone proteins. One co-chaperone, p23, binds to hsp90 only when hsp90 is in a conformation induced by the binding of ATP. Once formed, the p23-hsp90 complex is very stable upon the removal of ATP and dissipates at 30 degrees with a half-life of about 45 min. This was shown to be due to the high stability of the ATP-induced state of hsp90, not to the rate of p23 dissociation. Further stabilization of this ATP-induced state is achieved by including molybdate or by use of the ATP analogue ATPgammaS. This conformational state of hsp90 is correlated with the tight binding of ADP resulting from hydrolysis of bound ATP. Both p23 and molybdate enhance and stabilize the nucleotide-bound state of hsp90, and this state is maximized by the presence of both agents. These results can be explained in a model where the binding of ATP induces a conformational transition in hsp90 that traps the nucleotide and is committed to ATP hydrolysis. p23 specifically recognizes this state and may also facilitate subsequent steps in the chaperoning cycle.     

Are receptor-associated nuclear proteins associated with the earliest effects of steroid hormones?

The functional importance of the interaction of hsp90 with receptors for steroid hormones in the action of these hormones has been suggested. This hypothesis, although not yet proven, is supported by new data obtained in our laboratory and in those of others, whereas no conflicting experimental results have been presented. Our recent studies have dealt with the cloning of hsp90, transfection of normal and mutated receptors, the effects of the antihormone RU486 and immunohistochemistry.     

The involvement of p23, hsp90, and immunophilins in the assembly of progesterone receptor complexes

To better understand the assembly mechanism for the progesterone receptor (PR), we have developed cell-free systems for studying interactions of PR, hsp90, and other associated proteins. When PR is incubated in rabbit reticulocyte lysate, its association with hsp90, hsp70, the three immunophilins FKBP54, FKBP52 and CyP-40, and with p23 is observed. These interactions require ATP/Mg²⁺ and when ATP is limiting the PR complex is altered to one containing the proteins p60 and p48, but lacking immunophilins and p23. We have studied two pre-formed hsp90 complexes that may participate in the assembly of PR complexes. One contains hsp90 bound to hsp70 and p60 and this complex forms spontaneously in the absence of ATP. A second complex contains hsp90 bound to p23 plus the three immunophilins and some hsp70. The formation of this complex requires ATP. In further studies we have shown that purified hsp90 can bind to purified p23 and this interaction requires both ATP and molybdate. This explains, in part, the known effects of ATP and molybdate on assembly of PR complexes.     

Tosylcyclonovobiocic acids promote cleavage of the hsp90-associated cochaperone p23

The cochaperone p23 is required for the chaperoning cycle of hsp90 and to enhance the maturation of several client proteins. Tosylcyclonovobiocic acids (4TCNA and 7TCNA) are potent analogs of novobiocin and induce cell cycle arrest, apoptosis and degradation of hsp90 client proteins in a panel of cancer cells. In this study, Western blotting shows that 4TCNA and 7TCNA triggered processing of the hsp90 cochaperone p23 in a dose-dependent manner. Small interfering RNA (siRNA)-mediated reduction of p23 expression in MCF-7 breast cancer cells did not block 4TCNA-induced caspase activation as assessed by the cleavage of PARP. This result indicates that 4TCNA-mediated cell death is a p23-independent process. In HT29 colon cancer cells, 4TCNA and 7TCNA up-regulated GRP78 and GRP94 supporting involvement of ER stress in apoptosis.     

Aha,another regulator for hsp90 chaperones

A large number of key regulators controlling homeostasis and cell fate are chaperoned by the Hsp90 folding machine. In this issue of Molecular Cell, report the discovery of a new stress-regulated cochaperone, Aha1, which accelerates the dynamics of this machine.     

Inhibition of MDM2 by hsp90 contributes to mutant p53 stabilization

Stabilization and overexpression are hallmarks of mutant p53 found in nearly 50% of human tumors. Mutations in the conformation-sensitive core domain of p53 often lead to association with molecular chaperones such as hsp70 and hsp90. Inhibition of hsp90 function accelerates mutant p53 degradation. We recently found that expression of p53 core domain mutants inhibits MDM2 degradation, suggesting that mutant p53 can modulate MDM2 functions. In this report, we show that mutant p53 mediates formation of MDM2-p53-hsp90 complexes. Release of MDM2 from the p53-hsp90 complex after DNA damage restores MDM2 but not p53 turnover, whereas dissociation of hsp90 by geldanamycin increases the degradation of both MDM2 and mutant p53. Mutant p53 degradation after hsp90 inhibition requires MDM2 expression. The interaction between MDM2 and hsp90 is disrupted by the 2A10 antibody, which recognizes a site on MDM2 important for binding to alternative reading frame (ARF). Expression of mutant p53 prevents MDM2 from binding ARF and accumulating in the nucleolus in an hsp90-dependent fashion. These results suggest that hsp90 recruited by mutant p53 conceals the ARF-binding site on MDM2 and inhibits its ubiquitin-protein isopeptide ligase function, resulting in the stabilization of both mutant p53 and MDM2.