Adam M. Silverstein

Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.

Steroid receptors are recovered from hormone-free cells in multiprotein complexes containing hsp90, p23, an immunophilin, and often some hsp70. The immunophilin, which can be of the FK506- or cyclosporin A-binding class, binds to hsp90 via its tetratricopeptide repeat (TPR) domain, and different receptor heterocomplexes exist depending upon which immunophilin occupies the TPR-binding region of hsp90. We have recently reported that a protein serine/threonine phosphatase that is designated PP5 and contains four TPRs binds to hsp90 and is co-purified with the glucocorticoid receptor (GR) (Chen, M.-S., Silverstein, A. M., Pratt, W. B., and Chinkers, M. (1996) J. Biol. Chem. 271, 32315–32320). In this work, we show that PP5 is recovered with both GR that is nuclear and GR that is cytoplasmic in hormone-free cells. Approximately one-half of the GR·hsp90 heterocomplexes in L cell cytosol contains an immunophilin with high affinity FK506 binding activity, such as FKBP51 or FKBP52, and ∼35% contains PP5. Only a small (but undetermined) fraction of the native GR·hsp90 heterocomplexes contain the cyclosporin A-binding immunophilin CyP-40. PP5, FKBP52, and CyP-40 exist in separate heterocomplexes with hsp90, and competition binding experiments with the PP5 TPR domain suggest that the three proteins occupy a common binding site on hsp90. A 55-residue connecting region between the N-terminal TPR domain of human PP5 and its C-terminal phosphatase domain has 50% amino acid homology and 22% identity with the central portion of the peptidylprolyl isomerase domain of human FKBP52. Of the 9 residues in this portion of FKBP52 involved in high affinity interactions with FK506, 3 residues are retained and 4 have homologous substitutions in PP5. Although immunoadsorbed PP5 did not bind [3H]FK506, we found that both rabbit PP5 in reticulocyte lysate and purified rat PP5 were specifically retained by an FK506 affinity matrix. Thus, we propose that PP5 possesses properties of an immunophilin with low affinity FK506 binding activity and that it determines a major portion of the native GR heterocomplexes in L cell cytosol.

The Tetratricopeptide Repeat Domain of Protein Phosphatase 5 Mediates Binding to Glucocorticoid Receptor Heterocomplexes and Acts as a Dominant Negative Mutant

We previously identified a protein-serine phosphatase designated PP5, based on the binding of its tetratricopeptide repeat (TPR) domain to the atrial natriuretic peptide receptor (Chinkers, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11075-11079). We have now identified another protein complex to which PP5 is targeted through its TPR domain. A 90-kDa protein, identified as heat shock protein 90 (hsp90) by immunoblotting, specifically co-immunoprecipitated from COS-7 cell lysates with the FLAG-tagged TPR domain of PP5. hsp90 also co-immunoprecipitated with full-length FLAG-tagged PP5 overexpressed in COS-7 cells and with endogenous PP5 from untransfected COS-7 cells or rat brain. During gel filtration, PP5 and hsp90 comigrated in a high molecular weight complex. Since glucocorticoid receptors (GR) exist as large heterocomplexes containing hsp90 bound to TPR proteins, we hypothesized that PP5 might be associated with these complexes. Consistent with this hypothesis, PP5 specifically co-immunoprecipitated with GR from mouse L cell lysates. To test the functional importance of this TPR-mediated association in living cells, we used a dominant negative PP5 mutant consisting only of its TPR domain. The mutant inhibited GR-mediated transactivation by approximately 70% in transfected CV-1 cells. This is the first evidence that the TPR proteins in steroid receptor heterocomplexes may be required for signaling in vivo.

The hsp90-binding Antibiotic Geldanamycin Decreases Raf Levels and Epidermal Growth Factor Signaling without Disrupting Formation of Signaling Complexes or Reducing the Specific Enzymatic Activity of Raf Kinase

We have expressed the mitogenic signaling proteins Src, Ras, Raf-1, Mek (MAP kinase kinase), and Erk (MAP kinase) in baculovirus-infected Sf9 insect cells in order to study a potential role for the chaperone hsp90 in formation of multiprotein complexes. One such complex obtained by immunoadsorption with anti-Ras antibody of cytosol prepared from cells simultaneously expressing Ras, Raf, Mek, and Erk contained Ras, Raf, and Erk. To detect directly the protein-protein interactions involved in forming multiprotein complexes, we combined cytosols from single infections in vitro in all possible combinations of protein pairs. We detected complexes between Ras·Raf, Ras·Src, Raf·Mek, and Raf·Src, but no complex containing Erk was obtained by mixing cytosols. Thus, cellular factors appear to be required for assembly of the Erk-containing multiprotein complex. One cellular factor thought to be involved in signaling protein complex formation is the chaperone hsp90, and we show that Src, Raf, and Mek are each complexed with insect hsp90. Treatment of Sf9 cells with geldanamycin, a benzoquinone ansamycin that binds to hsp90 and disrupts its function, did not decrease coadsorption of either Raf or Erk with Ras, although it did decrease the level of cytosolic Raf. To study geldanamycin action, we treated rat 3Y1 fibroblasts expressing v-Raf and showed that the antibiotic blocked assembly of Raf·hsp90 complexes at an intermediate stage of assembly where Raf is still bound to the p60 and hsp70 components of the assembly mechanism. As in Sf9 cells, Raf levels decline with geldanamycin treatment of 3Y1 cells. To determine if geldanamycin affects mitogenic response, we treated HeLa cells with epidermal growth factor (EGF) and showed that geldanamycin treatment decreased EGF signaling and decreased the level of Raf protein without affecting the EGF-mediated increase in Raf kinase activity. We conclude that hsp90 is not required for forming complexes between the mitogenic signaling proteins or for Raf kinase activity and that EGF signaling is decreased indirectly by geldanamycin because the antibiotic increases degradation of Raf and perhaps other components of the signaling pathway.

Different Regions of the Immunophilin FKBP52 Determine Its Association with the Glucocorticoid Receptor, hsp90, and Cytoplasmic Dynein

FKBP52 is a high molecular mass immunophilin possessing peptidylprolyl isomerase (PPIase) activity that is inhibited by the immunosuppressant drug FK506. FKBP52 is a component of steroid receptor·hsp90 heterocomplexes, and it binds to hsp90 via a region containing three tetratricopeptide repeats (TPRs). Here we demonstrate by cross-linking of the purified proteins that there is one binding site for FKBP52/dimer of hsp90. This accounts for the common heterotetrameric structure of native receptor heterocomplexes being 1 molecule of receptor, 2 molecules of hsp90, and 1 molecule of a TPR domain protein. Immunoadsorption of FKBP52 from reticulocyte lysate also yields co-immunoadsorption of cytoplasmic dynein, and we show that co-immunoadsorption of dynein is competed by a fragment of FKBP52 containing its PPIase domain, but not by a TPR domain fragment that blocks FKBP52 binding to hsp90. Using purified proteins, we also show that FKBP52 binds directly to the hsp90-free glucocorticoid receptor. Because neither the PPIase fragment nor the TPR fragment affects the binding of FKBP52 to the glucocorticoid receptor under conditions in which they block FKBP52 binding to dynein or hsp90, respectively, different regions of FKBP52 must determine its association with these three proteins.

Neuronal Nitric-oxide Synthase Is Regulated by the hsp90-based Chaperone System in Vivo

It is established that the multiprotein heat shock protein 90 (hsp90)-based chaperone system acts on the ligand binding domain of the glucocorticoid receptor (GR) to form a GR·hsp90 heterocomplex and to convert the receptor ligand binding domain to the steroid-binding state. Treatment of cells with the hsp90 inhibitor geldanamycin inactivates steroid binding activity and increases the rate of GR turnover. We show here that a portion of neuronal nitric-oxide synthase (nNOS) exists as a molybdate-stabilized nNOS·hsp90 heterocomplex in the cytosolic fraction of human embryonic kidney 293 cells stably transfected with rat nNOS. Treatment of human embryonic kidney 293 cells with geldanamycin both decreases nNOS catalytic activity and increases the rate of nNOS turnover. Similarly, geldanamycin treatment of nNOS-expressing Sf9 cells partially inhibits nNOS activation by exogenous heme. Like the GR, purified heme-free apo-nNOS is activated by the DE52-retained fraction of rabbit reticulocyte lysate, which also assembles nNOS·hsp90 heterocomplexes. However, in contrast to the GR, heterocomplex assembly with hsp90 is not required for increased heme binding and nNOS activation in this cell-free system. We propose that, in vivo, where access by free heme is limited, the complete hsp90-based chaperone machinery is required for sustained opening of the heme binding cleft and nNOS activation, but in the heme-containing cell-free nNOS-activating system transient opening of the heme binding cleft without hsp90 is sufficient to facilitate heme binding.

A Model for the Cytoplasmic Trafficking of Signalling Proteins Involving the hsp90-Binding Immunophilins and p50cdc37

A number of transcription factors and protein kinases involved in signal transduction exist in heterocomplexes with the ubiquitous and essential protein chaperone hsp90. These signalling protein x hsp90 heterocomplexes are assembled by a multiprotein chaperone system comprising hsp90, hsp70, Hop, hsp40, and p23. In the case of transcription factors, the heterocomplexes with hsp90 also contain a high molecular weight immunophilin with tetratricopeptide repeat (TPR) motifs, such as FKBP52 or CyP-40. In the case of the protein kinases, the heterocomplexes contain p50cdc37. The immunophilins bind to a single TPR acceptor site on hsp90, and p50cdc37 binds to an adjacent site so that binding is exclusive for p50cdc37 or an immunophilin. Direct interaction of immunophilins with the transcription factors or p50cdc37 with the protein kinases leads to selection of different heterocomplexes after their assembly by a common mechanism. Studies with the glucocorticoid receptor, for which translocation from the cytoplasm to the nucleus is under hormonal control, suggest that dynamic assembly of the heterocomplexes is required for rapid movement of the receptor through the cytoplasm along cytoskeletal tracts. As for the similar short-range trafficking of vesicles along microtubules, there must be a mechanism for linking the signalling protein solutes to the molecular motors involved in movement. We present here a model in which the immunophilins and p50cdc37 target, respectively, the retrograde or anterograde direction of signalling protein movement by functioning as connectors that link the signalling proteins to the movement machinery.

p50 cdc37 Binds Directly to the Catalytic Domain of Raf as Well as to a Site on hsp90 That Is Topologically Adjacent to the Tetratricopeptide Repeat Binding Site

Several protein kinases (e.g.pp60 src , v-Raf) exist in heterocomplexes with hsp90 and a 50-kDa protein that is the mammalian homolog of the yeast cell cycle control protein Cdc37. In contrast, unliganded steroid receptors exist in heterocomplexes with hsp90 and a tetratricopeptide repeat (TPR) domain protein, such as an immunophilin. Although p50 cdc37 and TPR domain proteins bind directly to hsp90, p50 cdc37 is not present in native steroid receptor·hsp90 heterocomplexes. To obtain some insight as to how v-Raf selects predominantly hsp90·p50 cdc37 heterocomplexes, rather than hsp90·TPR protein heterocomplexes, we have examined the binding of p50 cdc37 to hsp90 and to Raf. We show that p50 cdc37 exists in separate hsp90 heterocomplexes from the TPR domain proteins and that intact TPR proteins compete for p50 cdc37 binding to hsp90 but a protein fragment containing a TPR domain does not. This suggests that the binding site for p50 cdc37 lies topologically adjacent to the TPR acceptor site on the surface of hsp90. Also, we show that p50 cdc37 binds directly to v-Raf, with the catalytic domain of Raf being sufficient. We propose that the combination of exclusive binding of p50 cdc37 versus a TPR domain protein to hsp90 plus direct binding of p50 cdc37 to Raf allows the protein kinase to select for the dominant hsp90·p50 cdc37 composition that is observed with a variety of protein kinase heterocomplexes immunoadsorbed from cytosols.

The Hsp Organizer Protein Hop Enhances the Rate of but Is Not Essential for Glucocorticoid Receptor Folding by the Multiprotein Hsp90-based Chaperone System

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)·Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90·Hop·Hsp70·Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR·Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358–7366). Hop has been considered essential for the formation of receptor·Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR·Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.

The seven amino acids (547-553) of rat glucocorticoid receptor required for steroid and hsp90 binding contain a functionally independent LXXLL motif that is critical for steroid binding.

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547–553) overlapping the amino-terminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in full-length GR and glutathioneS-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a “hinged pocket,” with the hinge being in the region of the trypsin digestion site at Arg651. The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550–554 acting as a hydrophobic clasp.

Use of the thiol-specific derivatizing agent N-iodoacetyl-3-[125I]iodotyrosine to demonstrate conformational differences between the unbound and hsp90-bound glucocorticoid receptor hormone binding domain

The hormone binding domain (HBD) of the glucocorticoid receptor (GR) contains five cysteine residues, with three of them being spaced close to one another in the steroid binding pocket. The HBD also contains the contact region for the chaperone protein hsp90, which must be bound to the GR for it to have a steroid binding conformation. Binding of hsp90 to the receptor through its HBD inactivates the DNA binding domain (DBD). The DBD contains a number of cysteines essential to its DNA binding activity. Here, we assess the effects of hsp90 binding on the accessibility of cysteine residues in both the HBD and DBD to derivatization by a thiol-specific reagent. We report that N-iodoacetyltyrosine (IAT) inactivates steroid binding activity of the immunopurified, untransformed GRhsp90 complex in a manner that is prevented by the sulfhydryl reagents cysteine and dithiothreitol but is not reversed by them. The I-labeled IAT derivative N-iodoacetyl-3-[I]iodotyrosine ([I]IAIT) covalently labels the immunopurified, hsp90-bound receptor in a thiol-specific manner. Dissociation of hsp90 leads to an 2-fold increase in [I]IAIT labeling of the full-length, 100-kDa GR. The increase in thiol labeling is related to the presence of hsp90 because it is blocked by molybdate, which prevents hsp90 dissociation. Cleavage of the [I]IAIT-labeled receptor with trypsin yields a 15-kDa labeled fragment containing the DBD and a 30-kDa labeled fragment containing all of the cysteines in the HBD and the contact region for hsp90. Dissociation of hsp90 from the GR results in a 2.3-fold increase in [I]IAIT labeling of the 15-kDa fragment and a 50% decrease in labeling of the 30-kDa fragment. These data are consistent with the proposal that dissociation of hsp90 from the GR produces a conformational change in the HBD such that some of the thiols that are exposed in the GRhsp90 complex become buried and are no longer accessible to the [I]IAIT probe. In contrast, binding of the GR to hsp90 restricts access of cysteines in the DBD to this small thiol-derivatizing agent, a restriction that is relieved as a result of unmasking or conformational change accompanying hsp90 dissociation.