Heat shock protein 90 (Hsp90), one of the most abundant chaperones in eukaryotes, participates in folding and stabilization of signal-transducing molecules including steroid hormone receptors and protein kinases. The amino terminus of Hsp90 contains a non-conventional nucleotide-binding site, related to the ATP-binding motif of bacterial DNA gyrase. The anti-tumor agents geldanamycin and radicicol bind specifically at this site and induce destabilization of Hsp90-dependent client proteins. We recently demonstrated that the gyrase inhibitor novobiocin also interacts with Hsp90, altering the affinity of the chaperone for geldanamycin and radicicol and causing in vitro and in vivo depletion of key regulatory Hsp90-dependent kinases including v-Src, Raf-1, and p185ErbB2 . In the present study we used deletion/mutation analysis to identify the site of interaction of novobiocin with Hsp90, and we demonstrate that the novobiocin-binding site resides in the carboxyl terminus of the chaperone. Surprisingly, this motif also recognizes ATP, and ATP and novobiocin efficiently compete with each other for binding to this region of Hsp90. Novobiocin interferes with association of the cochaperones Hsc70 and p23 with Hsp90. These results identify a second site on Hsp90 where the binding of small molecule inhibitors can significantly impact the function of this chaperone, and they support the hypothesis that both amino-and carboxyl-terminal domains of Hsp90 interact to modulate chaperone activity.
Steroid hormones produce a response in target cells by binding to hormone-specific soluble receptors, which undergo a transformational change, leading to their interaction with chromatin and to modified gene expression. In a previous paper, we described a monoclonal antibody, BF4, that specifically recognizes and binds the non-transformed '8S' form of chicken oviduct progesterone receptor (8S-PR). We now show that BF4 does not form an immune complex with the 4S transformed form of 3H-progestin-labelled progesterone receptor, but does interact with the 8S non-transformed forms of the oestrogen, androgen and glucocorticosteroid receptors. Our results suggest that the antigenic determinant recognized by BF4 is present on a non-hormone binding unit, which we identify as a polypeptide of molecular weight (MW) 90,000 in the case of the progesterone receptor, and that this unit is common to other 8S non-transformed chicken steroid receptors.
Non‐transformed steroid receptors have an approximately 8S sedimentation coefficient that corresponds to an oligomeric structure of 250‐300 kd which includes a non‐hormone binding 90‐kd protein. A monoclonal antibody BF4 raised against the purified, molybdate‐stabilized, 8S progesterone receptor (8S‐PR) from chick oviduct, recognizes 8S forms of all steroid hormone receptors. BF4 was found specific for a 90‐kd protein present in great abundance in all chicken tissues, including that present in 8S‐forms of steroid receptors. Here, using immunological and biochemical techniques, we demonstrate that this ubiquitous BF4‐positive 90‐kd protein is in fact the chicken 90 kd heat‐shock protein (hsp 90): it increased in heat‐shocked chick embryo fibroblasts, and displayed identical migration in two‐dimensional gel electrophoresis and the same V8 peptide map as the already described hsp 90. We discuss the possibility that the interaction between hsp 90 and steroid hormone‐binding subunits may play a role in keeping the receptor in an inactive form.
Heat shock protein (hsp)90 functions in a complex chaperoning pathway where its activity is modulated by ATP and by interaction with several co-chaperones. One co-chaperone, p23, binds selectively to the ATP-bound state of hsp90. However, the isolated ATP-binding domain of hsp90 does not bind p23. In an effort to identify the p23-binding domain, we have constructed a series of hsp90 deletion mutants fused with glutathione-S-transferase (GST). Full-length GST-hsp90 is able to bind p23, and also, to chaperone assembly of progesterone receptor complexes. Truncations from the C terminus of GST-hsp90 reveal a C-terminal boundary for the p23-binding domain at approximately residue 490. This fragment contains, in order, the ATP-binding domain, a highly charged region, and 203 residues beyond the charged region. p23 binding is unaffected by deletion of the charged region, indicating that two noncontiguous regions of hsp90 are involved in p23 binding. These regions are only effective when hsp90 is in a dimeric state as shown by loss of p23 binding upon removal of GST or as shown by use of FK506-binding protein12-hsp90 constructs that form dimers and bind p23 only in the presence of a bivalent drug. Thus, p23 binding requires an hsp90 dimer with close proximity between N-terminal regions of hsp90 and a conformation specified by ATP.H eat shock protein (hsp)90 is an abundant and ubiquitous molecular chaperone that is required to assist the conformational maturation of specific targets involved in many key functions of the cell such as cell-cycle regulation and signal transduction. Among hsp90 targets are several protein kinases such as v-Src, Weel, and c-Raf, transcriptional regulators such as p53 and steroid receptors, and two polymerases: that of hepatitis B virus and telomerase (for review, see refs. 1-4). To date, the best known maturation process driven by hsp90 is the assembly of steroid receptors into a high-affinity hormone-binding conformation. This complex, multistep process occurs in an ATPdependent manner and involves several other chaperones and co-chaperones (4). Three dynamic steps have been observed in this assembly process (5-7). The receptor initially associates with hsp70, assisted by hsp40. This association develops into an intermediate complex in which the co-chaperone, hsp organizing protein (Hop), associates with both hsp70 on the receptor and hsp90, while the protein Hip binds to hsp70. As the receptor complex matures, hsp90 bound to the receptor interacts with p23 and any one of four tetratricopeptide repeat-containing proteins, FK506-binding protein (FKBP)52, FKBP51, cyclophilin-40, or phosphoprotein phosphatase 5. The details of the interactions between these partner proteins are poorly understood and the mechanisms driving the transitions from one complex to another are still unresolved.hsp90 is a dimeric protein with multiple domains. Several proteins involved in the hsp90 chaperone pathway possess structurally related tetratricopeptide repeat motifs that mediated their interaction with hsp90 a...
FKBP52 (HSP56, p59, HBI) is the 59-kDa immunosuppressant FK506-binding protein and has peptidyl prolyl isomerase as well as a chaperone-like activity in vitro. FKBP52 associates with the heat shock protein HSP90 and is included in the steroid hormone receptor complexes in vivo. FKBP52 possesses a well conserved phosphorylation site for casein kinase II (CK2) that was previously shown to be associated with HSP90. Here we examined whether FKBP52 is phosphorylated by CK2 both in vivo and in vitro. Recombinant rabbit FKBP52 was phosphorylated by purified CK2. We expressed and purified deletion mutants of FKBP52 to determine the site(s) phosphorylated by CK2. Thr-143 in the hinge I region was identified as the major phosphorylation site for CK2. A synthetic peptide corresponding to this region was phosphorylated by CK2, and the peptide competitively inhibited the phosphorylation of other substrates by CK2. The [ 32 P]phosphate labeling of FKBP52-expressing cells revealed that the same site is also phosphorylated in vivo. FK506 binding to FKBP52 did not affect the phosphorylation by CK2 and, conversely, the FK506-binding activity of FKBP52 was not affected by the phosphorylation. Most importantly, CK2-phosphorylated FKBP52 did not bind to HSP90. These results indicate that CK2 phosphorylates FKBP52 both in vitro and in vivo and thus may regulate the protein composition of chaperone-containing complexes such as those of steroid receptors and certain protein kinases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.