Cdc37 goes beyond Hsp90 and kinases

Cdc37 is a relatively poorly conserved and yet essential molecular chaperone. It has long been thought to function primarily as an accessory factor for Hsp90, notably directing Hsp90 to kinases as substrates. More recent discoveries challenge this simplistic view. Cdc37 client proteins other than kinases have now been found, and Cdc37 displays a variety of Hsp90-independent activities both in vitro and in vivo. It can function as a molecular chaperone by itself, interact with other Hsp90 cochaperones in the absence of Hsp90, and even support yeast growth and protein folding without its Hsp90-binding domain. Thus, for many substrates, there may be many alternative chaperone pathways involving Cdc37, Hsp90, or both.     

Hsp90 and Cdc37 -- a chaperone cancer conspiracy

The Hsp90 molecular chaperone system is involved in the activation of an important set of cell regulatory proteins, including many whose disregulation drives cancer. Recruitment of protein kinases to the Hsp90 system is mediated by the co-chaperone adaptor Cdc37 -- an essential protein whose overexpression is itself, oncogenic. Current structural, biochemical and biological studies of Cdc37 are beginning to unravel the nature of its interactions with Hsp90 and protein kinase clients, and implicate it as a key permissive factor in cell transformation by disregulated protein kinases. The central role of the Hsp90-Cdc37 chaperone complex makes it an important target for future anti-cancer drug development.     

Characterization of Celastrol to Inhibit Hsp90 and Cdc37 Interaction

The molecular chaperone heat shock protein 90 (Hsp90) is required for the stabilization and conformational maturation of various oncogenic proteins in cancer. The loading of protein kinases to Hsp90 is actively mediated by the cochaperone Cdc37. The crucial role of the Hsp90-Cdc37 complex has made it an exciting target for cancer treatment. In this study, we characterize Hsp90 and Cdc37 interaction and drug disruption using a reconstituted protein system. The GST pull-down assay and ELISA assay show that Cdc37 binds to ADP-bound/nucleotide-free Hsp90 but not ATP-bound Hsp90. Celastrol disrupts Hsp90-Cdc37 complex formation, whereas the classical Hsp90 inhibitors (e.g. geldanamycin) have no effect. Celastrol inhibits Hsp90 ATPase activity without blocking ATP binding. Proteolytic fingerprinting indicates celastrol binds to Hsp90 C-terminal domain to protect it from trypsin digestion. These data suggest that celastrol may represent a new class of Hsp90 inhibitor by modifying Hsp90 C terminus to allosterically regulate its chaperone activity and disrupt Hsp90-Cdc37 complex.     

The Hsp90 kinase co-chaperone Cdc37 regulates tau stability and phosphorylation dynamics

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.     

Domain-mediated dimerization of the Hsp90 cochaperones Harc and Cdc37

Hsp90 is a highly conserved molecular chaperone that acts in concert with Hsp70 and a cohort of cochaperones to mediate the folding of client proteins into functional conformations. The novel Hsp90 cochaperone Harc was identified previously on the basis of its amino acid sequence similarity to Cdc37. Although the biochemical role of Harc has not been established, the structural similarities between Harc and Cdc37 suggest that it too may function to regulate the binding of client proteins to Hsp90. We report here that Harc forms dimers in vitro. Functional dissection of Harc revealed that both the N-terminal and middle domains contributed to its dimerization. Notably, dimerization of the middle domain of Harc was required for the binding of Hsp90, suggesting that dimerized Harc binds to Hsp90 dimers. The N-terminal domain of Harc made an important contribution to the dimerization of Harc by facilitating the interaction of Hsp70 with Harc-Hsp90 heterocomplexes. Harc was also found to heterodimerize with Cdc37 in vitro. Titration experiments revealed that Harc homodimerization was favored over heterodimerization with Cdc37 when both cochaperones were at similar levels. However, formation of Harc homodimers and heterodimers of Harc and Cdc37 was comparable when the level of Cdc37 was approximately 10-fold above that of Harc. Furthermore, homo- and heterodimerization of Harc and Cdc37 was a dynamic process. Thus Harc could potentially contribute to the regulation of the Hsp90-mediated folding of Cdc37-dependent protein kinases into functional conformations via dimerization with Cdc37.     

Independent regulation of Hsp70 and Hsp90 chaperones by Hsp70/Hsp90-organizing protein Sti1 (Hop1)

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.     

The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones

Hsp90 is a dimeric molecular chaperone required for the activation and stabilization of numerous client proteins many of which are involved in essential cellular processes like signal transduction pathways. This activation process is regulated by ATP-induced large conformational changes, co-chaperones and posttranslational modifications. For some co-chaperones, a detailed picture on their structures and functions exists, for others their contributions to the Hsp90 system is still unclear. Recent progress on the conformational dynamics of Hsp90 and how co-chaperones affect the Hsp90 chaperone cycle significantly increased our understanding of the gearings of this complex molecular machinery. This article is part of a Special Issue entitled: Heat Shock Protein 90 (Hsp90).     

Cdc37 interacts with the glycine-rich loop of Hsp90 client kinases

Recently, we identified a client-binding site of Cdc37 that is required for its association with protein kinases. Phage display technology and liquid chromatography-tandem mass spectrometry (which identifies a total of 33 proteins) consistently identify a unique sequence, GXFG, as a Cdc37-interacting motif that occurs in the canonical glycine-rich loop (GXGXXG) of protein kinases, regardless of their dependence on Hsp90 or Cdc37. The glycine-rich motif of Raf-1 (GSGSFG) is necessary for its association with Cdc37; nevertheless, the N lobe of Raf-1 (which includes the GSGSFG motif) on its own cannot interact with Cdc37. Chimeric mutants of Cdk2 and Cdk4, which differ sharply in their affinities toward Cdc37, show that their C-terminal portions may determine this difference. In addition, a nonclient kinase, the catalytic subunit of cyclic AMP-dependent protein kinase, interacts with Cdc37 but only when a threonine residue in the activation segment of its C lobe is unphosphorylated. Thus, although a region in the C termini of protein kinases may be crucial for accomplishing and maintaining their interaction with Cdc37, we conclude that the N-terminal glycine-rich loop of protein kinases is essential for physically associating with Cdc37.     

Biochemical and structural studies of the interaction of Cdc37 with Hsp90

The heat shock protein Hsp90 plays a key, but poorly understood role in the folding, assembly and activation of a large number of signal transduction molecules, in particular kinases and steroid hormone receptors. In carrying out these functions Hsp90 hydrolyses ATP as it cycles between ADP- and ATP-bound forms, and this ATPase activity is regulated by the transient association with a variety of co-chaperones. Cdc37 is one such co-chaperone protein that also has a role in client protein recognition, in that it is required for Hsp90-dependent folding and activation of a particular group of protein kinases. These include the cyclin-dependent kinases (Cdk) 4/6 and Cdk9, Raf-1, Akt and many others. Here, the biochemical details of the interaction of human Hsp90 beta and Cdc37 have been characterised. Small angle X-ray scattering (SAXS) was then used to study the solution structure of Hsp90 and its complexes with Cdc37. The results suggest a model for the interaction of Cdc37 with Hsp90, whereby a Cdc37 dimer binds the two N-terminal domain/linker regions in an Hsp90 dimer, fixing them in a single conformation that is presumably suitable for client protein recognition.     

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.     

Dynamics of the regulation of Hsp90 by the co-chaperone Sti1

In eukaryotic cells, Hsp90 chaperones assist late folding steps of many regulatory protein clients by a complex ATPase cycle. Binding of clients to Hsp90 requires prior interaction with Hsp70 and a transfer reaction that is mediated by the co-chaperone Sti1/Hop. Sti1 furthers client transfer by inhibiting Hsp90's ATPase activity. To better understand how Sti1 prepares Hsp90 for client acceptance, we characterized the interacting domains and analysed how Hsp90 and Sti1 mutually influence their conformational dynamics using hydrogen exchange mass spectrometry. Sti1 stabilizes several regions in all three domains of Hsp90 and slows down dissociation of the Hsp90 dimer. Our data suggest that Sti1 inhibits Hsp90's ATPase activity by preventing N-terminal dimerization and docking of the N-terminal domain with the middle domain. Using crosslinking and mass spectrometry we identified Sti1 segments, which are in close vicinity of the N-terminal domain of Hsp90. We found that the length of the linker between C-terminal dimerization domain and the C-terminal MEEVD motif is important for Sti1 association rates and propose a kinetic model for Sti1 binding to Hsp90.     

TNF-induced recruitment and activation of the IKK complex require Cdc37 and Hsp90

The IKK complex, containing two catalytic subunits IKKalpha and IKKbeta and a regulatory subunit NEMO, plays central roles in signal-dependent activation of NF-kappaB. We identify Cdc37 and Hsp90 as two additional components of the IKK complex. IKKalpha/IKKbeta/NEMO and Cdc37/Hsp90 form an approximately 900 kDa heterocomplex, which is assembled via direct interactions of Cdc37 with Hsp90 and with the kinase domain of IKKalpha/IKKbeta. Geldanamycin (GA), an antitumor agent that disrupts the formation of this heterocomplex, prevents TNF-induced activation of IKK and NF-kappaB. GA treatment reduces the size of the IKK complex and abolishes TNF-dependent recruitment of the IKK complex to TNF receptor 1 (TNF-R1). Therefore, heterocomplex formation with Cdc37/Hsp90 is a prerequisite for TNF-induced activation and trafficking of IKK from the cytoplasm to the membrane.     

Cdk2: a genuine protein kinase client of Hsp90 and Cdc37

Hsp90 and its cochaperone Cdc37 cooperate to provide requisite support to numerous protein kinases involved in cellular signal transduction. In this report, we studied the interactions of Hsp90 and Cdc37 with the cyclin-dependent kinase, Cdk2. Treatment of K562 cells with the Hsp90 inhibitor, geldanamycin, caused a 75% reduction in Cdk2 levels and reduced the levels of its activating kinase, Cdk7, by more than 60%, suggesting that both of these kinases may be Hsp90 clients. Using classical pull-down assays and the Hsp90 inhibitory agents geldanamycin and molybdate, Cdk2 is shown to be a genuine client of the Hsp90 chaperone complex. Subsequently, pull-down assays directed at helix alphaC of Cdk2 are shown to disrupt Hsp90 and Cdc37 binding and explain the initial difficulties in demonstrating these interactions. Mutant constructs containing deletions of secondary structural elements from the N- and C-termini of Cdk2 were prepared and assayed for their ability to coadsorb Hsp90 and Cdc37 in a salt-stable high-affinity manner with and without the addition of molybdate. Consistent with similar work done with the cyclin-dependent kinase relative Cdk4, the presence of the G-box motif of Cdk2 was shown to be critical for Cdc37 binding, whereas consistent with work done with the Src-family tyrosine kinase Lck, the presence of helix alphaC and the stabilization of helix alphaE were shown to be needed for Hsp90 binding.     

Exposure of protein kinase motifs that trigger binding of Hsp90 and Cdc37

Hsp90 and its co-chaperone Cdc37 are required for the activity of numerous eukaryotic protein kinases. c-Jun N-terminal kinases (JNKs) appear to be Hsp90-independent kinases, as their activity is unaffected by Hsp90 inhibition. It is currently unknown why some protein kinases are Hsp90- and Cdc37-dependent for their function, while others are not. Therefore, we investigated what structural motifs within JNKs confer or defer Hsp90 and Cdc37 interaction. Both Hsp90 and Cdc37 recognized structural features that were exposed or destabilized upon deletion of JNK1alpha1's N-terminal non-catalytic structural motif, while only Hsp90 bound JNK when its C-terminal non-catalytic structural motif was deleted. Mutations in JNK's activation loop that are known to constitutively activate or inactivate its kinase activity had no effect on JNK's lack of interaction with Hsp90 and Cdc37. Our findings suggest a model in which Hsp90 and Cdc37 each recognize distinct features within the catalytic domains of kinases.     

Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37

In vivo activation of client proteins by Hsp90 depends on its ATPase-coupled conformational cycle and on interaction with a variety of co-chaperone proteins. For some client proteins the co-chaperone Sti1/Hop/p60 acts as a "scaffold," recruiting Hsp70 and the bound client to Hsp90 early in the cycle and suppressing ATP turnover by Hsp90 during the loading phase. Recruitment of protein kinase clients to the Hsp90 complex appears to involve a specialized co-chaperone, Cdc37p/p50(cdc37), whose binding to Hsp90 is mutually exclusive of Sti1/Hop/p60. We now show that Cdc37p/p50(cdc37), like Sti1/Hop/p60, also suppresses ATP turnover by Hsp90 supporting the idea that client protein loading to Hsp90 requires a "relaxed" ADP-bound conformation. Like Sti1/Hop/p60, Cdc37p/p50(cdc37) binds to Hsp90 as a dimer, and the suppressed ATPase activity of Hsp90 is restored when Cdc37p/p50(cdc37) is displaced by the immunophilin co-chaperone Cpr6/Cyp40. However, unlike Sti1/Hop/p60, which can displace geldanamycin upon binding to Hsp90, Cdc37p/p50(cdc37) forms a stable complex with geldanamycin-bound Hsp90 and may be sequestered in geldanamycin-inhibited Hsp90 complexes in vivo.     

In vivo analysis of the Hsp90 cochaperone Sti1 (p60).

Hsp90 interacts with Sti1 (p60) in lysates of yeast and vertebrate cells. Here we provide the first analysis of their interaction in vivo. Saccharomyces cerevisiae mutations that eliminate Sti1 or reduce intracellular concentrations of Hsp90 individually have little or no effect on growth at normal temperatures. However, when combined, the mutations greatly reduce or eliminate growth. Furthermore, overexpression of Sti1 has allele-specific effects on cells carrying various hsp90ts point mutations. These genetic interactions provide strong evidence that Hsp90 and Sti1 interact in vivo and that their functions are closely allied. Indeed, deletion of STI1 reduces the in vivo activity of the Hsp90 target protein, glucocorticoid receptor (GR). Mutations in GR that eliminate interaction with Hsp90 also eliminate the effects of the sti1 deletion. Examination of GR protein complexes in the sti1 deletion mutant reveals a selective increase in the concentration of GR-Ydj1 complexes, supporting previous hypotheses that Ydj1 functions at an early step in the maturation of GR and that Sti1 acts at an intermediate step. Deletion of STI1 also reduces the in vivo activity of another, unrelated Hsp90 target protein, v-Src. Our data indicate that Sti1 is a general factor in the maturation of Hsp90 target proteins and support earlier suggestions that Hsp90 matures even very different target proteins by a similar mechanism.     

Functional interactions between Hsp90 and the co-chaperones Cns1 and Cpr7 in Saccharomyces cerevisiae

Hsp90 complexes contain a class of co-chaperones characterized by a tetratricopeptide repeat (TPR) domain, which mediates binding to a carboxyl-terminal EEVD region in Hsp90. Among Hsp90 TPR co-chaperones in Saccharomyces cerevisiae, only Cns1 is essential. The amino terminus of Cns1, which harbors the TPR domain, is sufficient for viability when overexpressed. In a screen for temperature-sensitive alleles of CNS1, we identified mutations resulting in substitutions of conserved residues in the TPR domain. Mutations in CNS1 disrupt in vitro and in vivo interaction with Hsp90 and reduce Hsp90 function, indicating that Cns1 is a bona fide co-chaperone. Genetic interactions between CNS1 and another Hsp90 co-chaperone, CPR7, suggest that the two co-chaperones share an essential role in the cell. Although both the TPR and the isomerase domains of the cyclophilin Cpr7 are required for viability of cns1 mutant cells, this requirement does not depend on the catalytic function of the isomerase domain. Instead, hydrophilic residues on the surface of this domain appear to be important for the common Cns1.Cpr7 function. Although both co-chaperones interact with Hsp90 primarily through the carboxyl terminus (EEVD), Cns1 and Cpr7 are mostly found in complexes distinct from Hsp90. EEVD is required for normal growth in cns1 mutant cells, demonstrating for the first time in vivo requirement for this conserved region of Hsp90. Overall, our findings reveal a considerable degree of complexity in the interactions not only between Hsp90 and its co-chaperones, but also among the co-chaperones themselves.     

Akt forms an intracellular complex with heat shock protein 90 (Hsp90) and Cdc37 and is destabilized by inhibitors of Hsp90 function

Hsp90 is a chaperone required for the conformational maturation of certain signaling proteins including Raf, cdk4, and steroid receptors. Natural products and synthetic small molecules that bind to the ATP-binding pocket in the amino-terminal domain of Hsp90 inhibit its function and cause the degradation of these client proteins. Inhibition of Hsp90 function in cells causes down-regulation of an Akt kinase-dependent pathway required for D-cyclin expression and retinoblastoma protein-dependent G(1) arrest. Intracellular Akt is associated with Hsp90 and Cdc37 in a complex in which Akt kinase is active and regulated by phosphatidylinositol 3-kinase. Functional Hsp90 is required for the stability of Akt in the complex. Occupancy of the ATP-binding pocket by inhibitors is associated with the ubiquitination of Akt and its targeting to the proteasome, where it is degraded. This results in a shortening of the half-life of Akt from 36 to 12 h and an 80% reduction in its expression. Akt and its activating kinase, PDK1, are the only members of the protein kinase A/protein kinase B/protein kinase C-like kinase family that are affected by Hsp90 inhibitors. Thus, transduction of growth factor signaling via the Akt and Raf pathways requires functional Hsp90 and can be coordinately blocked by its inhibition.