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.     

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.     

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.     

Definition of protein kinase sequence motifs that trigger high affinity binding of Hsp90 and Cdc37

Hsp90 cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, the crystal structure of the Src family tyrosine kinase Lck was used to guide the creation of kinase constructs to determine features recognized by Hsp90 and its "kinase-specific" co-chaperone Cdc37. Two parameters were assayed: the ability and extent to which the constructs bound to Hsp90 and Cdc37, and the ability of the constructs to trigger salt-resistant high affinity complexes with Hsp90 and Cdc37 independent of the presence of molybdate. Although Hsp90 interacted with both the N-terminal and C-terminal lobes (NL and CL, respectively) of the catalytic domains of the kinases, the lobes themselves were not sufficient to trigger the high affinity binding of Hsp90. Only constructs containing a complete N- or C-terminal lobe and part of the adjacent lobe bound to Hsp90 and Cdc37 in salt-stable complexes independent of molybdate. The two minimum constructs that bound Hsp90 and Cdc37 contained the alpha-C-helix and the beta4- and beta5-strands of the NL through to end of the CL and the NL through to the alpha-E-helix and the amino acids that cap the helix. Cdc37 interacted with only the NL and minimally required the alpha-C-helix and beta4- and beta5-strands of this lobe of Lck. The results indicate that the high affinity binding activity of Hsp90 is triggered through its interaction with adjacent subdomain structures of kinase catalytic domains. Furthermore, the alpha-C-helix and part of its adjoining loop connection to the beta4-strand appear to be the primary determinants recognized by Cdc37.     

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.     

The Schizosaccharomyces pombe Cdc7 protein kinase required for septum formation is a client protein of Cdc37

Cdc37 is an essential molecular chaperone found in fungi and metazoa whose main specificity is for certain protein kinases. Cdc37 can act as an Hsp90 cochaperone or alone; in yeasts, the interaction with Hsp90 is weak and appears not to be essential for Cdc37 function. Numerous genetic interactions between Cdc37 and likely client proteins have been observed in yeasts, but biochemical confirmation has been reported in only a few cases. We and others have generated and characterized temperature-sensitive cdc37 alleles in S. pombe and have used them to investigate the cellular roles of Cdc37: previous work has shown that mitotic Cdc2 is a major client. In this paper, we describe a screen for mutations synthetically lethal with a cdc37ts mutant with the aim of identifying genes encoding further client proteins of Cdc37. Ten such strains were isolated, and genomic libraries were screened for rescuing plasmids. In one case, a truncated cdc7 gene was identified. Further experiments showed that the mutation in this strain was indeed in cdc7. Cdc7 is a protein kinase required for septum initiation, and we show that its kinase activity is greatly reduced when Cdc37 function is impaired. Cdc7 normally locates to the spindle pole body during mitosis, and this appears to be unaffected in the cdc37ts mutant. Other evidence suggests that, in addition to mitosis and septum initiation, Cdc37 may also be required for septum cleavage.     

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.     

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.     

Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes.     

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.     

Cdc37/Hsp90 protein complex disruption triggers an autophagic clearance cascade for TDP-43 protein

The RNA-binding protein, trans-active response DNA-binding protein 43 (TDP-43), is normally found in the nucleus, but in amyotrophic lateral sclerosis, frontal temporal dementia, and some cases of Alzheimer disease it is cleaved and mislocalized to the cytosol, leading to accumulation. The mechanisms contributing to this are largely unknown. Here, we show that part of the normal clearance cascade for TDP-43 involves the Cdc37/Hsp90 complex. An Hsp90 inhibitor that disrupts the Cdc37/Hsp90 complex reduced TDP-43 levels to a greater extent than a standard Hsp90 ATPase inhibitor. When Cdc37 was depleted, TDP-43 underwent proteolytic clearance that was dependent on nuclear retrotranslocation and autophagic uptake. Accumulation of the microtubule-associated protein tau prevented the clearance of cleaved TDP-43, but not its production. This caused cleaved TDP-43 to accumulate, a feature observed in the brain of persons with Alzheimer disease. Clearance of cleaved TDP-43 was also prevented by knockdown of the autophagic inducer beclin1. Thus, in cells where TDP-43 clearance is normally needed, a system that employs manipulation of the Hsp90 complex and autophagy exists. But when tau accumulation is occurring, cleaved TDP-43 can no longer be cleared, perhaps explaining the emergence of these co-pathologies.     

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 Hsp90 co-chaperones Cdc37 and Sti1 interact physically and genetically

Cdc37 associates with the heat-shock protein 90 (Hsp90) molecular chaperone as one of several auxiliary proteins that are collectively referred to as Hsp90 co-chaperones. Cdc37 has been proposed to be a specificity factor for Hsp90, directing it notably towards kinases. It is not known whether Cdc37 is essential for viability in the budding yeast Saccharomyces cerevisiae because of Hsp90-dependent or -independent functions or both. Sti1 and Cpr7 are non-essential Hsp90 co-chaperones that bind to a common surface on Hsp90 through tetratricopeptide repeats (TPR). We have found that Sti1 is specifically retained from yeast extracts by immobilized Cdc37. Similarly, the endogenous proteins are also found in a complex. Moreover, purified recombinant Sti1 and Cdc37 interact in the complete absence of Hsp90. Complexes between Cdc37 and Sti1 are not unique to this TPR protein since endogenous Cdc37 can be co-purified with exogenously expressed Cpr7 fused to glutathione-S-transferase. The heterogeneity of Cdc37 complexes, both with and without Hsp90, may expand the functional diversity of Cdc37. Here we show that the combination of cdc37 and sti1 mutations is synthetically lethal, suggesting that direct contacts between Cdc37 and Sti1 may at least contribute to vital functions in yeast.     

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.     

Identification and characterization of Harc, a novel Hsp90-associating relative of Cdc37.

Although little is known about the precise mechanisms by which the molecular chaperone Hsp90 recognizes its client proteins, Cdc37 has been shown to play a critical role in the targeting of Hsp90 to client protein kinases. Described here is the identification and characterization of a novel 35-kDa human protein that is 31% identical to Cdc37. We have named this novel protein Harc (Hsp90-associating relative of Cdc37). Northern blot analysis revealed the presence of Harc mRNA in several human tissues, including liver, skeletal muscle, and kidney. Biochemical fractionation and immunofluorescent localization of epitope-tagged Harc (i.e. FLAG-Harc) indicated that it is present in the cytoplasm of cells. FLAG-Harc binds Hsp90 but unlike Cdc37 does not bind Src family kinases or Raf-1. Mapping experiments indicate that the central 120 amino acids of both Harc and Cdc37 constitute a Hsp90-binding domain not described previously. FLAG-Harc is basally serine-phosphorylated and hyperphosphorylated when co-expressed with an activated mutant of the Src family kinase Hck. Notably, FLAG-Harc forms complexes with Hsp90, Hsp70, p60Hop, immunophilins, and an unidentified p22 protein but not with the Hsp90 co-chaperone p23. Thus Harc likely represents a novel participant in Hsp90-mediated protein folding, potentially targeting Hsp90 to Hsp70-client protein heterocomplexes.     

Optimization and bioevaluation of Cdc37-derived peptides: An insight into Hsp90-Cdc37 protein-protein interaction modulators

Targeting Hsp90-Cdc37 protein-protein interaction (PPI) is becoming an alternative approach for future anti-cancer drug development. We previously reported the discovery of an eleven-residue peptide (Pep-1) with micromolar activity for the disruption of Hsp90-Cdc37 PPI. Efforts to improve upon the Pep-1 led to the discovery of more potent modulators for Hsp90-Cdc37 PPI. Through the analysis of peptides binding patterns, more peptides were designed for further verification which resulted in Pep-5, the shortest peptide targeting Hsp90-Cdc37, exerting the optimal structure and the most efficient binding mode. Subsequent MD simulation analysis also confirmed that Pep-5 could perform more stable binding ability and better ligand properties than Pep-1. Under the premise of retentive binding capacity, Pep-5 exhibited lower molecular weight and higher ligand efficiency with a K_d value of 5.99 μM (Pep-1 K_d = 6.90 μM) in both direct binding determination and biological evaluation. The optimal and shortest Pep-5 might provide a breakthrough and a better model for the future design of small molecule inhibitors targeting Hsp90-Cdc37 PPI.     

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.