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.     

Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding

Hsp90 and its co-chaperone Cdc37 facilitate the folding and activation of numerous protein kinases. In this report, we examine the structure-function relationships that regulate the interaction of Cdc37 with Hsp90 and with an Hsp90-dependent kinase, the heme-regulated eIF2alpha kinase (HRI). Limited proteolysis of native and recombinant Cdc37, in conjunction with MALDI-TOF mass spectrometry analysis of peptide fragments and peptide microsequencing, indicates that Cdc37 is comprised of three discrete domains. The N-terminal domain (residues 1-126) interacts with client HRI molecules. Cdc37's middle domain (residues 128-282) interacts with Hsp90, but does not bind to HRI. The C-terminal domain of Cdc37 (residues 283-378) does not bind Hsp90 or kinase, and no functions were ascribable to this domain. Functional assays did, however, suggest that residues S127-G163 of Cdc37 serve as an interdomain switch that modulates the ability of Cdc37 to sense Hsp90's conformation and thereby mediate Hsp90's regulation of Cdc37's kinase-binding activity. Additionally, scanning alanine mutagenesis identified four amino acid residues at the N-terminus of Cdc37 that are critical for high-affinity binding of Cdc37 to client HRI molecules. One mutation, Cdc37/W7A, also implicated this region as an interpreter of Hsp90's conformation. Results illuminate the specific Cdc37 motifs underlying the allosteric interactions that regulate binding of Hsp90-Cdc37 to immature kinase molecules.     

Hsp90 regulates p50(cdc37) function during the biogenesis of the activeconformation of the heme-regulated eIF2 alpha kinase

Recent studies indicate that p50(cdc37) facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50(cdc37) is required for the biogenesis of the heme-regulated eIF2 alpha kinase (HRI) in reticulocyte lysate. p50(cdc37) interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50(cdc37) stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50(cdc37) function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50(cdc37) interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50(cdc37) interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50(cdc37) to bind HRI and promote its activation, but did not disrupt the native association of p50(cdc37) with Hsp90. A C-terminal truncated mutant of p50(cdc37) inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50(cdc37) were detected in cultured K562 erythroleukemia cells. These results suggest that p50(cdc37) provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.     

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.     

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.     

Hsp90·Cdc37 Complexes with Protein Kinases Form Cooperatively with Multiple Distinct Interaction Sites

Protein kinases are the most prominent group of heat shock protein 90 (Hsp90) clients and are recruited to the molecular chaperone by the kinase-specific cochaperone cell division cycle 37 (Cdc37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of Caenorhabditis elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces, binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the mitogen-activated protein kinase extracellular signal-regulated kinase 2 (Erk2), we observe that certain features of the interaction with Cdc37·Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.     

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.     

A client-binding site of Cdc37

The molecular chaperone Hsp90 is distinct from Hsp70 and chaperonin in that client proteins are apparently restricted to a subset of proteins categorized as cellular signaling molecules. Among these, many specific protein kinases require the assistance of Hsp90 and its co-chaperone Cdc37/p50 for their biogenesis. A series of Cdc37 deletion mutants revealed that all mutants capable of binding Raf-1 possess amino acid residues between 181 and 200. The 20-residue region is sufficient and, in particular, a five-residue segment (residue 191-195) is essential for binding to Raf-1. These five residues are present in one alpha helix (residues 184-199) in the middle of Cdc37, which is unexpectedly nested within the Hsp90-interacting domain of Cdc37, which was recently determined by crystallography, but does not seem to contribute to direct contact with Hsp90. Furthermore, an N-terminally truncated mutant of Cdc37 composed of residues 181-378 was shown to bind the N-terminal portion of Raf-1 (subdomains I-IV). This mutant can bind not only other Hsp90 client protein kinases, Akt1, Aurora B and Cdk4, but also Cdc2 and Cdk2, which to date have not been shown to physically interact with Cdc37. These results suggest that a region of Cdc37 other than the client-binding site may be responsible for discriminating client protein kinases from others.     

p50(Cdc37) can buffer the temperature-sensitive properties of a mutant of Hck

Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast. We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50(Cdc37), the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50(Cdc37) in the folding and stabilization of Src family kinases. A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F. Significantly, overexpression of p50(Cdc37) rescues the catalytic activity of tsHck499F at 33 degrees C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39 degrees C). Hsp90 function is required for tsHck499F activity and its stabilization by p50(Cdc37), but overexpression of Hsp90 is not sufficient to stabilize tsHck499F. Overexpression of p50(Cdc37) promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50(Cdc37) might be the rate-limiting step in the association of tsHck499F with Hsp90. A truncation mutant of p50(Cdc37) that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90. This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50(Cdc37) may also act allosterically to enhance the association of tsHck499F with Hsp90. The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.     

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.     

Cdc37-Hsp90 complexes are responsive to nucleotide-induced conformational changes and binding of further cofactors

Hsp90 is an ATP-dependent molecular chaperone, which facilitates the activation and stabilization of hundreds of client proteins in cooperation with a defined set of cofactors. Many client proteins are protein kinases, which are activated and stabilized by Hsp90 in cooperation with the kinase-specific co-chaperone Cdc37. Other Hsp90 co-chaperones, like the ATPase activator Aha1, also are implicated in kinase activation, and it is not yet clear how Cdc37 is integrated into Hsp90 co-chaperone complexes. Here, we studied the interaction between Cdc37, Hsp90, and other Hsp90 co-chaperones from the nematode Caenorhabditis elegans. Nematode Cdc37 binds with high affinity to Hsp90 and strongly inhibits the ATPase activity. In contrast to the human Hsp90 system, we observed binding of Cdc37 to open and closed Hsp90 conformations, potentially reflecting two different binding modes. Using a novel ultracentrifugation setup, which allows accurate analysis of multifactorial protein complexes, we show that cooperative and competitive interactions exist between other co-chaperones and Cdc37-Hsp90 complexes in the C. elegans system. We observed strong competitive interactions between Cdc37 and the co-chaperones p23 and Sti1, whereas the binding of the phosphatase Pph5 and the ATPase activator Aha1 to Cdc37-Hsp90 complexes is possible. The ternary Aha1-Cdc37-Hsp90 complex is disrupted by the nucleotide-induced closing reaction at the N terminus of Hsp90. This implies a carefully regulated exchange process of cofactors during the chaperoning of kinase clients by Hsp90.     

Dynamic Tyrosine Phosphorylation Modulates Cycling of the HSP90-P50(CDC37)-AHA1 Chaperone Machine

Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1, which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining cochaperones.     

Physical interaction of Cdc28 with Cdc37 in Saccharomyces cerevisiae

The Cdc37 protein in Saccharomyces cerevisiae is thought to be a kinase-targeting subunit of the chaperone Hsp90. In a genetic screen, four protein kinases were identified as interacting with Cdc37 - Cdc5, Cdc7, Cdc15 and Cak1. This result underlines the importance of Cdc37 for the folding of protein kinases. In addition, we showed that Ydj1, a yeast DnaJ homolog belonging to the Hsp40 family of chaperones, genetically interacts with Cdc37. No physical interaction has so far been detected between Cdc37 and Cdc28, although genetic interactions (synthetic lethality and mutation suppression), and biochemical studies have suggested that these two proteins functionally interact. We found that, when separately expressed, the N-terminal lobe of Cdc28 interacted strongly with the C-terminal moiety of Cdc37 in a two-hybrid system. This was not the case for the full-length Cdc28 protein. We present models to explain these results.     

Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.     

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.     

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.     

Signal responsiveness of IkappaB kinases is determined by Cdc37-assisted transient interaction with Hsp90

The IkappaB kinase (IKK) holocomplex, containing the kinases IKKalpha, IKKbeta, and the scaffold NEMO (NF-kappaB essential modifier), mediates activation of NF-kappaB by numerous physiological stimuli. Heat shock protein 90 (Hsp90) and the co-chaperone Cdc37 have been indicated as additional subunits, but their specific functions in signal transduction are indistinct. Using an RNA interference approach, we demonstrate that Cdc37 recruits Hsp90 to the IKK complex in a transitory manner, preferentially via IKKalpha. Binding is conferred by N-terminal as well as C-terminal residues of Cdc37. Cdc37 is essential for the maturation of de novo synthesized IKKs into enzymatically competent kinases but not for assembly of an IKK holocomplex. Mature IKKs, T-loop-phosphorylated after stimulation either by receptor-mediated signaling or upon DNA damage, further require Hsp90-Cdc37 to generate an activated state. Thus, the present data denote Hsp90-Cdc37 as a transiently acting essential regulatory component of IKK signaling.     

Analysis of the CK2-dependent phosphorylation of serine 13 in Cdc37 using a phospho-specific antibody and phospho-affinity gel electrophoresis

The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829-2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117-38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065-4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg(2+) and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.     

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.     

Structure of an Hsp90-Cdc37-Cdk4 complex

Activation of many protein kinases depends on their interaction with the Hsp90 molecular chaperone system. Recruitment of protein kinase clients to the Hsp90 chaperone system is mediated by the cochaperone adaptor protein Cdc37, which acts as a scaffold, simultaneously binding protein kinases and Hsp90. We have now expressed and purified an Hsp90-Cdc37-Cdk4 complex, defined its stoichiometry, and determined its 3D structure by single-particle electron microscopy. Comparison with the crystal structure of Hsp90 allows us to identify the locations of Cdc37 and Cdk4 in the complex and suggests a mechanism by which conformational changes in the kinase are coupled to the Hsp90 ATPase cycle.