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.     

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.     

p50(cdc37) is a nonexclusive Hsp90 cohort which participates intimately in Hsp90-mediated folding of immature kinase molecules

Hsp90 and p50(cdc37) provide a poorly understood biochemical function essential to certain protein kinases, and recent models describe p50(cdc37) as an exclusive hsp90 cohort which links hsp90 machinery to client kinases. We describe here the recovery of p50(cdc37) in immunoadsorptions directed against the hsp90 cohorts FKBP52, cyp40, p60HOP, hsp70, and p23. Additionally, monoclonal antibodies against FKBP52 coadsorb maturation intermediates of the hsp90-dependent kinases p56(lck) and HRI, and the presence of these maturation intermediates significantly increases the representation of p50(cdc37) and hsp90 on FKPB52 machinery. Although the native heterocomplex between hsp90 and p50(cdc37) is salt-labile, their dynamic interactions with kinase substrates produce kinase-chaperone heterocomplexes which are highly salt-resistant. The hsp90 inhibitor geldanamycin does not directly disrupt the native association of hsp90 with p50(cdc37) per se, but does result in the formation of salt-labile hsp90-kinase heterocomplexes which lack the p50(cdc37) cohort. We conclude that p50(cdc37) does not simply serve as a passive structural bridge between hsp90 and its kinase substrates; instead, p50(cdc37) is a nonexclusive hsp90 cohort which responds to hsp90's nucleotide-regulated conformational switching during the generation of high-affinity interactions within the hsp90-kinase-p50(cdc37) heterocomplex.     

Hsp90 functions to balance the phosphorylation state of Akt during C2C12 myoblast differentiation

The function of the 90-kDa heat shock protein (Hsp90) is essential for the regulation of a myriad of signal transduction cascades that control all facets of a cell's physiology. Akt (PKB) is an Hsp90-dependent serine-threonine kinase that plays critical roles in the regulation of muscle cell physiology, including roles in the regulation of muscle differentiation and anti-apoptotic responses that modulate cell survival. In this report, we have examined the role of Hsp90 in regulating the activity of Akt in differentiating C2C12 myoblasts. While long-term treatment of differentiating C2C12 cells with the Hsp90 inhibitor geldanamycin led to the depletion of cellular Akt levels, pulse-chase analysis indicated that geldanamycin primarily enhanced the turnover rate of newly synthesized Akt. Hsp90 maintained an interaction with mature Akt, while Cdc37, Hsp90's kinase-specific co-chaperone, was lost from the chaperone complex upon Akt maturation. Geldanamycin partially disrupted the interaction of Cdc37 with Akt, but had a much less significant effect on the interaction of Hsp90 with Akt. Surprisingly, short-term treatment of differentiating C2C12 with geldanamycin increased the phosphorylation of Akt on Ser473, an effect mimicked by treatment of C2C12 cells with okadaic acid or the Hsp90 inhibitor novobiocin. Furthermore, Akt was found to interact directly with catalytic subunit of protein phosphatase 2A (PP2Ac) in C2C12 cells, and this interaction was not disrupted by geldanamycin. Thus, our findings indicate that Hsp90 functions to balance the phosphorylation state of Akt by modulating the ability of Akt to be dephosphorylated by PP2Ac during C2C12 myoblast differentiation.     

High affinity binding of Hsp90 is triggered by multiple discrete segments of its kinase clients

The 90 kDa heat shock protein (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, crystal structures of protein kinases were used to guide the dissection of two kinases [the Src-family tyrosine kinase, Lck, and the heme-regulated eIF2alpha kinase (HRI)], and the association of Hsp90 and Cdc37 with these constructs was assessed. Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the kinases' catalytic domains. In contrast, Cdc37 interacted only with the NL. The Hsp90 antagonist molybdate was necessary to stabilize the interactions between isolated subdomains and Hsp90 or Cdc37, but the presence of both lobes of the kinases' catalytic domain generated a stable salt-resistant chaperone-client heterocomplex. The Hsp90 co-chaperones FKBP52 and p23 interacted with the catalytic domain and the NL of Lck, whereas protein phosphatase 5 demonstrated unique modes of kinase binding. Cyp40 was a salt labile component of Hsp90 complexes formed with the full-length, catalytic domains, and N-terminal catalytic lobes of Lck and HRI. Additionally, dissections identify a specific kinase motif that triggers Hsp90's conformational switching to a high-affinity client binding state. Results indicate that the Hsp90 machine acts as a versatile chaperone that recognizes multiple regions of non-native proteins, while Cdc37 binds to a more specific kinase segment, and that concomitant recognition of multiple client segments is communicated to generate or stabilize high-affinity chaperone-client heterocomplexes.     

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.     

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.     

p50(cdc37) acting in concert with Hsp90 is required for Raf-1 function

Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.     

Phosphorylation of a heme-regulated eukaryotic initiation factor 2α kinase enhances the interaction with heat-shock protein 90 and substantially upregulates kinase activity

Heme-regulated eukaryotic initiation factor 2α kinase (HRI) functions under conditions of heme shortage caused by blood diseases such as erythropoietic protoporphyria and β-thalassemia, and retains the heme:globin ratio at 1:1 by sensing the heme concentration in reticulocytes. This HRI function is regulated by various factors including autophosphorylation and protein-protein interactions. A heat-shock protein controls HRI function, however, the molecular mechanism of catalytic regulation of HRI by the heat-shock protein is unclear. In the present study, we examined the interactions of HRI with a heat-shock protein, Hsp90, under various conditions, using a pull-down assay and measuring catalytic activity. It was found that [1] an interaction between Hsp90 and phosphorylated HRI was evident, whereas no interaction was observed between Hsp90 and HRI dephosphorylated by treatment with λ protein phosphatase; [2] Hsp90 enhanced the kinase activity of phosphorylated HRI but not dephosphorylated HRI, but this enhancement was not observed in the presence of heme; and, [3] autophosphorylation of HRI was not influenced by Hsp90. Therefore, we propose that autophosphorylation of HRI is critical for catalytic regulation by Hsp90 under heme-shortage conditions.     

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.     

Hsp90 regulates protein synthesis by activating the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase in rabbit reticulocyte lysates

Heat shock protein 90 (Hsp90), an abundant and ubiquitous cytoplasmic protein has recently been indicated to participate in the regulation of protein synthesis by interacting with the heme-regulated eukaryotic initiation factor 2α (eIF-2α) kinase, also known as the heme-regulated inhibitor (HRI). However, there exists an ambiguity on the exact nature of its action. In this investigation, the interaction of Hsp90 and HRI has been examined bothin vitro using purified proteins, andin situ in rabbit reticulocyte lysates subjected to heat shock and treatment with N-ethylmaleimide (NEM), a sulfhydryl reagent known to induce stress response. During heat shock or NEM-treatment of reticulocyte lysates, Hsp90 co-immunoprecipitated with activated HRI by anti-HRI monoclonal antibodies. Furthermore, the amount of Hsp90 being associated with HRI was a function of duration of heat shock and was correlated with the extent of HRI activation. Interestingly, simultaneous heat shock and NRM-treatment of reticulocyte lysates led to maximal association of HRI and Hsp90, leaving nearly no free HRI in the lysates.In vitro, with the purified proteins, the autokinase and the eIF-2α kinase activities of HRI were enhanced when HRI was pre-incubated with Hsp90, both in the presence and absence of hemin. These data, therefore, clearly demonstrate that Hsp90 interacts with HRI during stress, and that this association leads to activation of HRI and thereby inhibition of protein synthesis at the level of initiation. Considering the ubiquitous nature of Hsp90 and the presence of HRI or HRI-like eIF-2α kinase activity in a number of organisms, it is highly possible that Hsp90 may universally mediate down regulation of global protein synthesis during stress response.     

The Mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37)

Recruitment of protein kinase clients to the Hsp90 chaperone involves the cochaperone p50(cdc37) acting as a scaffold, binding protein kinases via its N-terminal domain and Hsp90 via its C-terminal region. p50(cdc37) also has a regulatory activity, arresting Hsp90's ATPase cycle during client-protein loading. We have localized the binding site for p50(cdc37) to the N-terminal nucleotide binding domain of Hsp90 and determined the crystal structure of the Hsp90-p50(cdc37) core complex. Dimeric p50(cdc37) binds to surfaces of the Hsp90 N-domain implicated in ATP-dependent N-terminal dimerization and association with the middle segment of the chaperone. This interaction fixes the lid segment in an open conformation, inserts an arginine side chain into the ATP binding pocket to disable catalysis, and prevents trans-activating interaction of the N domains.     

Interdomain interactions regulate the activation of the heme-regulated eIF 2 alpha kinase

The heme-regulated inhibitor of protein synthesis (HRI) regulates translation through the phosphorylation of the alpha-subunit of eukaryotic initiation factor-2 (eIF 2). While HRI is best known for its activation in response to heme-deficiency, we recently showed that the binding of NO and CO to the N-terminal heme-binding domain (NT-HBD) of HRI activated and suppressed its activity, respectively. Here, we examined the effect of hemin, NO, and CO on the interaction between the NT-HBD and the catalytic domain of HRI (HRI/Delta HBD). Hemin stabilized the interaction of NT-HBD with HRI/Delta HBD, and NO and CO disrupted and stabilized this interaction, respectively. Mutant HRI (Delta H-HRI), lacking amino acids 116-158 from the NT-HBD, was less sensitive to heme-induced inhibition, and mutant NT-HBD lacking these residues did not bind to HRI/Delta HBD. HRI/Delta HBD and Delta H-HRI also activated more readily than HRI in response to heme-deficiency. Thus, HRI's activity is regulated through the modulation of the interaction between its NT-HBD and catalytic domain.     

Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.     

Hsp90/p50cdc37 is required for mixed-lineage kinase (MLK) 3 signaling

Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH(2)-terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG.MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90alpha,beta (Hsp90) and its kinase-specific co-chaperone p50(cdc37). We show that endogenous MLK3 complexes with Hsp90 and p50(cdc37). Further experiments demonstrate that MLK3 associates with Hsp90/p50(cdc37) through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor alpha-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50(cdc37) regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50(cdc37) clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50(cdc37) also regulates protein kinases involved in apoptotic signaling.     

The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest

The molecular chaperone Cdc37 is thought to act in part as a targeting subunit of the heat-shock protein 90 (Hsp90) chaperone complex. We demonstrate here that Cdc37 is required for activity of the kinase Ste11 in budding yeast. A cdc37 mutant strain is defective in Ste11-mediated pheromone signaling and in accumulation and functional maturation of the constitutively active Ste11 version Ste11DeltaN. Moreover, Cdc37, Ste11DeltaN and Hsp90 coprecipitate pairwise. Thus, Hsp90 and Cdc37 may transiently associate with Ste11 to promote proper folding and/or association with additional regulatory factors. Our results establish Ste11 as the first endogenous Cdc37 client protein in yeast.     

Dual Role for Hsc70 in the Biogenesis and Regulation of the Heme-Regulated Kinase of the α Subunit of Eukaryotic Translation Initiation Factor 2

The heme-regulated kinase of the alpha subunit of eukaryotic initiation factor 2 (HRI) is activated in rabbit reticulocyte lysate (RRL) in response to a number of environmental conditions, including heme deficiency, heat shock, and oxidative stress. Activation of HRI causes an arrest of initiation of protein synthesis. Recently, we have demonstrated that the heat shock cognate protein Hsc70 negatively modulates the activation of HRI in RRL in response to these environmental conditions. Hsc70 is also known to be a critical component of the Hsp90 chaperone machinery in RRL, which plays an obligatory role for HRI to acquire and maintain a conformation that is competent to activate. Using de novo-synthesized HRI in synchronized pulse-chase translations, we have examined the role of Hsc70 in the regulation of HRI biogenesis and activation. Like Hsp90, Hsc70 interacted with nascent HRI and HRI that was matured to a state which was competent to undergo stimulus-induced activation (mature-competent HRI). Interaction of HRI with Hsc70 was required for the transformation of HRI, as the Hsc70 antagonist clofibric acid inhibited the folding of HRI into a mature-competent conformation. Unlike Hsp90, Hsc70 also interacted with transformed HRI. Clofibric acid disrupted the interaction of Hsc70 with transformed HRI that had been matured and transformed in the absence of the drug. Disruption of Hsc70 interaction with transformed HRI in heme-deficient RRL resulted in its hyperactivation. Furthermore, activation of HRI in response to heat shock or denatured proteins also resulted in a similar blockage of Hsc70 interaction with transformed HRI. These results indicate that Hsc70 is required for the folding and transformation of HRI into an active kinase but is subsequently required to negatively attenuate the activation of transformed HRI.     

G protein-coupled receptor kinase interaction with Hsp90 mediates kinase maturation

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine-specific protein kinase that mediates agonist-dependent phosphorylation of numerous G protein-coupled receptors. In an effort to identify proteins that regulate GRK2 function, we searched for interacting proteins by immunoprecipitation of endogenous GRK2 from HL60 cells. Subsequent analysis by gel electrophoresis and mass spectrometry revealed that GRK2 associates with heat shock protein 90 (Hsp90). GRK2 interaction with Hsp90 was confirmed by co-immunoprecipitation and was effectively disrupted by geldanamycin, an Hsp90-specific inhibitor. Interestingly, geldanamycin treatment of HL60 cells decreased the expression of endogenous GRK2 in a dose- and time-dependent manner, and metabolic labeling demonstrated that geldanamycin rapidly accelerated the degradation of newly synthesized GRK2. The use of various protease inhibitors suggested that GRK2 degradation induced by geldanamycin was predominantly through the proteasome pathway. To test whether Hsp90 plays a general role in regulating GRK maturation, additional GRKs were studied by transient expression in COS-1 cells and subsequent treatment with geldanamycin. These studies demonstrate that GRK3, GRK5, and GRK6 are also stabilized by interaction with Hsp90. Taken together, our work revealed that GRK interaction with heat shock proteins plays an important role in regulating GRK maturation.     

A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases

We report here the identification of CDC37, which encodes a putative Hsp90 co-chaperone, as a multicopy suppressor of a temperature-sensitive allele (cka2-13(ts)) of the CKA2 gene encoding the alpha' catalytic subunit of protein kinase CKII. Unlike wild-type cells, cka2-13 cells were sensitive to the Hsp90-specific inhibitor geldanamycin, and this sensitivity was suppressed by overexpression of either Hsp90 or Cdc37. However, only CDC37 was capable of suppressing the temperature sensitivity of a cka2-13 strain, implying that Cdc37 is the limiting component. Immunoprecipitation of metabolically labeled Cdc37 from wild-type versus cka2-13 strains revealed that Cdc37 is a physiological substrate of CKII, and Ser-14 and/or Ser-17 were identified as the most likely sites of CKII phosphorylation in vivo. A cdc37-S14,17A strain lacking these phosphorylation sites exhibited severe growth and morphological defects that were partially reversed in a cdc37-S14,17E strain. Reduced CKII activity was observed in both cdc37-S14A and cdc37-S17A mutants at 37 degrees C, and cdc37-S14A or cdc37-S14,17A overexpression was incapable of protecting cka2-13 mutants on media containing geldanamycin. Additionally, CKII activity was elevated in cells arrested at the G(1) and G(2)/M phases of the cell cycle, the same phases during which Cdc37 function is essential. Collectively, these data define a positive feedback loop between CKII and Cdc37. Additional genetic assays demonstrate that this CKII/Cdc37 interaction positively regulates the activity of multiple protein kinases in addition to CKII.     

Modulation of chaperone function and cochaperone interaction by novobiocin in the C-terminal domain of Hsp90: evidence that coumarin antibiotics disrupt Hsp90 dimerization

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An alpha-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50(cdc37) cochaperone, which targets the N-terminal nucleotide-binding domain, and is prevalent in Hsp90 complexes with protein kinase substrates. Novobiocin therefore acts locally and allosterically to induce conformational changes within multiple regions of the Hsp90 protein. We provide evidence that coumermycin A1, a coumarin structurally related to novobiocin, interferes with dimerization of the Hsp90 C-terminal domain. Coumarin-based inhibitors then may antagonize Hsp90 function by inducing a conformation favoring separation of the C-terminal domains and release of substrate.