Molecular chaperones and protein kinase quality control

The Hsp90-Cdc37 chaperone pair has special responsibility for folding of protein kinases. This function has made Hsp90 a target for new chemotherapeutic approaches, and several compounds are currently being tested for their ability to inhibit many different kinases simultaneously. Not all kinases are sensitive to these inhibitors, however, and this difference might depend on how each kinase interacts with Hsp90 and Cdc37 during folding of the nascent chain and thereafter. Indeed, several kinases require the persistent presence of both chaperones after initial folding and some of these kinases seem to be particularly sensitive to Hsp90 inhibitors. This requirement might relate to conformational changes that take place during the protein kinase activity cycle.     

Protein quality control of DYRK family protein kinases by the Hsp90-Cdc37 molecular chaperone

The DYRK (Dual-specificity tYrosine-phosphorylation Regulated protein Kinase) family consists of five related protein kinases (DYRK1A, DYRK1B, DYRK2, DYRK3, DYRK4). DYRKs show homology to Drosophila Minibrain, and DYRK1A in human chromosome 21 is responsible for various neuronal disorders including human Down syndrome. Here we report identification of cellular proteins that associate with specific members of DYRKs. Cellular proteins with molecular masses of 90, 70, and 50-kDa associated with DYRK1B and DYRK4. These proteins were identified as molecular chaperones Hsp90, Hsp70, and Cdc37, respectively. Microscopic analysis of GFP-DYRKs showed that DYRK1A and DYRK1B were nuclear, while DYRK2, DYRK3, and DYRK4 were mostly cytoplasmic in COS7 cells. Overexpression of DYRK1B induced nuclear re-localization of these chaperones with DYRK1B. Treatment of cells with specific Hsp90 inhibitors, geldanamycin and 17-AAG, abolished the association of Hsp90 and Cdc37 with DYRK1B and DYRK4, but not of Hsp70. Inhibition of Hsp90 chaperone activity affected intracellular dynamics of DYRK1B and DYRK4. DYRK1B and DYRK4 underwent rapid formation of cytoplasmic punctate dots after the geldanamycin treatment, suggesting that the chaperone function of Hsp90 is required for prevention of protein aggregation of the target kinases. Prolonged inhibition of Hsp90 by geldanamycin, 17-AAG, or ganetespib, decreased cellular levels of DYRK1B and DYRK4. Finally, DYRK1B and DYRK4 were ubiquitinated in cells, and ubiquitinated DYRK1B and DYRK4 further increased by Hsp90 inhibition with geldanamycin. Taken together, these results indicate that Hsp90 and Cdc37 discriminate specific members of the DYRK kinase family and play an important role in quality control of these client kinases in cells.     

UBR1 promotes protein kinase quality control and sensitizes cells to Hsp90 inhibition

UBR1 and UBR2 are N-recognin ubiquitin ligases that function in the N-end rule degradation pathway. In yeast, the UBR1 homologue also functions by N-end rule independent means to promote degradation of misfolded proteins generated by treatment of cells with geldanamycin, a small molecule inhibitor of Hsp90. Based on these studies we examined the role of mammalian UBR1 and UBR2 in the degradation of protein kinase clients upon Hsp90 inhibition. Our findings show that protein kinase clients Akt and Cdk4 are still degraded in mouse Ubr1(-)/(-) cells treated with geldanamycin, but that their levels recover much more rapidly than is found in wild type cells. These findings correlate with increased induction of Hsp90 expression in the Ubr1(-)/(-) cells compared with wild type cells. We also observed a reduction of UBR1 protein levels in geldanamycin-treated mouse embryonic fibroblasts and human breast cancer cells, suggesting that UBR1 is an Hsp90 client. Further studies revealed a functional overlap between UBR1 and the quality control ubiquitin ligase, CHIP. Our findings show that UBR1 function is conserved in controlling the levels of Hsp90-dependent protein kinases upon geldanamycin treatment, and suggest that it plays a role in determining the sensitivity of cancer cells to the chemotherapeutic effects of Hsp90 inhibitors.     

Hsp70 clears misfolded kinases that partitioned into distinct quality-control compartments

Hsp70 aids in protein folding and directs misfolded proteins to the cellular degradation machinery. We describe discrete roles of Hsp70,SSA1 as an important quality-control machinery that switches functions to ameliorate the cellular environment. SSA1 facilitates folding/maturation of newly synthesized protein kinases by aiding their phosphorylation process and also stimulates ubiquitylation and degradation of kinases in regular protein turnover or during stress when kinases are denatured or improperly folded. Significantly, while kinases accumulate as insoluble inclusions upon SSA1 inhibition, they form soluble inclusions upon Hsp90 inhibition or stress foci during heat stress. This suggests formation of inclusion-specific quality-control compartments under various stress conditions. Up-regulation of SSA1 results in complete removal of these inclusions by the proteasome. Elevation of the cellular SSA1 level accelerates kinase turnover and protects cells from proteotoxic stress. Upon overexpression, SSA1 targets heat-denatured kinases toward degradation, which could enable them to recover their functional state under physiological conditions. Thus active participation of SSA1 in the degradation of misfolded proteins establishes an essential role of Hsp70 in deciding client fate during stress.     

The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.     

Heat shock protein 90: The cancer chaperone

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the stability and function of a number of conditionally activated and/or expressed signalling proteins, as well as multiple mutated, chimeric, and/or over-expressed signalling proteins, that promote cancer cell growth and/or survival. Hsp90 inhibitors are unique in that, although they are directed towards a specific molecular target, they simultaneously inhibit multiple cellular signalling pathways. By inhibiting nodal points in multiple overlapping survival pathways utilized by cancer cells, combination of an Hsp90 inhibitor with standard chemotherapeutic agents may dramatically increase the in vivo efficacy of the standard agent. Hsp90 inhibitors may circumvent the characteristic genetic plasticity that has allowed cancer cells to eventually evade the toxic effects of most molecularly targeted agents. The mechanism-based use of Hsp90 inhibitors, both alone and in combination with other drugs, should be effective toward multiple forms of cancer. Further, because Hsp90 inhibitors also induce Hsf-1-dependent expression of Hsp70, and because certain mutated Hsp90 client proteins are neurotoxic, these drugs display ameliorative properties in several neurodegenerative disease models, suggesting a novel role for Hsp90 inhibitors in treating multiple pathologies involving neurodegeneration.     

Hsp90 recognizes a common surface on client kinases

Hsp90 is a highly abundant chaperone whose clientele includes hundreds of cellular proteins, many of which are central players in key signal transduction pathways and the majority of which are protein kinases. In light of the variety of Hsp90 clientele, the mechanism of selectivity of the chaperone toward its client proteins is a major open question. Focusing on human kinases, we have demonstrated that the chaperone recognizes a common surface in the amino-terminal lobe of kinases from diverse families, including two newly identified clients, NFkappaB-inducing kinase and death-associated protein kinase, and the oncoprotein HER2/ErbB-2. Surface electrostatics determine the interaction with the Hsp90 chaperone complex such that introduction of a negative charge within this region disrupts recognition. Compiling information on the Hsp90 dependence of 105 protein kinases, including 16 kinases whose relationship to Hsp90 is first examined in this study, reveals that surface features, rather than a contiguous amino acid sequence, define the capacity of the Hsp90 chaperone machine to recognize client kinases. Analyzing Hsp90 regulation of two major signaling cascades, the mitogen-activated protein kinase and phosphatidylinositol 3-kinase, leads us to propose that the selectivity of the chaperone to specific kinases is functional, namely that Hsp90 controls kinases that function as hubs integrating multiple inputs. These lessons bear significance to pharmacological attempts to target the chaperone in human pathologies, such as cancer.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Quantitative proteomics reveals that Hsp90 inhibition preferentially targets kinases and the DNA damage response

Despite the increasing importance of heat shock protein 90 (Hsp90) inhibitors as chemotherapeutic agents in diseases such as cancer, their global effects on the proteome remain largely unknown. Here we use high resolution, quantitative mass spectrometry to map protein expression changes associated with the application of the Hsp90 inhibitor, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG). In depth data obtained from five replicate SILAC experiments enabled accurate quantification of about 6,000 proteins in HeLa cells. As expected, we observed activation of a heat shock response with induced expression of molecular chaperones, which refold misfolded proteins, and proteases, which degrade irreparably damaged polypeptides. Despite the broad range of known Hsp90 substrates, bioinformatics analysis revealed that particular protein classes were preferentially affected. These prominently included proteins involved in the DNA damage response, as well as protein kinases and especially tyrosine kinases. We followed up on this observation with a quantitative phosphoproteomic analysis of about 4,000 sites, which revealed that Hsp90 inhibition leads to much more down- than up-regulation of the phosphoproteome (34% down versus 6% up). This study defines the cellular response to Hsp90 inhibition at the proteome level and sheds light on the mechanisms by which it can be used to target cancer cells.     

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.

The heat shock protein Hsp90 has been shown to associate with various cellular signalling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. We have identified mutations in the hsp83 gene, the Drosophila homologue of hsp90, in a search for dominant mutations that attenuate signalling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. We show that both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Our results indicate that Hsp83 is essential for Raf function in vivo.     

Hsp90 phosphorylation is linked to its chaperoning function. Assembly of the reovirus cell attachment protein.

Studies on Hsp90 have mainly focused on its involvement in the activation of several families of protein kinases and of steroid hormone receptors. Little is known regarding the role of Hsp90 in the folding of nascent proteins. We previously reported that Hsp90 plays an active role in the posttranslational assembly of the C-terminal globular head of the reovirus attachment protein final sigma1. We show here that Hsp90 becomes phosphorylated in this process. However, only the unphosphorylated form of Hsp90 is complexed with final sigma1, suggesting that Hsp90 phosphorylation is coupled to the release of the chaperone from the target protein. Geldanamycin, which blocks final sigma1 maturation by preventing the release of Hsp90 from final sigma1, also inhibits Hsp90 phosphorylation. Taken together, these results demonstrate that Hsp90 phosphorylation is linked to its chaperoning function.     

Blocking the chaperone kinome pathway: mechanistic insights into a novel dual inhibition approach for supra-additive suppression of malignant tumors

The chaperone Hsp90 is involved in regulating the stability and activation state of more than 200 ‘client’ proteins and takes part in the cancer diseased states. The major clientele-protein kinases depend on Hsp90 for their proper folding and functioning. Cdc37, a kinase targeting co-chaperone of Hsp90, mediates the interactions between Hsp90 and protein kinases. Targeting of Cdc37 has the prospect of delivering predominantly kinase-selective molecular responses as compared to the current pharmacologic Hsp90 inhibitors. The present work reports a bio-computational study carried out with the aim of exploring the dual inhibition of Hsp90/Cdc37 chaperone/co-chaperone association complex by the naturally occurring drug candidates withaferin A and 17-DMAG along with their possible modes of action. Our molecular docking studies reveal that withaferin A in combination with 17-DMAG can act as potent chaperone system inhibitors. The structural and thermodynamic stability of the ligands’ bound complex was also observed from molecular dynamics simulations in water. Our results suggest a novel tumor suppressive action mechanism of herbal ligands which can be looked forward for further clinical investigations for possible anticancer drug formulations.     

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.     

Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1.

Serine/threonine kinase Akt is thought to mediate many biological actions toward anti-apoptotic responses. Screening of drugs that could interfere with the Akt signaling pathway revealed that Hsp90 inhibitors (e.g. geldanamycin, radicicol, and its analogues) induced Akt dephosphorylation, which resulted in Akt inactivation and apoptosis of the cells. Hsp90 inhibitors did not directly affect Akt kinase activity in vitro. Thus, we examined the effects of Hsp90 inhibitors on upstream Akt kinases, phosphatidylinositide-3-OH kinase (PI3K) and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Hsp90 inhibitors had no effect on PI3K protein expression. In contrast, treatment of the cells with Hsp90 inhibitors decreased the amount of PDK1 without directly inhibiting PDK1 kinase activity. We found that the kinase domain of PDK1 was essential for complex formation with Hsp90 and that Hsp90 inhibitors suppressed PDK1 binding to Hsp90. PDK1 degradation mechanisms revealed that inhibition of PDK1 binding to Hsp90 caused proteasome-dependent degradation of PDK1. Treatment of proteasome inhibitors increased the amount of detergent-insoluble PDK1 in Hsp90 inhibitor-treated cells. Therefore, the association of PDK1 with Hsp90 regulates its stability, solubility, and signaling. Because Akt binding to Hsp90 is also involved in the maintenance of Akt kinase activity, Hsp90 plays an important role in PDK1-Akt survival signaling pathway.     

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.