Co-chaperone Cdc37 Research Articles

Holding kinases at the ready

Structural Biology About 60% of kinases only reach their active state in the presence of the molecular chaperone Hsp90 and its co-chaperone Cdc37. It is unclear how the chaperones facilitate kinase function or why only some kinases are chaperone-dependent. Verba et al. determined a 3.9 A cryo–electron microscopy structure of Hsp90:Cdc37 in complex with the kinase Cdk4. Together, Hsp90 and Cdc37 trap the kinase in an open, partially unfolded state. Taking on this state probably has direct functional benefits. Science , this issue p. [1542][1] [1]: /lookup/doi/10.1126/science.aaf5023

Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase.

The Hsp90 molecular chaperone and its Cdc37 cochaperone help stabilize and activate more than half of the human kinome. However, both the mechanism by which these chaperones assist their "client" kinases and the reason why some kinases are addicted to Hsp90 while closely related family members are independent are unknown. Our structural understanding of these interactions is lacking, as no full-length structures of human Hsp90, Cdc37, or either of these proteins with a kinase have been elucidated. Here we report a 3.9 angstrom cryo-electron microscopy structure of the Hsp90-Cdc37-Cdk4 kinase complex. Surprisingly, the two lobes of Cdk4 are completely separated with the β4-β5 sheet unfolded. Cdc37 mimics part of the kinase N lobe, stabilizing an open kinase conformation by wedging itself between the two lobes. Finally, Hsp90 clamps around the unfolded kinase β5 strand and interacts with exposed N- and C-lobe interfaces, protecting the kinase in a trapped unfolded state. On the basis of this structure and an extensive amount of previously collected data, we propose unifying conceptual and mechanistic models of chaperone-kinase interactions.

The co-chaperone Cdc37 regulates the rabies virus phosphoprotein stability by targeting to Hsp90AA1 machinery.

Cdc37, as a kinase-specific co-chaperone of the chaperone Hsp90AA1 (Hsp90), actively aids with the maturation, stabilization and activation of the cellular or viral kinase/kinase-like targets. Phosphoprotein (P) of rabies virus (RABV) is a multifunctional, non-kinase protein involved in interferon antagonism, viral transcription and replication. Here, we demonstrated that the RABV non-kinase P is chaperoned by Cdc37 and Hsp90 during infection. We found that Cdc37 and Hsp90 affect the RABV life cycle directly. Activity inhibition and knockdown of Cdc37 and Hsp90 increased the instability of the viral P protein. Overexpression of Cdc37 and Hsp90 maintained P’s stability but did not increase the yield of infectious RABV virions. We further demonstrated that the non-enzymatic polymerase cofactor P protein of all the genotypes of lyssaviruses is a target of the Cdc37/Hsp90 complex. Cdc37, phosphorylated or unphosphorylated on Ser13, aids the P protein to load onto the Hsp90 machinery, with or without Cdc37 binding to Hsp90. However, the interaction between Cdc37 and Hsp90 appears to have additional allosteric regulation of the conformational switch of Hsp90. Our study highlighted a novel mechanism in which Cdc37/Hsp90 chaperones a non-kinase target, which has significant implications for designing therapeutic targets against Rabies.

Mutations Y493G and K546D in human HSP90 disrupt binding of celastrol and reduce interaction with Cdc37.

Celastrol, a natural compound derived from the Chinese herb Tripterygium wilfordii Hook F, has been proven to inhibit heat shock protein 90 (HSP90) activity and has attracted much attention because of its promising effects in cancer treatment and in ameliorating degenerative neuron diseases. However, the HSP90 structure involved in celastrol interaction is not known. Here, we report a novel celastrol‐binding pocket in the HSP90 dimer, predicted by molecular docking. Mutation of the two key binding pocket amino acids (Lys546 and Tyr493) disrupted the binding of celastrol to HSP90 dimers, as detected by isothermal titration calorimetry (ITC). Interestingly, such mutations also reduced binding between HSP90 and the cochaperone Cdc37, thus providing a new explanation for reported findings that celastrol shows more obvious effects in disrupting binding between HSP90 and Cdc37 than between HSP90 and other cochaperones. In short, our work discloses a novel binding pocket in HSP90 dimer for celastrol and provides an explanation as to why celastrol has a strong effect on HSP90 and Cdc37 binding.

Molecular Mechanism of Protein Kinase Recognition and Sorting by the Hsp90 Kinome-Specific Cochaperone Cdc37

Despite the essential functions of Hsp90, little is known about the mechanism that controls substrate entry into its chaperone cycle. We show that the role of Cdc37 cochaperone reaches beyond that of an adaptor protein and find that it participates in the selective recruitment of only client kinases. Cdc37 recognizes kinase specificity determinants in both clients and nonclients and acts as a general kinase scanning factor. Kinase sorting within the client-to-nonclient continuum relies on the ability of Cdc37 to challenge the conformational stability of clients by locally unfolding them. This metastable conformational state has high affinity for Cdc37 and forms stable complexes through a multidomain cochaperone interface. The interaction with nonclients is not accompanied by conformational changes of the substrate and results in substrate dissociation. Collectively, Cdc37 performs a quality control of protein kinases, where induced conformational instability acts as a "flag" for Hsp90 dependence and stable cochaperone association.

Natural Product Kongensin A is a Non-Canonical HSP90 Inhibitor that Blocks RIP3-dependent Necroptosis

RIP3-dependent necroptosis has recently garnered significant interest because of the unique signaling mechanisms and pathologic functions involved in this process. Accordingly, a number of chemical screens have identified several effective small-molecule inhibitors that specifically block necroptosis. Here, we report the discovery that kongensin A (KA), a natural product isolated from Croton kongensis, is a potent inhibitor of necroptosis and an inducer of apoptosis. Using a new bioorthogonal ligation method (TQ ligation), we reveal that the direct cellular target of KA is heat shock protein 90 (HSP90). Further studies demonstrate that KA covalently binds to a previously uncharacterized cysteine 420 in the middle domain of HSP90 and dissociates HSP90 from its cochaperone CDC37, which leads to inhibition of RIP3-dependent necroptosis and promotion of apoptosis in multiple cancer cell lines. Collectively, our findings demonstrate that KA is an effective HSP90 inhibitor that has potential anti-necroptosis and anti-inflammation applications.

Withaferin A Regulates LRRK2 Levels by Interfering with the Hsp90- Cdc37 Chaperone Complex.

Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multi-domain protein that has been found to be mutated in patients with familial and sporadic Parkinson's disease, Alzheimer's disease and Crohn's disease. While the functions of LRRK2 are still largely unclear and mutations in LRRK2 are associated with adverse gain-of-function activities such as increased kinase activity, increased levels of LRRK2 alone are associated with toxicity in neurons. Consequently, exploring mechanisms to decrease levels of LRRK2 using pharmacological inhibitors would be highly advantageous. Previous work has shown that the chaperone heat shock protein 90 (Hsp90) and its co-chaperone Cdc37 interact with and stabilize LRRK2. In the current study, we explore the regulation of LRRK2 by withaferin A (WA), a potent inhibitor of the interaction between Hsp90 and Cdc37. We report that treatment of the microglial cell line N9 with WA causes a decrease in cellular levels of LRRK2 in a dose- and time-dependent manner. We also find that treatment with WA disrupts the interaction between Hsp90, its co-chaperone Cdc37 and LRRK2, which leads to the destabilization and decreased levels of LRRK2. Additionally, treatment with celastrol, which is also an inhibitor of the Hsp90-Cdc37 complex, decreased LRRK2 levels. Interestingly, treatment with WA in the presence of celastrol enhanced the clearance of LRRK2. Overall, our data suggest that LRRK2 levels can be regulated by targeting the Hsp90-Cdc37 complex, which may have implications in the search for therapeutic strategies for Alzheimer's disease, Parkinson's disease and other LRRK2 proteinopathies.

Structural identification of putative USPs in Catharanthus roseus

Nucleotide sequences of the C. roseus SRA database were assembled and translated in order to detect putative universal stress proteins (USPs). Based on the known conserved USPA domain, 24 Pfam putative USPA proteins in C. roseus were detected and arranged in six architectures. The USPA-like domain was detected in all architectures, while the protein kinase-like (or PK-like), tyrPK-like and/or U-box domains are shown downstream it. Three other domains were also shown to coexist with the USPA domain in C. roseus putative USPA sequences. These domains are tetratricopeptide repeat (or TPR), apolipophorin III (or apoLp-III) and Hsp90 co-chaperone Cdc37. Subsequent analysis divided USPA-like domains based on the ability to bind ATP. The multiple sequence alignment indicated the occurrence of eight C. roseus residues of known features of the bacterial 1MJH secondary structure. The data of the phylogenetic tree indicated several distinct groups of USPA-like domains confirming the presence of high level of sequence conservation between the plant and bacterial USPA-like sequences.

Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.

The protein kinase family includes attractive targets for drug development. Methods for screening of kinase inhibitors remain largely limited to in vitro catalytic assays. It has been shown that ATP-competitive inhibitors antagonize interaction between the target kinase and kinase-specific co-chaperone CDC37 in living cells. Here we show a cell-based method to screen kinase inhibitors using fusion protein of CDC37 with a mutated catalytic 19-kDa component of Oplophorus luciferase, nanoKAZ (CDC37-nanoKAZ). A dual-specificity kinase DYRK1A, an importance of which has been highlighted in Alzheimer’s disease, was targeted in this study. We established 293T cells stably expressing CDC37-nanoKAZ, and analyzed interaction between CDC37-nanoKAZ and DYRK1A. We revealed that DYRK1A interacted with CDC37-nanoKAZ. Importantly, point mutations that affect autophosphorylation strengthened the interaction, thus improving signal/noise ratio of the interaction relative to non-specific binding of CDC37-nanoKAZ. This high signal/noise ratio enabled screening of chemical library that resulted in identification of a potent inhibitor of DYRK1A, named CaNDY. CaNDY induced selective degradation of DYRK1A, and inhibited catalytic activity of recombinant DYRK1A with IC50 value of 7.9 nM by competing with ATP. This method based on a mutant target kinase and a bioluminescence-eliciting co-chaperone CDC37 could be applicable to evaluation and development of inhibitors targeting other kinases.

Abstract 1732: Effect of 19-substituted benzoquinone ansamycin Hsp90 inhibitors on Hsp90/Cdc37/co-chaperone complexes and casein kinase 2 (CK2) activity

Abstract Benzoquinone ansamycin (BQA) Hsp90 inhibitors such as 17-DMAG and 17-AAG have off target toxicities including hepatotoxicity. Mechanisms underlying the toxicity of BQAs are a function of their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. Therefore, we designed 19-substituted BQAs (19BQAs) to prevent conjugation with glutathione and protein thiols as an approach to reduce the hepatotoxicity and minimize off target effects of the BQA class of Hsp90 inhibitors. Our data demonstrated that 19BQAs had reduced toxicity to liver cells relative to their parent quinones while maintaining Hsp90 inhibitory activity, validating the overall approach. 19BQAs depend on tumor cell levels of NQO1 (NAD(P)H: quinone oxidoreductase 1) for optimal activity. We examined 19-phenyl-17-DMAG as a model compound in human isogenic breast cancer cell lines MDA468 (NQO1 null) and MDA468/NQ16 (NQO1 overexpressing) and in NQO1expressing BT474 breast cancer cells. 19-phenyl-17-DMAG induced growth inhibition, apoptosis and the molecular signature of Hsp90 inhibition including decreases in Hsp90 client protein levels and compensatory induction of Hsp70. In addition, treatment of MD468/NQ16 and BT474 cell lines with 19-phenyl-17-DMAG or 17-DMAG also caused the dissociation of Hsp90 from the co-chaperone Cdc37. In these studies we also observed that treatment with19-phenyl-17-DMAG or 17-DMAG resulted in inhibition of Cdc37 phosphorylation. A major kinase responsible for phosphorylation of Cdc37 is casein kinase 2 (CK2) and inhibitors of CK2 are currently under development as anticancer drugs. CK2 activity assays, immunoprecipitation and western blot analysis demonstrated that both 19-phenyl-17-DMAG and 17-DMAG inhibited CK2 kinase activity and reduced CK2α protein subunit expression in NQO1-expressing MD468/NQ16 and BT474 breast cancer cells. Decreased phosphorylation of Cdc37 led to dissociation of the Hsp90/Cdc37/client complex and resulted in the degradation of multiple kinase clients including Raf-1, Cdk4, Akt and HER2. These data suggest that inhibition of both Hsp90 and CK2 by Hsp90 inhibitors may play a role in their antitumor effects. (Supported by CA51210). Citation Format: Chuan-Hsin Chang, David Ross, David Siegel, Christopher J Moody, Russell Kitson. Effect of 19-substituted benzoquinone ansamycin Hsp90 inhibitors on Hsp90/Cdc37/co-chaperone complexes and casein kinase 2 (CK2) activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1732. doi:10.1158/1538-7445.AM2015-1732

Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.

A cytosolic heat shock protein 90 and cochaperone CDC37 complex is required for RIP3 activation during necroptosis

Receptor-interacting protein kinase 3, RIP3, and a pseudokinase mixed lineage kinase-domain like protein, MLKL, constitute the core components of the necroptosis pathway, which causes programmed necrotic death in mammalian cells. Latent RIP3 in the cytosol is activated by several upstream signals including the related kinase RIP1, which transduces signals from the tumor necrosis factor (TNF) family of cytokines. We report here that RIP3 activation following the induction of necroptosis requires the activity of an HSP90 and CDC37 cochaperone complex. This complex physically associates with RIP3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli. Moreover, an HSP90 inhibitor that is currently under clinical development as a cancer therapy was able to prevent systemic inflammatory response syndrome in rats treated with TNF-α. HSP90 and CDC37 cochaperone complex-mediated protein folding is thus an important part of the RIP3 activation process during necroptosis.

5-Azacytidine-induced Protein 2 (AZI2) Regulates Bone Mass by Fine-tuning Osteoclast Survival

5-Azacytidine-induced protein 2 (AZI2) is a TNF receptor (TNFR)-associated factor family member-associated NF-κB activator-binding kinase 1-binding protein that regulates the production of IFNs. A previous in vitro study showed that AZI2 is involved in dendritic cell differentiation. However, the roles of AZI2 in immunity and its pleiotropic functions are unknown in vivo. Here we report that AZI2 knock-out mice exhibit normal dendritic cell differentiation in vivo. However, we found that adult AZI2 knock-out mice have severe osteoporosis due to increased osteoclast longevity. We revealed that the higher longevity of AZI2-deficient osteoclasts is due to an augmented activation of proto-oncogene tyrosine-protein kinase Src (c-Src), which is a critical player in osteoclast survival. We found that AZI2 inhibits c-Src activity by regulating the activation of heat shock protein 90 (Hsp90), a chaperone involved in c-Src dephosphorylation. Furthermore, we demonstrated that AZI2 indirectly inhibits c-Src by interacting with the Hsp90 co-chaperone Cdc37. Strikingly, administration of a c-Src inhibitor markedly prevented bone loss in AZI2 knock-out mice. Together, these findings indicate that AZI2 regulates bone mass by fine-tuning osteoclast survival.

Abstract 1791: 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems

Abstract The 19-substituted benzoquinone ansamycin (BQA) class of Hsp90 inhibitors were developed to decrease the off-target toxicity of their parent unsubstituted BQAs, geldanamcyin, 17-AAG, and 17-DMAG. We have shown that 19-BQAs do not react with thiols and show decreased toxicity to liver cell systems relative to parent BQAs. As a class, Hsp90 inhibitors exhibit anti-cancer activity by decreasing the levels of Hsp90 client proteins critical to cell growth and survival. Some Hsp90 inhibitors also disrupt interaction with Hsp90 co-chaperones, including cdc37, contributing to inhibited growth. Upon inhibition, the transcription factor heat shock factor 1 (Hsf1) dissociates from Hsp90 leading to a compensatory induction of other heat shock proteins, including Hsp70 which is commonly used as a molecular biomarker of cellular Hsp90 inhibition. We undertook a detailed study of the mechanism of action of 19-BQAs in HER2-positive breast cancer cells (BT474) using 19-phenyl- and 19-methyl-DMAG as model compounds. Their ability to disrupt association of Hsp90 with the co-chaperones p23 and cdc37, the kinetics of decreased Hsp90-Hsf1 levels, nuclear Hsf1 accumulation and increases in Hsp70 levels were defined. Both 19-phenyl- and 19-methyl -DMAG disrupted Hsp90-p23 association, confirming binding of the compounds at the N-terminal ATPase site of Hsp90. 19-Phenyl-DMAG and 19-methyl-DMAG also disrupted association of Hsp90 with cdc37, although 19-phenyl-DMAG was more potent. A detailed time course of decreased levels of Hsp90 client proteins and Hsp induction showed that increased Hsp70 was a more sensitive cellular marker as it could be detected at lower drug concentrations. Increases in Hsp70 did not appear to be a generalized stress response, since 19-substituted-BQAs caused dissociation of the Hsp90-Hsf1 dimer, nuclear translocation of Hsf-1, and subsequent increases in Hsp70 levels in a temporal manner. These data indicate that 19-substituted BQAs may exhibit their growth inhibitory effects in breast cancer cells through disruption of Hsp90 and its co-chaperones, particularly cdc37. The N-terminal ATPase site binding and Hsp90 inhibitory activity of these compounds was confirmed through disruption of Hsp90-p23 and Hsp90-Hsf1 associations, respectively (Supported by NCI grant CA51210). Citation Format: Derek A. Drechsel, Chuan-Hsin Chang, Russell Kitson, David Siegel, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2014-1791

Suppressing the CDC37 cochaperone in hepatocellular carcinoma cells inhibits cell cycle progression and cell growth.

The molecular cochaperone CDC37 regulates the activities of multiple protein kinases, and is an attractive broad-spectrum target in many types of cancers in which it is over-expressed. This study investigates the antitumour effects of inhibiting CDC37 in human hepatocellular carcinoma (HCC). A total of 91 patients were enrolled for CDC37 mRNA detection by using quantitative real-time PCR. Cell proliferation, gene expression changes and tumourigenicity were determined by targeting CDC37 using RNA interference in human hepatoma cell lines. We confirmed the significant over-expression of CDC37 transcript and protein in HBV-associated HCC patients. Using a CDC37-specific small oligo-siRNA, we silenced CDC37 expression in HepG2 and Huh7 hepatoma cell lines, and observed inhibition of in vitro cell proliferation, cell cycle arrest at the G1 phase, and enhanced apoptosis. Specifically, we found concomitant down-regulation of Cyclin D1, CDK4, and pRB (S807/811 and S795) upon CDC37 suppression, which could mediate the arrest of cell cycle progression at the G1 phase. Gene expression profiling further identified several genes involved in cell proliferation, cell cycle progression, and apoptosis that are regulated by CDC37 suppression. Huh7 cells with stable knockdown of CDC37 showed decreased in vitro colony formation ability, and significantly slowed xenograft growth in vivo. On the basis of the observed antitumour effects of inhibiting CDC37 expression, we propose that CDC37 is a promising therapeutic target in HCC. Its ability to regulate multiple pathways makes it potentially valuable in treating the heterogeneous subtypes of this malignancy.

Novel celastrol derivatives inhibit the growth of hepatocellular carcinoma patient-derived xenografts.

The molecular co-chaperone CDC37 is over-expressed in hepatocellular carcinoma (HCC) cells, where it functions with HSP90 to regulate the activity of protein kinases in multiple oncogenic signaling pathways that contribute towards hepatocarcinogenesis. Disruption of these signaling pathways via inhibition of HSP90/CDC37 interaction is therefore a rational therapeutic approach. We evaluated the anti-tumor effects of celastrol, pristimerin, and two novel derivatives (cel-D2, and cel-D7) on HCC cell lines in vitro and on orthotopic HCC patient-derived xenografts in vivo. All four compounds preferentially inhibited viability of HCC cells in vitro,and significantly inhibited the growth of three orthotopic HCC patient-derived xenografts in vivo; with the novel derivatives cel-D2 and cel-D7 exhibiting lower toxicity. All four compounds also induced cell apoptosis; and promoted degradation and inhibited phosphorylation of protein kinases in the Raf/MEK/ERK and PI3K/AKT/mTOR signaling pathways. We demonstrated that HSP90/CDC37 antagonists are potentially broad spectrum agents that might be beneficial for treating the heterogeneous subtypes of HCC, either as monotherapy, or in combination with other chemotherapeutic agents.

Quantitative proteomics identifies unanticipated regulators of nitrogen- and glucose starvation.

The molecular mechanisms underlying how cells sense, respond, and adapt to alterations in nutrient availability have been studied extensively during the past years. While most of these studies have focused on the linear connections between signaling components, it is increasingly being recognized that signaling pathways are interlinked in molecular circuits and networks such that any metabolic perturbation will induce signaling-wide ripple effects. In the present study, we have used quantitative mass spectrometry (MS) to examine how the yeast Saccharomyces cerevisiae responds to nitrogen- or glucose starvation. We identify nearly 1400 phosphorylation sites of which more than 500 are regulated in a temporal manner in response to glucose- or nitrogen starvation. By bioinformatics and network analyses, we have identified the cyclin-dependent kinase (CDK) inhibitor Sic1, the Hsp90 co-chaperone Cdc37, and the Hsp90 isoform Hsp82 to putatively mediate some of the starvation responses. Consistently, quantitative expression analyses showed that Sic1, Cdc37, and Hsp82 are required for normal expression of nutrient-responsive genes. Collectively, we therefore propose that Sic1, Cdc37, and Hsp82 may orchestrate parts of the cellular starvation response by regulating transcription factor- and kinase activities.

Proteomic Analysis Identifies MaxiK Channel Intracellular Partners from Human Coronary Artery

The large conductance voltage and calcium-activated potassium (MaxiK) channel, a key modulator of vascular tone, interacts with several types of proteins (e.g. regulatory subunits, G-protein coupled receptors, signaling kinases). In this study, we searched for MaxiK channel partners in human coronary artery using a proteomic approach. A MaxiK polyclonal antibody and 3 coronary arteries from explanted hearts were used to immunoprecipitate MaxiK. IgG was used as negative control. Immunoprecipitated proteins were separated by 1-D SDS-PAGE; subsequently, gel bands were excised for trypsin digestion and LC/MS/MS analysis. As positive control, we searched for MaxiK peptides and found 7 peptides (93 amino acids) from MaxiK C-terminus. No peptides of MaxiK were found in IgG negative controls (n=3). Various groups of intracellular proteins that form complex with MaxiK channels were identified. A group of proteins are cytoskeleton proteins, which include coronin-1A, PACN2, tubulin beta-3 chain and beta-centractin. PACN2 plays a role in the formation of flask-shaped caveolae at the plasma membrane and coronin-1A is involved in cell locomotion. Another group of proteins are linked to transcription and mRNA-splicing. These proteins are TFIID subunit 9B, DDX1, and pre-mRNA-splicing factor SYF1. Mitochondria metabolic proteins were also identified as MaxiK partners including ADP/ATP translocase and PCCB (Propionyl-CoA carboxylase beta chain). In addition, we found that Hsp90 co-chaperone CDC37 (CDC37), WASL and WASL-interacting protein family member 1 (WIPF1) associate with MaxiK. These three proteins also form complex with Hsp90. WASL and WIPF1 are involved in the formation of actin filament and cell motility. Hsp90 binds to WASL regulating actin polymerization. CDC37 and Hsp90 assemble to stabilize protein kinases. In summary, proteomic analysis identified intracellular proteins forming complex with MaxiK channel and the underlying functions for these interactions need further investigation. Supported by NIH.

Restricting direct interaction of CDC37 with HSP90 does not compromise chaperoning of client proteins

The HSP90 molecular chaperone plays a key role in the maturation, stability and activation of its clients, including many oncogenic proteins. Kinases are a substantial and important subset of clients requiring the key cochaperone CDC37. We sought an improved understanding of protein kinase chaperoning by CDC37 in cancer cells. CDC37 overexpression in human colon cancer cells increased CDK4 protein levels, which was negated upon CDC37 knockdown. Overexpressing CDC37 increased CDK4 protein half-life and enhanced binding of HSP90 to CDK4, consistent with CDC37 promoting kinase loading onto chaperone complexes. Against expectation, expression of C-terminus truncated CDC37 (ΔC-CDC37) that lacks HSP90 binding capacity did not affect kinase client expression or activity; moreover, as with wildtype CDC37 overexpression, it augmented CDK4-HSP90 complex formation. However, although truncation blocked binding to HSP90 in cells, ΔC-CDC37 also showed diminished client protein binding and was relatively unstable. CDC37 mutants with single and double point mutations at residues M164 and L205 showed greatly reduced binding to HSP90, but retained association with client kinases. Surprisingly, these mutants phenocopied wildtype CDC37 overexpression by increasing CDK4-HSP90 association and CDK4 protein levels in cells. Furthermore, expression of the mutants was sufficient to protect kinase clients CDK4, CDK6, CRAF and ERBB2 from depletion induced by silencing endogenous CDC37, indicating that CDC37’s client stabilising function cannot be inactivated by substantially reducing its direct interaction with HSP90. However, CDC37 could not compensate for loss of HSP90 function, showing that CDC37 and HSP90 have their own distinct and non-redundant roles in maintaining kinase clients. Our data substantiate the important function of CDC37 in chaperoning protein kinases. Furthermore, we demonstrate that CDC37 can stabilise kinase clients by a mechanism that is not dependent on a substantial direct interaction between CDC37 and HSP90, but nevertheless requires HSP90 activity. These results have significant implications for therapeutic targeting of CDC37.

Expression and purification of recombinant NRL-Hsp90α and Cdc37-CRL proteins for in vitro Hsp90/Cdc37 inhibitors screening

Hsp90 has emerged as a promising target for cancer treatment. Hsp90 interacts with co-chaperone Cdc37 to mediate the conformational maturation of its kinase client proteins. Screening small molecule inhibitors targeting Hsp90/Cdc37 might be a promising strategy for further cancer therapeutic. In order to establish a recombinant protein system, the novel cloning and purification of full-length human Hsp90α and Cdc37 from BL21 (DE3) Escherichia coli is described here. In this work, we cloned and expressed recombinant NRL-Hsp90α and Cdc37-CRL that represent the full-length human Hsp90α and Cdc37 fused with the split Renilla luciferase (RL) protein fragments.We also expressed the full-length RL protein as a control for inhibitors screening. Moreover, we confirmed that the interaction proteins were able to complement split luciferase fragments and show the RL activity when substrate was added. In comparison, two mutations NRL-Hsp90α (Q133A) and Cdc37 (R167A)-CRL retained only 20% of the complemented RL activities. Six small molecule compounds were tested using this recombinant system. Very interestingly, Sulforaphane, Withaferin A, Celastrol and EGCG all decreased the complemented NRL-Hsp90α/Cdc37-CRL activities in the concentration-dependent manner. In addition, neither Sulforaphane nor Withaferin A showed non-specific inhibition on full length RL activity. However, Celastrol and EGCG showed different RL inhibition levels. The other two compounds LBH-589 and 17-AAG showed neither NRL-Hsp90α/Cdc37-CRL nor RL inhibition activities. These results indicate that purified NRL-Hsp90α and Cdc37-CRL appeared as pure, stable and active conformation, and can be used as an in vitro bioluminescence system for Hsp90/Cdc37 inhibitors screening.