Regulation of RAF family kinases: new insights from recent structural and biochemical studies.

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Cdc37 as a Co-chaperone to Hsp90.

The co-chaperone p50/Cdc37 is an important partner for Hsp90, assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Analysis of the structure of Hsp90-Cdc37-kinase complexes demonstrates the way in which Cdc37 interacts with and controls the folding of a large proportion of intracellular protein kinases. This co-chaperone thus stands at the hub of a multitude of intracellular signaling networks. Indeed, the influence of Cdc37 reaches beyond the housekeeping pathways of protein folding into the regulation of a wide range of cellular processes. This co-chaperone has attracted attention as a potential intermediate in carcinogenesis. Cdc37 is an attractive potential target in cancer due to (1) high expression in a number of tumor types and (2) control of multiple signaling pathways. These properties indicate (3) a potential for selectivity due to its elevated expression in malignant cells and (4) robustness, as the co-chaperone may control multiple growth signaling pathways and thus be less prone to evolution of resistance than less versatile oncoproteins. Cdc37 may also be involved in other aspects of pathophysiology and has been shown to be secreted in exosomes. Protein aggregation disorders have been linked to age-related declines in molecular chaperones and co-chaperones. Cdc37 also appears to be a potential agent in longevity due to its links to protein folding and autophagy, and it will be informative to study the role of Cdc37 maintenance/decline in aging organisms.

p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and µNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

HSP90-CDC37 functions as a chaperone for the oncogenic FGFR3-TACC3 fusion.

The FGFR3-TACC3 (F3-T3) fusion gene was discovered as an oncogenic molecule in glioblastoma and bladder cancers, and has subsequently been found in many cancer types. Notably, F3-T3 was found to be highly expressed in both untreated and matched recurrence glioblastoma under the concurrent radiotherapy and temozolomide (TMZ) treatment, suggesting that targeting F3-T3 is a valid strategy for treatment. Here, we show that the F3-T3 protein is a client of heat shock protein 90 (HSP90), forming a ternary complex with the cell division cycle 37 (CDC37). Deprivation of HSP90 or CDC37 disrupts the formation of the ternary complex, which destabilizes glycosylated F3-T3, and thereby suppresses F3-T3 oncogenic activity. Gliomas harboring F3-T3 are resistant to TMZ chemotherapy. HSP90 inhibitors sensitized F3-T3 glioma cells to TMZ via the inhibition of F3-T3 activation and potentiated TMZ-induced DNA damage. These results demonstrate that F3-T3 oncogenic function is dependent on the HSP90 chaperone system and suggests a new clinical option for targeting this genetic aberration in cancer.

TTT (Tel2-Tti1-Tti2) Complex, the Co-Chaperone of PIKKs and a Potential Target for Cancer Chemotherapy.

The heterotrimeric Tel2-Tti1-Tti2 or TTT complex is essential for cell viability and highly observed in eukaryotes. As the co-chaperone of ATR, ATM, DNA-PKcs, mTOR, SMG1, and TRRAP, the phosphatidylinositol 3-kinase-related kinases (PIKKs) and a group of large proteins of 300-500 kDa, the TTT plays crucial roles in genome stability, cell proliferation, telomere maintenance, and aging. Most of the protein kinases in the kinome are targeted by co-chaperone Cdc37 for proper folding and stability. Like Cdc37, accumulating evidence has established the mechanism by which the TTT interacts with chaperone Hsp90 via R2TP (Rvb1-Rvb2-Tah1-Pih1) complex or other proteins for co-translational maturation of the PIKKs. Recent structural studies have revealed the α-solenoid structure of the TTT and its interactions with the R2TP complex, which shed new light on the co-chaperone mechanism and provide new research opportunities. A series of mutations of the TTT have been identified that cause disease syndrome with neurodevelopmental defects, and misregulation of the TTT has been shown to contribute to myeloma, colorectal, and non-small-cell lung cancers. Surprisingly, Tel2 in the TTT complex has recently been found to be a target of ivermectin, an antiparasitic drug that has been used by millions of patients. This discovery provides mechanistic insight into the anti-cancer effect of ivermectin and thus promotes the repurposing of this Nobel-prize-winning medicine for cancer chemotherapy. Here, we briefly review the discovery of the TTT complex, discuss the recent studies, and describe the perspectives for future investigation.

Targeting the HSP90-CDC37-kinase chaperone cycle: A promising therapeutic strategy for cancer.

Heat shock protein 90 (HSP90) is an indispensable molecular chaperone that facilitates the maturation of numerous oncoproteins in cancer cells, including protein kinases, ribonucleoproteins, steroid hormone receptors, and transcription factors. Although over 30 HSP90 inhibitors have steadily entered clinical trials, further clinical advancement has been restricted by their limited efficacy, inevitable heat shock response, and multiple side-effects, likely induced via an ATP inhibition mechanism. Since both ATP and various co-chaperones play essential roles in the HSP90 chaperone cycle to achieve integrated function, optimal therapeutics require an understanding of the dynamic interactions among HSP90, ATP, and cochaperones. To date, continuous research has promoted the exploration of the cochaperone cell division cycle 37 (CDC37) as a kinase-specific recognizer and has shown that the HSP90-CDC37-kinase complex is particularly relevant in cancers. Indeed, disrupting the HSP90-CDC37-kinase complex, rather than totally blocking the ATP function of HSP90, is emerging as an alternative way to avoid the limitations of current inhibitors. In this review, we first briefly introduce the HSP90-CDC37-kinase cycle and present the currently available approaches for inhibitor development targeting this cycle and provide insights into selective regulation of the kinase clients of HSP90 by more directional ways.

Lipid droplets and autophagosomes together with chaperones fine-tune expression of SGK1.

Serum-glucocorticoid-induced kinase-1 (SGK1) regulates ion homeostasis and promotes survival under stress conditions. The expression of SGK1 is under transcriptional and post-translational regulations that are frequently altered in cancer and immune disorders. We report that an N-terminal amphipathic alpha-helix determines SGK1 expression levels through two distinct mechanisms. It tethers SGK1 to intracellular organelles generating a large pool of membrane-bound SGK1, which is differentially stabilized in lipid droplets (LD) in fed conditions or degraded in the endoplasmic reticulum by ER-phagy in starvation. Association of the α-helix to organelles does not depend on dedicated receptors or special phospholipids rather, it is intrinsic to its physicochemical properties and depends on the presence of bulky hydrophobic residues for attachment to LDs. The second mechanism is recruitment of protein-chaperones that recognize the α-helix as an unfolded protein promoting survival of the cytosolic SGK1 fraction. Together, the findings unveil an unexpected link between levels of energy storage and abundance of SGK1 and how changes in calorie intake could be used to modulate SGK1 expression, whereas the inhibition of molecular chaperones could serve as an additional enhancer in the treatment of malignancies and autoimmune disorders with high levels of SGK1 expression.

Okicamelliaside targets the N-terminal chaperone pocket of HSP90 disrupts the chaperone protein interaction of HSP90-CDC37 and exerts antitumor activity.

Heat shock protein 90 (HSP90) has been recognized as a crucial target in cancer cells. However, various toxic reactions targeting the ATP binding site of HSP90 may not be the best choice for HSP90 inhibitors. In this paper, an ellagic acid derivative, namely, okicamelliaside (OCS), with antitumor effects was found. To identify potential anti-cancer mechanisms, an OCS photosensitive probe was applied to target fishing and tracing. Chemical proteomics and protein-drug interaction experiments have shown that HSP90 is a key target for OCS, with a strong binding affinity (KD = 6.45 µM). Mutation analysis of the target protein and molecular dynamics simulation revealed that OCS could competitively act on the key Glu-47 site at the N-terminal chaperone pocket of HSP90, where the co-chaperone CDC37 binds to HSP90, affect its stability and reduce the ∆Gbind of HSP90-CDC37. It was demonstrated that OCS destroys the protein-protein interactions of HSP90-CDC37; selectively affects downstream kinase client proteins of HSP90, including CDK4, P-AKT473, and P-ERK1/2; and exerts antitumor effects on A549 cells. Furthermore, tumor xenograft experiments demonstrated high antitumor activity and low toxicity of OCS in the same way. Our findings identified a novel N-terminal chaperone pocket natural inhibitor of HSP90, that is, OCS, which selectively inhibits the formation of the HSP90-CDC37 protein complex, and provided further insight into HSP90 inhibitors for anti-cancer candidate drugs.

Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition.

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC-PCP-SILAC, we analyzed protein-protein interactions of hyperactive BRAFV600E and wild-type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600E resides in large complexes of higher molecular mass and activity, while BRAFWT is confined to smaller, slightly less active complexes. However, expression of oncogenic K-RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWT into large, active complexes, whereas pharmacological inhibition of BRAFV600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors.

AUY922 induces retinal toxicity through attenuating TRPM1.

BACKGROUND: Ocular adverse events are common dose-limiting toxicities in cancer patients treated with HSP90 inhibitors, such as AUY922; however, the pathology and molecular mechanisms that mediate AUY922-induced retinal toxicity remain undescribed. METHODS: The impact of AUY922 on mouse retinas and cell lines was comprehensively investigated using isobaric tags for relative and absolute quantitation (iTRAQ)­based proteomic profiling and pathway enrichment analysis, immunohistochemistry and immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, MTT assay, colony formation assay, and western blot analysis. The effect of AUY922 on the Transient Receptor Potential cation channel subfamily M member 1 (TRPM1)-HSP90 chaperone complex was characterized by coimmunoprecipitation. TRPM1-regulated gene expression was analyzed by RNAseq analysis and gene set enrichment analysis (GSEA). The role of TRPM1 was assessed using both loss-of-function and gain-of-function approaches. RESULTS: Here, we show that the treatment with AUY922 induced retinal damage and cell apoptosis, dysregulated the photoreceptor and retinal pigment epithelium (RPE) layers, and reduced TRPM1 expression. Proteomic profiling and functional annotation of differentially expressed proteins reveals that those related to stress responses, protein folding processes, regulation of apoptosis, cell cycle and growth, reactive oxygen species (ROS) response, cell junction assembly and adhesion regulation, and proton transmembrane transport were significantly enriched in AUY922-treated cells. We found that AUY922 triggered caspase-3-dependent cell apoptosis, increased ROS production and inhibited cell growth. We determined that TRPM1 is a bona fide HSP90 client and characterized that AUY922 may reduce TRPM1 expression by disrupting the CDC37-HSP90 chaperone complex. Additionally, GSEA revealed that TRPM1-regulated genes were associated with retinal morphogenesis in camera-type eyes and the JAK-STAT cascade. Finally, gain-of-function and loss-of-function analyses validated the finding that TRPM1 mediated the cell apoptosis, ROS production and growth inhibition induced by AUY922. CONCLUSIONS: Our study demonstrates the pathology of AUY922-induced retinal toxicity in vivo. TRPM1 is an HSP90 client, regulates photoreceptor morphology and function, and mediates AUY922-induced cytotoxicity.

Discovery of novel celastrol-triazole derivatives with Hsp90-Cdc37 disruption to induce tumor cell apoptosis.

To enhance the disruption of Hsp90-Cdc37, we designed and synthesized a series (27) of CEL-triazole derivatives. Most of the target compounds showed enhanced anti-proliferative activity on four cancer cell lines (MDA-MB-231, MCF-7, HepG2 and A459). Among them, compound 6 showed the best anti-proliferation (IC50 = 0.34 ± 0.01 µM) on MDA-MB-231. Pharmacological studies had found that compound 6 showed a higher ability to disrupt Hsp90-Cdc37 interaction in cells and inhibited the expression of the key Hsp90-Cdc37 clients in a concentration-dependent manner. Further studies indicated that an enhanced covalent binding between compound 6 and thiols (cysteine) might be one of the reasons for the increased activity. Furthermore, compound 6 arrested cells in the G0/G1 phase and induced tumor cell apoptosis significantly. Overall, for cancer treatment, compound 6 was worth further exploring.

(20S) Ginsenoside Rh2 Exerts Its Anti-Tumor Effect by Disrupting the HSP90A-Cdc37 System in Human Liver Cancer Cells.

(20S) ginsenoside Rh2 (G-Rh2), a major bioactive metabolite of ginseng, effectively inhibits the survival and proliferation of human liver cancer cells. However, its molecular targets and working mechanism remain largely unknown. Excitingly, we screened out heat shock protein 90 alpha (HSP90A), a key regulatory protein associated with liver cancer, as a potential target of (20S) G-Rh2 by phage display analysis and mass spectrometry. The molecular docking and thermal shift analyses demonstrated that (20S) G-Rh2 directly bound to HSP90A, and this binding was confirmed to inhibit the interaction between HSP90A and its co-chaperone, cell division cycle control protein 37 (Cdc37). It is well-known that the HSP90A-Cdc37 system aids in the folding and maturation of cyclin-dependent kinases (CDKs). As expected, CDK4 and CDK6, the two G0-G1 phase promoting kinases as well as CDK2, a key G1-S phase transition promoting kinase, were significantly downregulated with (20S) G-Rh2 treatment, and these downregulations were mediated by the proteasome pathway. In the same condition, the cell cycle was arrested at the G0-G1 phase and cell growth was inhibited significantly by (20S) G-Rh2 treatment. Taken together, this study for the first time reveals that (20S) G-Rh2 exerts its anti-tumor effect by targeting HSP90A and consequently disturbing the HSP90A-Cdc37 chaperone system. HSP90A is frequently overexpressed in human hepatoma cells and the higher expression is closely correlated to the poor prognosis of liver cancer patients. Thus, (20S) G-Rh2 might become a promising alternative drug for liver cancer therapy.

The Activity and Stability of p56Lck and TCR Signaling Do Not Depend on the Co-Chaperone Cdc37.

Lymphocyte-specific protein tyrosine kinase (Lck) is a pivotal tyrosine kinase involved in T cell receptor (TCR) signaling. Because of its importance, the activity of Lck is regulated at different levels including phosphorylation of tyrosine residues, protein-protein interactions, and localization. It has been proposed that the co-chaperone Cdc37, which assists the chaperone heat shock protein 90 (Hsp90) in the folding of client proteins, is also involved in the regulation of the activity/stability of Lck. Nevertheless, the available experimental data do not clearly support this conclusion. Thus, we assessed whether or not Cdc37 regulates Lck. We performed experiments in which the expression of Cdc37 was either augmented or suppressed in Jurkat T cells. The results of our experiments indicated that neither the overexpression nor the suppression of Cdc37 affected Lck stability and activity. Moreover, TCR signaling proceeded normally in T cells in which Cdc37 expression was either augmented or suppressed. Finally, we demonstrated that also under stress conditions Cdc37 was dispensable for the regulation of Lck activity/stability. In conclusion, our data do not support the idea that Lck is a Cdc37 client.

SUMOylation Is Required for ERK5 Nuclear Translocation and ERK5-Mediated Cancer Cell Proliferation.

The MAP kinase ERK5 contains an N-terminal kinase domain and a unique C-terminal tail including a nuclear localization signal and a transcriptional activation domain. ERK5 is activated in response to growth factors and stresses and regulates transcription at the nucleus by either phosphorylation or interaction with transcription factors. MEK5-ERK5 pathway plays an important role regulating cancer cell proliferation and survival. Therefore, it is important to define the precise molecular mechanisms implicated in ERK5 nucleo-cytoplasmic shuttling. We previously described that the molecular chaperone Hsp90 stabilizes and anchors ERK5 at the cytosol and that ERK5 nuclear shuttling requires Hsp90 dissociation. Here, we show that MEK5 or overexpression of Cdc37-mechanisms that increase nuclear ERK5-induced ERK5 Small Ubiquitin-related Modifier (SUMO)-2 modification at residues Lys6/Lys22 in cancer cells. Furthermore, mutation of these SUMO sites abolished the ability of ERK5 to translocate to the nucleus and to promote prostatic cancer PC-3 cell proliferation. We also show that overexpression of the SUMO protease SENP2 completely abolished endogenous ERK5 nuclear localization in response to epidermal growth factor (EGF) stimulation. These results allow us to propose a more precise mechanism: in response to MEK5 activation, ERK5 SUMOylation favors the dissociation of Hsp90 from the complex, allowing ERK5 nuclear shuttling and activation of the transcription.

Design of Disruptors of the Hsp90-Cdc37 Interface.

The molecular chaperone Hsp90 is a ubiquitous ATPase-directed protein responsible for the activation and structural stabilization of a large clientele of proteins. As such, Hsp90 has emerged as a suitable candidate for the treatment of a diverse set of diseases, such as cancer and neurodegeneration. The inhibition of the chaperone through ATP-competitive inhibitors, however, was shown to lead to undesirable side effects. One strategy to alleviate this problem is the development of molecules that are able to disrupt specific protein-protein interactions, thus modulating the activity of Hsp90 only in the particular cellular pathway that needs to be targeted. Here, we exploit novel computational and theoretical approaches to design a set of peptides that are able to bind Hsp90 and compete for its interaction with the co-chaperone Cdc37, which is found to be responsible for the promotion of cancer cell proliferation. In spite of their capability to disrupt the Hsp90-Cdc37 interaction, no important cytotoxicity was observed in human cancer cells exposed to designed compounds. These findings imply the need for further optimization of the compounds, which may lead to new ways of interfering with the Hsp90 mechanisms that are important for tumour growth.

Relationship between the Expression Level of PSMD11 and Other Proteasome Proteins with the Activity of Ricin and Viscumin.

The role of proteasome proteins and proteins of the ERAD system in the cytotoxicity of type II ribosome-inactivating proteins ricin and viscumin was investigated. For this, the cell line of colorectal adenocarcinoma HT29, as well as the HT29-sh002 line obtained on its basis, were used. On the basis on the proteome analysis of these lines and the estimation of the proportion of inactivated ribosomes, it was shown that the contribution of the proteasome to the degradation of the catalytic subunits of toxins is different. The role of the Cdc37 co-chaperone in maintaining the stability of A subunit of viscumin in the cytoplasm is shown.

Cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the CDK9 activation loop promotes P-TEFb assembly with Tat and proviral HIV reactivation.

The HIV trans-activator Tat recruits the host transcription elongation factor P-TEFb to stimulate proviral transcription. Phosphorylation of Thr-186 on the activation loop (T-loop) of cyclin-dependent kinase 9 (CDK9) is essential for its kinase activity and assembly of CDK9 and cyclin T1 (CycT1) to form functional P-TEFb. Phosphorylation of a second highly conserved T-loop site, Ser-175, alters the competitive binding of Tat and the host recruitment factor bromodomain containing 4 (BRD4) to P-TEFb. Here, we investigated the intracellular mechanisms that regulate these key phosphorylation events required for HIV transcription. Molecular dynamics simulations revealed that the CDK9/CycT1 interface is stabilized by intramolecular hydrogen bonding of pThr-186 by an arginine triad and Glu-96 of CycT1. Arginine triad substitutions that disrupted CDK9/CycT1 assembly accumulated Thr-186-dephosphorylated CDK9 associated with the cytoplasmic Hsp90/Cdc37 chaperone. The Hsp90/Cdc37/CDK9 complex was also present in resting T cells, which lack CycT1. Hsp90 inhibition in primary T cells blocked P-TEFb assembly, disrupted Thr-186 phosphorylation, and suppressed proviral reactivation. The selective CDK7 inhibitor THZ1 blocked CDK9 phosphorylation at Ser-175, and in vitro kinase assays confirmed that CDK7 activity is principally responsible for Ser-175 phosphorylation. Mutation of Ser-175 to Lys had no effect on CDK9 kinase activity or P-TEFb assembly but strongly suppressed both HIV expression and BRD4 binding. We conclude that the transfer of CDK9 from the Hsp90/Cdc37 complex induced by Thr-186 phosphorylation is a key step in P-TEFb biogenesis. Furthermore, we demonstrate that CDK7-mediated Ser-175 phosphorylation is a downstream nuclear event essential for facilitating CDK9 T-loop interactions with Tat.

Structural and functional basis of protein phosphatase 5 substrate specificity.

The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP-substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors.

Serine/Threonine Kinase Unc-51-like Kinase-1 (Ulk1) Phosphorylates the Co-chaperone Cell Division Cycle Protein 37 (Cdc37) and Thereby Disrupts the Stability of Cdc37 Client Proteins.

The serine/threonine kinase Unc-51-like kinase-1 (Ulk1) is thought to be essential for induction of autophagy, an intracellular bulk degradation process that is activated by various stresses. Although several proteins have been suggested as Ulk1 substrates during autophagic process, it still remains largely unknown about Ulk1's physiological substrates. Here, by performing in vitro and in vivo phosphorylation assay, we report that the co-chaperone cell division cycle protein 37 (Cdc37) is a Ulk1 substrate. Ulk1-mediated phosphorylation of Ser-339 in Cdc37 compromised the recruitment of client kinases to a complex comprising Cdc37 and heat shock protein 90 (Hsp90) but only modestly affected Cdc37 binding to Hsp90. Because the recruitment of protein kinase clients to the Hsp90 complex is essential for their stability and functions, Ser-339 phosphorylation of Cdc37 disrupts its ability as a co-chaperone to coordinate Hsp90. Hsp90 inhibitors are cancer chemotherapeutic agents by inducing depletion of clients, many of which are oncogenes. Upon treatment with an Hsp90 inhibitor in cancer cells, Ulk1 promoted the degradation of Hsp90-Cdc37 client kinases, resulting in increased cellular sensitivity to Hsp90 inhibitors. Thus, our study provides evidence for an anti-proliferative role of Ulk1 in response to Hsp90 inhibition in cancer cells.

Simvastatin functions as a heat shock protein 90 inhibitor against triple-negative breast cancer.

Acetylation plays an important role in regulating the chaperone activity of heat shock protein 90 (Hsp90) during malignant transformation through the stabilization and conformational maturation of oncogenic proteins. However, the functional acetylation sites, potential anticancer drug targets, are still emerging. We found that acetylation at K292 in Hsp90α is critical for the development and treatment of breast cancer. Acetylation at K292 not only augments the affinity of Hsp90 to ATP, cochaperones, and client proteins but it also promotes cancer cell colony formation, migration, and invasion in vitro as well as tumor growth in vivo. Importantly, K292-acetylated Hsp90 has been validated as an exciting anticancer drug target by interfering with the complex formation between K292-acetylated Hsp90 and cochaperone Cdc37, leading to diminishment of kinase client maturation and proteasome-dependent degradation of kinase substrates. Furthermore, we showed that simvastatin prevented, whereas LBH589 promoted, the progression of Hsp90 chaperone cycling and client maturation, resulting in an increment of cell apoptosis by the combination of simvastatin and LBH589 in a mouse xenograft model. These data suggest that simvastatin is a novel Hsp90 inhibitor to disrupt the formation of the K292-acetylated Hsp90/Cdc37 complex in triple-negative breast cancer cells. The combination of simvastatin with LBH589 could be used as a novel therapeutic strategy for triple-negative breast cancer.