A Mutation in the Catalytic Loop of Hsp90 Specifically Impairs ATPase Stimulation by Aha1p,But Not Hch1p

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a central role in maintaining cellular homeostasis by facilitating activation of a large number of client proteins. ATP-dependent client activation by Hsp90 is tightly regulated by a host of co-chaperone proteins that control progression through the activation cycle. ATPase stimulation of Hsp90 by Aha1p requires a conserved RKxK motif that interacts with the catalytic loop of Hsp90. In this study, we explore the role of this RKxK motif in the biological and biochemical properties of Hch1p. We found that this motif is required for Hch1p-mediated ATPase stimulation in vitro, but mutations that block stimulation do not impair the action of Hch1p in vivo. This suggests that the biological function of Hch1p is not directly linked to ATPase stimulation. Moreover, a mutation in the catalytic loop of Hsp90 specifically impairs ATPase stimulation by Aha1p but not by Hch1p. Our work here suggests that both Hch1p and Aha1p regulate Hsp90 function through interaction with the catalytic loop but do so in different ways.     

Disease Variants of FGFR3 Reveal Molecular Basis for the Recognition and Additional Roles for Cdc37 in Hsp90 Chaperone System

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Heat Shock Protein 90 Functions to Stabilize and Activate the Testis-specific Serine/Threonine Kinases,a Family of Kinases Essential for Male Fertility

Spermiogenesis is characterized by a profound morphological differentiation of the haploid spermatid into spermatozoa. The testis-specific serine/threonine kinases (TSSKs) comprise a family of post-meiotic kinases expressed in spermatids, are critical to spermiogenesis, and are required for male fertility in mammals. To explore the role of heat shock protein 90 (HSP90) in regulation of TSSKs, the stability and catalytic activity of epitope-tagged murine TSSKs were assessed in 293T and COS-7 cells. TSSK1, -2, -4, and -6 (small serine/threonine kinase) were all found to associate with HSP90, and pharmacological inhibition of HSP90 function using the highly specific drugs 17-AAG, SNX-5422, or NVP-AUY922 reduced TSSK protein levels in cells. The attenuation of HSP90 function abolished the catalytic activities of TSSK4 and -6 but did not significantly alter the specific activities of TSSK1 and -2. Inhibition of HSP90 resulted in increased TSSK ubiquitination and proteasomal degradation, indicating that HSP90 acts to control ubiquitin-mediated catabolism of the TSSKs. To study HSP90 and TSSKs in germ cells, a mouse primary spermatid culture model was developed and characterized. Using specific antibodies against murine TSSK2 and -6, it was demonstrated that HSP90 inhibition resulted in a marked decrease of the endogenous kinases in spermatids. Together, our findings demonstrate that HSP90 plays a broad and critical role in stabilization and activation of the TSSK family of protein kinases.     

Increased Membrane/Nuclear Translocation and Phosphorylation of p90 KD Ribosomal S6 Kinase in the Brain of Hypoxic Preconditioned Mice

Our previous studies have demonstrated that hypoxic precondition (HPC) increased membrane translocation of protein kinase C isoforms and decreased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the brain of mice. The goal of this study was to determine the involvement of p90 KD ribosomal S6 kinase (RSK) in cerebral HPC of mice. Using Western-blot analysis, we found that the levels of membrane/nuclear translocation, but not protein expression of RSK increased significantly in the frontal cortex and hippocampus of HPC mice. In addition, we found that the phosphorylation levels of RSK at the Ser227 site (a PDK1 phosphorylation site), but not at the Thr359/Ser363 sites (ERK1/2 phosphorylated sites) increased significantly in the brain of HPC mice. Similar results were confirmed by an immunostaining study of total RSK and phospho-Ser227 RSK. To further define the cellular populations to express phospho-Ser227 RSK, we found that the expression of phospho-Ser227 RSK co-localized with neurogranin, a neuron-specific marker, in cortex and hippocampus of HPC mice by using double-labeled immunofluorescent staining method. These results suggest that increased RSK membrane/nuclear translocation and PDK1 mediated neuron-specific phosphorylation of RSK at Ser227 might be involved in the development of cerebral HPC of mice.     

Autophagy is involved in regulating influenza A virus RNA and protein synthesis associated with both modulation of Hsp90 induction and mTOR/p70S6K signaling pathway

Influenza A virus (IAV) infection triggers autophagosome formation, but inhibits the fusion of autophagosomes with lysosomes. However, the role of autophagy in IAV replication is still largely unclarified. In this study, we aim to reveal the role of autophagy in IAV replication and the molecular mechanisms underlying the regulation. By using autophagy-deficient (Atg7^−/−) MEFs, we demonstrated that autophagy deficiency significantly reduced the levels of viral proteins, mRNA and genomic RNAs (vRNAs) without affecting viral entry. We further found that autophagy deficiency lead to a transient increase in phosphorylation of mTOR and its downstream targets including 4E-BP1 and S6 at a very early stage of IAV infection, and markedly suppressed p70S6K phosphorylation at the late stage of IAV infection. Furthermore, autophagy deficiency resulted in impairment of Hsp90 induction in response to IAV infection. These results indicate that IAV regulates autophagy to benefit the accumulation of viral elements (synthesis of viral proteins and genomic RNA) during IAV replication. This regulation is associated with modulation of Hsp90 induction and mTOR/p70S6K signaling pathway. Our results provide important evidence for the role of autophagy in IAV replication and the mechanisms underlying the regulation.     

Drosophila Spag Is the Homolog of RNA Polymerase II-associated Protein 3 (RPAP3) and Recruits the Heat Shock Proteins 70 and 90 (Hsp70 and Hsp90) during the Assembly of Cellular Machineries

The R2TP is a recently identified Hsp90 co-chaperone, composed of four proteins as follows: Pih1D1, RPAP3, and the AAA⁺-ATPases RUVBL1 and RUVBL2. In mammals, the R2TP is involved in the biogenesis of cellular machineries such as RNA polymerases, small nucleolar ribonucleoparticles and phosphatidylinositol 3-kinase-related kinases. Here, we characterize the spaghetti (spag) gene of Drosophila, the homolog of human RPAP3. This gene plays an essential function during Drosophila development. We show that Spag protein binds Drosophila orthologs of R2TP components and Hsp90, like its yeast counterpart. Unexpectedly, Spag also interacts and stimulates the chaperone activity of Hsp70. Using null mutants and flies with inducible RNAi, we show that spaghetti is necessary for the stabilization of snoRNP core proteins and target of rapamycin activity and likely the assembly of RNA polymerase II. This work highlights the strong conservation of both the HSP90/R2TP system and its clients and further shows that Spag, unlike Saccharomyces cerevisiae Tah1, performs essential functions in metazoans. Interaction of Spag with both Hsp70 and Hsp90 suggests a model whereby R2TP would accompany clients from Hsp70 to Hsp90 to facilitate their assembly into macromolecular complexes.     

A screening system for the identification of refolding conditions for a model protein kinase, p38alpha

Protein kinases are key drug targets involved in the regulation of a wide variety of cellular processes. To aid the development of drugs targeting these kinases, it is necessary to express recombinant protein in large amounts. The expression of these kinases in Escherichia coli often leads to the accumulation of the expressed protein as insoluble inclusion bodies. The refolding of these inclusion bodies could provide a route to soluble protein, but there is little reported success in this area. We set out to develop a system for the screening of refolding conditions for a model protein kinase, p38α, and applied this system to denatured p38α derived from natively folded and inclusion body protein. Clear differences were observed in the refolding yields obtained, suggesting differences in the folded state of these preparations. Using the screening system, we have established conditions under which soluble, folded p38α can be produced from inclusion bodies. We have shown that the refolding yields obtained in this screen are suitable for the economic large-scale production of refolded p38α protein kinase.     

A Novel C-Terminal Homologue of Aha1 Co-Chaperone Binds to Heat Shock Protein 90 and Stimulates Its ATPase Activity in Entamoeba histolytica

Cytosolic heat shock protein 90 (Hsp90) has been shown to be essential for many infectious pathogens and is considered a potential target for drug development. In this study, we have carried out biochemical characterization of Hsp90 from a poorly studied protozoan parasite of clinical importance, Entamoeba histolytica. We have shown that Entamoeba Hsp90 can bind to both ATP and its pharmacological inhibitor, 17-AAG (17-allylamino-17-demethoxygeldanamycin), with K_d values of 365.2 and 10.77 μM, respectively, and it has a weak ATPase activity with a catalytic efficiency of 4.12 × 10^− 4 min^− 1 μM^− 1. Using inhibitor 17-AAG, we have shown dependence of Entamoeba on Hsp90 for its growth and survival. Hsp90 function is regulated by various co-chaperones. Previous studies suggest a lack of several important co-chaperones in E. histolytica. In this study, we describe the presence of a novel homologue of co-chaperone Aha1 (activator of Hsp90 ATPase), EhAha1c, lacking a canonical Aha1 N-terminal domain. We also show that EhAha1c is capable of binding and stimulating ATPase activity of EhHsp90. In addition to highlighting the potential of Hsp90 inhibitors as drugs against amoebiasis, our study highlights the importance of E. histolytica in understanding the evolution of Hsp90 and its co-chaperone repertoire.     

A pair of putative protein kinase genes (YPK1 and YPK2) is required for cell growth in Saccharomyces cerevisiae

Summary Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast Saccharomyces cerevisiae. We reported previously the isolation of the yeast PKC1 gene, a homolog of the , , and subspecies of mammalian PKC. Here we report the isolation and genetic characterization of a pair of previously described genes (YPK1 and YPK2) which are predicted to encode protein kinases that share 90% amino acid identity with each other and 44–46% identity with various isozymes of PKC throughout their putative catalytic domains. Deletion of YPK2 resulted in no apparent phenotypic defect, but loss of YPK1 resulted in slow growth. Cells deleted for both YPK1 and YPK2 were defective in vegetative growth, indicating that the protein kinases predicted to be encoded by these genes are functionally overlapping and play an essential role in the proliferation of yeast cells. The YPK1 gene was mapped to the left arm of chromosome XI and YPK2 was mapped to the right arm of chromosome XIII.     

Trafficking of the myrosinase‐associated protein GLL23 requires NUC/MVP1/GOLD36/ERMO3 and the p24 protein CYB

Proteins detrimental to endoplasmic reticulum (ER) morphology need to be efficiently exported. Here, we identify two mechanisms that control trafficking of Arabidopsis thalianaGLL23, a 43 kDa GDSL‐like lipase implicated in glucosinolate metabolism through its association with the β‐glucosidase myrosinase. Using immunofluorescence, we identified two mutants that showed aberrant accumulation of GLL23: large perinuclear ER aggregates in the nuclear cage (nuc) mutant; and small compartments contiguous with the peripheral ER in the cytoplasmic bodies (cyb) mutant. Live imaging of fluorescently tagged GLL23 confirmed its presence in the nuc and cyb compartments, but lack of fluorescent signals in the wild‐type plants suggested that GLL23 is normally post‐translationally modified for ER export. NUC encodes the MVP1/GOLD36/ERMO3 myrosinase‐associated protein, previously shown to have vacuolar distribution. CYB is an ER and Golgi‐localized p24 type I membrane protein component of coat protein complex (COP) vesicles, animal and yeast homologues of which are known to be involved in selective cargo sorting for ER–Golgi export. Without NUC, GLL23 accumulates in the ER this situation suggests that NUC is in fact active in the ER. Without CYB, both GLL23 and NUC were found to accumulate in cyb compartments, consistent with a role for NUC in GLL23 processing and indicated that GLL23 is the likely sorting target of the CYB p24 protein.     

Molecular and Functional Basis for the Scaffolding Role of the p50/Dynamitin Subunit of the Microtubule-associated Dynactin Complex

p50/dynamitin (DM) is a major subunit of the microtubule-associated dynactin complex that is required for stabilization and attachment of its two distinct structural domains, namely the Arp1 rod and the shoulder/sidearm. Here, we define the determinants of p50/DM required for self-oligomerization of the protein and for interactions with other subunits of the dynactin complex. Whereas the N-terminal 1–91-amino acid region of the protein is required and sufficient for binding to the Arp1 rod, additional determinants contained within the first half of the protein are required for optimal recruitment of the p150^Glued subunit of the shoulder/sidearm. Overexpression experiments confirmed that the N-terminal 1–91-amino acid region of p50/DM is critical for dynactin functionality, because this fragment acts as a dominant negative to inhibit both dynein-dependent and -independent functions of the complex. Furthermore, the first two predicted coiled-coil motifs of p50/DM contain determinants that mediate self-association of the protein. Interestingly, p50/DM self-association does not contribute to p50/DM-induced disruption of the dynactin complex, but most likely participates in the stabilization of the complex.     

Characterization of the heat shock protein 90 gene in the plant parasitic nematode Meloidogyne artiellia and its expression as related to different developmental stages and temperature

The full-length cDNA and the corresponding gene of the heat shock protein 90, Mt-Hsp90, were isolated and characterized in the plant parasitic nematode Meloidogyne artiellia. The full-length Mt-Hsp90 cDNA contained a 5′ untranslated region (UTR) of 45 bp with the 22 bp trans-spliced leader SL1, an ORF of 2172 bp encoding a polypeptide of 723 amino acids and a 3′ UTR of 191 bp. The deduced amino acid sequence of Mt-hsp90 showed high similarity with other known Hsp90s. Five conserved amino acid signatures indicated that Mt-hsp90 is a cytosolic member of the Hsp90 family. The gene consists of 10 exons and 9 introns, a more expanded gene structure compared to the corresponding Caenorhabditis elegans gene, daf-21. Mt-hsp90 gene was constitutively expressed at high levels in all developmental stages of M. artiellia. Egg masses and second stage juveniles (J2s) were exposed at 5° and 30 °C for different periods of times in order to explore the impact of adverse temperature on Mt-hsp90 gene expression. Expression levels of Mt-hsp90 were examined by fluorescent real-time PCR. At 30 °C a burst of expression for Mt-hsp90 was observed in J2s after 2 h of heat shock treatment, then expression dropped with longer exposing times, although remaining still relatively high after 24 h. This temperature did not affect Mt-hsp90 gene expression in the egg masses. However, egg masses exposed at 5 °C showed a little but gradual increase in the mRNA level with time. By contrast, no significant changes in the Mt-hsp90 level were observed in J2s exposed to cold. These data show that egg masses and J2s exposed to cold and heat stresses have different expression profiles suggesting that Mt-Hsp90 may provide a link between environmental conditions and the life cycle of the nematode.     

A screen for upstream components of the yeast protein kinase C signal transduction pathway identifies the product of the SLG1 gene

We employed the constitutive BCK1-20 allele of the gene for the MAP kinase kinase kinase (MAPKKK) in the yeast Pkc signal transduction pathway to develop a genetic screen for mutants in genes encoding upstream components. Transposon mutagenesis yielded a mutant that was completely dependent on the active allele in the absence of osmotic stabilization. The transposon had integrated at the yeast SLG1 (HCS77) locus. This gene encodes a putative membrane protein. Haploid slg1 deletion strains are sensitive to caffeine, as expected for mutants in the Pkc pathway, as well as a variety of other drugs. The response to elevated temperatures and the dependence on osmotic stabilization depends on the genetic background. Thus, in the strain used for mutagenesis, disruption of SLG1 causes the cells to become non-viable in the absence of osmotic stabilization at both 30° C and 37° C. In a different genetic background this phenotype was not observed. Sensitivity of the haploid deletion mutants to caffeine can be partially suppressed by overexpression of genes for other components of the Pkc pathway, such as PKC1, SLT2, ROM2, and STE20. In addition, a SLG1-lacZ reporter construct shows higher expression in the presence of caffeine or magnesium chloride in a wild-type diploid background. Received: 2 December 1997 / Accepted: 15 December 1997     

Hsp90 Is Required for Transfer of the Cholera Toxin A1 Subunit from the Endoplasmic Reticulum to the Cytosol

Cholera toxin (CT) is an AB₅ toxin that moves from the cell surface to the endoplasmic reticulum (ER) by retrograde vesicular transport. In the ER, the catalytic A1 subunit dissociates from the rest of the toxin and enters the cytosol by exploiting the quality control system of ER-associated degradation (ERAD). The driving force for CTA1 dislocation into the cytosol is unknown. Here, we demonstrate that the cytosolic chaperone Hsp90 is required for CTA1 passage into the cytosol. Hsp90 bound to CTA1 in an ATP-dependent manner that was blocked by geldanamycin (GA), an established Hsp90 inhibitor. CT activity against cultured cells and ileal loops was also blocked by GA, as was the ER-to-cytosol export of CTA1. Experiments using RNA interference or N-ethylcarboxamidoadenosine, a drug that inhibits ER-localized GRP94 but not cytosolic Hsp90, confirmed that the inhibitory effects of GA resulted specifically from the loss of Hsp90 activity. This work establishes a functional role for Hsp90 in the ERAD-mediated dislocation of CTA1.     

Stanford A型主动脉夹层血管壁及外周血中热休克蛋白90的表达及意义

目的:探索热休克蛋白90(Heat shock protein 90, HSP90)在Stanford A型主动脉夹层(Aortic dissection, AD)血管壁组织中的表达及其与平滑肌细胞(Smooth muscle cells, SMCs)表型标志物的相关性。方法:收集本院急性Stanford A型主动脉夹层患者病变主动脉壁组织和外周血标本(AD组,20例),心脏移植供体等来源的正常主动脉壁组织和外周血标本(正常组,10例);ELISA法检测血浆HSP90含量;免疫组化染色检测HSP90的表达差异及定位;Western blot检测组织标本中蛋白的表达差异;应用Spearman分析HSP90和SMCs表型标志物表达的相关性。结果:AD患者血浆中HSP90的含量显著高于正常组(142.38±40.16ng/mL vs. 54.99±24.46 ng/mL,P<0.01)。相对于正常主动脉壁组织,主动脉夹层血管壁组织中HSP90表达明显升高,且主要分布在血管壁中层细胞的胞浆中,分泌型SMCs标志物OPN的表达明显增多,而收缩型标志物α-SMA表达则显著减少。HSP90和α-SMA的蛋白表达呈负相关(R2=0.677,P<0.01),和OPN呈正相关(R²=0.572,P<0.01)。结论:主动脉夹层患者的血管壁组织及外周血中的HSP90含量明显升高,参与了主动脉夹层的发生发展。     

Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr²⁰⁹, Ser²⁴⁷, Ser²⁷⁰, and Ser³⁰³ as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr²⁰⁹ and Ser²⁴⁷ on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr²⁰⁹ peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.