CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies.

The cochaperone BCL2-associated athanogene 3 (BAG3), in complex with the heat shock protein HSPB8, facilitates mitotic rounding, spindle orientation, and proper abscission of daughter cells. BAG3 and HSPB8 mitotic functions implicate the sequestosome p62/SQSTM1, suggesting a role for protein quality control. However, the interplay between this chaperone-assisted pathway and the mitotic machinery is not known. Here, we show that BAG3 phosphorylation at the conserved T285 is regulated by CDK1 and activates its function in mitotic cell shape remodeling. BAG3 phosphorylation exhibited a high dynamic at mitotic entry and both a non-phosphorylatable BAG3T285A and a phosphomimetic BAG3T285D protein were unable to correct the mitotic defects in BAG3-depleted HeLa cells. We also demonstrate that BAG3 phosphorylation, HSPB8, and CDK1 activity modulate the molecular assembly of p62/SQSTM1 into mitotic bodies containing K63 polyubiquitinated chains. These findings suggest the existence of a mitotically regulated spatial quality control mechanism for the fidelity of cell shape remodeling in highly dividing cells.

Chaperone-Assisted Mitotic Actin Remodeling by BAG3 and HSPB8 Involves the Deacetylase HDAC6 and Its Substrate Cortactin.

The fidelity of actin dynamics relies on protein quality control, but the underlying molecular mechanisms are poorly defined. During mitosis, the cochaperone BCL2-associated athanogene 3 (BAG3) modulates cell rounding, cortex stability, spindle orientation, and chromosome segregation. Mitotic BAG3 shows enhanced interactions with its preferred chaperone partner HSPB8, the autophagic adaptor p62/SQSTM1, and HDAC6, a deacetylase with cytoskeletal substrates. Here, we show that depletion of BAG3, HSPB8, or p62/SQSTM1 can recapitulate the same inhibition of mitotic cell rounding. Moreover, depletion of either of these proteins also interfered with the dynamic of the subcortical actin cloud that contributes to spindle positioning. These phenotypes were corrected by drugs that limit the Arp2/3 complex or HDAC6 activity, arguing for a role for BAG3 in tuning branched actin network assembly. Mechanistically, we found that cortactin acetylation/deacetylation is mitotically regulated and is correlated with a reduced association of cortactin with HDAC6 in situ. Remarkably, BAG3 depletion hindered the mitotic decrease in cortactin-HDAC6 association. Furthermore, expression of an acetyl-mimic cortactin mutant in BAG3-depleted cells normalized mitotic cell rounding and the subcortical actin cloud organization. Together, these results reinforce a BAG3's function for accurate mitotic actin remodeling, via tuning cortactin and HDAC6 spatial dynamics.

Adenoviral protein E4orf4 interacts with the polarity protein Par3 to induce nuclear rupture and tumor cell death.

The tumor cell-selective killing activity of the adenovirus type 2 early region 4 ORF4 (E4orf4) protein is poorly defined at the molecular level. Here, we show that the tumoricidal effect of E4orf4 is typified by changes in nuclear dynamics that depend on its interaction with the polarity protein Par3 and actomyosin contractility. Mechanistically, E4orf4 induced a high incidence of nuclear bleb formation and repetitive nuclear ruptures, which promoted nuclear efflux of E4orf4 and loss of nuclear integrity. This process was regulated by nucleocytoskeletal connections, Par3 clustering proximal to nuclear lamina folds, and retrograde movement of actin bundles that correlated with nuclear ruptures. Significantly, Par3 also regulated the incidence of spontaneous nuclear ruptures facilitated by the downmodulation of lamins. This work uncovered a novel role for Par3 in controlling the actin-dependent forces acting on the nuclear envelope to remodel nuclear shape, which might be a defining feature of tumor cells that is harnessed by E4orf4.

Validation of two Amanita species from eastern North America: A.rhacopus sp. nov. and A.variicolor sp. nov.

Members of the mushroom genus Amanita usually can easily be identified to the genus in the field, however, species circumscription and identification are often problematic. Several names have been misapplied and cryptic species exist. Here, we formally describe and validate two new species of Amanitasect.Vaginatae from eastern North America that were recognised under the umbrella European names A.ceciliae by past authors: Amanitarhacopus sp. nov. and Amanitavariicolor sp. nov.

HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

BCL2-associated athanogene (BAG)-3 is viewed as a platform that would physically and functionally link distinct classes of molecular chaperones of the heat shock protein (HSP) family for the stabilization and clearance of damaged proteins. In this study, we show that HSPB8, a member of the small heat shock protein subfamily, cooperates with BAG3 to coordinate the sequestration of harmful proteins and the cellular adaptive response upon proteasome inhibition. Silencing of HSPB8, like depletion of BAG3, inhibited targeting of ubiquitinated proteins to the juxtanuclear aggresome, a mammalian system of spatial quality control. However, aggresome targeting was restored in BAG3-depleted cells by a mutant BAG3 defective in HSPB8 binding, uncoupling HSPB8 function from its binding to BAG3. Depletion of HSPB8 impaired formation of ubiquitinated microaggregates in an early phase and interfered with accurate modifications of the stress sensor p62/sequestosome (SQSTM)-1. This impairment correlated with decreased coupling of BAG3 to p62/SQSTM1 in response to stress, hindering Kelch-like ECH-associated protein (KEAP)-1 sequestration and stabilization of nuclear factor E2-related factor (Nrf)-2, an important arm of the antioxidant defense. Notably, the myopathy-associated mutation of BAG3 (P209L), which lies within the HSPB8-binding motif, deregulated the association between BAG3 and p62/SQSTM1 and the KEAP1-Nrf2 signaling axis. Together, our findings support a so-far-unrecognized role for the HSPB8-BAG3 connection in mounting of an efficient stress response, which may be involved in BAG3-related human diseases.-Guilbert, S. M., Lambert, H., Rodrigue, M.-A., Fuchs, M., Landry, J., Lavoie, J. N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

Fine-tuning of actin dynamics by the HSPB8-BAG3 chaperone complex facilitates cytokinesis and contributes to its impact on cell division.

The small heat shock protein HSPB8 and its co-chaperone BAG3 are proposed to regulate cytoskeletal proteostasis in response to mechanical signaling in muscle cells. Here, we show that in dividing cells, the HSPB8-BAG3 complex is instrumental to the accurate disassembly of the actin-based contractile ring during cytokinesis, a process required to allow abscission of daughter cells. Silencing of HSPB8 markedly decreased the mitotic levels of BAG3 in HeLa cells, supporting its crucial role in BAG3 mitotic functions. Cells depleted of HSPB8 were delayed in cytokinesis, remained connected via a disorganized intercellular bridge, and exhibited increased incidence of nuclear abnormalities that result from failed cytokinesis (i.e., bi- and multi-nucleation). Such phenotypes were associated with abnormal accumulation of F-actin at the intercellular bridge of daughter cells at telophase. Remarkably, the actin sequestering drug latrunculin A, like the inhibitor of branched actin polymerization CK666, normalized F-actin during cytokinesis and restored proper cell division in HSPB8-depleted cells, implicating deregulated actin dynamics as a cause of abscission failure. Moreover, this HSPB8-dependent phenotype could be corrected by rapamycin, an autophagy-promoting drug, whereas it was mimicked by drugs impairing lysosomal function. Together, the results further support a role for the HSPB8-BAG3 chaperone complex in quality control of actin-based structure dynamics that are put under high tension, notably during cell cytokinesis. They expand a so-far under-appreciated connection between selective autophagy and cellular morphodynamics that guide cell division.

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments.

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3'UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into >75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

A Role for the Chaperone Complex BAG3-HSPB8 in Actin Dynamics, Spindle Orientation and Proper Chromosome Segregation during Mitosis.

The co-chaperone BAG3, in complex with the heat shock protein HSPB8, plays a role in protein quality control during mechanical strain. It is part of a multichaperone complex that senses damaged cytoskeletal proteins and orchestrates their seclusion and/or degradation by selective autophagy. Here we describe a novel role for the BAG3-HSPB8 complex in mitosis, a process involving profound changes in cell tension homeostasis. BAG3 is hyperphosphorylated at mitotic entry and localizes to centrosomal regions. BAG3 regulates, in an HSPB8-dependent manner, the timely congression of chromosomes to the metaphase plate by influencing the three-dimensional positioning of the mitotic spindle. Depletion of BAG3 caused defects in cell rounding at metaphase and dramatic blebbing of the cortex associated with abnormal spindle rotations. Similar defects were observed upon silencing of the autophagic receptor p62/SQSTM1 that contributes to BAG3-mediated selective autophagy pathway. Mitotic cells depleted of BAG3, HSPB8 or p62/SQSTM1 exhibited disorganized actin-rich retraction fibres, which are proposed to guide spindle orientation. Proper spindle positioning was rescued in BAG3-depleted cells upon addition of the lectin concanavalin A, which restores cortex rigidity. Together, our findings suggest the existence of a so-far unrecognized quality control mechanism involving BAG3, HSPB8 and p62/SQSTM1 for accurate remodelling of actin-based mitotic structures that guide spindle orientation.

Protein quantification by chemiluminescent Western blotting: elimination of the antibody factor by dilution series and calibration curve.

Quantification of chemiluminescent signals from a Western blot is routinely used to determine the increase or the decrease in protein expression or modification in cell or tissue extracts. However, although scientists readily agree that such a procedure is not quantitative, it is nonetheless used quantitatively in most publications without appropriate controls that would increase the accuracy of the measurement. Here we reexamined this aspect and found that the primary antibody itself influences the relation between the Western blot signal and the protein amount on the membrane. This relation is non-linear and varies from one antibody to another. In that context, we strongly encourage researchers to use dilution series and calibration curve when quantifying protein by Western blot using chemiluminescent signal.

Identification of the key structural motifs involved in HspB8/HspB6-Bag3 interaction.

The molecular chaperone HspB8 [Hsp (heat-shock protein) B8] is member of the B-group of Hsps. These proteins bind to unfolded or misfolded proteins and protect them from aggregation. HspB8 has been reported to form a stable molecular complex with the chaperone cohort protein Bag3 (Bcl-2-associated athanogene 3). In the present study we identify the binding regions in HspB8 and Bag3 crucial for their interaction. We present evidence that HspB8 binds to Bag3 through the hydrophobic groove formed by its strands beta4 and beta8, a region previously known to be responsible for the formation and stability of higher-order oligomers of many sHsps (small Hsps). Moreover, we demonstrate that two conserved IPV (Ile-Pro-Val) motifs in Bag3 mediate its binding to HspB8 and that deletion of these motifs suppresses HspB8 chaperone activity towards mutant Htt43Q (huntingtin exon 1 fragment with 43 CAG repeats). In addition, we show that Bag3 can bind to the molecular chaperone HspB6. The interaction between HspB6 and Bag3 requires the same regions that are involved in the HspB8-Bag3 association and HspB6-Bag3 promotes clearance of aggregated Htt43Q. Our findings suggest that the co-chaperone Bag3 might prevent the accumulation of denatured proteins by regulating sHsp activity and by targeting their substrate proteins for degradation. Interestingly, a mutation in one of Bag3 IPV motifs has recently been associated with the development of severe dominant childhood muscular dystrophy, suggesting a possible important physiological role for HspB-Bag3 complexes in this disease.

HspB8 participates in protein quality control by a non-chaperone-like mechanism that requires eIF2{alpha} phosphorylation.

Aggregation of mutated proteins is a hallmark of many neurodegenerative disorders, including Huntington disease. We previously reported that overexpression of the HspB8.Bag3 chaperone complex suppresses mutated huntingtin aggregation via autophagy. Classically, HspB proteins are thought to act as ATP-independent molecular chaperones that can bind unfolded proteins and facilitate their processing via the help of ATP-dependent chaperones such as the Hsp70 machine, in which Bag3 may act as a molecular link between HspB, Hsp70, and the ubiquitin ligases. However, here we show that HspB8 and Bag3 act in a non-canonical manner unrelated to the classical chaperone model. Rather, HspB8 and Bag3 induce the phosphorylation of the alpha-subunit of the translation initiator factor eIF2, which in turn causes a translational shut-down and stimulates autophagy. This function of HspB8.Bag3 does not require Hsp70 and also targets fully folded substrates. HspB8.Bag3 activity was independent of the endoplasmic reticulum (ER) stress kinase PERK, demonstrating that its action is unrelated to ER stress and suggesting that it activates stress-mediated translational arrest and autophagy through a novel pathway.

HspB8 chaperone activity toward poly(Q)-containing proteins depends on its association with Bag3, a stimulator of macroautophagy.

Mutations in HspB8, a member of the B group of heat shock proteins (Hsp), have been associated with human neuromuscular disorders. However, the exact function of HspB8 is not yet clear. We previously demonstrated that overexpression of HspB8 in cultured cells prevents the accumulation of aggregation-prone proteins such as the polyglutamine protein Htt43Q. Here we report that HspB8 forms a stable complex with Bag3 in cells and that the formation of this complex is essential for the activity of HspB8. Bag3 overexpression resulted in the accelerated degradation of Htt43Q, whereas Bag3 knockdown prevented HspB8-induced Htt43Q degradation. Additionally, depleting Bag3 caused a reduction in the endogenous levels of LC3-II, a key molecule involved in macroautophagy, whereas overexpressing Bag3 or HspB8 stimulated the formation LC3-II. These results suggested that the HspB8-Bag3 complex might stimulate the degradation of Htt43Q by macroautophagy. This was confirmed by the observation that treatments with macroautophagy inhibitors significantly decreased HspB8- and Bag3-induced degradation of Htt43Q. We conclude that the HspB8 activity is intrinsically dependent on Bag3, a protein that may facilitate the disposal of doomed proteins by stimulating macroautophagy.

Structural instability caused by a mutation at a conserved arginine in the alpha-crystallin domain of Chinese hamster heat shock protein 27.

Mutations in the alpha-crystallin domain of 4 of the small heat shock proteins (sHsp) (Hsp27/HspB1, alphaA-crystallin/ HspB4, alphaB-crystallin/HspB5, and HspB8) are responsible for dominant inherited diseases in humans. One such mutation at a highly conserved arginine residue was shown to cause major conformational defects and intracellular aggregation of alphaA- and alphaB-crystallins and HspB8. Here, we studied the effect of this Arg mutation on the structure and function of Hsp27. Chinese hamster Hsp27 with Arg148 replaced by Gly (Hsp27R148G) formed dimers in vitro and in vivo, which contrasted with the 12- or 24-subunit oligomers formed by the wild-type protein (Hsp27WT). Despite these alterations, Hsp27R148G had a chaperone activity almost as high as Hsp27WT. The dimers of Hsp27R148G did not further deoligomerize on phosphorylation and like the dimers formed by phosphorylated Hsp27WT were not affected by the deletion of the N-terminal WD/EPF (single letter amino acid code) motif, suggesting that mutation of Arg148, deletion of the N-terminal WD/EPF motif, and phosphorylation of Ser90 may produce similar structural perturbations. Nevertheless, the structure of Hsp27R148G appeared unstable, and the mutated protein accumulated as aggregates in many cells. Both a lower basal level of phosphorylation of Hsp27R148G and the coexpression of Hsp27WT could reduce the frequency of formation of these aggregates, suggesting possible mechanisms regulating the onset of the sHsp-mediated inherited diseases.

HspB8, a small heat shock protein mutated in human neuromuscular disorders, has in vivo chaperone activity in cultured cells.

The family of small heat shock proteins (sHsp) is composed of 10 members in mammals, four of which are found mutated in diseases associated with the accumulation of protein aggregates. Though many sHsp have demonstrated molecular chaperone activity in vitro in cell-free conditions, their activity in vivo in the normal cellular context remains unclear. In the present study, we investigated the capacity of the sHsp, HspB8/Hsp22, to prevent protein aggregation in the cells using the polyglutamine protein Htt43Q as a model. In control conditions, Htt43Q accumulated in perinuclear inclusions composed of SDS-insoluble aggregates. Co-transfected with Htt43Q, HspB8 became occasionally trapped within the inclusions; however, in most cells, HspB8 blocked inclusion formation. Biochemical analyses indicated that HspB8 inhibited the accumulation of SDS-insoluble Htt43Q as efficiently as Hsp40 which was taken as a positive control. Htt43Q then accumulated in the SDS-soluble fraction, provided that protein degradation was blocked by proteasome and autophagy inhibitors. In contrast, the other sHsp Hsp27/HspB1 and alphaB-crystallin/HspB5 had no effect. This suggested that HspB8 functions as a molecular chaperone, maintaining Htt43Q in a soluble state competent for rapid degradation. Analyses of Hsp27-HspB8 chimeric proteins indicated that the C-terminal domain of HspB8 contains the specific sequence necessary for chaperone activity. Missense mutations in this domain at lysine 141, which are found in human motor neuropathies, significantly reduced the chaperone activity of the protein. A decrease in the HspB8 chaperone activity may therefore contribute to the development of these diseases.

Essential role of the NH2-terminal WD/EPF motif in the phosphorylation-activated protective function of mammalian Hsp27.

Hsp27 is expressed at high levels after mild heat shock and contributes to making cells extremely resistant to subsequent treatments. The activity of the protein is regulated at the transcriptional level, but also by phosphorylation, which occurs rapidly during stress and is responsible for causing the dissociation of large 700-kDa Hsp27 oligomers into dimers. We investigated the mechanism by which phosphorylation and oligomerization modulate the protective activity of Chinese hamster Hsp27. In contrast to oligomer dissociation, which only required Ser90 phosphorylation, activation of Hsp27 thermoprotective activity required the phosphorylation of both Ser90 and Ser15. Replacement of Ser90 by Ala90, which prevented the dissociation of the oligomer upon stress, did cause a severe defect in the protective activity. Dissociation was, however, not a sufficient condition to activate the protein because replacement of Ser15 by Ala15, which caused little effect in the oligomeric organization of the protein, also yielded an inactive protein. Analyzes of mutants with short deletions in the NH2 terminus identified the Hsp27 WD/EPF or PF-rich domain as essential for protection, maintenance of the oligomeric structure, and in vitro chaperone activity of the protein. In light of a three-dimensional model of Hsp27 based on the crystallographic structure of wheat Hsp16.9, we propose that the conserved WD/EPF motif of mammalian Hsp27 mediates important intramolecular interactions with hydrophic surfaces of the alpha-crystallin domain of the protein. These interactions are destabilized by Ser90 phosphorylation, making the motif free to interact with heterologous molecular targets upon the additional phosphorylation of the nearby Ser15.

Distinct chaperone mechanisms can delay the formation of aggresomes by the myopathy-causing R120G alphaB-crystallin mutant.

A familial form of desmin-related myopathy (DRM) is associated with a missense mutation (R120G) in alphaB-crystallin (alphaB) and is characterized by intracellular desmin aggregation. Because alphaB is a molecular chaperone that participates in the assembly of desmin filaments, it has been suggested that the desmin aggregation might be due to the loss of alphaB function. We report here that alphaBR120G has indeed impaired in vivo function and structure as reflected by a highly reduced capacity to protect cells against heat shock and by an abnormal supramolecular organization even in cells not expressing desmin. In many cells, alphaBR120G accumulated in inclusion bodies that had characteristics of aggresomes concentrating around the centrosome following a microtubule-facilitated process. Three distinct chaperone mechanisms could reduce or even prevent the formation of the alphaBR120G aggresomes. Wild-type alphaB and Hsp27 prevented aggresome formation by co-oligomerizing with alphaBR120G. Hsp70 with its co-chaperone Hdj-1 or Chip-1 but not a mutant of Chip-1 lacking ubiquitin ligase activity, reduced the frequency of aggresome formation likely by targeting alphaBR120G for degradation. Finally, HspB8 interacted only transiently with alphaB but nonetheless rescued the alphaBR120G oligomeric organization, suggesting that it acted as a true chaperone assisting in the folding of the mutant protein. Hence, the formation of inclusion bodies in alphaBR120G-mediated DRM is probably due to the misfolding of alphaBR120G per se and can be delayed or prevented by expression of the wild type alphaB allele or other molecular chaperones, thereby explaining the adult onset of the disease.

The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27.

HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the alpha-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an alpha-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.

c-Myc potentiates the mitochondrial pathway of apoptosis by acting upstream of apoptosis signal-regulating kinase 1 (Ask1) in the p38 signalling cascade.

Cell transformation by growth-promoting oncoproteins renders cells extremely sensitive to apoptosis through an unknown mechanism affecting the mitochondrial pathway of apoptosis. We have shown previously that sensitization to apoptosis also correlated with the activation of the stress-activated protein kinase p38. In the present study, we investigated the role of p38 in c-Myc-dependent apoptosis induced by the anticancer agent cisplatin. Cisplatin treatment of Rat1 cells with deregulated expression of c-Myc resulted in nuclear fragmentation that was accompanied in all cells by the activation of Bax and the translocation of cytochrome c from the mitochondria to the cytoplasm. None of these features of apoptosis was induced in control Rat-1 cells. p38 was also activated by cisplatin only in cells with deregulated expression of c-Myc, but, in contrast with all features of apoptosis, this activation was not affected by Bcl-2. Remarkably, overexpression of an interfering mutant of the p38alpha isoform, but not p38beta, blocked cisplatin-induced Bax activation or cytochrome c release and nuclear fragmentation. Analysis of the kinase cascade upstream of p38 revealed a c-Myc-dependent activation by cisplatin of mitogen-activated protein kinase kinase (MKK) 3/6 and apoptosis signal-regulating kinase 1 (Ask1). Inhibition of Ask1 blocked p38 activation by cisplatin and all features of apoptosis. Several of these data were confirmed using other DNA-damaging agents. The findings indicated that c-Myc potentiation of the mitochondrial pathway of apoptosis results, at least in part, from a sensitization of Ask1 activation, allowing DNA-damaging agents to induce in cascade Ask1, p38alpha and Bax.

Activation of the p38 signaling pathway by heat shock involves the dissociation of glutathione S-transferase Mu from Ask1.

Despite the importance of the stress-activated protein kinase pathways in cell death and survival, it is unclear how stressful stimuli lead to their activation. In the case of heat shock, the existence of a specific mechanism of activation has been evidenced, but the molecular nature of this pathway is undefined. Here, we found that Ask1 (apoptosis signal-regulating kinase 1), an upstream activator of the stress-activated protein kinase p38 during exposure to oxidative stress and other stressful stimuli, was also activated by heat shock. Ask1 activity was required for p38 activation since overexpression of a kinase dead mutant of Ask1, Ask1(K709M), inhibited heat shock-induced p38 activation. The activation of Ask1 by oxidative stress involves the oxidation of thioredoxin, an endogenous inhibitor of Ask1. A different activation mechanism takes place during heat shock. In contrast to p38 induction by H(2)O(2), induction by heat shock was not antagonized by pretreatment with the antioxidant N-acetyl-l-cysteine or by overexpressing thioredoxin and was not accompanied by the dissociation of thioredoxin from Ask1. Instead, heat shock caused the dissociation of glutathione S-transferase Mu1-1 (GSTM1-1) from Ask1 and overexpression of GSTM1-1-inhibited induction of p38 by heat shock. We concluded that because of an alternative regulation by the two distinct repressors thioredoxin and GSTM1-1, Ask1 constitutes the converging point of the heat shock and oxidative stress-sensing pathways that lead to p38 activation.