Intracellular aggregation of polypeptides with expanded polyglutamine domain is stimulated by stress-activated kinase MEKK1.

Abnormal proteins, which escape chaperone-mediated refolding or proteasome-dependent degradation, aggregate and form inclusion bodies (IBs). In several neurodegenerative diseases, such IBs can be formed by proteins with expanded polyglutamine (polyQ) domains (e.g., huntingtin). This work studies the regulation of intracellular IB formation using an NH(2)-terminal fragment of huntingtin with expanded polyQ domain. We demonstrate that the active form of MEKK1, a protein kinase that regulates several stress-activated signaling cascades, stimulates formation of the IBs. This function of MEKK1 requires kinase activity, as the kinase-dead mutant of MEKK1 cannot stimulate this process. Exposure of cells to UV irradiation or cisplatin, both of which activate MEKK1, also augmented the formation of IBs. The polyQ-containing huntingtin fragment exists in cells in two distinct forms: (a) in a discrete soluble complex, and (b) in association with insoluble fraction. MEKK1 strongly stimulated recruitment of polyQ polypeptides into the particulate fraction. Notably, a large portion of the active form of MEKK1 was associated with the insoluble fraction, concentrating in discrete sites, and polyQ-containing IBs always colocalized with them. We suggest that MEKK1 is involved in a process of IB nucleation. MEKK1 also stimulated formation of IBs with two abnormal polypeptides lacking the polyQ domain, indicating that this kinase has a general effect on protein aggregation.

The molecular chaperone Ydj1 is required for the p34CDC28-dependent phosphorylation of the cyclin Cln3 that signals its degradation.

The G1 cyclin Cln3 of the yeast Saccharomyces cerevisiae is rapidly degraded by the ubiquitin-proteasome pathway. This process is triggered by p34CDC28-dependent phosphorylation of Cln3. Here we demonstrate that the molecular chaperone Ydj1, a DnaJ homolog, is required for this phosphorylation. In a ydj1 mutant at the nonpermissive temperature, both phosphorylation and degradation of Cln3 were deficient. No change was seen upon inactivation of Sis1, another DnaJ homolog. The phosphorylation defect in the ydj1 mutant was specific to Cln3, because no reduction in the phosphorylation of Cln2 or histone H1, which also requires p34CDC28, was observed. Ydj1 was required for Cln3 phosphorylation and degradation rather than for the proper folding of this cyclin, since Cln3 produced in the ydj1 mutant was fully active in the stimulation of p34CDC28 histone kinase activity. Moreover, Ydj1 directly associates with Cln3 in close proximity to the segment that is phosphorylated and signals degradation. Thus, binding of Ydj1 to this domain of Cln3 seems to be essential for the phosphorylation and breakdown of this cyclin. In a cell-free system, purified Ydj1 stimulated the p34CDC28-dependent phosphorylation of the C-terminal segment of Cln3 and did not affect phosphorylation of Cln2 (as was found in vivo). The reconstitution of this process with pure components provides evidence of a direct role for the chaperone in the phosphorylation of Cln3.

Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death.

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.

Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72.

Various stresses activate the c-Jun N-terminal kinase (JNK), which is involved in the regulation of many aspects of cellular physiology, including apoptosis. Here we demonstrate that in contrast to UV irradiation, heat shock causes little or no stimulation of the JNK-activating kinase SEK1, while knocking out the SEK1 gene completely blocks heat-induced JNK activation. Therefore, we tested whether heat shock activates JNK via inhibition of JNK dephosphorylation. The rate of JNK dephosphorylation in unstimulated cells was high, and exposure to UV irradiation, osmotic shock, interleukin-1, or anisomycin did not affect this process. Conversely, exposure of cells to heat shock and other protein-damaging conditions, including ethanol, arsenite, and oxidative stress, strongly reduced the rate of JNK dephosphorylation. Under these conditions, we did not observe any effects on dephosphorylation of the homologous p38 kinase, suggesting that suppression of dephosphorylation is specific to JNK. Together, these data indicate that activation of JNK by protein-damaging treatments is mediated primarily by inhibition of a JNK phosphatase(s). Elevation of cellular levels of the major heat shock protein Hsp72 inhibited a repression of JNK dephosphorylation by these stressful treatments, which explains recent reports of the suppression of JNK activation by Hsp72.

Role of Hsp70 in regulation of stress-kinase JNK: implications in apoptosis and aging.

Cell protection from stresses by the major heat shock protein Hsp72 was previously attributed to its ability to prevent aggregation and to accelerate refolding of damaged proteins. This repair function of Hsp72 may play an important role in cell survival after extremely harsh protein damaging treatments leading to necrotic cell death. On the other hand, protein repair function of Hsp72 cannot explain how it protects cells from stresses which do not cause direct protein damage, e.g. some genotoxic agents. These stresses kill cells through activation of apoptosis, and Hsp72 increases cell survival by interfering with the apoptotic program. Recently it has been found that Hsp72 mediates suppression of a stress-activated protein kinase, JNK, an early component of stress-induced apoptotic signalling pathway. This finding provides the basis for the anti-apoptotic activity of Hsp72. These observations can explain increased stress sensitivity of aged cells in which compromised inducibility of Hsp72 leads to a loss of control of JNK activation by stresses and subsequently to a higher rate of apoptotic death.

Heat shock protein Hsp72 plays an essential role in Her2-induced mammary tumorigenesis.

The major heat shock protein Hsp72 is expressed at elevated levels in many human cancers and its expression correlates with tumor progression. Here, we investigated the role of Hsp72 in Her2 oncogene-induced neoplastic transformation and tumorigenesis. Expression of Her2 in untransformed MCF10A mammary epithelial cells caused transformation, as judged by foci formation in culture and tumorigenesis in xenografts. However, expression of Her2 in Hsp72-depleted cells failed to induce transformation. The anti-tumorigenic effects of Hsp72 downregulation were associated with cellular senescence because of accumulation of p21 and depletion of survivin. Accordingly, either knockdown of p21 or expression of survivin reversed this senescence process. Further, we developed an animal model of Hsp72-dependent breast cancer associated with expression of Her2. Knockout (KO) of Hsp72 almost completely suppressed tumorigenesis in the MMTVneu breast cancer mouse model. In young Hsp72 KO mice, expression of Her2 instead of mammary tissue hyperplasia led to suppression of duct development and blocked alveolar budding. These effects were due to massive cell senescence in mammary tissue, which was associated with upregulation of p21 and downregulation of survivin. Therefore, Hsp72 has an essential role in Her2-induced tumorigenesis by regulating oncogene-induced senescence pathways.

HSP72 depletion suppresses gammaH2AX activation by genotoxic stresses via p53/p21 signaling.

Knockout of heat shock protein Hsp72 was shown to promote chromosomal instability and increase radiation sensitivity of mouse fibroblasts. Here, we report that downregulation of Hsp72 in human tumor cells leads to suppression of a specific branch of the DNA damage response (DDR) that facilitates DNA repair following genotoxic insults, that is, reduced accumulation of the phosphorylated form of histone H2AX (gammaH2AX). This inhibition was due to decreased expression of H2AX as well as higher rate of gammaH2AX dephosphorylation. Formation of gammaH2AX and MDC1 radiation-induced foci was impaired in Hsp72-depleted cells, which in turn enhanced DNA damage, resulting in sensitization of cells to gamma-radiation and doxorubicin. These effects of Hsp72 knockdown were dependent on activation of the p53/p21-signaling pathway. Overall, permanent activation of the p53/p21 signaling in Hsp72-depleted cells specifically impaired the gammaH2AX pathway of the DDR, enhanced DNA damage following genotoxic insults, and led to further stimulation of the p53/p21 pathway, thus creating a positive feedback loop. The resulting strong induction of p21 precipitated senescence following exposure to DNA-damaging agents, thus accounting for higher sensitivity of cells to genotoxic stresses.

The function of HSP72 in suppression of c-Jun N-terminal kinase activation can be dissociated from its role in prevention of protein damage.

Activation of the c-Jun N-terminal kinase (JNK) by a variety of stimuli is critical for regulation of many cellular processes including apoptosis. The major inducible heat shock protein Hsp72 has previously been demonstrated to inhibit activation of JNK in cells exposed to heat shock and other protein-damaging agents, thus suppressing apoptosis. Hsp72 can protect proteins from stress-induced damage. To test if this protective function of Hsp72 is involved in JNK suppression, we investigated whether Hsp72 can avert activation of JNK by stimuli that do not cause protein damage. We show that Hsp72 suppresses activation of JNK induced by non-protein-damaging stimuli, interleukin-1 and UV irradiation, as well as by constitutively active components of the JNK signaling cascade Cdc42 and MEKK1. Furthermore, Hsp72 strongly reduced activation of JNK by phosphatase inhibitors. We also demonstrate that an Hsp72 mutant that lacks the ATPase domain is still capable of JNK suppression, thus indicating that the protein refolding activity of Hsp72 is not critical for inhibition of JNK activation. Taken together these data suggest that Hsp72 plays a regulatory role in JNK signaling and that the function of Hsp72 in protein protection or refolding is not involved in JNK regulation.

Suppression of stress kinase JNK is involved in HSP72-mediated protection of myogenic cells from transient energy deprivation. HSP72 alleviates the stewss-induced inhibition of JNK dephosphorylation.

Since protection of cells from stress-induced apoptosis by the heat shock protein Hsp72 involves suppression of stress kinase JNK, we suggested that Hsp72-mediated JNK inhibition might also be critical for myocardial protection from ischemia/reperfusion. Transient energy deprivation of H9c2 myogenic cells, used as an in vitro model of myocardial ischemia, led to cell death that had morphological features of apoptosis and necrosis and was independent of caspases. Surprisingly, this unusual type of cell death was regulated by JNK and ERK kinases. In fact, specific inhibition of JNK increased cell survival; specific inhibition of ERKs enhanced deleterious consequences of energy deprivation, whereas inhibition of p38 kinase had no effect. Hsp72 suppressed activation of JNK and did not increase ERK activity, suggesting that inhibition of JNK is the important component of Hsp72-mediated protection. Upon transient energy deprivation, activation of JNK proceeds via two distinct pathways, stimulation of JNK phosphorylation by a protein kinase SEK1 and inhibition of JNK dephosphorylation. Remarkably, in cells exposed to transient energy deprivation, Hsp72 enhanced the rate of JNK dephosphorylation but did not affect SEK1 activity. Therefore, it appears that Hsp72 specifically down-regulates JNK by accelerating its dephosphorylation, which reduces the susceptibility of cardiac cells to simulated ischemia/reperfusion.