Stress-activated Mitogen-activated Protein Kinase Pathway Research Articles

Fungal plant pathogen "mutagenomics" reveals tagged and untagged mutations in Zymoseptoria tritici and identifies SSK2 as key morphogenesis and stress-responsive virulence factor.

"Mutagenomics" is the combination of random mutagenesis, phenotypic screening, and whole-genome re-sequencing to uncover all tagged and untagged mutations linked with phenotypic changes in an organism. In this study, we performed a mutagenomics screen on the wheat pathogenic fungus Zymoseptoria tritici for altered morphogenetic switching and stress sensitivity phenotypes using Agrobacterium-mediated "random" T-DNA mutagenesis (ATMT). Biological screening identified four mutants which were strongly reduced in virulence on wheat. Whole genome re-sequencing defined the positions of the T-DNA insertion events and revealed several unlinked mutations potentially affecting gene functions. Remarkably, two independent reduced virulence mutant strains, with similarly altered stress sensitivities and aberrant hyphal growth phenotypes, were found to have a distinct loss of function mutations in the ZtSSK2 MAPKKK gene. One mutant strain had a direct T-DNA insertion affecting the predicted protein's N-terminus, while the other possessed an unlinked frameshift mutation towards the C-terminus. We used genetic complementation to restore both strains' wild-type (WT) function (virulence, morphogenesis, and stress response). We demonstrated that ZtSSK2 has a non-redundant function with ZtSTE11 in virulence through the biochemical activation of the stress-activated HOG1 MAPK pathway. Moreover, we present data suggesting that SSK2 has a unique role in activating this pathway in response to specific stresses. Finally, dual RNAseq-based transcriptome profiling of WT and SSK2 mutant strains revealed many HOG1-dependent transcriptional changes in the fungus during early infection and suggested that the host response does not discriminate between WT and mutant strains during this early phase. Together these data define new genes implicated in the virulence of the pathogen and emphasise the importance of a whole genome sequencing step in mutagenomic discovery pipelines.

Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity.

Mitogen-activated protein kinase (MAPK) pathways represent ubiquitous signal transduction pathways that regulate all aspects of life and are frequently altered in disease. Here, we focus on the role of MAPK pathways in modulating drug sensitivity and resistance in cancer. We briefly discuss new findings in the extracellular signaling-regulated kinase (ERK) pathway, but mainly focus on the mechanisms how stress activated MAPK pathways, such as p38 MAPK and the Jun N-terminal kinases (JNK), impact the response of cancer cells to chemotherapies and targeted therapies. In this context, we also discuss the role of metabolic and epigenetic aberrations and new therapeutic opportunities arising from these changes.

RNA-Binding Protein Rnc1 Regulates Cell Length at Division and Acute Stress Response in Fission Yeast through Negative Feedback Modulation of the Stress-Activated Mitogen-Activated Protein Kinase Pathway

RNA-binding proteins (RBPs) play a major role during control of mRNA localization, stability, and translation and are central to most cellular processes. In the fission yeast Schizosaccharomyces pombe, the multiple K homology (KH) domain RBP Rnc1 downregulates the activity of the cell integrity pathway (CIP) via stabilization of pmp1 + mRNA, which encodes the Pmp1 phosphatase that inactivates Pmk1, the mitogen-activated protein kinase (MAPK) component of this signaling cascade. However, Rnc1 likely regulates the half-life/stability of additional mRNAs. We show that Rnc1 downregulates the activity of Sty1, the MAPK of the stress-activated MAPK pathway (SAPK), during control of cell length at division and recovery in response to acute stress. Importantly, this control strictly depends on Rnc1's ability to bind mRNAs encoding activators (Wak1 MAPKKK, Wis1 MAPKK) and downregulators (Atf1 transcription factor, Pyp1 and Pyp2 phosphatases) of Sty1 phosphorylation through its KH domains. Moreover, Sty1 is responsible for Rnc1 phosphorylation in vivo at multiple phosphosites during growth and stress, and these modifications trigger Rnc1 for proper binding and destabilization of the above mRNA targets. Phosphorylation by Sty1 prompts Rnc1-dependent mRNA destabilization to negatively control SAPK signaling, thus revealing an additional feedback mechanism that allows precise tuning of MAPK activity during unperturbed cell growth and stress.IMPORTANCE Control of mRNA localization, stability, turnover, and translation by RNA-binding proteins (RBPs) influences essential processes in all eukaryotes, including signaling by mitogen-activated protein kinase (MAPK) pathways. We describe that in the fission yeast Schizosaccharomyces pombe the RBP Rnc1 negatively regulates cell length at division during unperturbed growth and recovery after acute stress by reducing the activity of the MAPK Sty1, which regulates cell growth and differentiation during environmental cues. This mechanism relies on Rnc1 binding to specific mRNAs encoding both enhancers and negative regulators of Sty1 activity. Remarkably, multiple phosphorylation of Rnc1 by Sty1 favors RBP binding and destabilization of the above mRNAs. Thus, posttranscriptional modulation of MAP kinase signaling by RNA-binding proteins emerges as a major regulatory mechanism that dictates the growth cycle and cellular adaptation in response to the changing environment in eukaryotic organisms.

Quorum sensing and stress-activated MAPK signaling repress yeast to hypha transition in the fission yeast Schizosaccharomyces japonicus.

Quorum sensing (QS), a mechanism of microbial communication dependent on cell density, governs developmental decisions in many bacteria and in some pathogenic and non-pathogenic fungi including yeasts. In these simple eukaryotes this response is mediated by the release into the growth medium of quorum-sensing molecules (QSMs) whose concentration increases proportionally to the population density. To date the occurrence of QS is restricted to a few yeast species. We show that a QS mediated by the aromatic alcohols phenylethanol and tryptophol represses the dimorphic yeast to hypha differentiation in the fission yeast S. japonicus in response to an increased population density. In addition, the stress activated MAPK pathway (SAPK), which controls cell cycle progression and adaptation to environmental changes in this organism, constitutively represses yeast to hypha differentiation both at transcriptional and post-translational levels. Moreover, deletion of its main effectors Sty1 MAPK and Atf1 transcription factor partially suppressed the QS-dependent block of hyphal development under inducing conditions. RNAseq analysis showed that the expression of nrg1+, which encodes a putative ortholog of the transcription factor Nrg1 that represses yeast to hypha dimorphism in C. albicans, is downregulated both by QS and the SAPK pathway. Remarkably, Nrg1 may act in S. japonicus as an activator of hyphal differentiation instead of being a repressor. S. japonicus emerges as an attractive and amenable model organism to explore the QS mechanisms that regulate cellular differentiation in fungi.

Modulation of TOR complex 2 signaling by the stress-activated MAPK pathway in fission yeast.

Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Inhibition of GCN2 sensitizes ASNS-low cancer cells to asparaginase by disrupting the amino acid response

General control nonderepressible 2 (GCN2) plays a major role in the cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, the contribution of GCN2 to cancer cell survival and proliferation is poorly understood. In this study, we generated GCN2 inhibitors and demonstrated that inhibition of GCN2 sensitizes cancer cells with low basal-level expression of asparagine synthetase (ASNS) to the antileukemic agent l-asparaginase (ASNase) in vitro and in vivo. We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of ASNS, resulting in reduced levels of de novo protein synthesis. Comprehensive gene-expression profiling revealed that combined treatment with ASNase and GCN2 inhibitors induced the stress-activated MAPK pathway, thereby triggering apoptosis. By using cell-panel analyses, we also showed that acute myelogenous leukemia and pancreatic cancer cells were highly sensitive to the combined treatment. Notably, basal ASNS expression at protein levels was significantly correlated with sensitivity to combined treatment. These results provide mechanistic insights into the role of GCN2 in the amino acid response and a rationale for further investigation of GCN2 inhibitors for the treatment of cancer.

Abstract 3847: Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response

Abstract General control nonderepressible 2 (GCN2) plays a major role in cellular response to amino acid limitation. Although maintenance of amino acid homeostasis is critical for tumor growth, little is known concerning the contribution of GCN2 to cancer cell proliferation and its potential as a therapeutic target. In this study, we demonstrate that inhibition of GCN2 with small-molecule compounds sensitizes cancer cells to anti-leukemic agent asparaginase (ASNase). We first tested acute lymphoblastic leukemia (ALL) cells and showed that treatment with GCN2 inhibitors rendered ALL cells sensitive to ASNase by preventing the induction of asparagine synthetase (ASNS). The prevention of ASNS expression suppresses asparagine synthesis and thus inhibits protein translation. In addition, comprehensive gene expression profiling revealed that the combined treatment induced stress-activated MAPK pathway and thereby triggering apoptosis. Using cell-panel analyses, we also identified a clear synergy between GCN2 inhibitors and ASNase in acute myelogenous leukaemia (AML) and pancreatic cancer cells besides ALL. Notably, basal ASNS expression levels were significantly correlated with sensitivity to combinatorial treatment. Furthermore, the combination displayed robust antitumor activity in mouse xenograft models of ALL, AML, and pancreatic cancer. These results demonstrate a novel mechanistic insight into a role of GCN2 in amino acid response and provide a rationale for further investigating GCN2 inhibitors for the treatment of cancer. Citation Format: Akito Nakamura, Tadahiro Nambu, Shunsuke Ebara, Yuka Hasegawa, Kosei Toyoshima, Yasuko Tsuchiya, Daisuke Tomita, Jun Fujimoto, Osamu Kurasawa, Chisato Takahara, Ayumi Kawamura, Ryuichi Nishigaki, Yoshinori Satomi, Akito Hata, Takahito Hara. Inhibition of GCN2 sensitizes ASNS-downregulated cancer cells to asparaginase by disrupting amino acid response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3847.

RNA sequencing analysis shows that titanium dioxide nanoparticles induce endoplasmic reticulum stress, which has a central role in mediating plasma glucose in mice

Titanium dioxide nanoparticles (TiO2 NPs) constitute the top five NPs in use today. In this study, oral administration of 50, 100, and 200 mg/kg body weight (b.w.) TiO2 NPs increases plasma glucose in mice, whereas 10 and 20 mg/kg b.w. TiO2 NPs did not. RNA sequencing (RNA-seq) technology was used to investigate genome-wide effects of TiO2 NPs. Clustering analysis of the RNA-seq data showed the most significantly enriched gene ontology terms and KEGG pathways related to the endoplasmic reticulum (ER) and ER stress. Molecular biology verification showed that 50 mg/kg b.w. and higher doses TiO2 NPs activated a xenobiotic biodegradation response and increased expression of cytochrome P450 family genes in mouse livers, thus inducing ER stress in mice. ER stress-activated MAPK and NF-κB pathways and induced an inflammation response, resulting in phosphorylation of the insulin receptor substrate 1 and, consequently, insulin resistance. This was the main mechanism by which TiO2 NPs increased plasma glucose in mice. Meanwhile, ER stress disturbed the monooxygenase system, and thus generated reactive oxygen species (ROS). Relief of ER stress with 4-phenylbutyric acid inhibited all the above effects of TiO2 NPs, including the generation of ROS. Therefore, TiO2 NP-induced ER stress was a decisive factor with a central role in plasma glucose disturbance in mice.

Role of the Talaromyces marneffei (Penicillium marneffei) sakA gene in nitrosative stress response, conidiation and red pigment production.

Stress-activated MAPK pathways are systems used to regulate the stress adaptation of most fungi. It has been shown that in Talaromyces marneffei (Penicillium marneffei), a pathogenic dimorphic fungus, the sakA gene is involved, not only in tolerance against oxidative and heat stresses, but also in playing a role in asexual development, yeast cell generation in vitro and survival inside macrophage cell lines. In this study, the role of the T. marneffei sakA gene on the nitrosative stress response and the regulation of gene expression were investigated. The susceptibility of the sakA mutant to NaNO2 was investigated using drop dilution assay and the expression of genes of interest were determined by RT-PCR, comparing them to the wild-type and complemented strains. The results demonstrated that the T. marneffei sakA gene played a role in the stress response to NaNO2, the expression of genes functioning in conidial development (brlA, abaA and wetA) and red pigment biosynthesis (pks3, rp1, rp2 and rp3). These findings suggest that T. marneffei sakA is broadly involved in a wide variety of cell activities, including stress response, cell morphogenesis, asexual development and pigmentation.

A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

Cells possess stress-activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

AMD-Associated Genes Encoding Stress-Activated MAPK Pathway Constituents Are Identified by Interval-Based Enrichment Analysis

PurposeTo determine whether common DNA sequence variants within groups of genes encoding elements of stress-activated mitogen-activated protein kinase (MAPK) signaling pathways are, in aggregate, associated with advanced AMD (AAMD).MethodsWe used meta-regression and exact testing methods to identify AAMD-associated SNPs in 1177 people with AAMD and 1024 AMD-free elderly peers from 3 large-scale genotyping projects on the molecular genetics of AMD. SNPs spanning independent AAMD-associated genomic intervals were examined with a multi-locus-testing method (INRICH) for enrichment within five sets of genes encoding constituents of stress-activated MAPK signaling cascades.ResultsFour-of-five pathway gene sets showed enrichment with AAMD-associated SNPs; findings persisted after adjustment for multiple testing in two. Strongest enrichment signals (P = 0.006) existed in a c-Jun N-terminal kinase (JNK)/MAPK cascade (Science Signaling, STKE CMP_10827). In this pathway, seven independent AAMD-associated regions were resident in 6 of 25 genes examined. These included sequence variants in: 1) three MAP kinase kinase kinases (MAP3K4, MAP3K5, MAP3K9) that phosphorylate and activate the MAP kinase kinases MAP2K4 and MAP2K7 (molecules that phosphorylate threonine and tyrosine residues within the activation loop of JNK); 2) a target of MAP2K7 (JNK3A1) that activates complexes involved in transcriptional regulation of stress related genes influencing cell proliferation, apoptosis, motility, metabolism and DNA repair; and 3) NR2C2, a transcription factor activated by JNK1A1 (a drugable molecule influencing retinal cell viability in model systems). We also observed AAMD-related sequence variants resident in genes encoding PPP3CA (a drugable molecule that inactivates MAP3K5), and two genes (TGFB2, TGFBR2) encoding factors involved in MAPK sensing of growth factors/cytokines.ConclusionsLinkage disequilibrium (LD)-independent genomic enrichment analysis yielded associations of AAMD with aggregates of functionally related genes encoding constituents of the JNK MAPK signaling pathway. FDA-approved drugs now exist to target constituents of stress-activated MAPK pathways and may offer reasonable approaches to preventing or treating AAMD.

A Measurable Activation of the bZIP Transcription Factor Atf1 in a Fission Yeast Strain Devoid of Stress-activated and Cell Integrity Mitogen-activated Protein Kinase (MAPK) Activities

In Schizosaccharomyces pombe, the stress-activated Sty1 MAPK pathway is essential for cell survival under stress conditions. The Sty1 MAPK regulates Atf1 transcription factor to elicit stress responses in extreme conditions of osmolarity and reactive oxygen species-generating agents such as hydrogen peroxide, heat, low glucose, and heavy metal. Herein, using a newly developed Renilla luciferase reporter assay with enhanced detection sensitivity and accuracy, we show that distinct signaling pathways respond to cadmium and other reactive oxygen species-generating agents for the activation of Atf1. Also, surprisingly, a measurable activation of Atf1 transcription factor was still observed devoid of Sty1 MAPK activity. Further genetic and biological analyses revealed that the residual activation is caused by the activation of the cell wall integrity Pmk1 MAPK pathway and a redox-mediated activation of Atf1.

Molecular mechanisms of liver injury and hepatocarcinogenesis: focusing on the role of stress-activated MAPK.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality. Short-term prognosis of patients with HCC has improved recently due to advances in early diagnosis and treatment, but long-term prognosis is still unsatisfactory. Therefore, obtaining a further understanding of the molecular carcinogenic mechanisms and the unique pathogenic biology of HCC is important. The most characteristic process in hepatocarcinogenesis is underlying chronic liver injury, which leads to repeated cycles of hepatocyte death, inflammation, and compensatory proliferation and subsequently provides a mitogenic and mutagenic environment leading to the development of HCC. Recent in vivo studies have shown that the stress-activated mitogen-activated protein kinase (MAPK) cascade converging on c-Jun NH2-terminal kinase (JNK) and p38 plays a central role in these processes, and it has attracted considerable attention as a therapeutic target. However, JNK and p38 have complex functions and a wide range of cellular effects. In addition, crosstalk with each other and the nuclear factor-kappaB pathway further complicate these functions. A full understanding is essential to bring these observations into clinical settings. In this paper, we discuss the latest findings regarding the mechanisms of liver injury and hepatocarcinogenesis focusing on the role of the stress-activated MAPK pathway.

The Cell Surface Protein Ecm33 Is Involved in Negative Feedback Regulation of MAP Kinase Signalling and Development of the <i>In Vivo</i> Real-time Monitoring of MAP Kinase Signalling

The mitogen-activated protein kinase (MAPK) pathways are signal transduction mechanisms that regulate many cellular processes in eukaryotic organisms, from yeasts to mammals. Multiple MAPKs regulate eukaryotic gene expression in response to various extracellular stimuli through phosphorylation of transcription factors. We have been studying the Pmk1 MAPK, a homologue of the mammalian ERK/MAPK in fission yeast. The Pmk1 MAPK regulates cell integrity and cell morphology. We have previously demonstrated that Atf1, a transcription factor downstream of the stress-activated MAPK pathway, serves also as a target of the Pmk1 MAPK signaling in fission yeast. Here, we identified ecm33⁺ gene, encoding a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. The gene expression of ecm33⁺ is regulated by two transcription factors Atf1 and Mbx1. We also developed an in vivo real-time monitoring system of Atf1 or Mbx1 transcriptional activity, which enables to monitor the activation of the Pmk1 MAPK pathway by various stimuli. Finally, we demonstrated that Ecm33 is involved in the negative regulation of the Pmk1 MAPK signaling through the control of Ca²⁺ homeostasis. The ecm33 deleted cells displayed Ca²⁺ sensitivity and increased phosphorylation levels of Pmk1 MAPK. In addition, the Ecm33 overproducing cells displayed phenotypes closely similar to those of the pmk1 knockout cell. Collectively, Ecm33 plays a role in the negative feedback regulation of Pmk1 cell integrity signaling.

Photodynamic therapy (PDT) – Initiation of apoptosis via activation of stress-activated p38 MAPK and JNK signal pathway in H460 cell lines

The purpose of this study was to investigate the photoefficacies of protoporphyrin IX (PpIX) generated by drug precursor 5-aminolevulinic acid (ALA) and its hexyl ester (H-ALA) on two human non-small lung carcinoma cell lines (H460/Bcl-2 and H460/neo). Drug uptake and the photoefficacies of PpIX were measured by flow cytometry and MTT assay; while the mode of cell death and alternation of signal transduction pathways were studied with 4',6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis, respectively. The flow cytometric analysis of H-ALA (5μM) uptake revealed optimal fluorescent intensity at 8h incubation, while ALA (0.5mM) was still far from saturation. The LD(30) of H-ALA-PDT was 30μM, 2J/cm(2), while the LD(30) of ALA-PDT was 3mM, 2J/cm(2). The dark toxicities mediated by both pro-drug H-ALA and ALA were negligible. By DAPI staining, apoptotic cell death was observed. In addition, by Western blot analysis, H-ALA- and ALA-mediated PDT initiated apoptotic cell death via the up-regulation and activation of p38 mitogen activated protein kinase (MAPK), the stress-activated c-jun N-terminal kinases (JNK) and ERK. These results suggested that H-ALA and ALA mediated PDT displayed similar photocytotoxicities towards the two non-small lung cancer cells. Our present study also demonstrates H-ALA or ALA mediated PDT in H460 cells are closely related to the activation of p38 MAPK and JNK signalling pathway.

The Transcription Factor Atf1 Binds and Activates the APC/C Ubiquitin Ligase in Fission Yeast

Fission yeast Atf1 is a member of the ATF/CREB basic leucine zipper (bZIP) family of transcription factors with strong homology to mammalian ATF2. Atf1 regulates transcription in response to stress stimuli and also plays a role in controlling heterochromatin formation and recombination. However, its DNA binding independent role is poorly studied. Here, we report that Atf1 has a distinct role in regulating the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. We have identified atf1(+) as a dose-dependent suppressor of apc5-1, a mutation causing mitotic arrest. Remarkably, the suppression is not dependent upon the bZIP domain and is therefore independent of the ability of Atf1 to bind DNA. Interestingly, Atf1 physically binds the APC/C in vivo. Furthermore, we show that addition of purified Atf1 proteins into a cell-free system stimulates ubiquitylation of cyclin B and securin by the APC/C. These results reveal a novel role for Atf1 in cell cycle control through protein-protein interaction.

Talking About Stress

CELL BIOLOGY Cells encounter many different forms of stress and have evolved a variety of methods to deal with them. They tackle relatively minor stresses, such as excessive heat or insufficient oxygen (hypoxia), by forming cytoplasmic stress granules, which prevent the accumulation of defective proteins that can irreparably damage the cell. However, some stresses, including x-rays and DNA-damaging agents, are insurmountable, and the cell acknowledges defeat by killing itself in a process called apoptosis. This is triggered via the intracellular signaling cascades known as the stress-activated p38 and JNK MAPK (SAPK) pathways. Whether and how these two mechanisms of stress management are connected was unknown. Arimoto et al. find that the formation of stress granules in response to minor stresses specifically inhibits the SAPK-mediated cell death response, indicating a connection between the two pathways. They found that the signaling scaffold protein RACK1 is required for the apoptotic response by binding directly to a protein in the SAPK pathway. However, during minor stresses RACK1 becomes sequestered within the cytoplasmic stress granules, thereby inhibiting apoptosis. The authors also showed that when cells are exposed to both types of stresses simultaneously, SAPK-mediated apoptosis is blocked. This mechanism of cross talk between two stress-management pathways could explain in part why cancer cells, which live under the constant minor stress of hypoxia, are resistant to apoptosis induced by radiotherapy and chemotherapy. — HP [*][1] Nat. Cell Biol. , 10.1038/ncb1791 (2008). [1]: #fn-1

Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways

When confronted with environmental stress, cells either activate defence mechanisms to survive, or initiate apoptosis, depending on the type of stress. Certain types of stress, such as hypoxia, heatshock and arsenite (type 1 stress), induce cells to assemble cytoplasmic stress granules (SGs), a major adaptive defence mechanism. SGs are multimolecular aggregates of stalled translation pre-initiation complexes that prevent the accumulation of mis-folded proteins. Type 2 stress, which includes X-rays and genotoxic drugs, induce apoptosis through the stress-activated p38 and JNK MAPK (SAPK) pathways. A functional relationship between the SG and SAPK responses is unknown. Here, we report that SG formation negatively regulates the SAPK apoptotic response, and that the signalling scaffold protein RACK1 functions as a mediator between the two responses. RACK1 binds to the stress-responsive MTK1 MAPKKK and facilitates its activation by type 2 stress; however, under conditions of type 1 stress, RACK1 is sequestered into SGs. Thus, type 1 conditions suppress activation of the MTK1-SAPK pathway and apoptosis induced by type 2 stress. These findings may be relevant to the problem of hypoxia-induced resistance to cancer chemotherapy.

Expression of Apoptosis Signal-Regulating Kinase 1 in Mouse Spinal Cord Under Chronic Mechanical Compression

To examine apoptosis signal cascade in neurons and oligodendrocytes under the chronic spinal cord compression of tiptoe-walking Yoshimura (TWY) mouse, which is model of progressive cervical cord compression. To clarify the biologic mechanisms of apoptosis, which may produce destructive changes in the spinal cord under chronic mechanical compression, with a resulting irreversible neurologic deficit. The stress-activated mitogen-activated protein kinase pathways including ASK1 transmitted apoptosis signals after acute spinal cord injury. Apoptosis in acute spinal cord injury induced both secondary degeneration around the site of injury and chronic demyelination. Chronic spinal cord compression showed myelin destruction, loss of axons, and oligodendrocytes in white matter, and loss of neurons in gray matter. Apoptosis associated with chronic spinal cord compression contributes to these changes. However, the biologic mechanisms of apoptosis in the spinal cord under chronic mechanical compression remain unclear. We examined the expression of phosphorylated-apoptosis signal-regulating kinase 1 (ASK1), phosphorylated-c-Jun N-terminal kinase (JNK), phosphorylated-p38 mitogen-activated protein kinase (p38), and activated caspase-3 immunohistologically in TWY mice, an animal model of progressive cervical spinal cord compression, since the ASK1-JNK and -p38 signaling cascades participate in the signaling pathway leading to apoptosis in neural tissue and neuronal culture. Double immunohistochemistry for phosphorylated-ASK1, phosphorylated-JNK, phosphorylated-p38, activated-caspase3, and cell-specific markers confirmed the presence of apoptosis signals in both neurons and oligodendrocytes in compressed spinal cord cells. We found that mitogen-activated protein kinase pathways including ASK1, JNK, and p38 were activated in destructive spinal cord under chronic compression.

Fission Yeast Mitogen-Activated Protein Kinase Sty1 Interacts with Translation Factors

Signaling by stress-activated mitogen-activated protein kinase (MAPK) pathways influences translation efficiency in mammalian cells and budding yeast. We have investigated the stress-activated MAPK from fission yeast, Sty1, and its downstream protein kinase, Mkp1/Srk1, for physically associated proteins using tandem affinity purification tagging. We find Sty1, but not Mkp1, to bind to the translation elongation factor eukaryotic elongation factor 2 (eEF2) and the translation initiation factor eukaryotic initiation factor 3a (eIF3a). The Sty1-eIF3a interaction is weakened under oxidative or hyperosmotic stress, whereas the Sty1-eEF2 interaction is stable. Nitrogen deprivation causes a transient strengthening of both the Sty1-eEF2 and the Sty1-Mkp1 interactions, overlapping with the time of maximal Sty1 activation. Analysis of polysome profiles from cells under oxidative stress, or after hyperosmotic shock or nitrogen deprivation, shows that translation in sty1 mutant cells recovers considerably less efficiently than that in the wild type. Cells lacking the Sty1-regulated transcription factor Atf1 are deficient in maintaining and recovering translational activity after hyperosmotic shock but not during oxidative stress or nitrogen starvation. In cells lacking Sty1, eIF3a levels are decreased, and phosphorylation of eIF3a is reduced. Taken together, our data point to a central role in translational adaptation for the stress-activated MAPK pathway in fission yeast similar to that in other investigated eukaryotes, with the exception that fission yeast MAPK-activated protein kinases seem not to be directly involved in this process.