Response regulator-mediated MAPKKK heteromer promotes stress signaling to the Spc1 MAPK in fission yeast.
ABSTRACT: The Spc1 mitogen-activated protein kinase (MAPK) cascade in fission yeast is activated by two MAPK kinase kinase (MAPKKK) paralogues, Wis4 and Win1, in response to multiple forms of environmental stress. Previous studies identified Mcs4, a "response regulator" protein that associates with the MAPKKKs and receives peroxide stress signals by phosphorelay from the Mak2/Mak3 sensor histidine kinases. Here we show that Mcs4 has an unexpected, phosphorelay-independent function in promoting heteromer association between the Wis4 and Win1 MAPKKKs. Only one of the MAPKKKs in the heteromer complex needs to be catalytically active, but disturbing the integrity of the complex by mutations to Mcs4, Wis4, or Win1 results in reduced MAPKKK-MAPKK interaction and, consequently, compromised MAPK activation. The physical interaction among Mcs4, Wis4, and Win1 is constitutive and not responsive to stress stimuli. Therefore the Mcs4-MAPKKK heteromer complex might serve as a stable platform/scaffold for signaling proteins that convey input and output of different stress signals. The Wis4-Win1 complex discovered in fission yeast demonstrates that heteromer-mediated mechanisms are not limited to mammalian MAPKKKs.
Project description:Phosphorelay signaling of environmental stimuli by two-component systems is prevailing in bacteria and also utilized by fungi and plants. In the fission yeast Schizosaccharomyces pombe, peroxide stress signals are transmitted from the Mak2/3 sensor kinases to the Mpr1 histidine-containing phosphotransfer (HPt) protein and finally to the Mcs4 response regulator, which activates a MAP kinase cascade. Here we show that, unexpectedly, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) physically associates with the Mcs4 response regulator and stress-responsive MAP kinase kinase kinases (MAPKKKs). In response to H2O2 stress, Cys-152 of the Tdh1 GAPDH is transiently oxidized, which enhances the association of Tdh1 with Mcs4. Furthermore, Tdh1 is essential for the interaction between the Mpr1 HPt protein and the Mcs4 response regulator and thus for phosphorelay signaling. These results demonstrate that the glycolytic enzyme GAPDH plays an essential role in the phosphorelay signaling, where its redox-sensitive cysteine residue may provide additional input signals.
Project description:The Schizosaccharomyces pombe wis1(+) gene is essential for cell survival under stress conditions. The MAPKK homologue Wis1 is required for activation of the MAPK homologue Spc1, and integrity of the Wis1-Spc1 pathway is required for survival in extreme conditions of heat, osmolarity, oxidation or limited nutrition. We show here that Wis4, a protein kinase of a new MAPKKK class, phosphorylates Wis1 in vitro and activates it in vivo. Win1 is also required for full activation of Wis1, and Win1 rather than Wis4 mediates the osmotic stress signal. Surprisingly, the pathway can still be activated by heat or oxidative stress independently of the phosphorylation of two conserved Wis1 residues. Evidence is presented that the Pyp1 protein tyrosine phosphatase, which dephosphorylates Spc1, is central to this alternative activation mechanism.
Project description:In fission yeast, the Sty1/Spc1/Phh1 mitogen-activated protein kinase (MAPK) pathway is known to be involved in multiple-stress responses. It is currently thought that the Sty1 MAPK cascade is mediated by histidine kinases and phosphorelay proteins in response to oxidative stress signals. However, studies of the exact transduction mechanism of multiple-stress responses are lacking. Thus, in response to various stimuli, we monitored the Sty1 MAPK pathway through the downstream transcription factor Atf1 in living cells using a highly sensitive luciferase reporter gene. Surprisingly, in cadmium and low glucose (LG) medium, Atf1 activation was observed even in the absence of all of the four fission yeast MAPK kinase kinases (MAPKKKs); whereas in osmotic stress, Atf1 activation was abolished. Thus, the osmotic stress likely mediates the MAPK activation via MAPKKKs, whereas a cadmium or LG condition activates the MAPK in a MAPKKK-independent manner. On the other hand, knockout of tyrosine phosphatase gene pyp1(+) abolished the Atf1 response to cadmium and LG, but not to osmotic stress, suggesting that Pyp1 is a sensor for cadmium and LG.
Project description:The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize.
Project description:The RRM-type RNA-binding protein Mei2 is a master regulator of meiosis in fission yeast, in which it stabilizes meiosis-specific mRNAs by blocking their destruction. Artificial activation of Mei2 can provoke the entire meiotic process, and it is suspected that Mei2 may do more than the stabilization of meiosis-specific mRNAs. In our current study using a new screening system, we show that Mei2 genetically interacts with subunits of CTDK-I, which phosphorylates serine-2 residues on the C-terminal domain of RNA polymerase II (Pol II CTD). Phosphorylation of CTD Ser-2 is essential to enable the robust transcription of ste11, which encodes an HMG-type transcription factor that regulates the expression of mei2 and other genes necessary for sexual development. CTD Ser-2 phosphorylation increases under nitrogen starvation, and the stress-responsive MAP kinase pathway, mediated by Wis1 MAPKK and Sty1 MAPK, is critical for this stress response. Sty1 phosphorylates Lsk1, the catalytic subunit of CTDK-I. Furthermore, a feedback loop stemming from activated Mei2 to Win1 and Wis4 MAPKKKs operates in this pathway and eventually enhances CTD Ser-2 phosphorylation and ste11 transcription. Hence, in addition to starting meiosis, Mei2 functions to reinforce the commitment to it, once cells have entered this process. This study also demonstrates clearly that the stress-responsive MAP kinase pathway can modulates gene expression through phosphorylation of Pol II CTD.
Project description:The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily conserved signal transduction pathway that is involved in plant development and stress responses. As the first component of this phosphorelay cascade, mitogen-activated protein kinase kinase kinases (MAPKKKs) act as adaptors linking upstream signaling steps to the core MAPK cascade to promote the appropriate cellular responses; however, the functions of MAPKKKs in maize are unclear. Here, we identified 71 MAPKKK genes, of which 14 were novel, based on a computational analysis of the maize (Zea mays L.) genome. Using an RNA-seq analysis in the leaf, stem and root of maize under well-watered and drought-stress conditions, we identified 5,866 differentially expressed genes (DEGs), including 8 MAPKKK genes responsive to drought stress. Many of the DEGs were enriched in processes such as drought stress, abiotic stimulus, oxidation-reduction, and metabolic processes. The other way round, DEGs involved in processes such as oxidation, photosynthesis, and starch, proline, ethylene, and salicylic acid metabolism were clearly co-expressed with the MAPKKK genes. Furthermore, a quantitative real-time PCR (qRT-PCR) analysis was performed to assess the relative expression levels of MAPKKKs. Correlation analysis revealed that there was a significant correlation between expression levels of two MAPKKKs and relative biomass responsive to drought in 8 inbred lines. Our results indicate that MAPKKKs may have important regulatory functions in drought tolerance in maize. Overall design: Totally six samples, two treatments and three organs
Project description:The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1+ and wis4+, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1+ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.
Project description:Plant mitogen-activated protein kinase (MAPK) cascades play important roles in development and stress responses. In previous studies, we have systematically investigated the mitogen-activated protein kinase kinase (MKK) and MAPK gene families in cotton. However, the complete interactions between MAPK gene family members in MAPK signaling cascade is poorly characterized. Herein, we investigated the mitogen-activated protein kinase kinase kinase (MAPKKK) family members and identified a total of 89 MAPKKK genes in the Gossypium raimondii genome. We cloned 51 MAPKKKs in G. hirsutum and investigated the interactions between MKK and MAPKKK proteins through yeast-two hybrid assays. A total of 18 interactive protein pairs involved in 14 MAPKKKs and six MKKs were found. Among these, 13 interactive pairs had not been reported previously. Gene expression patterns revealed that 12 MAPKKKs were involved in diverse signaling pathways triggered by hormone treatments or abiotic stresses. By combining the MKK-MAPK and MKK-MAPKKK protein interactions with gene expression patterns, 38 potential MAPK signaling modules involved in the complicated cross-talks were identified, which provide a basis on elucidating biological function of the MAPK cascade in response to hormonal and/or stress responses. The systematic investigation in MAPK signaling cascades will lay a foundation for understanding the functional roles of different MAPK cascades in signal transduction pathways, and for the improvement of various defense responses in cotton.
Project description:Mitogen Activated Protein Kinase (MAPK) signaling cascades transduce information arising from events external to the cell, such as environmental stresses, to a variety of downstream effectors and transcription factors. The fission yeast stress activated MAP kinase (SAPK) pathway is conserved with the p38 and JNK pathways in humans, and comprises the MAPKKKs Win1, Wis4, the MAPKK Wis1, and the MAPK, Sty1. Sty1 and its main downstream effector Atf1 regulate a large set of core environmental stress response genes. The fission yeast genome encodes three other ATF proteins: Atf21, Atf31 and Pcr1. Among these, atf21 is specifically induced under conditions of high osmolarity. We have therefore instigated a programme to investigate the role played by non coding RNAs (ncRNAs) in response to osmotic stress challenge in wild type and atf21Δ cells. By integrating global proteomics and RNA sequencing data, we identified a systematic program in which elevated antisense RNAs arising both from ncRNAs and from 3'-overlapping convergent gene-pairs is directly associated with substantial reductions in protein levels throughout the fission yeast genome. We also found an extensive array of ncRNAs with trans associations that have the potential to influence different biological processes and stress responses in fission yeast, suggesting ncRNAs comprise additional components of the SAPK regulatory system. Overall design: Global transcription profiles of fission yeast wild type (WT) and atf21Δ (atf21del) strains over an osmotic stress time course following treatment with 1M sorbitol at 0, 15, 30, 60, 120 and 180 mins. Strand-specific single end sequencing of total RNA was performed in biological triplicates on the Applied Biosystems SOLiD 5500xl Genetic Analyzer System.
Project description:Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.