Pmk1 Pathway Research Articles

Aronia melanocarpa extract extends the lifespan and health-span of Caenorhabditis elegans via mitogen-activated protein kinase 1.

Aging is a highly complex process and one of the largest risk factors for many chronic diseases. Aronia melanocarpa (AM) is rich in bioactive phytochemicals with antioxidant, anti-inflammatory, and anticancer properties. However, little is known about its effects on aging. The objective of this study was to evaluate the effects of AM extract on lifespan and health-span using Caenorhabditis elegans as a representative model. The mechanisms of its effects were explored using transcriptomics and untargeted metabolomics. Results showed that the lifespan of C. elegans was significantly extended by 22.2% after high-dose AM treatment. AM improved the behavior and physiological functions of C. elegans by increasing the pharyngeal pumping rate, decreasing lipofuscin accumulation and the reactive oxygen species level, enhancing resistance to oxidative stress, and increasing the activities of superoxide dismutase and catalase. Transcriptome analysis showed that the pmk-1 gene (mitogen-activated protein kinase 1), which is involved in the MAPK signaling pathway, was the gene with the largest fold change after AM intervention. However, in the C. elegans pmk-1(km25) mutant, the beneficial effect of AM in improving nematode senescence disappeared. An untargeted metabolomics study showed that the levels of 4-hydroxyproline, rhamnose, and cysteine were increased after AM supplementation, and their extending effect on the lifespan and health-span of C. elegans were partly dependent on the pmk-1 gene. In conclusion, our results revealed that AM can promote the lifespan and health-span of C. elegans via the PMK-1 pathway, highlighting the potential of AM as a dietary supplement to delay aging.

Autophagy-related genes genetically interact with Pmk1 MAPK signaling in fission yeast.

Apart from the highly conserved role in the cellular degradation process, autophagy also appears to play a key role in cellular proliferation. Here, we describe the genetic interaction of autophagy-related genes and Pmk1 MAPK signaling in fission yeast. atg1 deletion cells (Δ atg1 ) exhibit the vic (viable in the presence of immunosuppressant and Cl - ) phenotype, indicative of Pmk1 signaling inhibition. Moreover, the Δ atg1 Δ pmk1 double mutant resembles the single Δ pmk1 mutant, suggesting that Atg1 functions in the Pmk1 pathway. In addition, the growth defect induced by overexpression of Pck2, an upstream activator of Pmk1 MAPK was alleviated by the deletion of atg1 + . Finally, the deletion of autophagy-related genes recapitulates Pmk1 MAPK signaling inhibition. Our data suggest a novel role for autophagy in MAPK signaling regulation.

DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans.

Aging is accompanied by a progressive decline in immune function termed “immunosenescence”. Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie age-dependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger C. elegans DAF-16 is inactive unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16-mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life.

Innate Immunity in the C. elegans Intestine Is Programmed by a Neuronal Regulator of AWC Olfactory Neuron Development

SUMMARYOlfactory neurons allow animals to discriminate nutritious food sources from potential pathogens. From a forward genetic screen, we uncovered a surprising requirement for the olfactory neuron gene olrn-1 in the regulation of intestinal epithelial immunity in Caenorhabditis elegans. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen, and resistance to bacterial infection. These data reveal an unexpected connection between olfactory receptor development and innate immunity and show that anti-pathogen defenses in the intestine are developmentally programmed.

Tpc1 is an important Zn(II)2Cys6 transcriptional regulator required for polarized growth and virulence in the rice blast fungus

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.

Activation of the signalling mucin MoMsb2 and its functional relationship with Cbp1 in Magnaporthe oryzae.

Various surface signals are recognized by Magnaporthe oryzae to activate the Pmk1 MAP kinase that is essential for appressorium formation and invasive growth. One of upstream sensors of the Pmk1 pathway is the MoMsb2 signalling mucin. However, the activation of MoMsb2 and its relationship with other sensors is not clear. In this study, we showed that the cleavage and transmembrane domains are essential for MoMsb2 functions. Cleavage of MoMsb2 was further confirmed by western blot analysis, and five putative cleavage sites were functionally characterized. Expression of the extracellular region alone partially rescued the defects of Momsb2 in appressorium formation and virulence. The cytoplasmic region of MoMsb2, although dispensable for appressorium formation, was more important for penetration and invasive growth. Interestingly, the Momsb2 cbp1 double mutant deleted of both mucin genes was blocked in Pmk1 activation. It failed to form appressoria on artificial surfaces and was non-pathogenic. In addition, we showed that MoMsb2 interacts with Ras2 but not with MoCdc42 in co-immunoprecipitation assays. Overall, results from this study indicated that the extracellular and cytoplasmic regions of MoMsb2 have distinct functions in appressorium formation, penetration and invasive growth, and MoMsb2 has overlapping functions with Cbp1 in recognizing environmental signals for Pmk1 activation.

PAF104, a Synthetic Peptide to Control Rice Blast Disease by Blocking Appressorium Formation in Magnaporthe oryzae

Magnaporthe oryzae is the most devastating pathogen of rice and the main cause of crop losses worldwide. The successful management of blast disease caused by this fungus is a clear necessity. The synthetic peptide PAF104 has been characterized by its inhibition of M. oryzae appressorium formation on hydrophobic surfaces. Growth and the ability of conidia to germinate was not affected by PAF104, indicating the lack of toxicity on fungal conidia. The addition of the cutin monomer 1,16-hexadecanediol does not interfere with the inhibitory effect of PAF104 on in vitro hydrophobic surfaces. On the other hand, inhibition of appressorium formation by PAF104 was nullified by the exogenous addition of cAMP. Our results suggest that PAF104 affects the Pmk1 pathway by repression of the gene expression of MoMSB2, which encodes a sensing surface protein, and the mitogen-activated protein/extracellular signal-regulated kinase kinase kinase MST11. The pathogenicity of M. oryzae was reduced after PAF104 treatment specifically blocking appressorium formation. Our results support PAF104 as a promising compound to control rice blast disease by blocking a specific target related to appressorium formation, a process essential for infection of rice leaves. Moreover, PAF104 is proposed as a lead compound to develop novel specific fungicides with improved properties.

Role of the fission yeast cell integrity MAPK pathway in response to glucose limitation

BackgroundGlucose is a signaling molecule which regulates multiple events in eukaryotic organisms and the most preferred carbon source in the fission yeast Schizosaccharomyces pombe. The ability of this yeast to grow in the absence of glucose becomes strongly limited due to lack of enzymes of the glyoxylate cycle that support diauxic growth. The stress-activated protein kinase (SAPK) pathway and its effectors, Sty1 MAPK and transcription factor Atf1, play a critical role in the adaptation of fission yeast to grow on alternative non-fermentable carbon sources by inducing the expression of fbp1+ gene, coding for the gluconeogenic enzyme fructose-1,6-bisphosphatase. The cell integrity Pmk1 pathway is another MAPK cascade that regulates various processes in fission yeast, including cell wall construction, cytokinesis, and ionic homeostasis. Pmk1 pathway also becomes strongly activated in response to glucose deprivation but its role during glucose exhaustion and ensuing adaptation to respiratory metabolism is currently unknown.ResultsWe found that Pmk1 activation in the absence of glucose takes place only after complete depletion of this carbon source and that such activation is not related to an endogenous oxidative stress. Notably, Pmk1 MAPK activation relies on de novo protein synthesis, is independent on known upstream activators of the pathway like Rho2 GTPase, and involves PKC ortholog Pck2. Also, the Glucose/cAMP pathway is required operative for full activation of the Pmk1 signaling cascade. Mutants lacking Pmk1 displayed a partial growth defect in respiratory media which was not observed in the presence of glucose. This phenotype was accompanied by a decreased and delayed expression of transcription factor Atf1 and target genes fbp1+ and pyp2+. Intriguingly, the kinetics of Sty1 activation in Pmk1-less cells was clearly altered during growth adaptation to non-fermentable carbon sources.ConclusionsUnknown upstream elements mediate Pck2-dependent signal transduction of glucose withdrawal to the cell integrity MAPK pathway. This signaling cascade reinforces the adaptive response of fission yeast to such nutritional stress by enhancing the activity of the SAPK pathway.

The p38 MAPK PMK-1 shows heat-induced nuclear translocation, supports chaperone expression, and affects the heat tolerance of Caenorhabditis elegans

The p38 mitogen-activated protein kinase PMK-1 of Caenorhabditis elegans has been associated with heavy metal, oxidative and pathogen stress. Pmk-1 is part of an operon comprising three p38 homologues, with pmk-1 expression suggested to be regulated by the operon promoter. There are contradictory reports about the cellular localization of PMK-1. We were interested to study principles of pmk-1 expression and to analyze the role of PMK-1 under heat stress. Using a translational GFP reporter, we found pmk-1 expression to be driven by a promoter in front of pmk-1. PMK-1 was detected in intestinal cells and neurons, with a cytoplasmic localization at moderate temperature. Increasing temperature above 32 °C, however, induced a nuclear translocation of PMK-1 as well as PMK-1 accumulation near to apical membranes. Testing survival rates revealed 34-35 °C as critical temperature range, where short-term survival severely decreased. Mutants of the PMK-1 pathway (pmk-1Δ, sek-1Δ, mek-1Δ) as well as a mutant of JNK pathway (jnk-1Δ) showed significantly lower survival rates than wild-type or mutants of other pathways (kgb-1Δ, daf-2Δ). Rescue and overexpression experiments verified the negative effects of pmk-1Δ on heat tolerance. Studying gene expression by RNA-seq and semi-quantitative reverse transcriptase polymerase chain reaction revealed positive effects of the PMK-1 pathway on the expression of genes for chaperones, protein biosynthesis, protein degradation, and other functional categories. Thus, the PMK-1 pathway is involved in the heat stress responses of C. elegans, possibly by a PMK-1-mediated activation of the transcription factor SKN-1 and/or an indirect or direct PMK-1-dependent activation (hyperphosphorylation) of heat-shock factor 1.

Survive an innate immune response through XBP1

Survive an innate immune response through XBP1

Rga4 Modulates the Activity of the Fission Yeast Cell Integrity MAPK Pathway by Acting as a Rho2 GTPase-activating Protein

Rho GTPase-activating proteins (GAPs) are responsible for the inactivation of Rho GTPases, which are involved in the regulation of critical biological responses in eukaryotic cells, ranging from cell cycle control to cellular morphogenesis. The genome of fission yeast Schizosaccharomyces pombe contains six genes coding for putative Rho GTPases, whereas nine genes code for predicted Rho GAPs (Rga1 to Rga9). One of them, Rga4, has been recently described as a Cdc42 GAP, involved in the control of cell diameter and symmetry in fission yeast. In this work we show that Rga4 is also a Rho2 GAP that negatively modulates the activity of the cell integrity pathway and its main effector, MAPK Pmk1. The DYRK-type protein kinase Pom1, which regulates both the localization and phosphorylation state of Rga4, is also a negative regulator of the Pmk1 pathway, but this control is not dependent upon the Rga4 role as a Rho2-GAP. Hence, two subsets of Rga4 negatively regulate Cdc42 and Rho2 functions in a specific and unrelated way. Finally, we show that Rga7, another Rho2 GAP, down-regulates the Pmk1 pathway in addition to Rga4. These results reinforce the notion of the existence of complex mechanisms determining the selectivity of Rho GAPs toward Rho GTPases and their functions.

The Cell Surface Protein Geneecm33+Is a Target of the Two Transcription Factors Atf1 and Mbx1 and Negatively Regulates Pmk1 MAPK Cell Integrity Signaling in Fission Yeast

The highly conserved fission yeast Pmk1 MAPK pathway plays a key role in cell integrity by regulating Atf1, which belongs to the ATF/cAMP-responsive element-binding (CREB) protein family. We identified and characterized ecm33(+), which encodes a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. We demonstrated that the gene expression of Ecm33 is regulated by two transcription factors Atf1 and a MADS-box-type transcription factor Mbx1. We identified a putative ATF/CREB-binding site and an RLM1-binding site in the ecm33(+) promoter region and monitored the transcriptional activity of Atf1 or Mbx1 in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of the CRE and six tandem repeats of the Rlm1-binding sequence, respectively. These reporter genes reflect the activation of the Pmk1 pathway by various stimuli, thereby enabling the real-time monitoring of the Pmk1 cell integrity pathway. Notably, the Deltaecm33 cells displayed hyperactivation of the Pmk1 signaling together with hypersensitivity to Ca(2+) and an abnormal morphology, which were almost abolished by simultaneous deletion of the components of the Rho2/Pck2/Pmk1 pathway. Our results suggest that Ecm33 is involved in the negative feedback regulation of Pmk1 cell integrity signaling and is linked to cellular Ca(2+) signaling.

Tissue-Specific Activities of an Immune Signaling Module Regulate Physiological Responses to Pathogenic and Nutritional Bacteria in C. elegans

Microbes represent both an essential source of nutrition and a potential source of lethal infection to the nematode Caenorhabditis elegans. Immunity in C. elegans requires a signaling module comprised of orthologs of the mammalian Toll-interleukin-1 receptor (TIR) domain protein SARM, the mitogen-activated protein kinase kinase kinase (MAPKKK) ASK1, and MAPKK MKK3, which activates p38 MAPK. We determined that the SARM-ASK1-MKK3 module has dual tissue-specific roles in the C. elegans response to pathogens--in the cell-autonomous regulation of innate immunity and the neuroendocrine regulation of serotonin-dependent aversive behavior. SARM-ASK1-MKK3 signaling in the sensory nervous system also regulates egg-laying behavior that is dependent on bacteria provided as a nutrient source. Our data demonstrate that these physiological responses to bacteria share a common mechanism of signaling through the SARM-ASK1-MKK3 module and suggest the co-option of ancestral immune signaling pathways in the evolution of physiological responses to microbial pathogens and nutrients.

Protein Kinase D Is an Essential Regulator of C. elegans Innate Immunity

Protein kinase D (PKD) mediates signal transduction downstream from phospholipase C and diacylglycerol (DAG). PKDs are activated by hormones and stress in cell lines, but little is known about PKD functions and regulation in vivo. Here, we show that DKF-2, a C. elegans PKD, regulates innate immunity. Animals lacking DKF-2 were hypersensitive to killing by bacteria that are pathogens of C. elegans and humans. DKF-2 induced 85 mRNAs, which encode antimicrobial peptides and proteins that sustain intestinal epithelium. Induction of immune effector mRNAs by DKF-2 proceeded via PMK-1 (p38 Map-kinase)-dependent and -independent pathways. TPA-1, a PKCdelta homolog, regulated activation and functions of DKF-2 in vivo. Therefore, DKF-2 provides a molecular link that couples DAG signaling to regulation of immunity. This intersection between DAG-TPA-1-DKF-2 and PMK-1 pathways enables integrated immune responses to multiple stimuli. Thus, a PKD mobilizes activation of host immune defenses against pathogens by previously unappreciated signaling pathways and mechanisms.

Atf1 Is a Target of the Mitogen-activated Protein Kinase Pmk1 and Regulates Cell Integrity in Fission Yeast

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.

P38 MAPK Regulates Expression of Immune Response Genes and Contributes to Longevity in C. elegans

The PMK-1 p38 mitogen-activated protein kinase pathway and the DAF-2–DAF-16 insulin signaling pathway control Caenorhabditis elegans intestinal innate immunity. pmk-1 loss-of-function mutants have enhanced sensitivity to pathogens, while daf-2 loss-of-function mutants have enhanced resistance to pathogens that requires upregulation of the DAF-16 transcription factor. We used genetic analysis to show that the pathogen resistance of daf-2 mutants also requires PMK-1. However, genome-wide microarray analysis indicated that there was essentially no overlap between genes positively regulated by PMK-1 and DAF-16, suggesting that they form parallel pathways to promote immunity. We found that PMK-1 controls expression of candidate secreted antimicrobials, including C-type lectins, ShK toxins, and CUB-like genes. Microarray analysis demonstrated that 25% of PMK-1 positively regulated genes are induced by Pseudomonas aeruginosa infection. Using quantitative PCR, we showed that PMK-1 regulates both basal and infection-induced expression of pathogen response genes, while DAF-16 does not. Finally, we used genetic analysis to show that PMK-1 contributes to the enhanced longevity of daf-2 mutants. We propose that the PMK-1 pathway is a specific, indispensable immunity pathway that mediates expression of secreted immune response genes, while the DAF-2–DAF-16 pathway appears to regulate immunity as part of a more general stress response. The contribution of the PMK-1 pathway to the enhanced lifespan of daf-2 mutants suggests that innate immunity is an important determinant of longevity.

The G-beta subunit MGB1 is involved in regulating multiple steps of infection-related morphogenesis in Magnaporthe grisea.

Trimeric G-proteins transmit extracellular signals to various downstream effectors (e.g. MAP kinases) in eukaryotes. In the rice blast fungus Magnaporthe grisea, the Pmk1 MAP kinase is essential for appressorium formation and infectious growth. The pmk1 deletion mutant fails to form appressoria but still responds to exogenous cAMP for tip deformation. Since gene disruption mutants of three Galpha subunits still form appressoria and are phenotypically different from pmk1 mutants, it is likely that the Pmk1 pathway is activated by Gbeta in M. grisea. In this study, we isolated and characterized the MGB1 gene that encodes the G subunit in M. grisea. Mutants disrupted in MGB1 were reduced in conidiation. Conidia from mgb1 mutants were defective in appressorium formation and failed to penetrate or grow invasively on rice leaves. Exogenous cAMP induced appressorium formation in mgb1 mutants, but these appressoria were abnormal in shape and could not penetrate. The intracellular cAMP level was reduced in mgb1 mutants and the defects in conidiation and hyphal growth were partially suppressed with 1 mM cAMP. Transformants expressing multiple copies of MGB1 were able to form appressoria on hydrophilic surfaces. Our results suggest that MGB1 may be involved in the cAMP signalling for regulating conidiation, surface recognition and appressorium formation. The Pmk1 pathway may be the downstream target of MGB1 for regulating penetration and infectious hyphae growth in M. grisea.

Feedback regulation of MAPK signalling by an RNA-binding protein

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes that convert extracellular signals into various outputs such as cell growth, differentiation and cell death. MAPK phosphatases selectively inactivate MAPKs by dephosphorylating critical phosphothreonine and phosphotyrosine residues. The transcriptional induction of MAPK phosphatase expression by various stimuli, including MAPK activation, has been well documented as a negative-feedback mechanism of MAPK signalling. Here we show that Rnc1, a novel K-homology-type RNA-binding protein in fission yeast, binds and stabilizes Pmp1 messenger RNA, the MAPK phosphatase for Pmk1 (refs 10, 11). Rnc1 therefore acts as a negative regulator of Pmk1 signalling. Notably, Pmk1 phosphorylates Rnc1, causing enhancement of the RNA-binding activity of Rnc1. Thus, Rnc1 is a component of a new negative-feedback loop that regulates the Pmk1 pathway through its binding to Pmp1 mRNA. Our findings--the post-transcriptional mRNA stabilization of a MAPK phosphatase mediated by an RNA-binding protein--provide an additional regulatory mechanism for fine-tuning of MAPK signalling pathways.