Ribosome profiling reveals ribosome stalling on tryptophan codons and ribosome queuing upon oxidative stress in fission yeast.

Translational control is essential in response to stress. We investigated the translational programmes launched by the fission yeast Schizosaccharomyces pombe upon five environmental stresses. We also explored the contribution of defence pathways to these programmes: The Integrated Stress Response (ISR), which regulates translation initiation, and the stress-response MAPK pathway. We performed ribosome profiling of cells subjected to each stress, in wild type cells and in cells with the defence pathways inactivated. The transcription factor Fil1, a functional homologue of the yeast Gcn4 and the mammalian Atf4 proteins, was translationally upregulated and required for the response to most stresses. Moreover, many mRNAs encoding proteins required for ribosome biogenesis were translationally downregulated. Thus, several stresses trigger a universal translational response, including reduced ribosome production and a Fil1-mediated transcriptional programme. Surprisingly, ribosomes stalled on tryptophan codons upon oxidative stress, likely due to a decrease in charged tRNA-Tryptophan. Stalling caused ribosome accumulation upstream of tryptophan codons (ribosome queuing/collisions), demonstrating that stalled ribosomes affect translation elongation by other ribosomes. Consistently, tryptophan codon stalling led to reduced translation elongation and contributed to the ISR-mediated inhibition of initiation. We show that different stresses elicit common and specific translational responses, revealing a novel role in Tryptophan-tRNA availability.

p38 Mitogen-activated protein kinase regulates chamber-specific perinatal growth in heart.

In the mammalian heart, the left ventricle (LV) rapidly becomes more dominant in size and function over the right ventricle (RV) after birth. The molecular regulators responsible for this chamber-specific differential growth are largely unknown. We found that cardiomyocytes in the neonatal mouse RV had lower proliferation, more apoptosis, and a smaller average size compared with the LV. This chamber-specific growth pattern was associated with a selective activation of p38 mitogen-activated protein kinase (MAPK) activity in the RV and simultaneous inactivation in the LV. Cardiomyocyte-specific deletion of both the Mapk14 and Mapk11 genes in mice resulted in loss of p38 MAPK expression and activity in the neonatal heart. Inactivation of p38 activity led to a marked increase in cardiomyocyte proliferation and hypertrophy but diminished cardiomyocyte apoptosis, specifically in the RV. Consequently, the p38-inactivated hearts showed RV-specific enlargement postnatally, progressing to pulmonary hypertension and right heart failure at the adult stage. Chamber-specific p38 activity was associated with differential expression of dual-specific phosphatases (DUSPs) in neonatal hearts, including DUSP26. Unbiased transcriptome analysis revealed that IRE1α/XBP1-mediated gene regulation contributed to p38 MAPK-dependent regulation of neonatal cardiomyocyte proliferation and binucleation. These findings establish an obligatory role of DUSP/p38/IRE1α signaling in cardiomyocytes for chamber-specific growth in the postnatal heart.

Mnk1 (Mitogen-Activated Protein Kinase-Interacting Kinase 1) Deficiency Aggravates Cardiac Remodeling in Mice.

Identifying the key factor involved in cardiac remodeling is critically important for developing novel strategies to protect against heart failure. Here, the role of Mnk1 (mitogen-activated protein kinase-interacting kinase 1) in cardiac remodeling was clarified. Cardiac remodeling was induced by transverse aortic constriction in Mnk1-knockout mice and their wild-type control mice. After 4 weeks of transverse aortic constriction, Mnk1-knockout mice developed exaggerated cardiac hypertrophy, fibrosis, dysfunction, and cardiomyocyte apoptosis and showed increased ERK1/2 (extracellular signal-regulated kinase 1/2) activation along with reduced sprouty2 expression. In line with the in vivo studies, Mnk1 knockdown by Mnk1 siRNA transfection induced exaggerated angiotensin II-induced cardiomyocyte hypertrophy in neonatal rat ventricular myocytes (NRVMs). Moreover, adenovirus-mediated overexpression of Mnk1 in NRVMs protected cardiomyocytes from angiotensin II-induced hypertrophy. In addition, overexpression of sprouty2 rescued NRVMs with Mnk1 knockdown from angiotensin II-induced hypertrophy. In accordance with the in vivo studies, as compared with the control group, Mnk1 knockdown led to hyperphosphorylation of ERK1/2 and suppression of the sprouty2 expression in angiotensin II-treated NRVMs; furthermore, Mnk1 overexpression led to hypophosphorylation of ERK1/2 in angiotensin II-treated NRVMs. In addition, sprouty2 overexpression suppressed the activation of ERK1/2 in angiotensin II-treated NRVMs with Mnk1 knockdown. Impressively, MnK1-knockout mice with overexpression of sprouty2 exhibited signs of a blunted cardiac hypertrophic response. Mnk1 likely carries out a suppressive function in cardiac hypertrophy via regulating the sprouty2/ERK1/2 pathway. It implicates Mnk1 in the development of cardiac remodeling.

Nemo-Like Kinase (NLK) Is a Pathological Signaling Effector in the Mouse Heart.

Nemo-like kinase (NLK) is an evolutionary conserved serine/threonine protein kinase implicated in development, proliferation and apoptosis regulation. Here we identified NLK as a gene product induced in the hearts of mice subjected to pressure overload or myocardial infarction injury, suggesting a potential regulatory role with pathological stimulation to this organ. To examine the potential functional consequences of increased NLK levels, cardiac-specific transgenic mice with inducible expression of this gene product were generated, as well as cardiac-specific Nlk gene-deleted mice. NLK transgenic mice demonstrated baseline cardiac hypertrophy, dilation, interstitial fibrosis, apoptosis and progression towards heart failure in response to two surgery-induced cardiac disease models. In contrast, cardiac-specific deletion of Nlk from the heart, achieved by crossing a Nlk-loxP allele containing mouse with either a mouse containing a ß-myosin heavy chain promoter driven Cre transgene or a tamoxifen inducible α-myosin heavy chain promoter containing transgene driving a MerCreMer cDNA, protected the mice from cardiac dysfunction following pathological stimuli. Mechanistically, NLK interacted with multiple proteins including the transcription factor Stat1, which was significantly increased in the hearts of NLK transgenic mice. These results indicate that NLK is a pathological effector in the heart.

Virus-induced gene silencing in plant MAPK research.

Virus-induced gene silencing (VIGS) technology has become more and more widely used in various plant species for rapid screening of gene functions. VIGS does not require time-consuming tissue culture steps that are needed for stable transformation in most plant species and it can be used for studying gene function even in plants that are very difficult to stably transform. Furthermore, VIGS technology provides high gene silencing efficiency (up to 95 %) and specificity. Here, we describe a VIGS protocol that can be used for studying the functions of MAPKs and other genes in a wild tobacco species, Nicotiana attenuata. This method is also suitable for other Nicotiana species and tomato with minor modifications.

RNA interference of plant MAPK cascades for functional studies.

Arabidopsis genome contains 20 genes encoding mitogen-activated protein kinases (MAPKs, or MPKs), and ten genes encoding MAPK kinases (MAPKKs, or MKKs), the upstream kinases that activate MAPKs in the signaling cascades. They play critical roles in many different biological processes ranging from growth/development to response to environmental stimuli and pathogen invasion. T-DNA knockout lines are not currently available for all these genes. There is also functional redundancy at both MAPK and MAPKK levels. In addition, embryo lethality is associated with some double mutant combinations, which makes it difficult to investigate their specific functions in plants. In such situation, the use of RNA interference technology by which mRNA of interested gene is targeted by double-stranded RNA (dsRNA) for degradation and gene silencing provides a powerful tool for loss-of-function analyses. In this chapter, we describe the hairpin-RNA interference (hpRNAi) method we employed to silence MPK3/MPK6 and their upstream MKK4/MKK5 in the model plant Arabidopsis, with particular emphasis on the generation of hpRNAi constructs for single gene RNAi, tandem RNAi of two MAPKK genes, and tissue-specific RNAi.

Control analysis of the eukaryotic cell cycle using gene copy-number series in yeast tetraploids.

BACKGROUND: In the model eukaryote, Saccharomyces cerevisiae, previous experiments have identified those genes that exert the most significant control over cell growth rate. These genes are termed HFC for high flux control. Such genes are overrepresented within pathways controlling the mitotic cell cycle. RESULTS: We postulated that the increase/decrease in growth rate is due to a change in the rate of progression through specific cell cycle steps. We extended and further developed an existing logical model of the yeast cell cycle in order elucidate how the HFC genes modulated progress through the cycle. This model can simulate gene dosage-variation and calculate the cycle time, determine the order and relative speed at which events occur, and predict arrests and failures to correctly execute a step. To experimentally test our model's predictions, we constructed a tetraploid series of deletion mutants for a set of eight genes that control the G2/M transition. This system allowed us to vary gene copy number through more intermediate levels than previous studies and examine the impact of copy-number variation on growth, cell-cycle phenotype, and response to different cellular stresses. CONCLUSIONS: For the majority of strains, the predictions agreed with experimental observations, validating our model and its use for further predictions. Where simulation and experiment diverged, we uncovered both novel tetraploid-specific phenotypes and a switch in the determinative execution point of a key cell-cycle regulator, the Cdc28 kinase, from the G1/S to the S/G2 boundaries.

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner.

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.

S100A9 interaction with TLR4 promotes tumor growth.

By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFß expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

A dominant allele of Arabidopsis pectin-binding wall-associated kinase induces a stress response suppressed by MPK6 but not MPK3 mutations.

The plant cell wall is composed of a matrix of cellulose fibers, flexible pectin polymers, and an array of assorted carbohydrates and proteins. The receptor-like Wall-Associated Kinases (WAKs) of Arabidopsis bind pectin in the wall, and are necessary both for cell expansion during development and for a response to pathogens and wounding. Mitogen Activated Protein Kinases (MPKs) form a major signaling link between cell surface receptors and both transcriptional and enzyme regulation in eukaryotes, and Arabidopsis MPK6 and MPK3 indeed have important roles in development and the response to stress and pathogens. A dominant allele of WAK2 requires kinase activity and activates a stress response that includes an increased ROS accumulation and the up-regulation of numerous genes involved in pathogen resistance, wounding, and cell wall biogenesis. This dominant allele requires a functional pectin binding and kinase domain, indicating that it is engaged in a WAK signaling pathway. A null mutant of the major plasma membrane ROS-producing enzyme complex, rbohd/f does not suppress the WAK2cTAP-induced phenotype. A mpk6, but not a mpk3, null allele is able to suppress the effects of this dominant WAK2 mutation, thus distinguishing MPK3 and MPK6, whose activity previously was thought to be redundant. Pectin activation of gene expression is abated in a wak2-null, but is tempered by the WAK-dominant allele that induces elevated basal stress-related transcript levels. The results suggest a mechanism in which changes to the cell wall can lead to a large change in cellular responses and help to explain how pathogens and wounding can have general effects on growth.

A "molecular evolution" approach for isolation of intrinsically active (MEK-independent) MAP kinases.

Mitogen-activated protein (MAP) kinases are a large family of enzymes composed of about four subfamilies, each containing several isoforms and splicing variants. Many MAP kinases are coexpressed in each eukaryotic cell and coactivated in response to various stimuli. It is, therefore, difficult to explore the specific downstream effects of each species of MAPK. Expression of an intrinsically active variant of a MAPK, while other MAPKs are not active, allows for tracking of a specific array of substrates, target genes, and biological/pathological effects corresponding to the expressed molecule. This chapter describes a method for obtaining such intrinsically active MAPKs. Because of the unique mode of MAPK activation, which is absolutely dependent on unconventional phosphorylation (on neighboring Thr + Tyr residues), a rational design of mutations that would render the kinase intrinsically active is currently unfeasible. Our method is based, therefore, on a "Molecular Evolution" approach that uses the power of yeast genetics and is unbiased toward the mutation sites. We describe in detail how to prepare a large population of randomly mutated molecules of the desired MAPK and how to screen this library in a yeast strain lacking the relevant MAPK kinase (MAPKK). The idea is to identify MAPK variants that are fulfilling all MAPK functions and allow growth of this strain - namely, MAPK molecules that function biologically in the complete absence of their upstream activator. We further describe the details of the "plasmid-loss" assay used for distinguishing between true positive and false positive clones. Finally, we report on a new yeast strain lacking four MAPKKs that could serve as a universal target for screening for active MAPK of all subfamilies.

The C-terminal acidic tail is responsible for the inhibitory effects of HMGB1 on efferocytosis.

HMGB1 was described originally as a nuclear protein involved in DNA binding and transcriptional regulation. However, HMGB1 also has an extracellular role as a potent mediator of inflammation and can diminish the uptake of apoptotic cells by phagocytes, a process called efferocytosis. To explore the mechanism responsible for the ability of HMGB1 to inhibit efferocytosis, we examined the role of the C-terminal acidic tail, a region of HMGB1 that has been shown to participate in specific intramolecular interactions. Deletion of the C-terminal tail abrogated the ability of HMGB1 to decrease murine macrophage ingestion of apoptotic neutrophils and to diminish phagocytosis-induced activation of Erk and Rac-1 in macrophages. We found that RAGE plays a major role in efferocytosis, and deletion of the C-terminal tail of HMGB1 prevented binding of HMGB1 to RAGE but not to other macrophage receptors involved in efferocytosis, such as the α(V)ß(3) integrin. Whereas HMGB1 decreased ingestion of apoptotic neutrophils significantly by alveolar macrophages under in vivo conditions in the lungs of mice, this effect was lost when the C-terminal acidic tail was absent from HMGB1. These results demonstrate that the HMGB1 C-terminal tail is responsible for the inhibitory effects of HMGB1 on phagocytosis of apoptotic neutrophils under in vitro and in vivo conditions.

RAGE and ICAM-1 cooperate in mediating leukocyte recruitment during acute inflammation in vivo.

The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.

RAGE-independent autoreactive B cell activation in response to chromatin and HMGB1/DNA immune complexes.

Increasing evidence suggests that the excessive accumulation of apoptotic or necrotic cellular debris may contribute to the pathology of systemic autoimmune disease. HMGB1 is a nuclear DNA-associated protein, which can be released from dying cells thereby triggering inflammatory processes. We have previously shown that IgG2a-reactive B cell receptor (BCR) transgenic AM14 B cells proliferate in response to endogenous chromatin immune complexes (ICs), in the form of the anti-nucleosome antibody PL2-3 and cell debris, in a TLR9-dependent manner, and that these ICs contain HMGB1. Activation of AM14 B cells by these chromatin ICs was inhibited by a soluble form of the HMGB1 receptor, RAGE-Fc, suggesting HMGB1-RAGE interaction was important for this response. To further explore the role of HMGB1 in autoreactive B cell activation, we assessed the capacity of purified calf thymus HMGB1 to bind dsDNA fragments and found that HMGB1 bound both CG-rich and CG-poor DNA. However, HMGB1-DNA complexes could not activate AM14 B cells unless HMGB1 was bound by IgG2a and thereby able to engage the BCR. To ascertain the role of RAGE in autoreactive B cell responses to chromatin ICs, we intercrossed AM14 and RAGE-deficient mice. We found that spontaneous and defined DNA ICs activated RAGE+ and RAGE(- ) AM14 B cells to a comparable extent. These results suggest that HMGB1 promotes B cell responses to endogenous TLR9 ligands through a RAGE-independent mechanism.

Abeta-dependent Inhibition of LTP in different intracortical circuits of the visual cortex: the role of RAGE.

Oligomeric amyloid-beta (Abeta) interferes with long-term potentiation (LTP) and cognitive processes, suggesting that Abeta peptides may play a role in the neuronal dysfunction which characterizes the early stages of Alzheimer's disease (AD). Multiple lines of evidence have highlighted RAGE (receptor for advanced glycation end-products) as a receptor involved in Abeta-induced neuronal and synaptic dysfunction. In the present study, we investigated the effect of oligomeric soluble Abeta1-42 on LTP elicited by the stimulation of different intracortical pathways in the mouse visual cortex. A variety of nanomolar concentrations (20-200 nM) of Abeta1-42 were able to inhibit LTP in cortical layer II-III induced by either white matter (WM-Layer II/III) or the layer II/III (horizontal pathway) stimulation, whereas the inhibition of LTP was more susceptible to Abeta1-42, which occurred at 20 nM of Abeta, when stimulating layer II-III horizontal pathway. Remarkably, cortical slices were resistant to nanomolar Abeta1-42 in the absence of RAGE (genetic deletion of RAGE) or blocking RAGE by RAGE antibody. These results indicate that nanomolar Abeta inhibits LTP expression in different neocortical circuits. Crucially, it is demonstrated that Abeta-induced reduction of LTP in different cortical pathways is mediated by RAGE.

The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling.

The kinases MSK1 and MSK2 are activated 'downstream' of the p38 and Erk1/2 mitogen-activated protein kinases. Here we found that MSK1 and MSK2 were needed to limit the production of proinflammatory cytokines in response to stimulation of primary macrophages with lipopolysaccharide. By inducing transcription of the mitogen-activated protein kinase phosphatase DUSP1 and the anti-inflammatory cytokine interleukin 10, MSK1 and MSK2 exerted many negative feedback mechanisms. Deficiency in MSK1 and MSK2 prevented the binding of phosphorylated transcription factors CREB and ATF1 to the promoters of the genes encoding interleukin 10 and DUSP1. Mice doubly deficient in MSK1 and MSK2 were hypersensitive to lipopolysaccharide-induced endotoxic shock and showed prolonged inflammation in a model of toxic contact eczema induced by phorbol 12-myristate 13-acetate. Our results establish MSK1 and MSK2 as key components of negative feedback mechanisms needed to limit Toll-like receptor-driven inflammation.

Transamidation by transglutaminase 2 transforms S100A11 calgranulin into a procatabolic cytokine for chondrocytes.

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.

Identification of Tup1 and Cyc8 mutations defective in the responses to osmotic stress.

In the yeast Saccharomyces cerevisiae, Tup1, in association with Cyc8 (Ssn6), functions as a general transcriptional corepressor. This repression is mediated by recruitment of the Tup1-Cyc8 complex to target promoters through sequence-specific DNA-binding proteins such as Sko1, which mediates the HOG pathway-dependent regulation. We identified tup1 and cyc8 mutant alleles as the suppressor of osmo-sensitivity of the hog1Delta strain. In these mutants, although the expression of the genes under the control of DNA-binding proteins other than Sko1 was apparently normal, the Sko1-regulated genes GRE2 and AHP1 were derepressed under non-stress conditions, suggesting that the Tup1 and Cyc8 mutant proteins were specifically defective in the repression of the Sko1-dependent genes. Chromatin immunoprecipitation analyses of the GRE2 promoter in the mutants demonstrated that the Sko1-Tup1-Cyc8 complex was localized to the promoter, together with Gcn5/SAGA, suggesting that the erroneous recruitment of SAGA to the promoter led to the derepression.

MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.

The mating pathway in Saccharomyces cerevisiae has been the focus of considerable research effort, yet many quantitative aspects of its regulation still remain unknown. Using an integrated approach involving experiments in microfluidic chips and computational modelling, we studied gene expression and phenotypic changes associated with the mating response under well-defined pheromone gradients. Here we report a combination of switch-like and graded pathway responses leading to stochastic phenotype determination in a specific range of pheromone concentrations. Furthermore, we show that these responses are critically dependent on mitogen-activated protein kinase (MAPK)-mediated regulation of the activity of the pheromone-response-specific transcription factor, Ste12, as well as on the autoregulatory feedback of Ste12. In particular, both the switch-like characteristics and sensitivity of gene expression in shmooing cells to pheromone concentration were significantly diminished in cells lacking Kss1, one of the MAP kinases activated in the mating pathway. In addition, the dynamic range of gradient sensing of Kss1-deficient cells was reduced compared with wild type. We thus provide unsuspected functional significance for this kinase in regulation of the mating response.

Characterization and complementation of a Fus3/Kss1 type MAPK from Tuber borchii, TBMK.

Mitogen-activated protein kinases (MAPK) are used by organisms to transduce extra cellular signals from the environment in cellular events such as proliferation and differentiation. In the present study, we have characterized the first MAPK from the ectomycorrhizal fungus Tuber borchii (TBMK) which belongs to the YERK1 (yeast extra cellular regulated kinase) subfamily. TBMK is present as a single copy in the genome and the codified protein was phosphorylated during the interaction with the host plant, Tilia americana. Complementation studies showed that TBMK restores pheromone signaling in Saccharomyces cerevisiae and partially restores invasive growth of Fusarium oxysporum that lack the fmk1 gene. This suggests a protein kinase activity and its involvement in the infection processes. Hence, TBMK could play an important role during the pre-symbiotic phase of T. borchii with its host plant in the modulation of genes necessary for the establishment of symbiosis leading to the synthesis of functional ectomycorrhizae.