A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C.

Mitogen-activated protein (MAP) kinases are activated in response to a variety of stimuli through a protein kinase cascade that results in their phosphorylation on tyrosine and threonine residues. The molecular nature of this cascade is just beginning to emerge. Here we report the isolation of a Saccharomyces cerevisiae gene encoding a functional analog of mammalian MAP kinases, designated MPK1 (for MAP kinase). The MPK1 gene was isolated as a dosage-dependent suppressor of the cell lysis defect associated with deletion of the BCK1 gene. The BCK1 gene is also predicted to encode a protein kinase which has been proposed to function downstream of the protein kinase C isozyme encoded by PKC1. The MPK1 gene possesses a 1.5-kb uninterrupted open reading frame predicted to encode a 53-kDa protein. The predicted Mpk1 protein (Mpk1p) shares 48 to 50% sequence identity with Xenopus MAP kinase and with the yeast mating pheromone response pathway components, Fus3p and Kss1p. Deletion of MPK1 resulted in a temperature-dependent cell lysis defect that was virtually indistinguishable from that resulting from deletion of BCK1, suggesting that the protein kinases encoded by these genes function in a common pathway. Expression of Xenopus MAP kinase suppressed the defect associated with loss of MPK1 but not the mating-related defects associated with loss of FUS3 or KSS1, indicating functional conservation between the former two protein kinases. Mutation of the presumptive phosphorylated tyrosine and threonine residues of Mpk1p individually to phenylalanine and alanine, respectively, severely impaired Mpk1p function. Additional epistasis experiments, and the overall architectural similarity between the PKC1-mediated pathway and the pheromone response pathway, suggest that Pkc1p regulates a protein kinase cascade in which Bck1p activates a pair of protein kinases, designated Mkk1p and Mkk2p (for MAP kinase-kinase), which in turn activate Mpk1p.     

Fig1p facilitates Ca2+ influx and cell fusion during mating of Saccharomyces cerevisiae

During the mating process of yeast cells, two Ca2+ influx pathways become activated. The resulting elevation of cytosolic free Ca2+ activates downstream signaling factors that promote long term survival of unmated cells, but the roles of Ca2+ in conjugation have not been described. The high affinity Ca2+ influx system is composed of Cch1p and Mid1p and sensitive to feedback inhibition by calcineurin, a Ca2+/calmodulin-dependent protein phosphatase. To identify components and regulators of the low affinity Ca2+ influx system (LACS), we screened a collection of pheromone-responsive genes that when deleted lead to defects in LACS activity but not high affinity Ca2+ influx system activity. Numerous factors implicated in polarized morphogenesis and cell fusion (Fus1p, Fus2p, Rvs161p, Bni1p, Spa2p, and Pea2p) were found to be necessary for LACS activity. Each of these factors was also required for activation of the cell integrity mitogen-activated protein kinase cascade during the response to alpha-factor. Interestingly a polytopic plasma membrane protein, Fig1p, was required for LACS activity but not required for activation of Mpk1p mitogen-activated protein kinase. Mpk1p was not required for LACS activity, suggesting Mpk1p and Fig1p define two independent branches in the pheromone response pathways. Fig1p-deficient mutants exhibit defects in the cell-cell fusion step of mating, but unlike other fus1 and fus2 mutants the fusion defect of fig1 mutants can be largely suppressed by high Ca2+ conditions, which bypass the requirement for LACS. These findings suggest Fig1p is an important component or regulator of LACS and provide the first evidence for a role of Ca2+ signals in the cell fusion step of mating.     

MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants

The Arabidopsis (Arabidopsis thaliana) gene MEKK1 encodes a mitogen-activated protein kinase kinase kinase that has been implicated in the activation of the map kinases MPK3 and MPK6 in response to the flagellin elicitor peptide flg22. In this study, analysis of plants carrying T-DNA knockout alleles indicated that MEKK1 is required for flg22-induced activation of MPK4 but not MPK3 or MPK6. Experiments performed using a kinase-impaired version of MEKK1 (K361M) showed that the kinase activity of MEKK1 may not be required for flg22-induced MPK4 activation or for other macroscopic FLS2-mediated responses. MEKK1 may play a structural role in signaling, independent of its protein kinase activity. mekk1 knockout mutants display a severe dwarf phenotype, constitutive callose deposition, and constitutive expression of pathogen response genes. This dwarf phenotype was largely rescued by introduction into mekk1 knockout plants of either the MEKK1 (K361M) construct or a nahG transgene that degrades salicylic acid. When treated with pathogenic bacteria, the K361M plants were slightly more susceptible to an avirulent strain of Pseudomonas syringae and showed a delayed hypersensitive response, suggesting a role for MEKK1 kinase activity in this aspect of plant disease resistance. Our results indicate that MEKK1 acts upstream of MPK4 as a negative regulator of pathogen response pathways, a function that may not require MEKK1's full kinase activity.     

MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis

Innate immunity signaling pathways in both animals and plants are regulated by mitogen-activated protein kinase (MAPK) cascades. An Arabidopsis MAPK cascade (MEKK1, MKK4/MKK5, and MPK3/MPK6) has been proposed to function downstream of the flagellin receptor FLS2 based on biochemical assays using transient overexpression of candidate components. To genetically test this model, we characterized two mekk1 mutants. We show here that MEKK1 is not required for flagellin-triggered activation of MPK3 and MPK6. Instead, MEKK1 is essential for activation of MPK4, a MAPK that negatively regulates systemic acquired resistance. We also showed that MEKK1 negatively regulates temperature-sensitive and tissue-specific cell death and H(2)O(2) accumulation that are partly dependent on both RAR1, a key component in resistance protein function, and SID2, an isochorismate synthase required for salicylic acid production upon pathogen infection.     

Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast

Endoplasmic reticulum (ER) stress in the budding yeast Saccharomyces cerevisiae triggers Ca2+ influx through a plasma membrane channel composed of Cch1 and Mid1. This response activates calcineurin, which helps to prevent cell death during multiple forms of ER stress, including the response to azole-class antifungal drugs. Herein, we show that ER stress activates the cell integrity mitogen-activate protein kinase cascade in yeast and that the activation of Pkc1 and Mpk1 is necessary for stimulation of the Cch1-Mid1 Ca2+ channel independent of many known targets of Mpk1 (Rlm1, Swi4, Swi6, Mih1, Hsl1, and Swe1). ER stress generated in response to miconazole, tunicamycin, or other inhibitors also triggered a transient G2/M arrest that depended upon the Swe1 protein kinase. Calcineurin played little role in the Swe1-dependent cell cycle arrest and Swe1 had little effect on calcineurin-dependent avoidance of cell death. These findings help to clarify the interactions between Mpk1, calcineurin, and Swe1 and suggest that the calcium cell survival pathway promotes drug resistance independent of both the unfolded protein response and the G2/M cell cycle checkpoint.     

Toxicity of Linoleic Acid Hydroperoxide to Saccharomyces cerevisiae: Involvement of a Respiration-Related Process for Maximal Sensitivity and Adaptive Response

Linoleic acid hydroperoxide (LoaOOH) formed during free radical attack on long-chain unsaturated fatty acids is an important source of biomembrane damage and is implicated in the onset of atherosclerosis, hepatic diseases, and food rancidity. LoaOOH is toxic to wild-type Saccharomyces cerevisiae at a very low concentration (0.2 mM) relative to other peroxides. By using isogenic mutant strains, the possible roles of glutathione (gsh1 and gsh2), glutathione reductase (glr1), respiratory competence ([rho⁰] petite), and yAP-1p-mediated expression (yap1) in conferring LoaOOH resistance have been examined. Respiration-related processes were essential for maximal toxicity and adaptation, as evidenced by the fact that the [rho⁰] petite mutant was most resistant to LoaOOH but could not adapt. Furthermore, when respiration was blocked by using inhibitors of respiration and mutants defective in respiratory-chain components, cells became more resistant. An important role for reduced glutathione and yAP-1 in the cellular response to LoaOOH was shown, since the yap1 and glr1 mutants were more sensitive than the wild type. In addition, total glutathione peroxidase activity increased following treatment with LoaOOH, indicating a possible detoxification role for this enzyme. Yeast also showed an adaptive response when pretreated with a nonlethal dose of LoaOOH (0.05 mM) and subsequently treated with a lethal dose (0.2 mM), and de novo protein synthesis was required, since adaptation was abolished upon treatment of cells with cycloheximide (25 μg ml⁻¹). The wild-type adaptive response to LoaOOH was independent of those for the superoxide-generating agents paraquat and menadione and also of those for the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide. Pretreatment with LoaOOH induced resistance to hydrogen peroxide, while pretreatment of cells with malondialdehyde (a lipid peroxidation product) and heat shock (37°C) gave cross-adaptation to LoaOOH, indicating that yeast has effective overlapping defense systems that can detoxify fatty acid hydroperoxides directly or indirectly.     

The fission yeast pmk1+ gene encodes a novel mitogen-activated protein kinase homolog which regulates cell integrity and functions coordinately with the protein kinase C pathway.

We have isolated a gene, pmk1+, a third mitogen-activated protein kinase (MAPK) gene homolog from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence shows the most homology (63 to 65% identity) to those of budding yeast Saccharomyces Mpk1 and Candida Mkc1. The Pmk1 protein contains phosphorylated tyrosines, and the level of tyrosine phosphorylation was increased in the dsp1 mutant which lacks an attenuating phosphatase for Pmk1. The level of tyrosine phosphorylation appears constant during hypotonic or heat shock treatment. The cells with pmk1 deleted (delta pmk1) are viable but show various defective phenotypes, including cell wall weakness, abnormal cell shape, a cytokinesis defect, and altered sensitivities to cations, such as hypersensitivity to potassium and resistance to sodium. Consistent with a high degree of conservation of amino acid sequence, multicopy plasmids containing the MPK1 gene rescued the defective phenotypes of the delta pmk1 mutant. The frog MAPK gene also suppressed the pmk1 disruptant. The results of genetic analysis indicated that Pmk1 lies on a novel MAPK pathway which does not overlap functionally with the other two MAPK pathways, the Spk1-dependent mating signal pathway and Sty1/Spc1/Phh1-dependent stress-sensing pathway. In Saccharomyces cerevisiae, Mpk1 is involved in cell wall integrity and functions downstream of the protein kinase C homolog. In contrast, in S. pombe, Pmk1 may not act in a linear manner with respect to fission yeast protein kinase C homologs. Interestingly, however, these two pathways are not independent; instead, they regulate cell integrity in a coordinate manner.     

Multiple signaling pathways regulate yeast cell death during the response to mating pheromones

Mating pheromones promote cellular differentiation and fusion of yeast cells with those of the opposite mating type. In the absence of a suitable partner, high concentrations of mating pheromones induced rapid cell death in approximately 25% of the population of clonal cultures independent of cell age. Rapid cell death required Fig1, a transmembrane protein homologous to PMP-22/EMP/MP20/Claudin proteins, but did not require its Ca2+ influx activity. Rapid cell death also required cell wall degradation, which was inhibited in some surviving cells by the activation of a negative feedback loop involving the MAP kinase Slt2/Mpk1. Mutants lacking Slt2/Mpk1 or its upstream regulators also underwent a second slower wave of cell death that was independent of Fig1 and dependent on much lower concentrations of pheromones. A third wave of cell death that was independent of Fig1 and Slt2/Mpk1 was observed in mutants and conditions that eliminate calcineurin signaling. All three waves of cell death appeared independent of the caspase-like protein Mca1 and lacked certain "hallmarks" of apoptosis. Though all three waves of cell death were preceded by accumulation of reactive oxygen species, mitochondrial respiration was only required for the slowest wave in calcineurin-deficient cells. These findings suggest that yeast cells can die by necrosis-like mechanisms during the response to mating pheromones if essential response pathways are lacking or if mating is attempted in the absence of a partner.     

Regulation of the cell integrity pathway by rapamycin-sensitive TOR function in budding yeast

The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that in Saccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G(0).     

Silencing of the mitogen-activated protein kinase MPK6 compromises disease resistance in Arabidopsis

Here, we use a loss-of-function approach to demonstrate that the Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) MPK6 plays a role in resistance to certain pathogens. MPK6-silenced Arabidopsis showed no apparent morphological phenotype or reduced fertility, indicating MPK6 is not required for development. However, resistances to an avirulent strain of Peronospora parasitica and avirulent and virulent strains of Pseudomonas syringae were compromised, suggesting that MPK6 plays a role in both resistance gene-mediated and basal resistance. Furthermore, this result demonstrates that MPK6's function cannot be fully complemented by other endogenous MAPKs. Although MPK6-silenced plants exhibited enhanced disease susceptibility, their ability to develop systemic acquired resistance or induced systemic resistance was unaffected. Expression of the pathogen-inducible gene VEGETATIVE STORAGE PROTEIN1 (VSP1) in MPK6-silenced plants was severalfold lower than in control plants, but the expression of other defense genes was comparable to the level observed in control plants. Taken together, these results provide direct evidence that a specific MAPK positively regulates VSP1 expression and resistance to a primary infection by certain pathogens, whereas systemic resistance and expression of several other defense genes appears to be mediated either by a functionally redundant MAPK(s) or independently from MPK6-dependent resistance.     

Murine protein serine/threonine kinase 38 activates apoptosis signal-regulating kinase 1 via Thr 838 phosphorylation

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.     

YIL113w encodes a functional dual-specificity protein phosphatase which specifically interacts with and inactivates the Slt2/Mpk1p MAP kinase in S. cerevisiae

We show here that the YIL113w gene of Saccharomyces cerevisiae encodes a functional protein phosphatase. Yil113p shows no activity in vitro towards either phosphorylated casein or myelin basic protein. However, Yil113p dephosphorylates activated extracellular signal-regulated kinase 2 MAP kinase indicating that it is a dual-specificity MAP kinase phosphatase. In support of this we find that Yil113p specifically interacts with the stress-activated Slt2/Mpk1p MAP kinase of S. cerevisiae. Furthermore, expression of Yil113p causes the dephosphorylation of Slt2/Mpk1p in vivo, while expression of an inactive mutant of Yil113p causes the accumulation of phosphorylated Slt2/Mpk1p. We conclude that the physiological target of YIL113p is Slt2/Mpk1p.     

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.