Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS1)

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophoresis and gel-staining with ProQ Diamond and the protein was digested by either trypsin or chymotrypsin for maximum sequence coverage to facilitate identification of phosphorylated positions. Prior to analysis by mass spectrometry, samples were either desalted, passed over TiO(2) or both for improved phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine residues (Ser72, Ser108, Ser120) in the phosphorylated form.     

An S-locus receptor-like kinase plays a role as a negative regulator in plant defense responses

Plant cells often use cell surface receptors to sense environmental changes and then transduce external signals via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, and some of these are induced by pathogen infection, suggesting a possible role in plant defense responses. We previously characterized an S-locus RLK (CBRLK1) at the biochemical level. In this study, we examined the physiological function of CBRLK1 in defense responses. CBRLK1 mutant and CBRLK1-overexpressing transgenic plants showed enhanced and reduced resistance against a virulent bacterial pathogen, respectively. The altered pathogen resistances of the mutant and overexpressing transgenic plants were associated with increased and reduced induction of the pathogenesis-related gene PR1, respectively. These results suggest that CBRLK1 plays a negative role in the disease resistance signaling pathway in Arabidopsis.     

Sef is a spatial regulator for Ras/MAP kinase signaling

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is among the key mechanisms for regulating a wide variety of cellular processes. In this study, we report that human Sef (hSef), a recently identified inhibitor whose action mechanism has not been fully defined, acts as a molecular switch for ERK signaling by specifically blocking ERK nuclear translocation without inhibiting its activity in the cytoplasm. Thus, hSef binds to activated forms of MEK, inhibits the dissociation of the MEK-ERK complex, and blocks nuclear translocation of activated ERK. Consequently, hSef inhibits phosphorylation and activation of the nuclear ERK substrate Elk-1, while it does not affect phosphorylation of the cytoplasmic ERK substrate RSK2. Downregulation of endogenous hSef by hSef siRNA enhances the stimulus-induced ERK nuclear translocation and the activity of Elk-1. These results thus demonstrate that hSef acts as a spatial regulator for ERK signaling by targeting ERK to the cytoplasm.     

The common metabolite glycerol-3-phosphate is a novel regulator of plant defense signaling

Conversion of glycerol to glycerol-3-phosphate (G3P) is one of the highly conserved steps of glycerol metabolism in evolutionary diverse organisms. In plants, G3P is produced either via the glycerol kinase (GK)-mediated phosphorylation of glycerol, or via G3P dehydrogenase (G3Pdh)-mediated reduction of dihydroxyacetone phosphate (DHAP). We have recently shown that G3P levels contribute to basal resistance against the hemibiotrophic pathogen, Colletotrichum higginsianum. Since a mutation in the GLY1-encoded G3Pdh conferred more susceptibility compared to a mutation in the GLI1-encoded GK, we proposed that GLY1 is the major contributor of the total G3P pool that participates in defense against C. higginsianum.Key words: glycerol-3-phosphate, glycerol metabolism, defense, signalingGlycerol and its metabolites are involved in a variety of physiopathological processes in both prokaryotes and eukaryotes, most of which appear to be highly conserved,¹ signifying the fundamental importance of these molecules. Glycerol-3-phosphate (G3P), an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis, participates in the disease-related physiologies of many organisms. In humans, deficiencies in glycerol kinase activity (catalyzing the phosphorylation of glycerol to G3P) result in a variety of metabolic and neurological disorders, while mutations in G3P dehydrogenase (G3Pdh, catalyzing the oxidation of dihydroxyacetone phosphate, DHAP, to G3P) have been linked to sudden infant death syndrome and decreased cardiac Na²⁺ current resulting in ventricular arrhythmias and sudden death.^2,3 Given the fact that glycerol metabolism is conserved between plants and animals, it is conceivable that glycerol and/or G3P might also participate in disease physiology of plants. However, such a role for glycerol and/or G3P remains unexplored.Previous work from our laboratory and others has shown that GLY1-encoded G3Pdh plays an important role in plastidal oleic acid-mediated signaling^4–7 and systemic acquired resistance.⁸ This group of enzymes also plays an important role in fungi and it was recently shown that the disruption of a G3Pdh gene in Colletotrichum gloeosporioides eliminated the ability of the mutant fungus to grow on most carbon sources in vitro, including amino acids and glucose.⁹ However, the G3Pdh knockout (KO) fungus grew normally in the presence of glycerol. The G3Pdh KO fungus also developed normally in its plant host (the round-leaved mallow), prompting the suggestion that glycerol, rather than glucose or sucrose, was the primary transferred source of carbon in planta. This was an unexpected finding, but direct analysis of infected host leaves revealed that their glycerol content did decrease by 40% within 48 hours of infection with C. gloeosporioides.⁹ Since the hemibiotroph C. gloeosporioides appears to be able to utilize glycerol for growth and conidiation in planta, it was possible that glycerol metabolism and associated pathways in the host played an important role in the establishment of infections by Colletotrichum fungi. Furthermore, it was possible that the host had evolved to sense these pathogen-mediated changes in glycerol levels and utilize them as signal(s) to initiate defense.We tested these possibilities by characterizing the role of glycerol metabolism in the Arabidopsis—C. higginsianum interaction (Fig. 1). Infection with C. higginsianum reduced the glycerol content while concomitantly increasing the G3P content in Arabidopsis plants.¹⁰ Mutations in G3P-synthesizing genes gly1 (a G3Pdh) and gli1 (a glycerol kinase),¹¹ resulted in enhanced susceptibility to C. higginsianum. The gly1 plants were much more susceptible than the gli1 plants, suggesting that GLY1-encoded G3Pdh played a more important role in basal resistance to C. higginsianum. Conversely, the act1 mutant, which is impaired in the acylation of G3P with oleic acid (18:1) (Fig. 1), was more resistant to the fungus. The phenotypes seen in the infected gly1 and act1 plants correlated with pathogen-induced G3P levels; C. higginsianum inoculation induced ∼2-fold higher accumulation of G3P in the act1 plants, and ∼2-fold lower G3P in the susceptible gly1 plants, as compared to wild-type plants.¹⁰ To test the hypothesis that G3P synthesized via GLY1 entered the plastidial glycerolipid pathway via the ACT1 catalyzed reaction, we generated act1 gly1 plants. The results supported the hypothesis, as act1 gly1 plants were as susceptible to C. higginsianum as gly1 plants.Open in a separate windowFigure 1A condensed scheme of glycerol metabolism in plants. Glycerol is phosphorylated to glycerol-3-phosphate (G3P) by glycerol kinase (GK; GLI1). G3P can also be generated by G3P dehydrogenase (G3Pdh) via the reduction of dihydroxyacetone phosphate (DHAP) in both the cytosol and the plastids (represented by the oval). G3P generated by this reaction can be transported between the cytosol and plastid stroma. In the plastids G3P is acylated with oleic acid (18:1) by the ACT1-encoded G3P acyltransferase. This ACT1-utilized 18:1 is derived from the stearoyl-acyl carrier protein (ACP)-desaturase (SSI2)-catalyzed desaturation of stearic acid (18:0). The 18:1-ACP generated by SSI2 either enters the prokaryotic lipid biosynthetic pathway through acylation of G3P, or is exported out of the plastids as a coenzyme A (CoA)-thioester to enter the eukaryotic lipid biosynthetic pathway. Other abbreviations used are: PA, phosphatidic acid; Lyso-PA, acyl-G3P; PG, phosphatidylglycerol; MGD, monogalactosyldiacylglycerol; DGD, digalactosyl-diacylglycerol; SL, sulfolipid; DAG, diacylglycerol; DHA, dihydroxyacetone; Gl-3-P, glyceraldehyde-3-phosphate; TCA, tricarboxylic acid cycle. Enzymes as abbreviated as: ACT1, G3P acyltransferase; SSI2, stearoyl acyl carrier protein desaturase; GK, glycerol kinase; G3Pdh, G3P dehydrogenase; TPI, triose phosphate isomerase; DHAK, dihydroxyacetone kinase; F1,6-A, fructose 1,6-biphosphate aldolase; PF6P-P, pyrophosphate fructose-6-phosphate phosphotransferase; G6P-I, glucose-6-phosphate isomerase.More supporting evidence for the role of G3P in defense against C. higginsianum was obtained by overexpressing GLY1 in wild-type plants (Fig. 2A). Similar to act1, overexpression of GLY1 led to a ∼2-fold increase in G3P levels after pathogen inoculation, and these plants were also more resistant to C. higginsianum (Fig. 2B–D). Furthermore, plants overexpressing GLY1 or carrying a mutation in ACT1 exhibited enhanced resistance to C. higginsianum in the pad3 mutant background (Fig. 3).¹⁰ The pad3 plants are compromised in camalexin synthesis, and are hypersusceptible to necrotrophic pathogens.Open in a separate windowFigure 2Pathogen response and G3P levels in transgenic lines overexpressing GLY1. (A) Expression of the GLY1 gene in wild-type or 35S-GLY1 transgenic plant. RNA gel blot analysis was performed on ∼7 µg of total RNA. Ethidium bromide staining of rRNA was used as a loading control. (B) Disease symptoms in C. higginsianum-inoculated Col-0, gly1 or 35S-GLY1 plants at 5 dpi. The plants were spray-inoculated with 10⁶ spores/ml of C. higginsianum. (C) Lesion size in spot-inoculated genotypes. The plants were spot-inoculated with water or 10⁶ spores/ml and the lesion size was measured from 20–30 independent leaves at 6 dpi. Statistical significance was determined using Students t-test. Asterisks indicate data that is statistically significant from that of control (Col-0) (p < 0.05). Error bars indicate SD. (D) G3P levels in Col-0 and 35S-GLY1 plants at 0 and 72 h post-inoculation.Open in a separate windowFigure 3Pathogen response in C. higginsianum-inoculated 35S-GLY1 plants in pad3 background. (A) Disease symptoms on Col-0, pad3 or 35S-GLY1 or 35S-GLY1 pad3 plants spot-inoculated with 10⁶ spores/ml of C. higginsianum. The leaves were photographed at 7 dpi. (B) Lesion size in spot-inoculated Col-0, pad3 or 35S-GLY1 or 35S-GLY1 pad3 plants. The lesion size was measured from 20–30 independent leaves at 7 dpi. Asterisks indicate data that is statistically significant from that of control (Col-0) (p < 0.05). Error bars indicate SD.Exogenous glycerol application increased endogenous G3P and significantly enhanced the ability of the host to resist C. higginsianum.¹⁰ Glycerol-triggered synthesis of G3P also caused a decrease in 18:1 levels, which is known to induce defense signaling, resulting in enhanced basal resistance.^4–7 However, the glycerol-triggered increase in G3P precedes the reduction in 18:1 levels and confers resistance even at time points when low 18:1-mediated signaling is not induced, suggesting that the enhanced resistance after glycerol treatment was due to elevated G3P levels and not to the reduction in 18:1.Understanding the precise roles of G3P will require in-depth analysis of real-time alterations in its levels on a cellular level during pathogenesis. This is complicated by the presence of multiple isoforms of G3Pdh that contribute to the total G3P pool, and by the lack of appropriate tools for monitoring precise changes in intracellular G3P. Systematic analysis of various G3Pdh mutants, in combination with each other and with gli1, should yield novel insights into pathway(s) and steps regulating levels of G3P in the cell.     

A Caenorhabditis elegans MAP kinase kinase, MEK-1, is involved in stress responses

The c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, was shown to be involved in the response to various stresses in cultured cells. However, there is little in vivo evidence indicating a role for a JNK pathway in the stress response of an organism. We identified the Caenorhabditis elegans mek-1 gene, which encodes a 347 amino acid protein highly homologous to mammalian MKK7, an activator of JNK. Mek-1 reporter fusion proteins are expressed in pharyngeal muscle, uterus, a portion of intestine, and neurons. A mek-1 deletion mutant is hypersensitive to copper and cadmium ions and to starvation. A wild-type mek-1 transgene rescued the hypersensitivity to the metal ions. Double mutants of mek-1 with an eat-5, eat-11 or eat-18 mutation, which are characterized by a limited feeding defect, showed distinct growth defects under normal conditions. Expression of an activated form of MEK-1 in the whole animal or specifically in the pharynx inhibited pharyngeal pumping. These results suggest a role for mek-1 in stress responses, with a focus in the pharynx and/or intestine.     

Arabidopsis MAP kinase phosphatase 1 is phosphorylated and activated by its substrate AtMPK6

Arabidopsis MAP kinase phosphatase 1 (AtMKP1) is a member of the mitogen-activated protein kinase (MPK) phosphatase family, which negatively regulates AtMPKs. We have previously shown that AtMKP1 is regulated by calmodulin (CaM). Here, we examined the phosphorylation of AtMKP1 by its substrate AtMPK6. Intriguingly, AtMKP1 was phosphorylated by AtMPK6, one of AtMKP1 substrates. Four phosphorylation sites were identified by phosphoamino acid analysis, TiO(2) chromatography and mass spectrometric analysis. Site-directed mutation of these residues in AtMKP1 abolished the phosphorylation by AtMPK6. In addition, AtMKP1 interacted with AtMPK6 as demonstrated by the yeast two-hybrid system. Finally, the phosphatase activity of AtMKP1 increased approximately twofold following phosphorylation by AtMPK6. By in-gel kinase assays, we showed that AtMKP1 could be rapidly phosphorylated by AtMPK6 in plants. Our results suggest that the catalytic activity of AtMKP1 in plants can be regulated not only by Ca(2+)/CaM, but also by its physiological substrate, AtMPK6.     

The plant homologue of MAP kinase is expressed in a cell cycle-dependent and organ-specific manner

In animals, MAP kinase plays a key role in growth factor-stimulated signalling and in mitosis. The isolation of a Medicago sativa cDNA clone MsK7 which shows 52% identity to animal MAP kinases is reported. The deduced protein sequence shows all the important structural features of MAP kinases and also contains the highly conserved Thr-183 and Tyr-185 residues. Northern analysis of synchronized alfalfa cells showed that the MsK7 kinase gene is expressed at low levels in G1 phase but at higher levels in S and G2 phases of the cell cycle. In the plant, only stems and roots were found to contain MAP kinase MsK7 mRNA. Southern and PCR analyses indicated that alfalfa contains at least four highly related MAP kinase genes.     

MAP Kinase analyser: A tool for plant kinase and substrate analysis

MAPK (Mitogen Activated Protein Kinase) is a Ser/Thr kinase, which plays a crucial role in plant growth and development, transferring the extra cellular stimuli into intracellular response etc. Manual identification of these MAPK in the plant genome is tedious and time taking process. There are number of online servers which predict the P-site (phosphorylation site), find the motifs and domain but there is no specific tool which can identify all them together. In order to identify the P-Site, phosphorylation site consensus sequences and domain of the MAPK in plant genome, we developed a tool, MAP Kinase analyzer. MAP kinase analyzer take protein sequence as input in the fasta format and the output of tool includes: 1) The prediction of the phosphorylation site viz., Serine (S), Threonine (T), and Tyrosine (Y), Contex, Position, Score and phosphorylating kinase as well as the graphical output; 2) Phosphorylation site consensus sequence pattern for different kinases and 3) Domain information about the MAPK's. The MAP kinase analyser tool and supplementary files can be downloaded from http://www.bioinfogbpuat/mapk_OWN_1/.     

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3.     

Trihydrophobin 1 is a new negative regulator of A-Raf kinase

Our previous work indicated that instead of binding to B-Raf or C-Raf, trihydrophobin 1 (TH1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1-372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase-dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G(0)/G(1) phase-arrested. Taken together, our data provide a clue to understanding the cellular function of TH1 on Raf isoform-specific regulation.     

MAP kinase phosphorylation of plant profilin

Profilin is a small actin-binding protein and is expressed at high levels in mature pollen where it is thought to regulate actin filament dynamics upon pollen germination and tube growth. The majority of identified plant profilins contain a MAP kinase phosphorylation motif, P-X-T-P, and a MAP kinase interaction motif (KIM). In in vitro kinase assays, the tobacco MAP kinases p45(Ntf4) and SIPK, when activated by the tobacco MAP kinase kinase NtMEK2, can phosphorylate the tobacco profilin NtProf2. Mutagenesis of the threonine residue in this motif identified it as the site of MAP kinase phosphorylation. Fractionation of tobacco pollen extracts showed that p45(Ntf4) is found exclusively in the high-speed pellet fraction while SIPK and profilin are predominantly cytosolic. These data identify one of the first substrates to be directly phosphorylated by MAP kinases in plants.     

B1-phytoprostanes trigger plant defense and detoxification responses

Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B(1)-phytoprostanes (PPB(1)) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB(1)-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB(1) regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB(1) considerably prevented cell death caused by severe CuSO(4) poisoning. Several Arabidopsis genes induced by PPB(1), such as those coding for adenylylsulfate reductase, tryptophan synthase beta-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB(1) treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB(1). In three different plant species, PPB(1)-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB(1)-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB(1) represent stress signals that improve plants capacity to cope better with a variety of stresses.     

PSKR1 and PSY1R-mediated regulation of plant defense responses

Plant peptide signaling is an upcoming topic in many areas of plant research. Our recent findings show that the tyrosine sulfated peptide receptors PSKR1 and PSY1R are not only involved in growth and development but also in plant defense. They modulate salicylate- and jasmonate-dependent defense pathways in an antagonistic manner and this phenomenon might be dependent on the age and developmental stage of the plant. Here we discuss how the endogenous peptides might integrate growth, wounding, senescence and the opposing defense pathways against biotrophic and necrotrophic pathogens for increased fitness of the plant.