Casein Kinase 2 Dependent Phosphorylation of Neprilysin Regulates Receptor Tyrosine Kinase Signaling to Akt

Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling.     

Salicylic Acid Regulates Plasmodesmata Closure during Innate Immune Responses in Arabidopsis

In plants, mounting an effective innate immune strategy against microbial pathogens involves triggering local cell death within infected cells as well as boosting the immunity of the uninfected neighboring and systemically located cells. Although not much is known about this, it is evident that well-coordinated cell–cell signaling is critical in this process to confine infection to local tissue while allowing for the spread of systemic immune signals throughout the whole plant. In support of this notion, direct cell-to-cell communication was recently found to play a crucial role in plant defense. Here, we provide experimental evidence that salicylic acid (SA) is a critical hormonal signal that regulates cell-to-cell permeability during innate immune responses elicited by virulent bacterial infection in Arabidopsis thaliana. We show that direct exogenous application of SA or bacterial infection suppresses cell–cell coupling and that SA pathway mutants are impaired in this response. The SA- or infection-induced suppression of cell–cell coupling requires an ENHANCED DESEASE RESISTANCE1– and NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1–dependent SA pathway in conjunction with the regulator of plasmodesmal gating PLASMODESMATA-LOCATED PROTEIN5. We discuss a model wherein the SA signaling pathway and plasmodesmata-mediated cell-to-cell communication converge under an intricate regulatory loop.     

AtNUDX6, an ADP-Ribose/NADH Pyrophosphohydrolase in Arabidopsis,Positively Regulates NPR1-Dependent Salicylic Acid Signaling

Here, we investigated the physiological role of Arabidopsis (Arabidopsis thaliana) AtNUDX6, the gene encoding ADP-ribose (Rib)/NADH pyrophosphohydrolase, using its overexpressor (Pro_35S:AtNUDX6) or disruptant (KO-nudx6). The level of NADH in Pro_35S:AtNUDX6 and KO-nudx6 plants was decreased and increased, respectively, compared with that of the control plants, while the level of ADP-Rib was not changed in either plant. The activity of pyrophosphohydrolase toward NADH was enhanced and reduced in the Pro_35S:AtNUDX6 and KO-nudx6 plants, respectively. The decrease in the activity of NADH pyrophosphohydrolase and the increase in the level of NADH were observed in the rosette and cauline leaves, but not in the roots, of the KO-nudx6 plants. Notably, the expression level of AtNUDX6 and the activity of NADH pyrophosphohydrolase in the control plants, but not in the KO-nudx6 plants, were increased by the treatment with salicylic acid (SA). The expression of SA-induced genes (PR1, WRKY70, NIMIN1, and NIMIN2) depending on NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key component required for pathogen resistance, was significantly suppressed and enhanced in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively, under the treatment with SA. Induction of thioredoxin h5 (TRX-h5) expression, which catalyzes a SA-induced NPR1 activation, was suppressed and accelerated in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively. The expression of isochorismate synthase1, required for the regulation of SA synthesis through the NPR1-mediated feedback loop, was decreased and increased in the KO-nudx6 and Pro_35S:AtNUDX6 plants, respectively. Judging from seed germination rates, the KO-nudx6 plants had enhanced sensitivity to the toxicity of high-level SA. These results indicated that AtNUDX6 is a modulator of NADH rather than ADP-Rib metabolism and that, through induction of TRX-h5 expression, AtNUDX6 significantly impacts the plant immune response as a positive regulator of NPR1-dependent SA signaling pathways.Nudix (nucleoside diphosphates linked to some moiety X) hydrolases are a phylogenetically widespread enzyme family and are widely distributed among all classes of organisms, such as bacteria, yeast, algae, nematodes, vertebrates, and plants (Bessman et al., 1996; Xu et al., 2004; Kraszewska, 2008). The enzymes catalyze, with varying degrees of substrate specificity, the hydrolysis of a variety of nucleoside diphosphate derivatives: nucleoside diphosphates and triphosphates and their oxidized forms, dinucleoside polyphosphates, nucleotide sugars, NADH, CoA, and the mRNA caps (McLennan, 2006; Kraszewska, 2008; Gunawardana et al., 2009). Since these compounds are often toxic to cells, Nudix hydrolases seem to play protective, regulatory, and signaling roles in metabolism by hydrolytically removing such compounds (Bessman et al., 1996; Xu et al., 2004).We reported the molecular and enzymatic characteristics of Nudix hydrolases (AtNUDX1–AtNUDX27) in Arabidopsis (Arabidopsis thaliana) plants (Ogawa et al., 2005, 2008). Notably, among 27 types of AtNUDXs, cytosolic AtNUDX2, AtNUDX6, AtNUDX7, and AtNUDX10 had pyrophosphohydrolase activity toward both ADP-Rib and NADH in vitro. Recent studies have shown that the actions of NADH and/or ADP-Rib pyrophosphohydrolases are closely related to defense systems in response to biotic and abiotic stresses in higher plants.It has been reported that the expression of AtNUDX7 is induced by avirulent pathogenic attacks. Knockout AtNUDX7 mutants (KO-nudx7) showed enhanced resistance against both virulent and avirulent bacterial strains (Bartsch et al., 2006; Jambunathan and Mahalingam, 2006; Adams-Phillips et al., 2008). In addition, it was revealed that AtNUDX7 functions as a negative regulator on ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) signaling required for basal resistance to invasive pathogens (Bartsch et al., 2006); EDS1 regulates accumulation of the phenolic defense molecule, salicylic acid (SA), and other as yet unidentified signal intermediates and controls the defense activation and programmed cell death by collaborating with its interaction partner PHYTOALEXIN-DEFICIENT4 in cells surrounding pathogen infection foci. Furthermore, Ge et al. (2007) reported that AtNUDX7 functions to prevent excessive stimulation of the defense response, which is dependent on and independent of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a master regulator of SA-induced defense genes (SAIGs), and SA accumulation.On the other hand, we recently demonstrated the roles of Arabidopsis NADH/ADP-Rib pyrophosphohydrolases (AtNUDX2 and AtNUDX7) in tolerance to oxidative stress using the respective overexpressors (Pro_35S:AtNUDX2 and Pro_35S:AtNUDX7) or disruptants (KO-nudx7; Ishikawa et al., 2009; Ogawa et al., 2009). Interestingly, overexpression of AtNUDX2 and AtNUDX7 in Arabidopsis plants was responsible for an enhanced tolerance to oxidative stress derived from the treatment with paraquat (an agent producing O₂⁻) and salinity. Taken together, these results revealed that both AtNUDX2 and AtNUDX7 function in accelerating nucleotide recycling from ADP-Rib produced by poly(ADP-Rib) metabolism, leading to suppression of the overconsumption of NAD⁺ and ATP in Arabidopsis cells under stressful conditions. In addition, AtNUDX7 served to balance between NADH and NAD⁺ by NADH turnover and to regulate the defense mechanisms against DNA damage by modulation of the poly(ADP-ribosyl)ation (PAR) reaction through NADH metabolism in response to oxidative stress (Ishikawa et al., 2009; Ogawa et al., 2009). These findings clearly indicated that the regulation of NADH and/or ADP-Rib metabolism via Nudix hydrolases is involved in the responses to both biotic and abiotic stresses in higher plants.The question that we must consider next is whether the other AtNUDXs (AtNUDX6 and AtNUDX10) with pyrophosphohydrolase activities toward ADP-Rib and NADH are involved in the defense systems against oxidative stress and pathogen attack. The expression of AtNUDX6 has been reported to be induced by pathogenic attacks and treatment with the SA analogs 2,6-dichloroisonicotinic acid and acibenzolar-S-methyl benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH; Bartsch et al., 2006; Qiu et al., 2008; Knoth et al., 2009). Furthermore, the expression of AtNUDX6 was strongly dependent on EDS1 (Bartsch et al., 2006). However, the functional significance of AtNUDX6 is still unclear, since a loss-of-function mutant of AtNUDX6 has not yet been found.In this paper, to assess the physiological function of AtNUDX6, we identified an Arabidopsis mutant in which T-DNA is inserted into AtNUDX6 and subsequently studied the levels of ADP-Rib and NAD(H), PAR activity, expression of genes related to SA signaling, and SA tolerance in the AtNUDX6 overexpressors and disruptants in comparison with the AtNUDX7 disruptants. The results obtained here indicated that AtNUDX6 positively regulates NPR1-dependent SA signaling via modulation of NADH metabolism in the plant immune response.     

Autophagy Negatively Regulates Cell Death by Controlling NPR1-Dependent Salicylic Acid Signaling during Senescence and the Innate Immune Response in Arabidopsis

Autophagy is an evolutionarily conserved intracellular process for vacuolar degradation of cytoplasmic components. In higher plants, autophagy defects result in early senescence and excessive immunity-related programmed cell death (PCD) irrespective of nutrient conditions; however, the mechanisms by which cells die in the absence of autophagy have been unclear. Here, we demonstrate a conserved requirement for salicylic acid (SA) signaling for these phenomena in autophagy-defective mutants (atg mutants). The atg mutant phenotypes of accelerated PCD in senescence and immunity are SA signaling dependent but do not require intact jasmonic acid or ethylene signaling pathways. Application of an SA agonist induces the senescence/cell death phenotype in SA-deficient atg mutants but not in atg npr1 plants, suggesting that the cell death phenotypes in the atg mutants are dependent on the SA signal transducer NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1. We also show that autophagy is induced by the SA agonist. These findings imply that plant autophagy operates a novel negative feedback loop modulating SA signaling to negatively regulate senescence and immunity-related PCD.     

The Pim-1 Protein Kinase Is an Important Regulator of MET Receptor Tyrosine Kinase Levels and Signaling

MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology.     

Chloroplast-to-Nucleus Signaling Regulates MicroRNA Biogenesis in Arabidopsis

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GLP-1 Receptor Signaling in Astrocytes Regulates Fatty Acid Oxidation,Mitochondrial Integrity,and Function

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Lysophosphatidic Acid Acyltransferase Beta Regulates mTOR Signaling

Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.     

Receptor Tyrosine Kinase Signaling and Primordial Germ Cell Development

Primordial germ cells (PGCs) give rise to sperms and eggs. Their development is crucial to species propagation and has to be precisely controlled. Studies in several model organisms have identified many genes involved in the specification and guided migration of PGCs. However, the mechanisms governing the behaviors of this unique type of cells remain to be investigated. Interestingly, PGCs share certain cellular properties with metastasizing cancer cells including proliferation, invasion of other tissues, survival, and migration. Recently we have shown that in Drosophila the receptor tyrosine kinase Torso activates both STAT and Ras during the early phase of PGC development. In later stages, activation of both STAT and Ras, likely by other molecules, is required continuously for PGC migration. The requirement for RTK suggests molecular conservation between flies and mice in PGC development and also suggests that germ cells and cancer cells share certain intracellular signaling strategies.     

Salicylic Acid Levels in Thermogenic and Non-Thermogenic Plants

The natural trigger for heat production in the thermogenic inflorescencesof Sauromatum guttatum Schott (voodoo lily) was recently identifiedas salicylic acid (SA), which induced heat production at levelsas low as 13 ng g f. wt^–1. Since then the levels of SAwere determined in other thermogenic and non-thermogenic plantspecies. In thermogenic inflorescences of five aroid species,and in male cones of at least four thermogenic cycads SA levelsduring heat production exceeded 1 µg g f. wt^–1.SA was not detected in the thermogenic flowers of a water lily,Victoria regia Lindl. (Nymphaeaceae), and Bactris major Jacq.(Palmae). Levels of salicylic acid varied substantially in thefloral parts of seven non-thermogenic species and in the leavesof 27 non-thermogenic species. Amorphophallus campanulatus Blume ex Decne, Arum italicum Mill., Arum dioscoridis Sibth. & Son., Philodendron selloum Koch, Monstera deliciosa Liebm., Encephalartosferox Bertol. f., Encephalartos hildebrandtii A. Br. & Bouché, Encephalartos gratus Prain, Dioon edule Lindl. cv. edule, Dioon edule Lindl. cv angustifolium, Sauromatum guttatum Schott, voodoo lily, Victoria regia Lindl., Bactris major Jack, salicylic acid, thermogenicity, heat production     

Ligand Mobility Regulates B Cell Receptor Clustering and Signaling Activation

Antigen binding to the B cell receptor (BCR) induces receptor clustering, cell spreading, and the formation of signaling microclusters, triggering B cell activation. Although the biochemical pathways governing early B cell signaling have been well studied, the role of the physical properties of antigens, such as antigen mobility, has not been fully examined. We study the interaction of B cells with BCR ligands coated on glass or tethered to planar lipid bilayer surfaces to investigate the differences in B cell response to immobile and mobile ligands. Using high-resolution total internal reflection fluorescence (TIRF) microscopy of live cells, we followed the movement and spatial organization of BCR clusters and the associated signaling. Although ligands on either surface were able to cross-link BCRs and induce clustering, B cells interacting with mobile ligands displayed greater signaling than those interacting with immobile ligands. Quantitative analysis revealed that mobile ligands enabled BCR clusters to move farther and merge more efficiently than immobile ligands. These differences in physical reorganization of receptor clusters were associated with differences in actin remodeling. Perturbation experiments revealed that a dynamic actin cytoskeleton actively reorganized receptor clusters. These results suggest that ligand mobility is an important parameter for regulating B cell signaling.     

Ligand Mobility Regulates B Cell Receptor Clustering and Signaling Activation

Antigen binding to the B cell receptor (BCR) induces receptor clustering, cell spreading, and the formation of signaling microclusters, triggering B cell activation. Although the biochemical pathways governing early B cell signaling have been well studied, the role of the physical properties of antigens, such as antigen mobility, has not been fully examined. We study the interaction of B cells with BCR ligands coated on glass or tethered to planar lipid bilayer surfaces to investigate the differences in B cell response to immobile and mobile ligands. Using high-resolution total internal reflection fluorescence (TIRF) microscopy of live cells, we followed the movement and spatial organization of BCR clusters and the associated signaling. Although ligands on either surface were able to cross-link BCRs and induce clustering, B cells interacting with mobile ligands displayed greater signaling than those interacting with immobile ligands. Quantitative analysis revealed that mobile ligands enabled BCR clusters to move farther and merge more efficiently than immobile ligands. These differences in physical reorganization of receptor clusters were associated with differences in actin remodeling. Perturbation experiments revealed that a dynamic actin cytoskeleton actively reorganized receptor clusters. These results suggest that ligand mobility is an important parameter for regulating B cell signaling.