NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H(2)O(2)-mediated mitogen-activated protein kinase signaling in plants. H(2)O(2) stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H(2)O(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H(2)O(2)-activated A. thaliana mitogen-activated protein kinases. In the absence of H(2)O(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H(2)O(2)-mediated MAPK signaling in plants.     

MARCKS protein mediates hydrogen peroxide regulation of endothelial permeability

Impairment of endothelial barrier function is implicated in many vascular and inflammatory disorders. One prevalent mechanism of endothelial dysfunction is an increase in reactive oxygen species under oxidative stress. Previous reports have demonstrated that hydrogen peroxide (H(2)O(2)), a highly stable reactive oxygen species that modulates physiological signaling pathways, also enhances endothelial permeability, but the mechanism of this effect is unknown. Here, we identify the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) as a key mediator of the H(2)O(2)-induced permeability change in bovine aortic endothelial cells. MARCKS knockdown and H(2)O(2) treatment alter the architecture of the actin cytoskeleton in endothelial cells, and H(2)O(2) induces the phosphorylation and translocation of MARCKS from the cell membrane to the cytosol. Using pharmacological inhibitors and small interference RNA constructs directed against specific proteins, we uncover a signaling cascade from Rac1 to Abl1, phospholipase Cγ1, and PKCδ that is triggered by H(2)O(2) and leads to MARCKS phosphorylation. Our findings establish a distinct role for MARCKS in the regulation of H(2)O(2)-induced permeability change in endothelial cells, and suggest potential new therapeutic targets for the treatment of disorders involving oxidative stress and altered endothelial permeability.     

Responses of cardiac protein kinase C isoforms to distinct pathological stimuli are differentially regulated.

Currently at least 11 protein kinase C (PKC) isoforms have been identified and may play different roles in cell signaling pathways leading to changes in cardiac contractility, the hypertrophic response, and tolerance to myocardial ischemia. The purpose of the present study was to test the hypothesis that responses of individual PKC isoforms to distinct pathological stimuli were differentially regulated in the adult guinea pig heart. Isolated hearts were perfused by the Langendorff method and were exposed to ischemia, hypoxia, H(2)O(2), or angiotensin II. Hypoxia and ischemia induced translocation of PKC isoforms alpha, beta(2), gamma, and zeta, and H(2)O(2) translocated PKC isoforms alpha, beta(2), and zeta. Angiotensin II produced translocation of alpha, beta(2), epsilon, gamma, and zeta isoforms. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) blocked hypoxia-induced (alpha, beta(2), and zeta) and angiotensin II-induced (alpha, beta(2), gamma, and zeta) translocation of PKC isoforms. Inhibition of tyrosine kinase with genistein blocked translocation of PKC isoforms by hypoxia (beta(2) and zeta) and by angiotensin II (beta(2)). By contrast, neither D609 nor genistein blocked H(2)O(2)-induced translocation of any PKC isoform. We conclude that hypoxia-induced activation of PKC isoforms is mediated through pathways involving phospholipase C and tyrosine kinase, but oxidative stress may activate PKC isoforms independently of Galphaq-phospholipase C coupling and tyrosine kinase signaling. Because oxidative stress may directly activate PKC, and PKC activation appears to be involved in human heart failure, selective inhibition of the PKC isoforms may provide a novel therapeutic strategy for the prevention and treatment of this pathological process.     

MEK kinase 1 is critically required for c-Jun N-terminal kinase activation by proinflammatory stimuli and growth factor-induced cell migration

Exposure of eukaryotic cells to extracellular stimuli results in activation of mitogen-activated protein kinase (MAPK) cascades composed of MAPKs, MAPK kinases (MAP2Ks), and MAPK kinase kinases (MAP3Ks). Mammals possess a large number of MAP3Ks, many of which can activate the c-Jun N-terminal kinase (JNK) MAPK cascade when overexpressed, but whose biological function is poorly understood. We examined the function of the MAP3K MEK kinase 1 (MEKK1) in proinflammatory signaling. Using MEKK1-deficient embryonic stem cells prepared by gene targeting, we find that, in addition to its function in JNK activation by growth factors, MEKK1 is required for JNK activation by diverse proinflammatory stimuli, including tumor necrosis factor alpha, IL-1, double-stranded RNA, and lipopolysaccharide. MEKK1 is also essential for induction of embryonic stem cell migration by serum factors, but is not required for activation of other MAPKs or the IkappaB kinase signaling cascade.     

SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana

Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in Arabidopsis thaliana that can significantly impact drought tolerance of Arabidopsis plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in Arabidopsis roots. Additionally, transgenic plants with CaMV35S promoter::SRK2C-GFP displayed higher overall drought tolerance than control plants. Whereas stomatal regulation in 35S::SRK2C-GFP plants was not altered, microarray analysis revealed that their drought tolerance coincided with up-regulation of many stress-responsive genes, for example, RD29A, COR15A, and DREB1A/CBF3. From these results, we concluded that SRK2C is capable of mediating signals initiated during drought stress, resulting in appropriate gene expression. Our present study reveals new insights around signal output from osmotic-stress-activated SnRK2 protein kinase as well as supporting feasibility of manipulating SnRK2 toward improving plant osmotic-stress tolerance.     

Betaine suppresses proinflammatory signaling during aging: the involvement of nuclear factor-kappaB via nuclear factor-inducing kinase/IkappaB kinase and mitogen-activated protein kinases

Betaine is an important human nutrient obtained from various foods. In the present study, we assessed the anti-inflammatory effect of betaine on nuclear factor-kappaB (NF-kappaB) during aging. Sprague-Dawley (SD) rats, ages 7 and 21 months, were used in this study. The older rats were fed betaine. To elucidate the effect of betaine on oxidative stress-induced NF-kappaB and its signaling pathway at molecular levels, YPEN-1 cells were used. Results showed that betaine suppressed NF-kappaB and its related gene expressions of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule-1 (VCAM-1), and intracellular cell adhesion molecule-1 (ICAM-1) in aged kidney. Furthermore, betaine attenuated oxidative stress-induced NF-kappaB via nuclear factor-inducing kinase/IkappaB kinase (NIK/IKK) and mitogen-activated protein kinases (MAPKs) in the YPEN-1 cells. On the basis of these results, we concluded that betaine suppressed the age-related NF-kappaB activities associated with upregulated NIK/IKK and MAPKs that were induced by oxidative stress. Thus, betaine might be useful as a preventive agent against the activation of NF-kappaB induced during inflammation and aging.     

Phosphorylation and activation of PINOID by the phospholipid signaling kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1) in Arabidopsis

Activity of the serine-threonine protein kinase PINOID (PID) has been implicated in the asymmetrical localization of the membrane-associated PINFORMED (PIN) family of auxin transport facilitators. However, the means by which PID regulates PIN protein distribution is unknown. We have used recombinant PID protein to dissect the regulation of PID activity in vitro. We demonstrate that intramolecular PID autophosphorylation is required for the ability of PID to phosphorylate an exogenous substrate. PID-like mammalian AGC kinases act in a phosphorylation cascade initiated by the phospholipid-associated kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1), which binds to the C-terminal hydrophobic PDK1-interacting fragment (PIF) domain found in PDK1 substrates. We find that Arabidopsis PDK1 interacts with PID, and that transphosphorylation by PDK1 increases PID autophosphorylation. We show that a PID activation loop serine is required for PDK1-dependent PID phosphorylation. This activation is rapid and requires the PIF domain. Cell extracts from flowers and seedling shoots dramatically increase PID phosphorylation in a tissue-specific manner. A PID protein variant in which the PIF domain was mutated failed to be activated by the seedling shoot extracts. PID immunoprecipitated from Arabidopsis cells in which PDK1 expression was inhibited by RNAi showed a dramatic reduction in transphosphorylation of myelin basic protein substrate. These results indicate that AtPDK1 is a potent enhancer of PID activity and provide evidence that phospholipid signaling may play a role in the signaling processes controlling polar auxin transport.     

Phosphorylation of a mitotic kinesin-like protein and a MAPKKK by cyclin-dependent kinases (CDKs) is involved in the transition to cytokinesis in plants

Cytokinesis in eukaryotes involves specific arrays of microtubules (MTs), which are known as the "central spindle" in animals, the "anaphase spindle" in yeasts, and the "phragmoplast" in plants. Control of these arrays, which are composed mainly of bundled nonkinetochore MTs, is critically important during cytokinesis. In plants, an MAPK cascade stimulates the turnover of phragmoplast MTs, and a crucial aspect of the activation of this cascade is the interaction between the MAPKKK, nucleus- and phragmoplast-localized protein kinase 1 (NPK1) and the NPK1-activating kinesin-like protein 1 (NACK1), a key regulator of plant cytokinesis. However, little is known about the control of this interaction at the molecular level during progression through the M phase. We demonstrated that cyclin-dependent kinases (CDKs) phosphorylate both NPK1 and NACK1 before metaphase in tobacco cells, thereby inhibiting the interaction between these proteins, suggesting that such phosphorylation prevents the transition to cytokinesis. Failure to inactivate CDKs after metaphase prevents dephosphorylation of these two proteins, causing incomplete mitosis. Experiments with Arabidopsis NACK1 (AtNACK1/HINKEL) revealed that phosphorylated NACK1 fails to mediate cytokinesis. Thus, timely and coordinated phosphorylation by CDKs and dephosphorylation of cytokinetic regulators from prophase to anaphase appear to be critical for the appropriate onset and/or progression of cytokinesis.     

GSTT1 is upregulated by oxidative stress through p38-MK2 signaling pathway in human granulosa cells: possible association with mitochondrial activity

We previously reported that GSTT1 was upregulated in human granulosa cells during aging and that activation and localization of p38 MAPK was changed in parallel. Although oxidative stress is responsible for these changes, the age-associated expression of GSTT1 regulated by MAPKs and the role of GSTT1 in aged granulosa cells remain unclear. Therefore, we examined the relationship between the expression of GSTT1 and MAPK signaling pathways using human granulosa-like KGN cells stimulated with H(2)O(2) in the presence or absence of various MAPK inhibitors. Interestingly, H(2)O(2)-induced GSTT1 was only inhibited by a p38 inhibitor. An inhibitor of MK2, a downstream regulator of p38, also diminished H(2)O(2)-induced GSTT1 upregulation. Notably, both p38 and MK2 were significantly inactivated in cells carrying an shRNA construct of GSTT1 (?GSTT1 cells), suggesting that the p38-MK2 pathway is essential for age-associated upregulation of GSTT1. The relevance of GSTT1 in mitochondrial activity was then determined. ?GSTT1 cells displayed enhanced polarization of mitochondrial membrane potential without increasing the apoptosis, suggesting that the age-associated upregulation of GSTT1 may influence the mitochondrial activity of granulosa cells.     

Activation of mitogen activated protein kinases in post-infarcted patients

Activation of mitogen-activated protein kinases (MAPKs) signaling cascade are important pathophysiologic regulators during the development of acute myocardial infarction (AMI). In present study, we designed to monitor the activity of these MAPKs in Iranian patients with AMI comparing with controls. The degree of activation (phosphorylation) of p38 kinase, p44/42 extracellular regulated kinase, and c-Jun N-terminal kinase (JNK1/2) and their corresponding activity levels were analyzed in 258 patients with AMI and 250 normal subjects. The expression of p38α mRNA was determined. These analysis were carried out immediately and 12 h after AMI. Activity of p38 and JNK1/2 MAPKs were significantly increased in patients with AMI than controls immediately after infarction. These activities were reduced during 12 h after AMI. However, there were no statistically differences in activation and activity of p44/42 in the patients and controls. The mRNA expression of p38α was increased in the patients comparing with controls. Results of this study indicate that these MAPKs signaling pathway might be activated by AMI which signal transduction involves kinase phosphorylation and play important roles in their activity. Elevated activity of p38 and JNK1/2 MAPKs suggests that they may potentially play significant roles in AMI.     

Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana

Upon a dark-to-light shift, the conditional fluorescent (flu) mutant of Arabidopsis releases singlet oxygen (1O2) within the plastid compartment. Distinct sets of nuclear genes are activated that are different from those induced by superoxide (O2*-)) and/or hydrogen peroxide (H2O2), suggesting that different types of reactive oxygen species activate distinct signaling pathways. It is not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX). The overexpression of the H2O2-specific scavenger reduced strongly the activation of nuclear genes in plants treated with the herbicide paraquat that in the light leads to the enhanced generation of O2*- and H2O2. In the flu mutant overexpressing tAPX, the intensity of 1O2-mediated cell death and growth inhibition was increased when compared with the flu parental line. Also, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX did not lead to visible stress responses and had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions.     

Carotenoid oxidation products are stress signals that mediate gene responses to singlet oxygen in plants

(1)O(2) (singlet oxygen) is a reactive O(2) species produced from triplet excited chlorophylls in the chloroplasts, especially when plants are exposed to excess light energy. Similarly to other active O(2) species, (1)O(2) has a dual effect: It is toxic, causing oxidation of biomolecules, and it can act as a signal molecule that leads to cell death or to acclimation. Carotenoids are considered to be the main (1)O(2) quenchers in chloroplasts, and we show here that light stress induces the oxidation of the carotenoid β-carotene in Arabidopsis plants, leading to the accumulation of different volatile derivatives. One such compound, β-cyclocitral, was found to induce changes in the expression of a large set of genes that have been identified as (1)O(2) responsive genes. In contrast, β-cyclocitral had little effect on the expression of H(2)O(2) gene markers. β-Cyclocitral-induced reprogramming of gene expression was associated with an increased tolerance to photooxidative stress. The results indicate that β-cyclocitral is a stress signal produced in high light that is able to induce defense mechanisms and represents a likely messenger involved in the (1)O(2) signaling pathway in plants.     

Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco

Hypersensitive response (HR), a form of programmed cell death, is frequently associated with plant disease resistance. It has been proposed that mitogen-activated protein kinase (MAPK) cascades regulate HR cell death based on pharmacological studies by using kinase inhibitors. However, direct evidence is lacking. Here, we demonstrate that NtMEK2, a MAPK kinase, is upstream of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco MAPKs that are activated by various pathogens or pathogen-derived elicitors. Expression of a constitutively active mutant of NtMEK2 induces HR-like cell death in tobacco, which is preceded by the activation of endogenous SIPK and WIPK. In addition, NtMEK2-SIPK/WIPK cascade appears to control the expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and l-phenylalanine ammonia lyase (PAL), two defense genes encoding key enzymes in the phytoalexin and salicylic acid biosynthesis pathways. These results demonstrate that a plant MAPK cascade controls multiple defense responses against pathogen invasion.     

Mechanisms of endotoxin-induced NO, IL-6, and TNF-alpha production in activated rat hepatic stellate cells: role of p38 MAPK

Compelling experimental evidence indicates that the interactions between endotoxin and hepatic stellate cells (HSCs) can play a significant role in the pathogenesis of liver disease. Endotoxin-induced release of a multifunctional mediator NO (via inducible NO synthase) and the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6 by HSCs could be an important mechanism of pathological changes in the liver. However, the signaling mechanisms of these effects are poorly understood. In this study, we found that endotoxin causes activation of mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated protein kinase [ERK] 1 and 2, p38, and c-Jun NH2-terminal kinase [JNK]) and nuclear factor kappaB (NF-kappaB) and production of H(2)O(2) in culture-activated HSCs. However, only p38 and NF-kappaB were found to be responsible for the synthesis of NO, IL-6, and TNF-alpha. Exogenous H(2)O(2) caused modest stimulation of TNF-alpha synthesis, did not affect the synthesis of NO or IL-6, and did not activate NF-kappaB or MAPKs. Inhibition of p38 and NF-kappaB activation by SB203580 and pyrrolidine dithiocarbamate, respectively, blocked endotoxin-induced H(2)O(2), NO, TNF-alpha, and IL-6 synthesis. Inhibition of ERK1/2 and JNK phosphorylation did not alter these effects of endotoxin. Whereas SB203580 inhibited endotoxin-induced NF-kappaB activation, pyrrolidine dithiocarbamate did not affect p38 phosphorylation in endotoxin-stimulated cells. In conclusion, endotoxin-induced synthesis of NO, TNF-alpha, and IL-6 in HSCs is mediated by p38 and NF-kappaB, with involvement of H(2)O(2) in TNF-alpha production.     

Peroxynitrite activates ERK via Raf-1 and MEK, independently from EGF receptor and p21Ras in H9C2 cardiomyocytes

Peroxynitrite is a potent oxidant and nitrating species proposed as a direct effector of myocardial damage in a wide range of cardiac diseases. Whether peroxynitrite also acts indirectly, by modulating cell signal transduction pathways in the myocardium, has not been investigated. Here, we examined the ability of peroxynitrite to activate extracellular signal-related kinase (ERK), a MAP kinase which has been linked with hypertrophic and anti-apoptotic responses in the heart, in cultured H9C2 cardiomyocytes. Peroxynitrite elicited a concentration- and time-dependent activation of ERK, secondary to the upstream activation of MEK 1 (ERK kinase). Activation of MEK-ERK by peroxynitrite was related to the upstream activation of Raf-1 kinase, as ERK and MEK phosphorylation were prevented by the Raf-1 inhibitor BAY43-9006. These effects of peroxynitrite were not associated with the activation of p21(Ras), known as a common signaling target of cellular oxidative stress. In contrast to ERK activation mediated by the epidermal growth factor (EGF), ERK activation by peroxynitrite was not prevented by AG1478 (EGF receptor inhibitor). Peroxynitrite acted through oxidative, but not nitrative chemistry, as ERK remained activated while nitration was prevented by the flavanol epicatechin. In addition to ERK, peroxynitrite also potently activated two additional members of the MAP kinase family of signaling proteins, JNK and p38. Thus, peroxynitrite activates ERK in cardiomyocytes through an unusual signaling cascade involving Raf-1 and MEK 1, independently from EGFR and P21(Ras), and also acts as a potent activator of JNK and p38. These results provide the novel concept that peroxynitrite may represent a previously unrecognized signaling molecule in various cardiac pathologies.