Genome-Wide Analysis of Mitogen-Activated Protein Kinase Gene Family in Maize

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. As the last component of the MAPK cascade (MAPKKK–MAPKK–MAPK), MAPK plays important roles in linking upstream kinases and downstream substrates. The MAPK proteins belong to a complex gene family in plants, with 20 MAPK genes in the Arabidopsis genome, 17 in the rice genome, and 21 in the poplar genome. Although the maize genome sequencing has been completed, no comprehensive study has been reported thus far for the MAPK gene family in maize. In this study, we identified 19 MAPK genes in maize. These ZmMPK genes belong to four groups (A–D) found in other plants. The phylogeny, chromosomal location, gene structure, and the functional relevancy of ZmMPK genes were analyzed. Moreover, we discuss the evolutionary divergence of MAPK genes in maize. Furthermore, we analyzed the expression profiles of ZmMPKs using the public microarray data and performed expression analyses in maize seedlings and adult plants. The data obtained from our study contribute to a better understanding of the complexity of MAPKs in plants and provide a useful reference for further functional analysis of MAPK genes in maize.     

Genome-Wide Identification and Analysis of Expression Profiles of Maize Mitogen-Activated Protein Kinase Kinase Kinase

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction model in animals, yeast and plants. Plant MAPK cascades have been implicated in development and stress responses. Although MAPKKKs have been investigated in several plant species including Arabidopsis and rice, no systematic analysis has been conducted in maize. In this study, we performed a bioinformatics analysis of the entire maize genome and identified 74 MAPKKK genes. Phylogenetic analyses of MAPKKKs from maize, rice and Arabidopsis have classified them into three subgroups, which included Raf, ZIK and MEKK. Evolutionary relationships within subfamilies were also supported by exon-intron organizations and the conserved protein motifs. Further expression analysis of the MAPKKKs in microarray databases revealed that MAPKKKs were involved in important signaling pathways in maize different organs and developmental stages. Our genomics analysis of maize MAPKKK genes provides important information for evolutionary and functional characterization of this family in maize.     

Manipulation of Mitogen-Activated Protein Kinase Kinase Signaling in the Arabidopsis Stomatal Lineage Reveals Motifs That Contribute to Protein Localization and Signaling Specificity

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four “stomatal” MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.     

Tryptase Activation of Immortalized Human Urothelial Cell Mitogen-Activated Protein Kinase

The pathogenesis of interstitial cystitis/painful bladder syndrome (IC/PBS) is multifactorial, but likely involves urothelial cell dysfunction and mast cell accumulation in the bladder wall. Activated mast cells in the bladder wall release several inflammatory mediators, including histamine and tryptase. We determined whether mitogen-activated protein (MAP) kinases are activated in response to tryptase stimulation of urothelial cells derived from human normal and IC/PBS bladders. Tryptase stimulation of normal urothelial cells resulted in a 2.5-fold increase in extracellular signal regulated kinase 1/2 (ERK 1/2). A 5.5-fold increase in ERK 1/2 activity was observed in urothelial cells isolated from IC/PBS bladders. No significant change in p38 MAP kinase was observed in tryptase-stimulated normal urothelial cells but a 2.5-fold increase was observed in cells isolated from IC/PBS bladders. Inhibition of ERK 1/2 with PD98059 or inhibition of p38 MAP kinase with SB203580 did not block tryptase-stimulated iPLA₂ activation. Incubation with the membrane phospholipid-derived PLA₂ hydrolysis product lysoplasmenylcholine increased ERK 1/2 activity, suggesting the iPLA₂ activation is upstream of ERK 1/2. Real time measurements of impedance to evaluate wound healing of cell cultures indicated increased healing rates in normal and IC/PBS urothelial cells in the presence of tryptase, with inhibition of ERK 1/2 significantly decreasing the wound healing rate of IC/PBS urothelium. We conclude that activation of ERK 1/2 in response to tryptase stimulation may facilitate wound healing or cell motility in areas of inflammation in the bladder associated with IC/PBS.     

Direct Activation of Mitogen-Activated Protein Kinase Kinase Kinase MEKK1 by the Ste20p Homologue GCK and the Adapter Protein TRAF2

Mitogen-activated protein kinase (MAPK) pathways coordinate critical cellular responses to mitogens, stresses, and developmental cues. The coupling of MAPK kinase kinase (MAP3K) --> MAPK kinase (MEK) --> MAPK core pathways to cell surface receptors remains poorly understood. Recombinant forms of MAP3K MEK kinase 1 (MEKK1) interact in vivo and in vitro with the STE20 protein homologue germinal center kinase (GCK), and both GCK and MEKK1 associate in vivo with the adapter protein tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2). These interactions may couple TNF receptors to the SAPK/JNK family of MAPKs; however, a molecular mechanism by which these proteins might collaborate to recruit the SAPKs/JNKs has remained elusive. Here we show that endogenous GCK and MEKK1 associate in vivo. In addition, we have developed an in vitro assay system with which we demonstrate that purified, active GCK and TRAF2 activate MEKK1. The RING domain of TRAF2 is necessary for optimal in vitro activation of MEKK1, but the kinase domain of GCK is not. Autophosphorylation within the MEKK1 kinase domain activation loop is required for activation. Forced oligomerization also activates MEKK1, and GCK elicits enhanced oligomerization of coexpressed MEKK1 in vivo. These results represent the first activation of MEKK1 in vitro using purified proteins and suggest a mechanism for MEKK1 activation involving induced oligomerization and consequent autophosphorylation mediated by upstream proteins.     

Activation of p42 Mitogen-Activated Protein Kinase by Glutamate in Cultured Radial Glia

The effect of L-glutamate (Glu) and its structural analogs N-methyl-D-aspartate (NMDA), kainate (KA) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), on the activation of p42 mitogen activated protein kinase (MAPK) was examined in cultured chick radial glia cells, namely retinal Müller cells and cerebellar Bergmann cells. Glu, NMDA, AMPA and KA evoked a dose and time dependent increase in MAPK activity. AMPA and KA responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas NMDA responses were sensitive to 3-[(RS)-2-carboxypiperazin-4-yl)]-propyl-1-phosphonate (CPP) indicating that the increase in MAPK activity is mediated by AMPA/low affinity KA and NMDA subtypes of Glu receptors. The present findings open the possibility of a MAPK cascade involvement in the regulation of Glu-induced gene expression in radial glia.     

Direct Phosphorylation and Activation of a Mitogen-Activated Protein Kinase by a Calcium-Dependent Protein Kinase in Rice

The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase, MAPK kinase (MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a TXY motif by an MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK), CPK18, was identified as an upstream kinase of MAPK (MPK5) in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its TXY motif. Instead, CPK18 was found to predominantly phosphorylate two Thr residues (Thr-14 and Thr-32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKK-independent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery.     

Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cdelta (PKCdelta), whereas ERK activation in response to the mitogenic EGF is independent of PKCdelta. Antisense PKCdelta oligonucleotides or the PKCdelta-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCdelta functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCdelta also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCdelta in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCdelta requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCdelta in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCdelta contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling.     

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation.     

Okadaic Acid Induces Hyperphosphorylation of τ Independently of Mitogen-Activated Protein Kinase Activation

Abstract: Hyperphosphorylation of the microtubule-associated protein τ is a characteristic of Alzheimer brain tissue. Recent in vitro data suggest that mitogen-activated protein kinase (MAPK), a proline-directed protein kinase, phosphorylates the sites on τ common to Alzheimer's disease. Using an okadaic acid-induced τ hyperphosphorylation model, we have tested the requirement for MAPK activity, using a specific inhibitor {PD098059 [2-(2'-amino-3'-methoxyphenyl)oxanaphthalen-4-one]} of the MAPK activator Mek1. Mobility shift, phosphoepitope analysis, and direct measurement of kinase activity indicated that the Mek1 inhibitor dose-dependently blocked basal and okadaic acid-induced MAPK activation. Despite a block of MAPK activation by this inhibitor, robust τ hyperphosphorylation was observed in response to okadaic acid. In addition, activation of MAPK by phorbol 12-myristate 13-acetate did not result in τ phosphorylation, indicating that in primary cultures of cortical neurons elevated MAPK activity is not sufficient to induce τ hyperphosphorylation.     

Activation of Salicylic Acid–Induced Protein Kinase, a Mitogen-Activated Protein Kinase, Induces Multiple Defense Responses in Tobacco

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2^DD, in tobacco induces the expression of defense genes and hypersensitive response–like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid–induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response–like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.     

番茄NAC转录因子家族的鉴定及生物信息学分析

NAC转录因子家族是植物特有的一类转录因子,在植物的生长发育、器官建成及逆境胁迫和激素信号应答中均发挥重要作用。本研究在基因组范围内,利用生物信息学方法对番茄的NAC转录因子家族成员、分布及结构和功能等进行分析。预测结果显示番茄NAC家族包含102个蛋白质,分为12亚族,其中茄属特有的TNAC亚族中成员最多,具有25个,其他NAC转录因子与拟南芥NAc家族具有相似分类。保守基序分析,在番茄NAC结构域中包含7个保守的NAM基序,主要分布在序列的N端,表明这些基序的存在对NAC蛋白质功能的执行是必需的。理化性质和结构分析表明,番茄NAC蛋白质绝大多数是亲水蛋白质,主要以无规则卷曲构成,而α-螺旋、β-折叠和β-转角则散布于整个蛋白质中,在各亚族中没有规律。     

Genome-Wide Identification and Analysis of the TIFY Gene Family in Grape

Background

The TIFY gene family constitutes a plant-specific group of genes with a broad range of functions. This family encodes four subfamilies of proteins, including ZML, TIFY, PPD and JASMONATE ZIM-Domain (JAZ) proteins. JAZ proteins are targets of the SCF^COI1 complex, and function as negative regulators in the JA signaling pathway. Recently, it has been reported in both Arabidopsis and rice that TIFY genes, and especially JAZ genes, may be involved in plant defense against insect feeding, wounding, pathogens and abiotic stresses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant TIFY family members is limited, especially in a woody species such as grape.

Methodology/Principal Findings

A total of two TIFY, four ZML, two PPD and 11 JAZ genes were identified in the Vitis vinifera genome. Phylogenetic analysis of TIFY protein sequences from grape, Arabidopsis and rice indicated that the grape TIFY proteins are more closely related to those of Arabidopsis than those of rice. Both segmental and tandem duplication events have been major contributors to the expansion of the grape TIFY family. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologues of several grape TIFY genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of lineages that led to grape and Arabidopsis. Analyses of microarray and quantitative real-time RT-PCR expression data revealed that grape TIFY genes are not a major player in the defense against biotrophic pathogens or viruses. However, many of these genes were responsive to JA and ABA, but not SA or ET.

Conclusion

The genome-wide identification, evolutionary and expression analyses of grape TIFY genes should facilitate further research of this gene family and provide new insights regarding their evolutionary history and regulatory control.     

Activation of Mitogen-Activated Protein Kinase in Cultured Rat Hippocampal Neurons by Stimulation of Glutamate Receptors

Abstract: Mitogen-activated protein kinase (MAP kinase) was activated by stimulation of glutamate receptors in cultured rat hippocampal neurons. Ten micromolar glutamate maximally stimulated MAP kinase activity, which peaked during 10 min and decreased to the basal level within 30 min. Experiments using glutamate receptor agonists and antagonists revealed that glutamate stimulated MAP kinase through NMDA and metabotropic glutamate receptors but not through non-NMDA receptors. Glutamate and its receptor agonists had no apparent effect on MAP kinase activation in cultured cortical astrocytes. Addition of calphostin C, a protein kinase C (PKC) inhibitor, or down-regulation of PKC activity partly abolished the stimulatory effect by glutamate, but the MAP kinase activation by treatment with ionomycin, a Ca²⁺ ionophore, remained intact. Lavendustin A, a tyrosine kinase inhibitor, was without effect. In experiments with ³²P-labeled hippocampal neurons, MAP kinase activation by glutamate was associated with phosphorylation of the tyrosine residue located on MAP kinase. However, phosphorylation of Raf-1, the c- raf protooncogene product, was not stimulated by treatment with glutamate. Our observations suggest that MAP kinase activation through glutamate receptors in hippocampal neurons is mediated by both the PKC-dependent and the Ca²⁺-dependent pathways and that the activation of Raf-1 is not involved.