Arabidopsis thaliana MAP65-1 and MAP65-2 function redundantly with MAP65-3/PLEIADE in cytokinesis downstream of MPK4

Plant cytokinesis occurs by the growth of cell plates from the interior to the periphery of the cell. These dynamic events in cytokinesis are mediated by a plant-specific microtubule (MT) array called the phragmoplast, which consists of bundled antiparallel MTs between the two daughter nuclei. The NACK-PQR pathway, a NACK1 kinesin-like protein and mitogen activated protein kinase (MAPK) cascade, is a key regulator of plant cytokinesis through the regulation of phragmoplast MTs. The MT-associated protein MAP65 has been identified as one of the structural components of MT assays involved in cell division, and we recently showed that Arabidopsis AtMAP65-3/PLEIADE (PLE) is a substrate of MPK4 that is a component of the NACK-PQR pathway in Arabidopsis. Here we show that AtMAP65-1 and AtMAP65-2 are also phosphorylated by MPK4. AtMAP65-1 and AtMAP65-2 that localize to the phragmoplast were phosphorylated by MPK4 in vitro. Although mutants of the Arabidopsis AtMAP65-1 and AtMAP65-2 genes exhibited a wild-type phenotype, double mutations of AtMAP65-3 and AtMAP65-1 or AtMAP65-2 caused more severe growth and cytokinetic defects than the single atmap65-3/ple mutation. These results suggest that AtMAP65-1 and AtMAP65-2 also function in cytokinesis downstream of MPK4.Key words: MAP65, microtubule-associated protein, MAPK, cytokinesis, phragmoplast, microtubule, arabidopsisMitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes, and are involved in various signaling processes in plant development, cell division and responses to endogenous or exogenous stimuli.¹ The NACK-PQR pathway, one of the best-characterized MAPK cascades in plants, has been identified as a key regulator of plant cytokinesis in tobacco. This pathway is composed of NPK1 MAPK kinase kinase (MAPKKK), NQK1/NtMEK1 MAPK kinase (MAPKK), NRK1/NTF6 MAPK and NACK1 kinesin-like protein, an activator of NPK1 MAPKKK.^2–5 During cytokinesis, all these components are localized on the equator of the phragmoplast, which is the plant-specific cytokinetic apparatus organized by microtubules (MTs). Downstream of this pathway, tobacco MAP65-1, an MT-associated protein, is phosphorylated by NRK1/NTF6 MAPK and phosphorylated MAP65-1 is localized to the equator of the phragmoplast.⁶ Phosphorylation of MAP65-1 by NRK1/NTF6 decreases the ability of MAP65-1 to bundle MTs, suggesting that the NACK-PQR pathway regulates expansion of the phragmoplast through the phosphorylation of MAP65.⁶The NACK-PQR pathway also seems to be conserved in Arabidopsis and rice. Several orthologs of components of the NACK-PQR pathway except for MAPK have been identified independently as regulators of cytokinesis in these plants.^3,5,7–14 Recently we reported that ANP MAPKKKs, MPK6/ANQ MAPKK and MPK4 MAPK biochemically constitute the MAPK pathway and HINKEL/AtNACK1 functions as an activator of ANP MAPKKKs.¹⁵ In addition, we revealed that MPK4 MAPK is localized to cell plates during cytokinesis, is required for cytokinesis in Arabidopsis and phosphorylates AtMAP65-3.¹⁶ Although AtMAP65-3 is proposed to be involved in cytokinesis,^17,18 and AtMAP65-1 is supposed to be a substrate of MPK4 based on a series of experiments,^6,19,20 the involvement in cytokinesis of other closely related members of the Arabidopsis MAP65 family, AtMAP65-1 and AtNAP65-2, has yet to be tested. In this report, we suggest redundant functions of these MAP65 molecules in cytokinesis of Arabidopsis.     

MAPK级联途径调控植物细胞胞质分裂

胞质分裂(cytokinesis)是细胞分裂的最后关键一步,产生2个含有完整的遗传物质和胞质细胞器的子细胞．植物胞质分裂包括细胞板的形成,这一过程是在成膜体的牵引下由一些植物特有的步骤完成的．促分裂原活化蛋白激酶(MAPK)级联途径在真核生物中是高度保守的,由MAPKs,MAPKKs,MAPKKKs组成,通过MAPKKK→ MAPKK → MAPK的逐级磷酸化传递细胞信号．近来的研究表明, NACK-MAPKKK→MAPKK→MAPK→MAP65构成的信号途径调控植物细胞的胞质分裂．本文就这一信号途径,总结了植物胞质分裂机制的研究进展,并对其中的问题进行了讨论与展望．     

NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase,regulates innate immunity and development in plants

Mitogen-activated protein kinase (MAPK) cascades are rapidly activated upon plant recognition of invading pathogens. Here, we describe the use of virus-induced gene silencing (VIGS) to study the role of candidate plant MAP kinase kinase kinase (MAPKKK) homologs of human MEKK1 in pathogen-resistance pathways. We demonstrate that silencing expression of a tobacco MAPKKK, Nicotiana Protein Kinase 1 (NPK1), interferes with the function of the disease-resistance genes N, Bs2, and Rx, but does not affect Pto- and Cf4-mediated resistance. Further, NPK1-silenced plants also exhibit reduced cell size, defective cytokinesis, and an overall dwarf phenotype. Our results provide evidence that NPK1 functions in the regulation of N-, Bs2-, and Rx-mediated resistance responses and may play a role in one or more MAPK cascades, regulating multiple cellular processes.     

Control of plant cytokinesis by an NPK1-mediated mitogen-activated protein kinase cascade

Cytokinesis is the last essential step in the distribution of genetic information to daughter cells and partition of the cytoplasm. In plant cells, various proteins have been found in the phragmoplast, which corresponds to the cytokinetic apparatus, and in the cell plate, which corresponds to a new cross wall, but our understanding of the functions of these proteins in cytokinesis remains incomplete. Reverse genetic analysis of NPK1 MAPKKK (nucleus- and phragmoplast-localized protein kinase 1 mitogen-activated protein kinase kinase kinase) and investigations of factors that might be functionally related to NPK1 have helped to clarify new aspects of the mechanisms of cytokinesis in plant cells. In this review, we summarize the evidence for the involvement of NPK1 in cytokinesis. We also describe the characteristics of a kinesin-like protein and the homologue of a mitogen-activated protein kinase that we identified recently, and we discuss possible relationships among these proteins in cytokinesis.     

Signal control through Raf: in sickness and in health

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade is the prototype mammalian mitogen-activated protein kinase (MAPK) signaling cascade that regulates a number of processes, including proliferation, differentiation, survival, migration, stress responses and apoptosis. How this seemingly linear cascade is modulated to achieve a specific cellular function has been a main focus of the field. In this review, we describe new as well as old findings in the regulation of the ERK1/2 pathway in normal and disease states via MAP3Ks.     

Signaling through MAP kinase networks in plants

Protein phosphorylation is the most important mechanism for controlling many fundamental cellular processes in all living organisms including plants. A specific class of serine/threonine protein kinases, the mitogen-activated protein kinases (MAP kinases) play a central role in the transduction of various extra- and intracellular signals and are conserved throughout eukaryotes. These generally function via a cascade of networks, where MAP kinase (MAPK) is phosphorylated and activated by MAPK kinase (MAPKK), which itself is activated by MAPKK kinase (MAPKKK). Signaling through MAP kinase cascade can lead to cellular responses including cell division, differentiation as well as response to various stresses. In plants, MAP kinases are represented by multigene families and are organized into a complex network for efficient transmission of specific stimuli. Putative plant MAP kinase cascades have been postulated based on experimental analysis of in vitro interactions between specific MAP kinase components. These cascades have been tested in planta following expression of epitope-tagged kinases in protoplasts. It is known that signaling for cell division and stress responses in plants are mediated through MAP kinases and even auxin, ABA and possibly ethylene and cytokinin also utilize a MAP kinase pathway. Most of the biotic (pathogens and pathogen-derived elicitors) including wounding and abiotic stresses (salinity, cold, drought, and oxidative) can induce defense responses in plants through MAP kinase pathways. In this article we have covered the historical background, biochemical assay, activation/inactivation, and targets of MAP kinases with emphasis on plant MAP kinases and the responses regulated by them. The cross-talk between plant MAP kinases is also discussed to bring out the complexity within this three-component module.     

植物MAP激酶级联途径研究进展

MAP激酶(促分裂原活化蛋白激酶)级联途径可以将不同的细胞膜感受器与细胞应答联系起来,响应各种生物以及非生物胁迫,在植物激素信号以及细胞分裂和发育过程中发挥着重要的作用.为有效地传递各种特异信号,MAP激酶级联相互交叉形成复杂的信号传递网络.近年来,随着功能获得型突变体、功能缺失型突变体的获得以及其它一些新技术的应用,进一步阐明了MAP激酶级联途径在信号传导过程中的功能和作用.本文主要对植物MAPK级联途径在信号传导过程中交叉串通以及复杂性的最新研究结果进行综述.     

A tobacco protein kinase, NPK2, has a domain homologous to a domain found in activators of mitogen-activated protein kinases (MAPKKs)

A cDNA (cNPK2) that encodes a protein of 518 amino acids was isolated from a library prepared from poly(A)⁺ RNAs of tobacco cells in suspension culture. The N-terminal half of the predicted NPK2 protein is similar in amino acid sequence to the catalytic domains of kinases that activate mitogen-activated protein kinases (designated here MAPKKs) from various animals and to those of yeast homologs of MAPKKs. The N-terminal domain of NPK2 was produced as a fusion protein in Escherichia coli, and the purified fusion protein was found to be capable of autophosphorylation of threonine and serine residues. These results indicate that the N-terminal domain of NPK2 has activity of a serine/threonine protein kinase. Southern blot analysis showed that genomic DNAs from various plant species, including Arabidopsis thaliana and sweet potato, hybridized strongly with cNPK2, indicating that these plants also have genes that are closely related to the gene for NPK2. The structural similarity between the catalytic domain of NPK2 and those of MAPKKs and their homologs suggests that tobacco NPK2 corresponds to MAPKKs of other organisms. Given the existence of plant homologs of an MAP kinase and tobacco NPK1, which is structurally and functionally homologous to one of the activator kinases of yeast homologs of MAPKK (MAPKKKs), it seems likely that a signal transduction pathway mediated by a protein kinase cascade that is analogous to the MAP kinase cascades proposed in yeasts and animals, is also conserved in plants.     

Effect of the MAPK cascade structure, nuclear translocation and regulation of transcription factors on gene expression

The mitogen activated protein kinase (MAP kinase) cascade system represents a highly conserved prototype of signal transduction by enzyme cascades. One of the best-studied properties of the MAPK system is its ability to convert graded input stimulus to switch-like all-or-none responses. Previous theoretical studies have centered on quantifying dual phosphorylated MAPK as a final output response and have not incorporated its influence on the regulation of gene expression. The main objective of the current work is to understand the regulatory effect of positive feedback loop embedded in the MAPK cascade, nuclear translocation of active MAPK, phosphorylation and activation of nuclear target proteins on the regulation of specific gene expression. To achieve this objective, we have simulated the MAPK cascade system, which resembles Hog1p activation pathway in yeast, at steady state. Thus, the input signal to the MAPK system is correlated with gene expression as a final system-level output response. The steady state simulation results suggest that other than regulating the signal propagation through cascades, the nuclear translocation of activated MAPK and subsequent regulation of gene expression represent one of the key modes to control the threshold level of response. This work proposes that, it is essential to consider the compartmental distributions of signaling species and the corresponding regulatory mechanisms of gene expression to study the system-level performance of signaling modules such as the MAPK cascade. Such an analysis will relate the extracellular cues to the final phenotypic response by capturing the mechanistic details of the signaling pathway.     

Stressing the role of MAP kinases in mitogenic stimulation

In yeast and animal cells, distinct subfamilies of mitogen-activated protein kinases (MAPKs) have evolved for transmitting different types of signals, such as the extracellular signal-regulated kinase (ERK) for mitogenic stimuli and differentiation, p38 and JUN kinase (JNK) for stress factors. Based on sequence analysis, the presently known plant MAPKs are most similar to ERKs, even though compelling evidence implies a role in various forms of biotic and abiotic stress responses. However, knowledge of their involvement in controlling proliferation is just emerging. A subgroup of the plant MAPKs, containing the alfalfa MMK3 and tobacco NTF6, are only active in mitotic cells and their localisation to the cell plate suggests a role in cytokinesis. An upstream regulator of MAPKs, the tobacco NPK1, appears to be also activated during mitosis. NPK1 might be associated and regulated by a microtubule motor protein. The localisation of NPK1 to the cell plate and its mitosis-specific activation suggest that together with NTF6 it could constitute a mitotic MAPK signalling module in tobacco. NPK1 appears to have a second role in repression of auxin-induced gene expression. MAPKs might also be involved in signalling within the meristems as suggested by the recruitement of a small G-protein to the CLAVATA 1 receptor-like protein kinase upon activation. In animal and yeast cells some of the small G-proteins relay signals from receptors to MAPK pathways.     

Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes.     

AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades have been implicated in regulating various aspects of plant development, including somatic cytokinesis. The evolution of expanded plant MAPK gene families has enabled the diversification of potential MAPK cascades, but functionally overlapping components are also well documented. Here we report that Arabidopsis MPK4, an MAPK that was previously described as a regulator of disease resistance, can interact with and be phosphorylated by the cytokinesis-related MAP kinase kinase, AtMKK6. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to form a normal intersporal callose wall after male meiosis, and thus cannot complete meiotic cytokinesis. Nevertheless, the multinucleate mpk4 microspores subsequently proceed through mitotic cytokinesis, resulting in enlarged mature pollen grains that possess increased sets of the tricellular structure. This pollen development phenotype is reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants, and protein-protein interaction analysis defines a putative signalling module linking AtNACK2/TES/STUD, AtANP3, AtMKK6 and AtMPK4 together as a cascade that facilitates male-specific meiotic cytokinesis in Arabidopsis.     

环境胁迫下植物MAPK多叠级联响应(英文)

Plant mitogen-activated protein kinases(MAPKs) are involved in growth,evelopment and responses to endogenous and environmental cues．which link stimuli that areactivated by external sensors to cellular responses．In Arabidopsis,as amodel,all of MAP kinase genes have been listed and classified．Based on the Arabidopsis MAPK families．a number of MAPk inase genes in other plant species have been recently isolated and classified．Most of the cloned MAPk inase genes can be activated by avariety of stresss timuli including pathogen infection．wounding．temperature,drought．salinity．osmolarity．UV irradiation．ozone and reactive oxygen species．Some tools and strategies are used to investigate their functions and signal pathways under different environmental stresses．indicating complexity and crosstalk of plant MAPk inase signaling pathways．It is still necessary to explore more novel tools and strategies to clarify MAPK signaling pathways,and how to apply the MAPK cascade to improve the resistance of crop to abiotic and biotic stress     

Regulation of MAP kinase-dependent apoptotic pathway: implication of reactive oxygen and nitrogen species

Mitogen-activated protein (MAP) kinase signaling cascades are multi-functional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase that triggers apoptogenic kinase cascade leading to the phosphorylation/activation of c-Jun N-terminal kinases and p38-MAP kinase, which are responsible for inducing apoptotic cell death. This pathway plays a pivotal role in transduction of signals from different apoptotic stimuli. In the present review, we summarized the recent evidence concerning MAP kinase-dependent apoptotic pathway and its regulation in the mammalian cells and organism in vivo. We have shown that the key messengers of regulation of this pathway are the reactive oxygen and nitrogen species. The role of protein oxidation and S-nitrosation in induction of apoptotic cell death via ASK1 is discussed. Also we have outlined other recently discovered signal transduction processes involved in the regulation of ASK1 activity and downstream pathway.     

EDR1 Physically Interacts with MKK4/MKK5 and Negatively Regulates a MAP Kinase Cascade to Modulate Plant Innate Immunity

Mitogen-activated protein (MAP) kinase signaling cascades play important roles in the regulation of plant defense. The Raf-like MAP kinase kinase kinase (MAPKKK) EDR1 negatively regulates plant defense responses and cell death. However, how EDR1 functions, and whether it affects the regulation of MAPK cascades, are not well understood. Here, we showed that EDR1 negatively regulates the MKK4/MKK5-MPK3/MPK6 kinase cascade in Arabidopsis. We found that edr1 mutants have highly activated MPK3/MPK6 kinase activity and higher levels of MPK3/MPK6 proteins than wild type. EDR1 physically interacts with MKK4 and MKK5, and this interaction requires the N-terminal domain of EDR1. EDR1 also negatively affects MKK4/MKK5 protein levels. In addition, the mpk3, mkk4 and mkk5 mutations suppress edr1-mediated resistance, and over-expression of MKK4 or MKK5 causes edr1-like resistance and mildew-induced cell death. Taken together, our data indicate that EDR1 physically associates with MKK4/MKK5 and negatively regulates the MAPK cascade to fine-tune plant innate immunity.     

环境胁迫下植物MAPK多叠级联响应

植物MAP(mitogen-activated protein)激酶涉及植物的生长发育、对内源和外界环境刺激的反应.MAP激酶能将胞外感受器引起的刺激传递到胞内引起细胞的反应.拟南芥(Arabidopsis thaliana)作为模式植物,其全部的MAP激酶已经列出并进行了分类.根据已分类的拟南芥MAP激酶家族,已经分离出大量的MAP激酶基因,并将它们进行分类,发现它们大多能被包括病原、创伤、温度、干旱、盐、渗透、紫外线辐射、臭氧和活性氧等胁迫刺激激活.通过研究在不同环境胁迫下的功能和信号路径,发现植物MAP激酶信号传递系统是复杂且相互交错的.需要开发一些新的工具和策略去阐明MAPK信号传递路径,以及如何利用MAPK系统去改善农作物对生物和非生物胁迫的抗性.