MAP Kinase Modules: The Excursion Model and the Steps that Count

MAP kinase modules propagate diverse extracellular signals to downstream effectors. The two dual phosphorylation reactions catalyzed by the modules are thought to control the switch behavior of the pathway. Here we review recent approaches to understand these pathways through signal-to-response studies in cells and in vitro. These data are reconciled with physical models as well as predictions made on mathematical and theoretical grounds. Biochemical analysis has shown recently that the dual phosphorylation reactions catalyzed by MAP kinase modules are sequential at both levels of the cascade. The observed order of phosphorylation events suggests an excursion from the Ser/Thr kinase activity of the MAP3K into Tyr kinase activity of the central dual specificity MAP2K. How the order of events might be encoded in the structures and interactions is discussed. The ordered mechanism confirms predictions that reactions should be sequential to generate the steep signal-to-response curves and delayed responses observed in cells.     

MKK6重组腺病毒的构建与制备

构建腺病毒穿梭载体pAd RSV ,并将p3 8MAPK (mitogen activatedproteinkinase)的上游激酶MKK6(mitogen activatedproteinkinasekinase 6)及其持续激活和无活性的突变体基因亚克隆到该穿梭载体 .通过与腺病毒DNA(pJM17)在能够表达E1的HEK 2 93细胞同源重组生成了能够表达MKK6信号分子的重组腺病毒 .PCR结果表明 ,这些重组腺病毒DNA的插入片段大小是正确的 .而且 ,通过感染COS 7细胞 ,用免疫激酶活性测定证实这些重组的腺病毒能够表达具有功能的蛋白质 .     

ZAK: a MAP3Kinase that transduces Shiga toxin- and ricin-induced proinflammatory cytokine expression

Shiga toxins (Stxs) and ricin initiate damage to host cells by cleaving a single adenine residue on the α-sarcin loop of the 28S ribosomal RNA. This molecular insult results in a cascade of intracellular events termed the ribotoxic stress response (RSR). Although Stxs and ricin have been shown to cause the RSR, the mitogen-activated protein kinase kinase kinase (MAP3K) that transduces the signal from intoxicated ribosomes to activate SAPKinases has remained elusive. We show in vitro that DHP-2 (7-[3-fluoro-4-aminophenyl-(4-(2-pyridin-2-yl-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl))]-quinoline), a zipper sterile-α-motif kinase (ZAK)-specific inhibitor, blocks Stx2/ricin-induced SAPKinase activation. Treatment of cells with DHP-2 also blocks Stx2/ricin-mediated upregulation of the proinflammatory cytokine interleukin-8 and results in a modest but statistically significant improvement in cell viability following Stx2/ricin treatment. Finally we show that siRNA directed against the N-terminus of ZAK diminishes Stx2/Ricin-induced SAPKinase activation. Together, these data demonstrate that a ZAK isoform(s) is the MAP3Kinase that transduces the RSR. Therefore, ZAKα and/or β isoforms may act as potential therapeutic target(s) for treating Stx/ricin-associated illnesses. Furthermore, a small molecule inhibitor like DHP-2 may prove valuable in preventing the Stx/ricin-induced proinflammatory and/or apoptotic effects that are thought to contribute to pathogenesis by Stx-producing Escherichia coli and ricin.     

The Fission Yeast Mitotic Regulator win1+ Encodes an MAP Kinase Kinase Kinase That Phosphorylates and Activates Wis1 MAP Kinase Kinase in Response to High Osmolarity

The Schizosaccharomyces pombe win1-1 mutant has a defect in the G2-M transition of the cell cycle. Although the defect is suppressed by wis1⁺ and wis4⁺, which are components of a stress-activated MAP kinase pathway that links stress response and cell cycle control, the molecular identity of Win1 has not been known. We show here that win1⁺ encodes a polypeptide of 1436 residues with an apparent molecular size of 180 kDa and demonstrate that Win1 is a MAP kinase kinase kinase that phosphorylates and activates Wis1. Despite extensive similarities between Win1 and Wis4, the two MAP kinase kinase kinases have distinct functions. Wis4 is able to compensate for loss of Win1 only under unstressed conditions to maintain basal Wis1 activity, but it fails to suppress the osmosignaling defect conferred by win1 mutations. The win1-1 mutation is a spontaneous duplication of 16 nucleotides, which leads to a frameshift and production of a truncated protein lacking the kinase domain. We discuss the cell cycle phenotype of the win1-1 cdc25-22 wee1-50 mutant and its suppression by wis genes.     

The MAP1B case: An old MAP that is new again

The functions of microtubule‐associated protein 1B (MAP1B) have historically been linked to the development of the nervous system, based on its very early expression in neurons and glial cells. Moreover, mice in which MAP1B is genetically inactivated have been used extensively to show its role in axonal elongation, neuronal migration, and axonal guidance. In the last few years, it has become apparent that MAP1B has other cellular and molecular functions that are not related to its microtubule‐stabilizing properties in the embryonic and adult brain. In this review, we present a systematic review of the canonical and novel functions of MAP1B and propose that, in addition to regulating the polymerization of microtubule and actin microfilaments, MAP1B also acts as a signaling protein involved in normal physiology and pathological conditions in the nervous system. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 953–971, 2014     

MAP Kinase Pathways in the Yeast Saccharomyces cerevisiae

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.     

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.     

p38丝裂原活化蛋白激酶的功能与调控机制

丝裂原活化蛋白激酶家族可以在一系列细胞外刺激下调控细胞的行为。作为该家族的四个亚家族之一,p38亚族在许多生理过程中扮演着重要角色。p38信号通路可以在紫外照射、热击、高渗透压、炎症因子、生长因子等细胞外刺激时被激活,调控细胞分化、细胞周期、炎症反应等多种生理过程。文章重点讨论了p38亚族各个成员的特性、该信号通路的组成部分、调控机制以及生物学功能。另外,还分析了p38与其他信号通路的联系以及对一些生理过程的影响。     

MAP Kinase Pathways: Straight and narrow or tortuous and intersecting?

Stress and mitogens stimulate overlapping sets of MAP kinases in mammalian cells; MAP kinase pathways appear more distinct in yeast, but differences between the two systems may be less than is presently evident.     

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/.     

ZmMPK6, a Novel Maize MAP Kinase that Interacts with 14-3-3 Proteins

Although an increasing body of evidence indicates that plant MAP kinases are involved in a number of cellular processes, such as cell cycle regulation and cellular response to abiotic stresses, hormones and pathogen attack, very little is known about their biochemical properties and regulation mechanism. In this paper we report on the identification and characterization of a novel member of the MAP kinase family from maize, ZmMPK6. The amino acid sequence reveals a high degree of identity with group D plant MAP kinases. Recombinant ZmMPK6, expressed in Escherichia coli, is an active enzyme able to autophosphorylate. Remarkably, ZmMPK6 interacts in vitro with GF14-6, a maize 14-3-3 protein and the interaction is dependent on autophosphorylation. The interacting domain of ZmMPK6 is on the C-terminus and is comprised between amino acid 337 and amino acid 467. Our results represent the first evidence of an interaction between a plant MAP kinase and a 14-3-3 protein. Possible functional roles of this association in vivo are discussed.     

Mathematical Modelling of the MAP Kinase Pathway Using Proteomic Datasets

The advances in proteomics technologies offer an unprecedented opportunity and valuable resources to understand how living organisms execute necessary functions at systems levels. However, little work has been done up to date to utilize the highly accurate spatio-temporal dynamic proteome data generated by phosphoprotemics for mathematical modeling of complex cell signaling pathways. This work proposed a novel computational framework to develop mathematical models based on proteomic datasets. Using the MAP kinase pathway as the test system, we developed a mathematical model including the cytosolic and nuclear subsystems; and applied the genetic algorithm to infer unknown model parameters. Robustness property of the mathematical model was used as a criterion to select the appropriate rate constants from the estimated candidates. Quantitative information regarding the absolute protein concentrations was used to refine the mathematical model. We have demonstrated that the incorporation of more experimental data could significantly enhance both the simulation accuracy and robustness property of the proposed model. In addition, we used the MAP kinase pathway inhibited by phosphatases with different concentrations to predict the signal output influenced by different cellular conditions. Our predictions are in good agreement with the experimental observations when the MAP kinase pathway was inhibited by phosphatase PP2A and MKP3. The successful application of the proposed modeling framework to the MAP kinase pathway suggests that our method is very promising for developing accurate mathematical models and yielding insights into the regulatory mechanisms of complex cell signaling pathways.     

The Evolution of the MAP Kinase Pathways: Coduplication of Interacting Proteins Leads to New Signaling Cascades

The MAP-kinase pathways are intracellular signaling modules that are likely to exist in all eukaryotes. We provide an evolutionary model for these signaling pathways by focusing on the gene duplications that have occurred since the divergence of animals from yeast. Construction of evolutionary trees with confidence assessed by bootstrap clearly shows that the mammalian JNK and p38 pathways arose from an ancestral hyperosmolarity pathway after the split from yeast and before the split from C. elegans. These coduplications of interacting proteins at the MAPK and MEK levels have since evolved toward substrate specificity, thus giving distinct pathways. Mammalian duplications since the split from C. elegans are often associated with divergent tissue distribution but do not appear to confer detectable substrate specificity. The yeast kinase cascades have undergone similar fundamental functional changes since the split from mammals, with duplications giving rise to central signaling components of the filamentous and hypoosmolarity pathways. Experimentally defined cross-talk between yeast pheromone and hyperosmolarity pathways is mirrored with corresponding cross-talk in mammalian pathways, suggesting the existence of ancient orthologous cross-talk; our analysis of gene duplications at all levels of the cascade is consistent with this model but does not always provide significant bootstrap support. Our data also provide insights at different levels of the cascade where conflicting experimental evidence exists. Received: 2 December 1998 / Accepted: 9 June 1999     

Interfering with MAP Kinase Docking Interactions: Implications and Perspectives for the p38 Route

Docking interactions are key to understand the dynamic assembly of signal transductioncomplexes in the cell. In particular, the docking domain (D domain)-dependentinteractions described so far for several MAPK routes are essential to specify theupstream regulators, downstream mediators and also inactivators that complex with thep38, JNK and ERK proteins. In addition to contributing to the maintenance of thelinearity and specificity of these pathways, novel data have revealed that dockingcontacts also regulate the activity, subcellular distribution and substrate selection ofeach MAPK. Moreover, phosphorylation inside or around a docking domain isemerging as a novel mechanism of regulation of MAPK association with cellularpartners, suggesting new potential strategies for the design of selective MAPKinhibitors. Here, we discuss these novel data and the biochemical and cellularimplications they may have with specific emphasis on the p38 route.     

The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster''s clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila''s most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential “clock kinase” phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.