Scaffold proteins in mammalian MAP kinase cascades

The mitogen-activated protein kinase (MAPK) signaling pathway, which is conserved from yeast to humans, is activated in response to a variety of extra- and intracellular stimuli, and plays key roles in multiple cellular processes, including proliferation, differentiation, and apoptosis. The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinase to MAPK kinase to MAPK. Specific and efficient activation of the MAPK cascades is crucial for proper cellular responses to stimuli. As shown in yeast, the mammalian MAPK signaling system may also employ scaffold proteins, in part, to organize the MAPK signaling components into functional MAPK modules, thereby enabling the efficient activation of specific MAPK pathways. This review article describes recent advances in the study of potential mammalian scaffold proteins that may help us understand the complex regulation, including the spatial and temporal control, of the mammalian MAPK signaling pathways.     

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.     

MAPK级联途径参与ABA信号转导调节的植物生长发育过程

植物激素ABA参与调控植物生长发育和生理代谢以及多种胁迫应答过程,促分裂原活化蛋白激酶（MAPK）级联途径应答于多种生物和非生物胁迫,广泛参与调控植物的生长发育。MAPK级联途径与ABA信号转导协同作用参与调控植物种子萌发、气孔运动和生长发育,本文主要归纳了植物中受ABA调控激活的MAPK级联途径成员,阐述了它们参与ABA信号转导调控植物生理反应和生长发育的过程,并对MAPK级联途径与ABA信号转导的研究方向作出了展望,指出对MAPK下游底物的筛选是完善MAPK级联途径的重要组成部分。     

Negative feedback and ultrasensitivity can bring about oscillations in the mitogen-activated protein kinase cascades.

Functional organization of signal transduction into protein phosphorylation cascades, such as the mitogen-activated protein kinase (MAPK) cascades, greatly enhances the sensitivity of cellular targets to external stimuli. The sensitivity increases multiplicatively with the number of cascade levels, so that a tiny change in a stimulus results in a large change in the response, the phenomenon referred to as ultrasensitivity. In a variety of cell types, the MAPK cascades are imbedded in long feedback loops, positive or negative, depending on whether the terminal kinase stimulates or inhibits the activation of the initial level. Here we demonstrate that a negative feedback loop combined with intrinsic ultrasensitivity of the MAPK cascade can bring about sustained oscillations in MAPK phosphorylation. Based on recent kinetic data on the MAPK cascades, we predict that the period of oscillations can range from minutes to hours. The phosphorylation level can vary between the base level and almost 100% of the total protein. The oscillations of the phosphorylation cascades and slow protein diffusion in the cytoplasm can lead to intracellular waves of phospho-proteins.     

Membrane localization of scaffold proteins promotes graded signaling in the yeast MAP kinase cascade

BACKGROUND: Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or whether it must be enforced by separate mechanisms. It has been hypothesized that scaffold proteins promote graded behavior. RESULTS: Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust in that it resists perturbation by loss of several negative-feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. CONCLUSIONS: The results suggest that the MAPK cascade module is inherently ultrasensitive but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context.     

Regulation of cross-talk in yeast MAPK signaling pathways

MAP kinase (MAPK) modules are conserved three-kinase cascades that serve central roles in intracellular signal transduction in eukaryotic cells. MAPK pathways of different inputs and outputs use overlapping sets of signaling components. In yeast, for example, three MAPK pathways (pheromone response, filamentous growth response, and osmostress adaptation) all use the same Ste11 MAPK kinase kinase (MAPKKK). How undesirable leakage of signal, or cross-talk, is prevented between these pathways has been a subject of intensive study. This review discusses recent findings from yeast that indicate that there is no single mechanism, but that a combination of four general strategies (docking interactions, scaffold proteins, cross-pathway inhibition, and kinetic insulation) are utilized for the prevention of cross-talk between any two MAPK modules.     

The role of radical burst via MAPK signaling in plant immunity

Plants rely on the innate immune system to defend themselves from pathogen attacks. Reactive oxygen species (ROS) and nitric oxide (NO) play key roles in the activation of disease resistance mechanisms in plants. The evolutionarily conserved mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes and have been implicated in the plant innate immunity. There have been many disputations about the relationship between the radicals (ROS and NO) and MAPK cascades. Recently, we found that MAPK cascades participate in the regulation of the radical burst. Here, we discuss the regulatory mechanisms of the oxidative and NO bursts in response to pathogen attacks, and crosstalk between MAPK signaling and the radical burst.Key words: oxidative burst, MAPK, NADPH oxidase, NO burst, plant immunity     

Mitogen-activated protein kinase cascades in plant signaling

Mitogen-activated protein kinase (MAPK) cascades are key signaling modules downstream of receptors/sensors that perceive either endogenously produced stimuli such as peptide ligands and damage-associated molecular patterns (DAMPs) or exogenously originated stimuli such as pathogen/microbe-associated molecular patterns (P/MAMPs), pathogen-derived effectors, and environmental factors. In this review, we provide a historic view of plant MAPK research and summarize recent advances in the establishment of MAPK cascades as essential components in plant immunity, response to environmental stresses, and normal growth and development. Each tier of the MAPK cascades is encoded by a small gene family, and multiple members can function redundantly in an MAPK cascade. Yet, they carry out a diverse array of biological functions in plants. How the signaling specificity is achieved has become an interesting topic of MAPK research. Future investigations into the molecular mechanism(s) underlying the regulation of MAPK activation including the activation kinetics and magnitude in response to a stimulus, the spatiotemporal expression patterns of all the components in the signaling pathway, and functional characterization of novel MAPK substrates are central to our understanding of MAPK functions and signaling specificity in plants.     

Differential regulation of M3/6 (DUSP8) signaling complexes in response to arsenite-induced oxidative stress

Mitogen-activated protein kinase (MAPK) cascades are involved in the regulation of cellular proliferation, differentiation, survival, apoptosis, as well as in inflammatory responses. Signal intensity and duration have been recognized as crucial parameters determining MAPK signaling output. Phosphatases play a particularly important role in this respect, by tightly controlling MAPK phosphorylation and activation. M3/6 (DUSP8) is a dual-specificity phosphatase implicated in the dephosphorylation and inactivation of JNK and, to a lesser extent, p38 MAPKs and is found in a complex with these kinases, along with other pathway components, held together by scaffold proteins. The JNK family consists of three genes, giving rise to at least ten different splice variants. Some functional differences between these gene products have been demonstrated, but the underlying molecular mechanisms and the roles of individual splice variants are still incompletely understood. We have investigated the interaction of M3/6 with JNK isoforms, as well as scaffold proteins of the JNK interacting protein (JIP) family, in order to elucidate the contribution of M3/6 to the regulation of distinct JNK signaling modules. M3/6 exhibited stronger binding towards JNK1β and JNK2α isoforms and this was reflected in higher enzymatic activity towards JNK2α2 when compared to JNK1α1 in vitro. After activation of the pathway by exposure of cells to arsenite, the interaction of M3/6 with JNK1α and JNK3 was enhanced, whereas that with JNK1β or JNK2α decreased. The modulation of binding affinities was found to be independent of JNK-mediated M3/6 phosphorylation. Furthermore, arsenite treatment resulted in an inducible recruitment of M3/6 to JNK-interacting protein 3 (JIP3) scaffold complexes, while its interaction with JIP1 or JIP2 was constitutive. The presented data suggest an isoform-specific role for the M3/6 phosphatase and the dynamic targeting of M3/6 towards distinct JNK-containing signaling complexes.     

Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo

While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.     

The rice MAPKK–MAPK interactome: the biological significance of MAPK components in hormone signal transduction

Mitogen-activated protein kinase (MAPK) signaling cascades are evolutionarily conserved fundamental signal transduction pathways. A MAPK cascade consists of many distinct MAPKKK–MAPKK–MAPK modules linked to various upstream receptors and downstream targets through sequential phosphorylation and activation of the cascade components. These cascades collaborate in transmitting a variety of extracellular signals and in controlling cellular responses and processes such as growth, differentiation, cell death, hormonal signaling, and stress responses. Although MAPK proteins play central roles in signal transduction pathways, our knowledge of MAPK signaling in hormonal responses in rice has been limited to a small subset of specific upstream and downstream interacting targets. However, recent studies revealing direct MAPK and MAPKK interactions have provided the basis for elucidating interaction specificities, functional divergence, and functional modulation during hormonal responses. In this review, we highlight current insights into MAPKK–MAPK interaction patterns in rice, with emphasis on the biological significance of these interacting pairs in SA (salicylic acid), JA (jasmonic acid), ET (ethylene), and ABA (abscisic acid) responses, and discuss the challenges in understanding functional signal transduction networks mediated by these hormones.     

MNAR plays an important role in ERa activation of Src/MAPK and PI3K/Akt signaling pathways

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.     

A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway

Mitogen-activated protein kinase (MAPK) cascades are conserved signaling modules composed of three sequentially activated kinases (MAPKKK, MAPKK and MAPK). Because individual cells contain multiple MAPK cascades, mechanisms are required to ensure the fidelity of signal transmission. In yeast, external high osmolarity activates the HOG (high osmolarity glycerol) MAPK pathway, which consists of two upstream branches (SHO1 and SLN1) and common downstream elements including the Pbs2 MAPKK and the Hog1 MAPK. The Ssk2/Ssk22 MAPKKKs in the SLN1 branch, when activated, exclusively phosphorylate the Pbs2 MAPKK. We found that this was due to an Ssk2/Ssk22-specific docking site in the Pbs2 N-terminal region. The Pbs2 docking site constitutively bound the Ssk2/Ssk22 kinase domain. Docking site mutations drastically reduced the Pbs2-Ssk2/Ssk22 interaction and hampered Hog1 activation by the SLN1 branch. Fusion of the Pbs2 docking site to a different MAPKK, Ste7, allowed phosphorylation of Ste7 by Ssk2/Ssk22. Thus, the docking site contributes to both the efficiency and specificity of signaling. During these analyses, we also found a nuclear export signal and a possible nuclear localization signal in Pbs2.     

Integrated analysis of co-expressed MAP kinase substrates in Arabidopsis thaliana

MAP kinase (MAPK) signal transduction cascades are conserved eukaryotic pathways that modulate stress responses and developmental processes. In a recent report we have identified novel Arabidopsis MAPKK/MAPK/Substrate signaling pathways using microarrays containing 2,158 unique Arabidopsis proteins. Subsequently, several WRKY and TGA targets phosphorylated by MAPKs were verified in planta. We have also reported that specific MAPKK/MAPK modules expressed in Nicotiana benthamiana induced a cell death phenotype related to the immune response. We have generated a MAPK phosphorylation network based on our protein microarray experimental data. Here we further analyze our network by integrating phosphorylation and gene expression information to identify biologically relevant signaling modules. We have identified 108 phosphorylation events that occur among 96 annotated genes with highly similar pairwise expression profiles. Our analysis brings a new perspective on MAPK signaling by revealing new relationships between components of signaling pathways.Key words: MAPK, protein microarray, network, cell death, co-expression, signaling     

Quantitative effect of scaffold abundance on signal propagation

Protein scaffolds bring together multiple components of a signalling pathway, thereby promoting signal propagation along a common physical ‘backbone’. Scaffolds play a prominent role in natural signalling pathways and provide a promising platform for synthetic circuits. To better understand how scaffolding quantitatively affects signal transmission, we conducted an in vivo sensitivity analysis of the yeast mating pathway to a broad range of perturbations in the abundance of the scaffold Ste5. Our measurements show that signal throughput exhibits a biphasic dependence on scaffold concentration and that altering the amount of scaffold binding partners reshapes this biphasic dependence. Unexpectedly, the wild‐type level of Ste5 is ～10‐fold below the optimum needed to maximize signal throughput. This sub‐optimal configuration may be a tradeoff as increasing Ste5 expression promotes baseline activation of the mating pathway. Furthermore, operating at a sub‐optimal level of Ste5 may provide regulatory flexibility as tuning Ste5 expression up or down directly modulates the downstream phenotypic response. Our quantitative analysis reveals performance tradeoffs in scaffold‐based modules and defines engineering challenges for implementing molecular scaffolds in synthetic pathways.     

Effects of sequestration on signal transduction cascades

The building blocks of most signal transduction pathways are pairs of enzymes, such as kinases and phosphatases, that control the activity of protein targets by covalent modification. It has previously been shown [Goldbeter A & Koshland DE (1981) Proc Natl Acad Sci USA 78, 6840-6844] that these systems can be highly sensitive to changes in stimuli if their catalysing enzymes are saturated with their target protein substrates. This mechanism, termed zero-order ultrasensitivity, may set thresholds that filter out subthreshold stimuli. Experimental data on protein abundance suggest that the enzymes and their target proteins are present in comparable concentrations. Under these conditions a large fraction of the target protein may be sequestrated by the enzymes. This causes a reduction in ultrasensitivity so that the proposed mechanism is unlikely to account for ultrasensitivity under the conditions present in most in vivo signalling cascades. Furthermore, we show that sequestration changes the dynamics of a covalent modification cycle and may account for signal termination and a sign-sensitive delay. Finally, we analyse the effect of sequestration on the dynamics of a complex signal transduction cascade: the mitogen-activated protein kinase (MAPK) cascade with negative feedback. We show that sequestration limits ultrasensitivity in this cascade and may thereby abolish the potential for oscillations induced by negative feedback.     

Regulation of signal duration and the statistical dynamics of kinase activation by scaffold proteins

Scaffolding proteins that direct the assembly of multiple kinases into a spatially localized signaling complex are often essential for the maintenance of an appropriate biological response. Although scaffolds are widely believed to have dramatic effects on the dynamics of signal propagation, the mechanisms that underlie these consequences are not well understood. Here, Monte Carlo simulations of a model kinase cascade are used to investigate how the temporal characteristics of signaling cascades can be influenced by the presence of scaffold proteins. Specifically, we examine the effects of spatially localizing kinase components on a scaffold on signaling dynamics. The simulations indicate that a major effect that scaffolds exert on the dynamics of cell signaling is to control how the activation of protein kinases is distributed over time. Scaffolds can influence the timing of kinase activation by allowing for kinases to become activated over a broad range of times, thus allowing for signaling at both early and late times. Scaffold concentrations that result in optimal signal amplitude also result in the broadest distributions of times over which kinases are activated. These calculations provide insights into one mechanism that describes how the duration of a signal can potentially be regulated in a scaffold mediated protein kinase cascade. Our results illustrate another complexity in the broad array of control properties that emerge from the physical effects of spatially localizing components of kinase cascades on scaffold proteins.     

Synergistic interaction of MEK kinase 2, c-Jun N-terminal kinase (JNK) kinase 2, and JNK1 results in efficient and specific JNK1 activation

Mitogen-activated protein kinases (MAPKs) are activated through cascades or modules consisting of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Investigating the molecular basis of activation of the c-Jun N-terminal kinase (JNK) subgroup of MAPK by the MAPKKK MEKK2, we found that strong and specific JNK1 activation by MEKK2 was mediated by the MAPKK JNK kinase 2 (JNKK2) rather than by JNKK1 through formation of a tripartite complex consisting of MEKK2, JNKK2, and JNK1. No scaffold protein was required for the MEKK2-JNKK2-JNK1 tripartite-complex formation. Expression of JNK1, JNKK2, and MEKK2 significantly augmented the coprecipitation of, respectively, MEKK2-JNKK2, MEKK2-JNK1, and JNKK2-JNK1, indicating that the interaction of MEKK2, JNKK2, and JNK1 is synergistic. Finally, the JNK1 was activated more efficiently in the MEKK2-JNKK2-JNK1 complex than was the JNK1 excluded from the complex. Thus, formation of a signaling complex through synergistic interaction of a MAPKKK, a MAPKK, and a MAPK molecule like MEKK2-JNKK2-JNK1 is likely to be responsible for the efficient, specific flow of information via MAPK cascades.