Organization and regulation of mitogen-activated protein kinase signaling pathways

Mitogen-activated protein kinases (MAPKs) are components of a three kinase regulatory cascade. There are multiple members of each component family of kinases in the MAPK module. Specificity of regulation is achieved by organization of MAPK modules, in part, by use of scaffolding and anchoring proteins. Scaffold proteins bring together specific kinases for selective activation, sequestration and localization of signaling complexes. The recent elucidation of scaffolding mechanisms for MAPK pathways has begun to solve the puzzle of how specificity in signaling can be achieved for each MAPK pathway in different cell types and in response to different stimuli. As new MAPK members are defined, determining their organization in kinase modules will be critical in understanding their select role in cellular regulation.     

Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae

Scaffold proteins are believed to enhance specificity in cell signaling when different pathways share common components. The prototype scaffold Ste5 binds to multiple components of the Saccharomyces cerevisiae mating pheromone response pathway, thereby conducting the mating signal to the Fus3 mitogen-activated protein kinase (MAPK). Some of the kinases that Ste5 binds to, however, are also shared with other pathways. Thus, it has been presumed that Ste5 prevents its bound kinases from transgressing into other pathways and protects them from intrusions from those pathways. Here we found that Fus3MAPK required Ste5 scaffolding to receive legitimate signals from the mating pathway as well as misdirected signals leaking from other pathways. Furthermore, increasing the cellular concentration of active Ste5 enhanced the channeling of inappropriate stimuli to Fus3. This aberrant signal crossover resulted in the erroneous induction of cell cycle arrest and mating. In contrast to Fus3, the Kss1 MAPK did not require Ste5 scaffolding to receive either authentic or leaking signals. Furthermore, the Ste11 kinase, once activated via Ste5, was able to signal to Kss1 independently of Ste5 scaffolding. These results argue that Ste5 does not act as a barrier that actively prevents signal crossover to Fus3 and that Ste5 may not effectively sequester its activated kinases away from other pathways. Rather, we suggest that specificity in this network is promoted by the selective activation of Ste5 and the distinct requirements of the MAPKs for Ste5 scaffolding.     

ATGs: Scaffolds for MAPK/ERK signaling

Autophagy maintains cellular homeostasis by sequestering unwanted material within autophagosomes and transferring these to lysosomes for degradation. Several signaling cascades activate or suppress autophagy in response to diverse environmental cues. However, whether autophagic structures per se regulate cell signaling was not known. The MAPK/ERK (mitogen-activated protein kinase) pathway controls several functions in the cell, and studies have identified the importance of scaffold proteins in modulating MAPK signaling through the spatial coordination of the RAF1-MAP2K/MEK-MAPK cascade. Growth factors increase the nuclear localization and activity of MAPK, and since the nucleus has been reported to contain LC3, an autophagy-related protein, we asked whether autophagic structures could serve as cytosolic and nuclear scaffolds for growth factor-induced MAPK phosphorylation.     

ATGs

Autophagy maintains cellular homeostasis by sequestering unwanted material within autophagosomes and transferring these to lysosomes for degradation. Several signaling cascades activate or suppress autophagy in response to diverse environmental cues. However, whether autophagic structures per se regulate cell signaling was not known. The MAPK/ERK (mitogen-activated protein kinase) pathway controls several functions in the cell, and studies have identified the importance of scaffold proteins in modulating MAPK signaling through the spatial coordination of the RAF1-MAP2K/MEK-MAPK cascade. Growth factors increase the nuclear localization and activity of MAPK, and since the nucleus has been reported to contain LC3, an autophagy-related protein, we asked whether autophagic structures could serve as cytosolic and nuclear scaffolds for growth factor-induced MAPK phosphorylation.     

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.     

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.     

Localized feedback phosphorylation of Ste5p scaffold by associated MAPK cascade

Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p Gbeta subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation.     

Protein phosphatases in MAPK signalling: we keep learning from yeast

Because of their key role in cell signalling, a rigorous regulation of mitogen-activated protein kinases (MAPKs) is essential in eukaryotic physiology. Whereas the use of binding motifs and scaffold proteins guarantees the selective activation of a specific MAPK pathway, activating kinases and downregulating phosphatases control the appropriate intensity and timing of MAPK activation. Tyrosine, serine/threonine and dual-specificity phosphatases co-ordinately dephosphorylate and thereby inactivate MAPKs. In budding yeast, enzymes that belong to these three types of phosphatases have been shown to counteract the MAPKs that govern the cellular response to varied extracellular stimuli. Studies carried out with these yeast phosphatases have expanded our knowledge of essential key aspects of the biology of these negative regulators, such as their function, the mechanisms that operate in their modulation by MAPK pathways and their binding to MAPK substrates. Furthermore, yeast MAPK phosphatases have been shown to play additional and essential roles in MAPK-mediated signalling, controlling MAPK localization or cross-talk among pathways. This review stresses the importance of these negative regulators in eukaryotic signalling by discussing the recent developments and perspectives in the study of yeast MAPK phosphatases.     

Structural Conservation of Components in the Amino Acid Sensing Branch of the TOR Pathway in Yeast and Mammals

The highly conserved Rag family GTPases have a role in reporting amino acid availability to the TOR (target of rapamycin) signaling complex, which regulates cell growth and metabolism in response to environmental cues. The yeast Rag proteins Gtr1p and Gtr2p were shown in multiple independent studies to interact with the membrane-associated proteins Gse1p (Ego3p) and Gse2p (Ego1p). However, mammalian orthologs of Gse1p and Gse2p could not be identified. We determined the crystal structure of Gse1p and found it to match the fold of two mammalian proteins, MP1 (mitogen-activated protein kinase scaffold protein 1) and p14, which form a heterodimeric complex that had been assigned a scaffolding function in mitogen-activated protein kinase pathways. The significance of this structural similarity is validated by the recent identification of a physical and functional association between mammalian Rag proteins and MP1/p14. Together, these findings reveal that key components of the TOR signaling pathway are structurally conserved between yeast and mammals, despite divergence of sequence to a degree that thwarts detection through simple homology searches.     

Acetylcholine receptor clustering is required for the accumulation and maintenance of scaffolding proteins

The maintenance of a high density of postsynaptic receptors is essential for proper synaptic function. At the neuromuscular junction, acetylcholine receptor (AChR) aggregation is induced by nerve-clustering factors and mediated by scaffolding proteins. Although the mechanisms underlying AChR clustering have been extensively studied, the role that the receptors themselves play in the clustering process and how they are organized with scaffolding proteins is not well understood. Here, we report that the exposure of AChRs labeled with Alexa 594 conjugates to relatively low-powered laser light caused an effect similar to chromaphore-assisted light inactivation (CALI) , which resulted in the unexpected dissipation of the illuminated AChRs from clusters on cultured myotubes. This technique enabled us to demonstrate that AChR removal from illuminated regions induced the removal of scaffolding proteins and prevented the accumulation of new AChRs and associated scaffolding proteins. Further, the dissipation of clustered AChRs and scaffold was spatially restricted to the illuminated region and had no effect on neighboring nonilluminated AChRs. These results provide direct evidence that AChRs are essential for the local maintenance and accumulation of intracellular scaffolding proteins and suggest that the scaffold is organized into distinct modular units at AChR clusters.     

Targeted trafficking of neurotransmitter receptors to synaptic sites

Emerging data are sheding light on the critical task for synapses to locally control the production of neurotransmitter receptors ultimately leading to receptor accumulation and modulation at postsynaptic sites. By analogy with the epithelial-cell paradigm, the postsynaptic compartment may be regarded as a polarized domain favoring the selective recruitment and retention of newly delivered receptors at synaptic sites. Targeted delivery of receptors to synaptic sites is facilitated by a local organization of the exocytic pathway, likely resulting from spatial cues triggered by the nerve. This review focuses on the various mechanisms responsible for regulation of receptor assembly and trafficking. A particular emphasis is given to the role of synaptic anchoring and scaffolding proteins in the sorting and routing of their receptor companion along the exocytic pathway. Other cellular components such as lipidic microdomains, the docking and fusion machinery, and the cytoskeleton also contribute to the dynamics of receptor trafficking at the synapse.     

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.     

Differential input by Ste5 scaffold and Msg5 phosphatase route a MAPK cascade to multiple outcomes

Pathway specificity is poorly understood for mitogen-activated protein kinase (MAPK) cascades that control different outputs in response to different stimuli. In yeast, it is not known how the same MAPK cascade activates Kss1 MAPK to promote invasive growth (IG) and proliferation, and both Fus3 and Kss1 MAPKs to promote mating. Previous work has suggested that the Kss1 MAPK cascade is activated independently of the mating G protein (Ste4)-scaffold (Ste5) system during IG. Here we demonstrate that Ste4 and Ste5 activate Kss1 during IG and in response to multiple stimuli including butanol. Ste5 activates Kss1 by generating a pool of active MAPKKK (Ste11), whereas additional scaffolding is needed to activate Fus3. Scaffold-independent activation of Kss1 can occur at multiple steps in the pathway, whereas Fus3 is strictly dependent on the scaffold. Pathway specificity is linked to Kss1 immunity to a MAPK phosphatase that constitutively inhibits basal activation of Fus3 and blocks activation of the mating pathway. These findings reveal the versatility of scaffolds and how a single MAPK cascade mediates different outputs.     

Counteractive control of polarized morphogenesis during mating by mitogen-activated protein kinase Fus3 and G1 cyclin-dependent kinase

Cell polarization in response to external cues is critical to many eukaryotic cells. During pheromone-induced mating in Saccharomyces cerevisiae, the mitogen-activated protein kinase (MAPK) Fus3 induces polarization of the actin cytoskeleton toward a landmark generated by the pheromone receptor. Here, we analyze the role of Fus3 activation and cell cycle arrest in mating morphogenesis. The MAPK scaffold Ste5 is initially recruited to the plasma membrane in random patches that polarize before shmoo emergence. Polarized localization of Ste5 is important for shmooing. In fus3 mutants, Ste5 is recruited to significantly more of the plasma membrane, whereas recruitment of Bni1 formin, Cdc24 guanine exchange factor, and Ste20 p21-activated protein kinase are inhibited. In contrast, polarized recruitment still occurs in a far1 mutant that is also defective in G1 arrest. Remarkably, loss of Cln2 or Cdc28 cyclin-dependent kinase restores polarized localization of Bni1, Ste5, and Ste20 to a fus3 mutant. These and other findings suggest Fus3 induces polarized growth in G1 phase cells by down-regulating Ste5 recruitment and by inhibiting Cln/Cdc28 kinase, which prevents basal recruitment of Ste5, Cdc42-mediated asymmetry, and mating morphogenesis.     

Molecular recognitions in the MAP kinase cascades

The mitogen-activated protein kinase (MAPK) cascades play a pivotal role in many aspects of cellular functions, and are evolutionarily conserved from yeast to mammals. In mammals, there are four subfamily members in the MAPKs. Each MAPK has its own activators, substrates and inactivators. In order to achieve normal cellular functions, the MAPK cascades should transduce signals with high efficiency and fidelity. However, the molecular basis for the mechanism underlying the specific reactions in the MAPK cascades has not been fully understood. The MAPKs form a globular structure without a distinct domain specific for protein-protein interactions. Recent studies revealed two mechanisms regulating the signalling, the docking interaction and the scaffolding. The docking interaction is achieved through the common docking domain (the CD domain) on MAPKs, and is different from a transient enzyme-substrate interaction through the active centre of the enzymes. Almost all the MAPK-interacting molecules have a conserved motif interacting with the CD domain. The scaffolding usually utilizes a third molecule to tether several components of the MAPK cascades. Both of them are thought to regulate the enzymatic specificity and efficiency.     

In vivo co-localization of enzymes on RNA scaffolds increases metabolic production in a geometrically dependent manner

Co-localization of biochemical processes plays a key role in the directional control of metabolic fluxes toward specific products in cells. Here, we employ in vivo scaffolds made of RNA that can bind engineered proteins fused to specific RNA binding domains. This allows proteins to be co-localized on RNA scaffolds inside living Escherichia coli. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in one or two dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of substrates.     

Beta-arrestins as regulators of signal termination and transduction: how do they determine what to scaffold?

Over the last decade β-arrestins have emerged as pleiotropic scaffold proteins, capable of mediating numerous diverse responses to multiple agonists. Most well characterized are the G-protein-coupled receptor (GPCR) stimulated β-arrestin signals, which are sometimes synergistic with, and sometimes independent of, heterotrimeric G-protein signals. β-arrestin signaling involves the recruitment of downstream signaling moieties to β-arrestins; in many cases specific sites of interaction between β-arrestins and the downstream target have been identified. As more information unfolds about the nature of β-arrestin scaffolding interactions, it is evident that these proteins are capable of adopting multiple conformations which in turn reveal a specific set of interacting domains. Recruitment of β-arrestin to a specific GPCR can promote formation of a specific subset of available β-arrestin scaffolds, allowing for a higher level of specificity to given agonists. This review discusses recent advances in β-arrestin signaling, discussing the molecular details of a subset of known β-arrestin scaffolds and the significance of specific binding interactions on the ultimate cellular response.