Dose-to-duration encoding and signaling beyond saturation in intracellular signaling networks

The cellular response elicited by an environmental cue typically varies with the strength of the stimulus. For example, in the yeast Saccharomyces cerevisiae, the concentration of mating pheromone determines whether cells undergo vegetative growth, chemotropic growth, or mating. This implies that the signaling pathways responsible for detecting the stimulus and initiating a response must transmit quantitative information about the intensity of the signal. Our previous experimental results suggest that yeast encode pheromone concentration as the duration of the transmitted signal. Here we use mathematical modeling to analyze possible biochemical mechanisms for performing this “dose-to-duration” conversion. We demonstrate that modulation of signal duration increases the range of stimulus concentrations for which dose-dependent responses are possible; this increased dynamic range produces the counterintuitive result of “signaling beyond saturation” in which dose-dependent responses are still possible after apparent saturation of the receptors. We propose a mechanism for dose-to-duration encoding in the yeast pheromone pathway that is consistent with current experimental observations. Most previous investigations of information processing by signaling pathways have focused on amplitude encoding without considering temporal aspects of signal transduction. Here we demonstrate that dose-to-duration encoding provides cells with an alternative mechanism for processing and transmitting quantitative information about their surrounding environment. The ability of signaling pathways to convert stimulus strength into signal duration results directly from the nonlinear nature of these systems and emphasizes the importance of considering the dynamic properties of signaling pathways when characterizing their behavior. Understanding how signaling pathways encode and transmit quantitative information about the external environment will not only deepen our understanding of these systems but also provide insight into how to reestablish proper function of pathways that have become dysregulated by disease.     

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.     

Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other''s activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways.     

The regulation of filamentous growth in yeast

Filamentous growth is a nutrient-regulated growth response that occurs in many fungal species. In pathogens, filamentous growth is critical for host-cell attachment, invasion into tissues, and virulence. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth, which provides a genetically tractable system to study the molecular basis of the response. Filamentous growth is regulated by evolutionarily conserved signaling pathways. One of these pathways is a mitogen activated protein kinase (MAPK) pathway. A remarkable feature of the filamentous growth MAPK pathway is that it is composed of factors that also function in other pathways. An intriguing challenge therefore has been to understand how pathways that share components establish and maintain their identity. Other canonical signaling pathways-rat sarcoma/protein kinase A (RAS/PKA), sucrose nonfermentable (SNF), and target of rapamycin (TOR)-also regulate filamentous growth, which raises the question of how signals from multiple pathways become integrated into a coordinated response. Together, these pathways regulate cell differentiation to the filamentous type, which is characterized by changes in cell adhesion, cell polarity, and cell shape. How these changes are accomplished is also discussed. High-throughput genomics approaches have recently uncovered new connections to filamentous growth regulation. These connections suggest that filamentous growth is a more complex and globally regulated behavior than is currently appreciated, which may help to pave the way for future investigations into this eukaryotic cell differentiation behavior.     

Mathematical models of specificity in cell signaling

Cellular signaling pathways transduce extracellular signals into appropriate responses. These pathways are typically interconnected to form networks, often with different pathways sharing similar or identical components. A consequence of this connectedness is the potential for cross talk, some of which may be undesirable. Indeed, experimental evidence indicates that cells have evolved insulating mechanisms to partially suppress "leaking" between pathways. Here we characterize mathematical models of simple signaling networks and obtain exact analytical expressions for two measures of cross talk called specificity and fidelity. The performance of several insulating mechanisms--combinatorial signaling, compartmentalization, the inhibition of one pathway by another, and the selective activation of scaffold proteins--is evaluated with respect to the trade-off between the specificity they provide and the constraints they place on the network. The effects of noise are also examined. The insights gained from this analysis are applied to understanding specificity in the yeast mating and invasive growth MAP kinase signaling network.     

The Ustilago maydis ubc4 and ubc5 genes encode members of a MAP kinase cascade required for filamentous growth

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this we have taken a forward genetic approach. Earlier, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cyclic AMP (cAMP) to grow in the budding morphology. Complementation of a subset of these suppressor mutants led to the identification of the ubc4 and ubc5 genes, which are required for filamentous growth and encode a MAP (mitogen-activated protein) kinase kinase kinase and a MAP kinase kinase, respectively. Evidence suggests that they are important in the pheromone response pathway and in pathogenicity. These results further support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.     

Modeling specificity in the yeast MAPK signaling networks

Cells sense several kinds of stimuli and trigger corresponding responses through signaling pathways. As a result, cells must process and integrate multiple signals in parallel to maintain specificity and avoid erroneous cross-talk. In this study, we focus our theoretical effort on understanding specificity of a model network system in yeast, Saccharomyces cerevisiae, which contains three mitogen-activated protein kinase (MAPK) signal transduction cascades that share multiple signaling components. The cellular response to the pheromone, the filamentous growth and osmotic pressure stimuli in yeast is described and an integrative mathematical model for the three MAPK cascades is developed using available literature and experimental data. The theoretical framework for analyzing the specificity of signaling networks [Bardwell, L., Zou, X.F., Nie, Q., Komarova, N.L., 2007. Mathematical models of specificity in cell signaling. Biophys. J. 92, 3425-3441] is extended to include multiple interacting pathways with shared components. Simulations are also performed with any one stimulus, with any two simultaneous stimuli, and with the simultaneous application of the three stimuli. The interactions between the three pathways are systematically investigated. Moreover, the specificity and fidelity of this model system are calculated using our newly developed concept under different stimuli or with specific mutants. Our simulated and calculated results demonstrate that the yeast MAPK signaling network can achieve specificity and fidelity by filtering out spurious cross-talk between the relevant pathways through different mechanisms, such as scaffolding, cross-inhibiting, and feedback control. Proof that Pbs2 and Hog1 are essential for the maintenance of signaling specificity is presented. Our studies provide novel insights into integration of relevant signaling pathways in a biological system and the mechanisms conferring specificity in cellular signaling networks.     

Integrating signals between cAMP and the RAS/RAF/MEK/ERK signalling pathways. Based on the anniversary prize of the Gesellschaft für Biochemie und Molekularbiologie Lecture delivered on 5 July 2003 at the Special FEBS Meeting in Brussels

One of the hallmarks of cAMP is its ability to inhibit proliferation in many cell types, but stimulate proliferation in others. Clearly cAMP has cell type specific effects and the outcome on proliferation is largely attributed to crosstalk from cAMP to the RAS/RAF/mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway. We review the crosstalk between these two ancient and conserved pathways, describing the molecular mechanisms underlying the interactions between these pathways and discussing their possible biological importance.     

Global Regulation of a Differentiation MAPK Pathway in Yeast

Cell differentiation requires different pathways to act in concert to produce a specialized cell type. The budding yeast Saccharomyces cerevisiae undergoes filamentous growth in response to nutrient limitation. Differentiation to the filamentous cell type requires multiple signaling pathways, including a mitogen-activated protein kinase (MAPK) pathway. To identify new regulators of the filamentous growth MAPK pathway, a genetic screen was performed with a collection of 4072 nonessential deletion mutants constructed in the filamentous (Σ1278b) strain background. The screen, in combination with directed gene-deletion analysis, uncovered 97 new regulators of the filamentous growth MAPK pathway comprising 40% of the major regulators of filamentous growth. Functional classification extended known connections to the pathway and identified new connections. One function for the extensive regulatory network was to adjust the activity of the filamentous growth MAPK pathway to the activity of other pathways that regulate the response. In support of this idea, an unregulated filamentous growth MAPK pathway led to an uncoordinated response. Many of the pathways that regulate filamentous growth also regulated each other’s targets, which brings to light an integrated signaling network that regulates the differentiation response. The regulatory network characterized here provides a template for understanding MAPK-dependent differentiation that may extend to other systems, including fungal pathogens and metazoans.     

Crosstalk and Competition in Signaling Networks

Signaling networks have evolved to transduce external and internal information into critical cellular decisions such as growth, differentiation, and apoptosis. These networks form highly interconnected systems within cells due to network crosstalk, where an enzyme from one canonical pathway acts on targets from other pathways. It is currently unclear what types of effects these interconnections can have on the response of networks to incoming signals. In this work, we employ mathematical models to characterize the influence that multiple substrates have on one another. These models build off of the atomistic motif of a kinase/phosphatase pair acting on a single substrate. We find that the ultrasensitive, switch-like response these motifs can exhibit becomes transitive: if one substrate saturates the enzymes and responds ultrasensitively, then all substrates will do so regardless of their degree of saturation. We also demonstrate that the phosphatases themselves can induce crosstalk even when the kinases are independent. These findings have strong implications for how we understand and classify crosstalk, as well as for the rational development of kinase inhibitors aimed at pharmaceutically modulating network behavior.     

Crosstalk between cAMP-PKA and MAP kinase pathways is a key regulatory design necessary to regulate FLO11 expression

Signal transduction pathways crosstalk with one another and play a central role in regulation of cellular events. Crosstalk brings complexity to the system, and hence, a systematic analysis of these crosstalks helps in relating the signaling network structure to its function. Here, we present a modular steady state approach to quantify the network comprising of cAMP-PKA and MAP kinase pathways involved in the regulation of FLO11, a gene which is required for pseudohyphae growth in Saccharomyces cerevisiae under nitrogen starvation. These two pathways crosstalk by converging on the same target, i.e., FLO11 and through Ras2p, an upstream activator of both cAMP and MAPK pathway. Analysis of crosstalk at the gene level revealed that cAMP-PKA and MAPK pathways are indispensable to FLO11 expression. The dose response was highly sensitive and primarily controlled by cAMP-PKA pathway. We demonstrate that the highly sensitive response in the cAMP-PKA pathway was due to crosstalk and inhibitor ultrsensitivity, key regulatory designs present at the downstream of cAMP-PKA pathway. The analysis of the role of Ras2p in the crosstalk between the cAMP-PKA and MAPK pathways indicated that crosstalk essentially helped in amplification of the Gpa2p signal, another upstream activator of the cAMP-PKA pathway. However, the effect of crosstalk due to Ras2p on FLO11 expression was minimal under normal activation levels of Ras2p. Whereas, the crosstalk itself can bring about FLO11 expression under the hyperactivated Ras2p conditions thereby eliminating the requirement for the other activator Gpa2p. We also observed the presence of system level properties such as amplification, inhibitor ultrasensitvity and bistability, which can be attributed to the regulatory design present in the FLO11 expression system. These system level properties might help the organism to respond to varying nutritional status.     

Model-based analysis of interferon-beta induced signaling pathway

MOTIVATION: Interferon-beta induced JAK-STAT signaling pathways contribute to mucosal immune recognition and an anti-viral state. Though the main molecular mechanisms constituting these pathways are known, neither the detailed structure of the regulatory network, nor its dynamics has yet been investigated. The objective of this work is to build a mathematical model for the pathway that would serve two purposes: (1) to reproduce experimental results in simulation of both early and late response to Interferon-beta stimulation and (2) to explain experimental phenomena generating new hypotheses about regulatory mechanisms that cannot yet be tested experimentally. RESULTS: Experimentally determined time dependent changes in the major components of this pathway were used to build a mathematical model describing pathway dynamics in the form of ordinary differential equations. The experimental results suggested existence of unknown negative control mechanisms that were tested numerically using the model. Together, experimental and numerical data show that the epithelial JAK-STAT pathway might be subjected to previously unknown dynamic negative control mechanisms: (1) activation of dormant phosphatases and (2) inhibition of nuclear import of IRF1.     

The osmoregulatory pathway represses mating pathway activity in Saccharomyces cerevisiae: isolation of a FUS3 mutant that is insensitive to the repression mechanism.

Mitogen-activated protein (MAP) kinase cascades are conserved signal transduction pathways that are required for eukaryotic cells to respond to a variety of stimuli. Multiple MAP kinase pathways can function within a single cell type; therefore, mechanisms that insulate one MAP kinase pathway from adventitious activations by parallel pathways may exist. We have studied interactions between the mating pheromone response and the osmoregulatory (high-osmolarity glycerol response [HOG]) pathways in Saccharomyces cerevisiae which utilize the MAP kinases Fus3p and Hog1p, respectively. Inactivating mutations in HOG pathway kinases cause an increase in the phosphotyrosine content of Fus3p, greater expression of pheromone-responsive genes, and increased sensitivity to growth arrest by pheromone. Therefore, the HOG pathway represses mating pathway activity. In a HOG1+ strain, Fus3p phosphotyrosine increases modestly and transiently following an increase in the extracellular osmolarity; however, it increases to a greater extent and for a sustained duration in a hog1-delta strain. Thus, the HOG-mediated repression of mating pathway activity may insulate the mating pathway from activation by osmotic stress. A FUS3 allele whose gene product is resistant to the HOG-mediated repression of its phosphotyrosine content has been isolated. This mutant encodes an amino acid substitution in the highly conserved DPXDEP motif in subdomain XI. Other investigators have shown that the corresponding amino acid is also mutated in a gain-of-function allele of the MAP kinase encoded by the rolled locus in Drosophila melanogaster. These data suggest that the DPXDEP motif plays a role in the negative regulation of MAP kinases.     

Multiple Signals Converge on a Differentiation MAPK Pathway

An important emerging question in the area of signal transduction is how information from different pathways becomes integrated into a highly coordinated response. In budding yeast, multiple pathways regulate filamentous growth, a complex differentiation response that occurs under specific environmental conditions. To identify new aspects of filamentous growth regulation, we used a novel screening approach (called secretion profiling) that measures release of the extracellular domain of Msb2p, the signaling mucin which functions at the head of the filamentous growth (FG) MAPK pathway. Secretion profiling of complementary genomic collections showed that many of the pathways that regulate filamentous growth (RAS, RIM101, OPI1, and RTG) were also required for FG pathway activation. This regulation sensitized the FG pathway to multiple stimuli and synchronized it to the global signaling network. Several of the regulators were required for MSB2 expression, which identifies the MSB2 promoter as a target “hub” where multiple signals converge. Accessibility to the MSB2 promoter was further regulated by the histone deacetylase (HDAC) Rpd3p(L), which positively regulated FG pathway activity and filamentous growth. Our findings provide the first glimpse of a global regulatory hierarchy among the pathways that control filamentous growth. Systems-level integration of signaling circuitry is likely to coordinate other regulatory networks that control complex behaviors.     

Crosstalk between the Tor and Gcn2 pathways in response to different stresses

Regulating growth and the cell cycle in response to environmental fluctuations is important for all organisms in order to maintain viability. Two major pathways for translational regulation are found in higher eukaryotes: the Tor signaling pathway and those operating through the eIF2α kinases. Studies from several organisms indicate that the two pathways are interlinked, in that Tor complex 1 (TORC1) negatively regulates the Gcn2 kinase. Furthermore, inactivation of TORC1 may be required for activation of Gcn2 in response to stress. Here, we use the model organism Schizosaccharomyces pombe to investigate this crosstalk further. We find that the relationship is more complex than previously thought. First, in response to UV irradiation and oxidative stress, Gcn2 is fully activated in the presence of TORC1 signaling. Second, during amino-acid starvation, activation of Gcn2 is dependent on Tor2 activity, and Gcn2 is required for timely inactivation of the Tor pathway. Our data show that the crosstalk between the two pathways varies with the actual stress applied.     

Phosphoproteomic analyses reveal novel cross‐modulation mechanisms between two signaling pathways in yeast

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen‐activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time‐resolved phosphorylation site dynamics after pathway co‐stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone‐induced down‐regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.     

How can yeast cells decide between three activated MAP kinase pathways? A model approach

In yeast (Saccharomyces cerevisiae), the regulation of three MAP kinase pathways responding to pheromones (Fus3 pathway), carbon/nitrogen starvation (Kss1 pathway), and high osmolarity/osmotic stress (Hog1 pathway) is the subject of intensive research. We were interested in the question how yeast cells would respond when more than one of the MAP kinase pathways are activated simultaneously. Here, we give a brief overview over the regulatory mechanisms of the yeast MAP kinase pathways and investigate a kinetic model based on presently known molecular interactions and feedbacks within and between the three mitogen-activated protein kinases (MAPK) pathways. When two pathways are activated simultaneously with the osmotic stress response as one of them, the model predicts that the osmotic stress response (Hog1 pathway) is turned on first. The same is true when all three pathways are activated at the same time. When testing simultaneous stimulations by low nitrogen and pheromones through the Kss1 and Fus3 pathways, respectively, the low nitrogen response dominates over the pheromone response. Due to its autocatalytic activation mechanism, the pheromone response (Fus3 pathway) shows typical sigmoid response kinetics and excitability. In the presence of a small but sufficient amount of activated Fus3, a stimulation by pheromones will lead to a rapid self-amplification of the pheromone response. This ‘excitability’ appears to be a feature of the pheromone pathway that has specific biological significance.     

G protein signaling governing cell fate decisions involves opposing Galpha subunits in Cryptococcus neoformans

Communication between cells and their environments is often mediated by G protein-coupled receptors and cognate G proteins. In fungi, one such signaling cascade is the mating pathway triggered by pheromone/pheromone receptor recognition. Unlike Saccharomyces cerevisiae, which expresses two Galpha subunits, most filamentous ascomycetes and basidiomycetes have three Galpha subunits. Previous studies have defined the Galpha subunit acting upstream of the cAMP-protein kinase A pathway, but it has been unclear which Galpha subunit is coupled to the pheromone receptor and response pathway. Here we report that in the pathogenic basidiomycetous yeast Cryptococcus neoformans, two Galpha subunits (Gpa2, Gpa3) sense pheromone and govern mating. gpa2 gpa3 double mutants, but neither gpa2 nor gpa3 single mutants, are sterile in bilateral crosses. By contrast, deletion of GPA3 (but not GPA2) constitutively activates pheromone response and filamentation. Expression of GPA2 and GPA3 is differentially regulated: GPA3 expression is induced by nutrient-limitation, whereas GPA2 is induced during mating. Based on the phenotype of dominant active alleles, Gpa2 and Gpa3 signal in opposition: Gpa2 promotes mating, whereas Gpa3 inhibits. The incorporation of an additional Galpha into the regulatory circuit enabled increased signaling complexity and facilitated cell fate decisions involving choice between yeast growth and filamentous asexual/sexual development.