Proper Protein Glycosylation Promotes Mitogen-Activated Protein Kinase Signal Fidelity

The ability of cells to sense and respond appropriately to changing environmental conditions is often mediated by signal transduction pathways that employ mitogen-activated protein kinases (MAPKs). In the yeast Saccharomyces cerevisiae, the high osmolarity glycerol (HOG) and the filamentous growth (FG) pathways are activated following hyperosmotic stress and nutrient deprivation, respectively. Whereas the HOG pathway requires the MAPK Hog1, the FG pathway employs the MAPK Kss1. We conducted a comprehensive screen of nearly 5,000 gene deletion strains for mutants that exhibit inappropriate cross-talk between the HOG and FG pathways. We identified two novel mutants, mnn10Δ and mnn11Δ, that allow activation of Kss1 under conditions that normally stimulate Hog1. MNN10 and MNN11 encode mannosyltransferases that are part of the N-glycosylation machinery within the Golgi apparatus; deletion of either gene results in N-glycosylated proteins that have shorter mannan chains. Deletion of the cell surface mucin Msb2 suppressed the mnn11Δ phenotype, while mutation of a single glycosylation site within Msb2 was sufficient to confer inappropriate activation of Kss1 by salt stress. These findings reveal new components of the N-glycosylation machinery needed to ensure MAPK signaling fidelity

Positive roles for negative regulators in the mating response of yeast

Modeling and experimental analysis of the yeast mating pathway reveals that transcriptional repressor proteins protect a key transcriptional activator from degradation, ensuring that the system is poised to respond rapidly to pheromones and providing a novel mechanism for perfect adaptation.We combine experimentation and mathematical modeling to study the complex interplay between positive and negative regulation of gene expression in the yeast pheromone pathway.The transcriptional repressors Dig1 and Dig2 are shown to have a positive role in mating differentiation by stabilizing the transcriptional activator Ste12. This result was predicted by mathematical modeling and confirmed experimentally.The model also predicts that Ste12 degradation follows saturable kinetics. Again, this observation is confirmed experimentally.The model revealed that stabilization through protein–protein interactions provides a mechanism for robust perfect adaptation and allows the transcriptional response to occur on a time scale that is distinct from upstream signaling events.All cells must detect and respond to changes in their environment, often through changes in gene expression. The yeast pheromone pathway has been extensively characterized, and is an ideal system for studying transcriptional regulation. Here we combine computational and experimental approaches to study transcriptional regulation mediated by Ste12, the key transcription factor in the pheromone response. Our mathematical model is able to explain multiple counterintuitive experimental results and led to several novel findings. First, we found that the transcriptional repressors Dig1 and Dig2 positively affect transcription by stabilizing Ste12. This stabilization through protein–protein interactions creates a large pool of Ste12 that is rapidly activated following pheromone stimulation. Second, we found that protein degradation follows saturating kinetics, explaining the long half-life of Ste12 in mutants expressing elevated amounts of Ste12. Finally, our model reveals a novel mechanism for robust perfect adaptation through protein–protein interactions that enhance complex stability. This mechanism allows the transcriptional response to act on a shorter time scale than upstream pathway activity

Signal inhibition by a dynamically regulated pool of monophosphorylated MAPK

MAPKs are activated by dual phosphorylation. Much of the MAPK Fus3 is monophosphorylated and acts to inhibit signaling in vivo. Computational models reveal how a kinase scaffold and phosphatase act together to dynamically regulate dual-phosphorylated and monophosphorylated MAPKs and the downstream signal.Protein kinases regulate a broad array of cellular processes and do so through the phosphorylation of one or more sites within a given substrate. Many protein kinases are themselves regulated through multisite phosphorylation, and the addition or removal of phosphates can occur in a sequential (processive) or a stepwise (distributive) manner. Here we measured the relative abundance of the monophosphorylated and dual-phosphorylated forms of Fus3, a member of the mitogen-activated protein kinase (MAPK) family in yeast. We found that upon activation with pheromone, a substantial proportion of Fus3 accumulates in the monophosphorylated state. Introduction of an additional copy of Fus3 lacking either phosphorylation site leads to dampened signaling. Conversely, cells lacking the dual-specificity phosphatase (msg5Δ) or that are deficient in docking to the MAPK-scaffold (Ste5ND) accumulate a greater proportion of dual-phosphorylated Fus3. The double mutant exhibits a synergistic, or “synthetic,” supersensitivity to pheromone. Finally, we present a predictive computational model that combines MAPK scaffold and phosphatase activities and is sufficient to account for the observed MAPK profiles. These results indicate that the monophosphorylated and dual-phosphorylated forms of the MAPK act in opposition to one another. Moreover, they reveal a new mechanism by which the MAPK scaffold acts dynamically to regulate signaling

Combined computational and experimental analysis reveals mitogen-activated protein kinase-mediated feedback phosphorylation as a mechanism for signaling specificity

A series of mathematical models was used to quantitatively characterize pheromone-stimulated kinase activation and determine how mitogen-activated protein (MAP) kinase specificity is achieved. The findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity.Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The mating and invasive growth pathways in yeast each employ a mitogen-activated protein (MAP) kinase cascade that includes Ste20, Ste11, and Ste7. Whereas proper mating requires Ste7 activation of the MAP kinase Fus3, invasive growth requires activation of the alternate MAP kinase Kss1. To determine how MAP kinase specificity is achieved, we used a series of mathematical models to quantitatively characterize pheromone-stimulated kinase activation. In accordance with the computational analysis, MAP kinase feedback phosphorylation of Ste7 results in diminished activation of Kss1, but not Fus3. These findings reveal how feedback phosphorylation of a common pathway component can limit the activity of a competing MAP kinase through feedback phosphorylation of a common activator, and thereby promote signal fidelity

Regulation of Cell Signaling Dynamics by the Protein Kinase-Scaffold Ste5

Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic-growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase

Flexibility of a Eukaryotic Lipidome – Insights from Yeast Lipidomics

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism

Checkpoints in a Yeast Differentiation Pathway Coordinate Signaling during Hyperosmotic Stress

All eukaryotes have the ability to detect and respond to environmental and hormonal signals. In many cases these signals evoke cellular changes that are incompatible and must therefore be orchestrated by the responding cell. In the yeast Saccharomyces cerevisiae, hyperosmotic stress and mating pheromones initiate signaling cascades that each terminate with a MAP kinase, Hog1 and Fus3, respectively. Despite sharing components, these pathways are initiated by distinct inputs and produce distinct cellular behaviors. To understand how these responses are coordinated, we monitored the pheromone response during hyperosmotic conditions. We show that hyperosmotic stress limits pheromone signaling in at least three ways. First, stress delays the expression of pheromone-induced genes. Second, stress promotes the phosphorylation of a protein kinase, Rck2, and thereby inhibits pheromone-induced protein translation. Third, stress promotes the phosphorylation of a shared pathway component, Ste50, and thereby dampens pheromone-induced MAPK activation. Whereas all three mechanisms are dependent on an increase in osmolarity, only the phosphorylation events require Hog1. These findings reveal how an environmental stress signal is able to postpone responsiveness to a competing differentiation signal, by acting on multiple pathway components, in a coordinated manner

Constitutively active Hog1 dampens Fus3 activation and induction.

<p>(A) Activation kinetics of Fus3 with constitutively active Hog1; wild-type cells transformed with vector control or plasmid-borne <i>GAL1</i>-<i>SSK2^ΔN</i> were grown in SC media with 2% raffinose (Raf). Ssk2^ΔN expression was induced by addition of 2% galactose for 60 min followed by addition of 3 µM α factor for 30 min. Cell lysates were resolved by 12.5% SDS-PAGE. P-Fus3 and P-Kss1 were detected with phospho-p44/p42 antibodies. P-Hog1 was detected with phospho-p38 antibodies. Total Fus3 and Hog1 were detected with Fus3 and Hog1 antibodies. G6PDH served as a loading control. All primary antibodies were recognized by fluorescently labeled secondary antibody, detected by fluorescence scanner (Typhoon Trio) and quantified by scanning densitometry (ImageJ). The panels to the right show averaged scanning densitometry of four individual experiments. Error bars represent ± SEM. P-Hog1 reduced P-Fus3 by 49.4%±6.7% at 120 min. (B) Wild-type, <i>hog1</i>Δ, and, <i>hog1^K52R</i> cells transformed with <i>GAL1</i>-<i>SSK2^ΔN</i> or parent vector control were grown in SC and 2% galactose for 60 min followed by addition of 3 µM α factor or left untreated for 30 min. (C) <i>fus3</i>Δ cells transformed with <i>ADH1</i>-<i>FUS3</i> and <i>GAL1</i>-<i>SSK2^ΔN</i> or vector were grown and stimulated as in B. P-Hog1 (<i>SSK2^ΔN</i>) reduced P-Fus3 by 30.7%±3.2%. (D) <i>rck2</i>Δ cells transformed with <i>GAL1</i>-<i>SSK2^ΔN</i> or vector were grown and stimulated as in B. P-Hog1 reduced P-Fus3 by 31.0%±6.2%.</p

Hog1 dampens Fus3 activation by targeting Ste50.

<p>Constitutive activators of mating pathway highlighted in black: (A) Ste5^CTM, a C-terminal transmembrane domain (CTM) tethers Ste5 to the plasma membrane allowing MAPK activation without receptor or G-protein. (B) Wild-type cells transformed with <i>GAL1</i>-<i>STE5^CTM</i>, <i>GAL1</i>-<i>SSK2^ΔN</i> or parent vector controls were grown in 2% galactose for 60 min followed by addition of 3 µM α factor or left untreated for 30 min. Cell lysates were resolved by 12.5% SDS-PAGE. Statistical significance was calculated using two-way ANOVA. ***, p<0.001. (C) Ste11^ΔN, constitutively active amino-terminus truncation mutant of Ste11, allowing activation without binding the upstream activator Ste20, scaffold Ste5, or adaptor Ste50. (D) Wild-type cells transformed with <i>GAL1</i>-<i>STE11^ΔN</i>, <i>GAL1</i>-<i>SSK2^ΔN</i> or vector were grown in 2% galactose for 2.5 h followed by addition of 3 µM α factor or left untreated for 30 min. Statistical significance was calculated using two-way ANOVA, ns – not significant, p>0.05. (E) Wild-type and <i>ste50^5A</i> cells grown and treated as in B. (F) <i>ste50^5A rck2</i>Δ cells grown and treated as in B. (G) Quantitative mating assay, indicated strains were mated with wild-type <i>MAT</i>α strain for 4 h on YPD or YPD+0.5 M KCl. Statistical significance was calculated using two-way ANOVA. **, p<0.01.</p

Hyperosmotic stress delays and dampens mating transcription.

<p>Transcriptional activation (β-galactosidase activity) was measured spectrofluorometrically every 30 min in (A) wild-type, (B) <i>hog1</i>Δ, and (C) <i>hog1^K52R</i> cells transformed with a plasmid containing a pheromone-inducible reporter (<i>FUS1</i>-lacZ). Transcription was induced by the addition of 10 µM α factor, 10 µM α factor+0.5 M KCl, 10 µM α factor+0.75 M KCl, or 10 µM α factor+1 M KCl. Data are the mean ± SE of four individual colonies measured in quadruplicate and presented as percentage of wild-type maximum. Transcriptional activation (GFP expression) was measured by fluorescence microscopy in individual wild-type cells with an integrated pheromone-inducible reporter (<i>FUS1-GFP</i>). (D) Representative images of GFP expression in G1 cells stimulated by the addition of 10 µM α factor or 10 µM α factor+0.5 M KCl. Color spectrum indicates GFP pixel intensity as calculated using ImageJ. (E) Scatter plot of GFP fluorescence (average pixel intensity/cell area) in individual cells stimulated with 10 µM α factor or 10 µM α factor+0.5 M KCl. Insert is the average GFP intensity from the population of individual cells in (E), error bars indicate 95% CI.</p