Thematic Minireview Series: Complexities of Cellular Signaling Revealed by Simple Model Organisms*

All cells discriminate environmental signals and generate appropriate intracellular responses. Our understanding of these signal transduction mechanisms has benefitted from studies across the kingdoms of life, from fungi and fish to mice and men. This thematic minireview series examines lessons learned from three of the simplest (and best understood) eukaryotic model organisms. The first article focuses on the mating pheromone pathway in budding yeast Saccharomyces cerevisiae. The second describes stress-mediated signaling in the roundworm Caenorhabditis elegans. The third outlines some of the signaling pathways that dictate growth and development in the fruit fly Drosophila melanogaster. Each system has provided unique insights into hormone and neurotransmitter signaling mechanisms, in particular those mediated by the MAPKs. The advances described in these articles will continue to improve our understanding of human physiology and pharmacology

Pheromone-regulated Sumoylation of Transcription Factors That Mediate the Invasive to Mating Developmental Switch in Yeast

A fundamental question in biology is how different signaling pathways use common signaling proteins to attain different developmental outcomes. The yeast transcription factor Ste12 is required in at least two distinct signaling processes, each regulated by many of the same protein kinases. Whereas Ste12-Ste12 homodimers promote transcription of genes required for mating, Ste12-Tec1 heterodimers activate genes required for invasive growth. We report that Ste12 and Tec1 undergo covalent modification by the ubiquitin-related modifier SUMO. Stimulation by mating pheromone promotes sumoylation of Ste12 and diminishes the sumoylation of Tec1. In the absence of sumoylation Tec1 is more rapidly degraded. We propose that pheromone-regulated sumoylation of Ste12 and Tec1 promotes a developmental switch from the invasive to the mating differentiation program

Buried ionizable networks are an ancient hallmark of G protein-coupled receptor activation

In the early 1980s, scientists began searching for cell-surface receptors that bind to hormones and neurotransmitters. Among the first was the β-adrenergic receptor, a G protein-coupled receptor (GPCR) that is activated by norepinephrine and epinephrine. Recent breakthroughs have provided more than 100 new GPCR structures, including several in activated conformations. This new structural information presents an opportunity to identify features that distinguish unactivated and activated receptors. Here we use a computational approach to identify structural signatures unique to activated GPCRs. Remarkably, we find that these signatures also are present in distantly related receptors from archaea and bacteria. We propose that these new structural indicators are central to GPCR function and are indicative of GPCR activation

Illuminating G␤ 5 Signaling

ABSTRACT G proteins are key intermediates in cellular signaling and act in response to a variety of extracellular stimuli. The prevailing paradigm is that G protein subunits form a heterotrimeric complex and function principally at the plasma membrane. However, there is growing evidence for localization at, and signaling by, G proteins at intracellular compartments. Moreover, different cellular pools of G proteins may be composed of distinct subunit subtypes, including some binding partners that function in the place of G protein ␥ subunits. An article in this issue of Molecular Pharmacology (Yost et al., p. 812) describes the use of an innovative fluorescent cell imaging technique to study interactions of the G protein ␤ 5 subunit with a panel of G␥ subunits as well as regulator of G protein signaling (RGS) proteins that contain a G␥-like subdomain. The approach used here provides a new strategy to elucidate the spatial and temporal properties of G proteins, including a growing number of atypical G␤␥ pairings. Heterotrimeric G proteins normally consist of ␣, ␤, and ␥ subunits and are coupled to seven transmembrane receptors at the plasma membrane. Agonist binding to the receptor induces a conformational change of the G␣ subunit promoting the release of GDP and binding to GTP. This exchange triggers G␤␥ disassociation from the G␣, freeing both components to modulate downstream signals. Hydrolysis of GTP to GDP by the G␣ results in reassociation of the heterotrimer and termination of the signal (Sprang, 1997). So far, 23 G␣, 5 G␤, and 12 G␥ subunits have been identified in the mammalian genome. Of the G␤ isoforms, types 1 to 4 are highly conserved, sharing 80% sequence identity, but G␤ 5 is divergent, sharing only 50% identity. Like other ␤ isoforms, G␤ 5 interacts with G␥ subunits; unlike the others, G␤ 5 can also interact with RGS proteins from the R7 family (RGS6, RGS7, RGS9, and RGS11) The RGS/G␤ 5 complex could be thought of as a highly atypical G␤␥ pair. Others are likely to exist (see below). With the identification of such atypical subunit complexes, new techniques are needed to ascertain their function within the cell. Bimolecular fluorescence complementation (BiFC) is one promising technique In this issue of Molecular Pharmacology

Coactivation of G Protein Signaling by Cell-Surface Receptors and an Intracellular Exchange Factor

G protein-coupled receptors (GPCRs) mediate responses to a broad range of chemical and environmental signals. In yeast a pheromone-binding GPCR triggers events leading to the fusion of haploid cells. In general, GPCRs function as guanine nucleotide exchange factors (GEFs); upon agonist binding the receptor induces a conformational change in the G protein α subunit, resulting in exchange of GDP for GTP and in signal initiation. Signaling is terminated when GTP is hydrolyzed to GDP [1]. This well-established paradigm has in recent years been revised to include new components that alter the rates of GDP release, GTP binding [2-8], and GTP hydrolysis [9, 10]. Here we report the discovery of a non-receptor GEF, Arr4. Like receptors, Arr4 binds directly to the G protein, accelerates guanine nucleotide exchange, and stabilizes the nucleotide-free state of the α subunit. Moreover, Arr4 promotes G protein-dependent cellular responses including mitogen-activated protein kinase (MAPK) phosphorylation, new gene transcription and mating. In contrast to known GPCRs, however, Arr4 is not a transmembrane receptor, but rather a soluble intracellular protein. Our data suggest that intracellular proteins function in cooperation with mating pheromones to amplify G protein signaling, thereby leading to full pathway activation

Dynamic Ubiquitination of the Mitogen-activated Protein Kinase Kinase (MAPKK) Ste7 Determines Mitogen-activated Protein Kinase (MAPK) Specificity

Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCFCdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCFCdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCFCdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCFCdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK

The RACK1 Ortholog Asc1 Functions as a G-protein β Subunit Coupled to Glucose Responsiveness in Yeast

According to the prevailing paradigm, G-proteins are composed of three subunits, an alpha subunit with GTPase activity and a tightly associated betagamma subunit complex. In the yeast Saccharomyces cerevisiae there are two known Galpha proteins (Gpa1 and Gpa2) but only one Gbetagamma, which binds only to Gpa1. Here we show that the yeast ortholog of RACK1 (receptor for activated protein kinase C1) Asc1 functions as the Gbeta for Gpa2. As with other known Gbeta proteins, Asc1 has a 7-WD domain structure, interacts directly with the Galpha in a guanine nucleotide-dependent manner, and inhibits Galpha guanine nucleotide exchange activity. In addition, Asc1 binds to the effector enzyme adenylyl cyclase (Cyr1), and diminishes the production of cAMP in response to glucose stimulation. Thus, whereas Gpa2 promotes glucose signaling through elevated production of cAMP, Asc1 has opposing effects on these same processes. Our findings reveal the existence of an unusual Gbeta subunit, one having multiple functions within the cell in addition to serving as a signal transducer for cell surface receptors and intracellular effectors

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

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

Systematic Analysis of Essential Genes Reveals Important Regulators of G Protein Signaling

The yeast pheromone pathway consists of a canonical heterotrimeric G protein and MAP kinase cascade. To identify new signaling components we systematically evaluated 870 essential genes using a library of repressible-promoter strains. Quantitative transcription-reporter and MAPK activity assays were used to identify strains that exhibit altered pheromone sensitivity. Of the 92 newly identified essential genes required for proper G protein signaling, those involved with protein degradation were most highly-represented. Included in this group are members of the SCF (Skp-Cullin-F-Box) ubiquitin ligase complex. Further genetic and biochemical analysis reveals that SCFCdc4 acts together with the Cdc34 ubiquitin conjugating enzyme at the level of the G protein, promotes degradation of the G protein α subunit, Gpa1, in vivo and catalyzes Gpa1 ubiquitination in vitro. These new insights to the G protein signaling network reveal the essential-genome as an untapped resource for identifying new components and regulators of signal transduction pathways