Pheromone signaling mechanisms in yeast: a prototypical sex machine

The actions of many extracellular stimuli are elicited by complexes of cell surface receptors, heterotrimeric guanine nucleotide-binding proteins (G proteins), and mitogen-activated protein (MAP) kinase complexes. Analysis of haploid yeast cells and their response to peptide mating pheromones has produced important advances in our understanding of G protein and MAP kinase signaling mechanisms. Many of the components, their interrelationships, and their regulators were first identified in yeast. Current analysis of the pheromone response pathway (see the Connections Maps at Science's Signal Transduction Knowledge Environment) will benefit from new and powerful genomic, proteomic, and computational approaches that will likely reveal additional general principles that are applicable to more complex organisms.     

A seven-transmembrane RGS protein that modulates plant cell proliferation

G protein-coupled receptors (GPCRs) at the cell surface activate heterotrimeric G proteins by inducing the G protein alpha (Galpha) subunit to exchange guanosine diphosphate for guanosine triphosphate. Regulators of G protein signaling (RGS) proteins accelerate the deactivation of Galpha subunits to reduce GPCR signaling. Here we identified an RGS protein (AtRGS1) in Arabidopsis that has a predicted structure similar to a GPCR as well as an RGS box with GTPase accelerating activity. Expression of AtRGS1 complemented the pheromone supersensitivity phenotype of a yeast RGS mutant, sst2Delta. Loss of AtRGS1 increased the activity of the Arabidopsis Galpha subunit, resulting in increased cell elongation in hypocotyls in darkness and increased cell production in roots grown in light. These findings suggest that AtRGS1 is a critical modulator of plant cell proliferation.     

Rewiring MAP kinase pathways using alternative scaffold assembly mechanisms

How scaffold proteins control information flow in signaling pathways is poorly understood: Do they simply tether components, or do they precisely orient and activate them? We found that the yeast mitogen-activated protein (MAP) kinase scaffold Ste5 is tolerant to major stereochemical perturbations; heterologous protein interactions could functionally replace native kinase recruitment interactions, indicating that simple tethering is largely sufficient for scaffold-mediated signaling. Moreover, by engineering a scaffold that tethers a unique kinase set, we could create a synthetic MAP kinase pathway with non-natural input-output properties. These findings demonstrate that scaffolds are highly flexible organizing factors that can facilitate pathway evolution and engineering.     

Autophagy genes are essential for dauer development and life-span extension in C. elegans

Both dauer formation (a stage of developmental arrest) and adult life-span in Caenorhabditis elegans are negatively regulated by insulin-like signaling, but little is known about cellular pathways that mediate these processes. Autophagy, through the sequestration and delivery of cargo to the lysosomes, is the major route for degrading long-lived proteins and cytoplasmic organelles in eukaryotic cells. Using nematodes with a loss-of-function mutation in the insulin-like signaling pathway, we show that bec-1, the C. elegans ortholog of the yeast and mammalian autophagy gene APG6/VPS30/beclin1, is essential for normal dauer morphogenesis and life-span extension. Dauer formation is associated with increased autophagy and also requires C. elegans orthologs of the yeast autophagy genes APG1, APG7, APG8, and AUT10. Thus, autophagy is a cellular pathway essential for dauer development and life-span extension in C. elegans.     

G protein signaling from activated rat frizzled-1 to the beta-catenin-Lef-Tcf pathway

The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.     

A cell's sense of direction

In eukaryotic cells directional sensing is mediated by heterotrimeric guanine nucleotide-binding protein (G protein)-linked signaling pathways. In Dictyostelium discoideum amoebae and mammalian leukocytes, the receptors and G-protein subunits are uniformly distributed around the cell perimeter. Chemoattractants induce the transient appearance of binding sites for several pleckstrin homology domain-containing proteins on the inner face of the membrane. In gradients of attractant these sites are persistently present on the side of the cell facing the higher concentration, even in the absence of a functional actin cytoskeleton or cell movement. Thus, the cell senses direction by spatially regulating the activity of the signal transduction pathway.     

泛素化修饰在RLR信号通路中的研究进展

病毒入侵后被细胞的模式识别受体RIG-I样受体（RIG-I-like receptor, RLR）识别从而启动抗病毒RLR信号通路的激活,先天免疫反应的异常激活将导致慢性炎症和免疫器官损伤,甚至引起自身免疫性疾病。为了防止抗病毒信号过早激活或过度激活,机体建立了完善的调节系统防止信号传导过程发生紊乱。蛋白的翻译后修饰（Post-translational modification, PTM）是调节模式识别受体及其下游信号蛋白稳定性和活性的关键机制,而泛素化（Ubiquitination, UB）作为蛋白质翻译后修饰的重要部分在抗病毒信号通路中被广泛研究。其中K48和K63连接的泛素化最为常见,通过K48连接的泛素链能够引起靶蛋白通过蛋白酶体途径降解,而K63连接的泛素链能够促进蛋白激活和细胞信号转导。RIG-Ⅰ、MAVS、TBK1以及TRAF家族相关蛋白作为RLR通路的信号传递分子,其蛋白的泛素化修饰也成为研究的重点。本文讨论了K48和K63泛素化在抗病毒免疫信号通路中的研究进展,特别是RIG-I样受体引发的信号传导途径中蛋白的泛素化修饰。     

G proteins Go green: a plant G protein signaling FAQ sheet

Plants, like animals, use signal transduction pathways based on heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate many aspects of development and cell signaling. Some components of G protein signaling are highly conserved between plants and animals and some are not. This Viewpoint compares key aspects of G protein signal transduction in plants and animals and describes the current knowledge of this system in plants, the questions that still await exploration, and the value of research on plant G proteins to scientists who do not study plants. Pathways in Science's Signal Transduction Knowledge Environment Connections Maps database provide details about the emerging roles of G proteins in several cellular processes of plants.     

Variations on conserved signaling pathways in biocontrol and development:G protein and MAPK genes of Trichoderma. atroviride and T. virens

Filamentous fungi employ conserved eukaryotic signaling pathway to detect and respond to environmental signals, including the presence of the host. Genetic experiment in which a particular signaling protein is lost, or its activity enhanced, have defined some of the function of heterotrimeric G proteins and MAP kinases in development and virulence. A hallmark of these studies is that orthologs in different species may have different functions. Antagonistic fungal-fungal interactions form …     

Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics

Scaffold proteins link signaling molecules into linear pathways by physically assembling them into complexes. Scaffolds may also have a higher-order role as signal-processing hubs, serving as the target of feedback loops that optimize signaling amplitude and timing. We demonstrate that the Ste5 scaffold protein can be used as a platform to systematically reshape output of the yeast mating MAP kinase pathway. We constructed synthetic positive- and negative-feedback loops by dynamically regulating recruitment of pathway modulators to an artificial binding site on Ste5. These engineered circuits yielded diverse behaviors: ultrasensitive dose response, accelerated or delayed response times, and tunable adaptation. Protein scaffolds provide a flexible platform for reprogramming cellular responses and could be exploited to engineer cells with novel therapeutic and biotechnological functions.     

Regulatory role for GTP-binding proteins in endocytosis

Guanosine 5'-triphosphate (GTP)-binding proteins have been implicated in the transport of newly synthesized proteins along the secretory pathway of yeast and mammalian cells. Early vesicle fusion events that follow receptor-mediated endocytosis as measured by three in vitro assays were blocked by guanosine 5'-O-(3-thiotriphosphate) and aluminum fluoride. The effect was specific for guanosine nucleotides and depended on the presence of cytosolic factors. Thus, GTP-binding proteins may also have a role in the transport of molecules along the endocytic pathway.     

A network of control mediated by regulator of calcium/calmodulin-dependent signaling

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.     

53BP1, a mediator of the DNA damage checkpoint

53BP1 binds to the tumor suppressor protein p53 and has a potential role in DNA damage responses. We used small interfering RNA (siRNA) directed against 53BP1 in mammalian cells to demonstrate that 53BP1 is a key transducer of the DNA damage checkpoint signal. 53BP1 was required for p53 accumulation, G2-M checkpoint arrest, and the intra-S-phase checkpoint in response to ionizing radiation. 53BP1 played a partially redundant role in phosphorylation of the downstream checkpoint effector proteins Brca1 and Chk2 but was required for the formation of Brca1 foci in a hierarchical branched pathway for the recruitment of repair and signaling proteins to sites of DNA damage.     

Evolution of key cell signaling and adhesion protein families predates animal origins

The evolution of animals from a unicellular ancestor involved many innovations. Choanoflagellates, unicellular and colonial protozoa closely related to Metazoa, provide a potential window into early animal evolution. We have found that choanoflagellates express representatives of a surprising number of cell signaling and adhesion protein families that have not previously been isolated from nonmetazoans, including cadherins, C-type lectins, several tyrosine kinases, and tyrosine kinase signaling pathway components. Choanoflagellates have a complex and dynamic tyrosine phosphoprotein profile, and cell proliferation is selectively affected by tyrosine kinase inhibitors. The expression in choanoflagellates of proteins involved in cell interactions in Metazoa demonstrates that these proteins evolved before the origin of animals and were later co-opted for development.     

Regulation of MAPK function by direct interaction with the mating-specific Galpha in yeast

The mating response of the budding yeast Saccharomyces cerevisiae is mediated by a prototypical heterotrimeric GTP-binding protein (G protein) and mitogen-activated protein kinase (MAPK) cascade. Although signal transmission by such pathways has been modeled in detail, postreceptor down-regulation is less well understood. The pheromone-responsive G protein alpha subunit (Galpha) of yeast down-regulates the mating signal, but its targets are unknown. We have found that Galpha binds directly to the mating-specific MAPK in yeast cells responding to pheromone. This interaction contributes both to modulation of the mating signal and to the chemotropic response, and it demonstrates direct communication between the top and bottom of a Galpha-MAPK pathway.     

Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis

How cyclooxygenase-2 (COX-2) and its proinflammatory metabolite prostaglandin E2 (PGE2) enhance colon cancer progression remains poorly understood. We show that PGE2 stimulates colon cancer cell growth through its heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor, EP2, by a signaling route that involves the activation of phosphoinositide 3-kinase and the protein kinase Akt by free G protein betagamma subunits and the direct association of the G protein alphas subunit with the regulator of G protein signaling (RGS) domain of axin. This leads to the inactivation and release of glycogen synthase kinase 3beta from its complex with axin, thereby relieving the inhibitory phosphorylation of beta-catenin and activating its signaling pathway. These findings may provide a molecular framework for the future evaluation of chemopreventive strategies for colorectal cancer.     

Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit

To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.     

拟南芥NiNJA基因酵母双杂诱饵载体构建及互作蛋白的筛选

JAZ蛋白是植物茉莉酸信号途径的重要负调控因子,JAZ与NiNJA形成蛋白复合体抑制茉莉酸下游转录因子的转录活性,NiNJA蛋白是联系JAZ蛋白与下游转录因子的重要因子.为了研究NiNJA调控的下游基因,首先构建了NiNJA基因的诱饵载体pGBKT7-NiNJA,然后转化酵母Y2H Gold感受态,通过自转录激活实验,发现诱饵载体pGBKT7-NiNJA没有自转录激活活性.在此基础上,从拟南芥“Mate&PlateTM”Library 进行酵母双杂筛选,获得若干个与NiNJA互作的蛋白,为下一步鉴定NiNJA的互作蛋白及茉莉酸的信号调控途径打下基础.