Paradigm Of Signal Transduction Research Articles

Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

EnvZ/OmpR Two-Component Signaling: An Archetype System That Can Function Noncanonically.

Two-component regulatory systems represent the major paradigm for signal transduction in prokaryotes. The simplest systems are composed of a sensor kinase and a response regulator. The sensor is often a membrane protein that senses a change in environmental conditions and is autophosphorylated by ATP on a histidine residue. The phosphoryl group is transferred onto an aspartate of the response regulator, which activates the regulator and alters its output, usually resulting in a change in gene expression. In this review, we present a historical view of the archetype EnvZ/OmpR two-component signaling system, and then we provide a new view of signaling based on our recent experiments. EnvZ responds to cytoplasmic signals that arise from changes in the extracellular milieu, and OmpR acts canonically (requiring phosphorylation) to regulate the porin genes and noncanonically (without phosphorylation) to activate the acid stress response. Herein, we describe how insights gleaned from stimulus recognition and response in EnvZ are relevant to nearly all sensor kinases and response regulators.

Plant Osmotic Stress Signaling: MAPKKKs Meet SnRK2s.

Osmotic stress signaling in higher plants is crucial to cope with abiotic stress. RAF-like MAPKKKs are activated by hyperosmotic stress and activate downstream ABA-unresponsive and ABA-activated SnRK2s, integrating early osmotic stress and ABA signaling cascades. The connection of B2/B3/B4 RAF-like MAPKKKs with SnRK2s is a new paradigm in signal transduction.

SnapShot: Jak-STAT Signaling II

The JAK-STAT pathway is an evolutionarily conserved signal transduction paradigm, providing mechanisms for rapid receptor-to-nucleus communication and transcription control. Discoveries in this field provided insights into primary immunodeficiencies, inherited autoimmune and autoinflammatory diseases, and hematologic and oncologic disorders, giving rise to a new class of drugs, JAK inhibitors (or Jakinibs).

A blue light receptor that mediates RNA binding and translational regulation.

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 μM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL:RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way towards hitherto inaccessible optoribogenetic modalities.

LytTR Regulatory Systems: A potential new class of prokaryotic sensory system.

The most commonly studied prokaryotic sensory signal transduction systems include the one-component systems, phosphosignaling systems, extracytoplasmic function (ECF) sigma factor systems, and the various types of second messenger systems. Recently, we described the regulatory role of two separate sensory systems in Streptococcus mutans that jointly control bacteriocin gene expression, natural competence development, as well as a cell death pathway, yet they do not function via any of the currently recognized signal transduction paradigms. These systems, which we refer to as LytTR Regulatory Systems (LRS), minimally consist of two proteins, a transcription regulator from the LytTR Family and a transmembrane protein inhibitor of this transcription regulator. Here, we provide evidence suggesting that LRS are a unique uncharacterized class of prokaryotic sensory system. LRS exist in a basal inactive state. However, when LRS membrane inhibitor proteins are inactivated, an autoregulatory positive feedback loop is triggered due to LRS regulator protein interactions with direct repeat sequences located just upstream of the -35 sequences of LRS operon promoters. Uncharacterized LRS operons are widely encoded by a vast array of Gram positive and Gram negative bacteria as well as some archaea. These operons also contain unique direct repeat sequences immediately upstream of their operon promoters indicating that positive feedback autoregulation is a globally conserved feature of LRS. Despite the surprisingly widespread occurrence of LRS operons, the only characterized examples are those of S. mutans. Therefore, the current study provides a useful roadmap to investigate LRS function in the numerous other LRS-encoding organisms.

Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C(+) cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling by a receptor at the cell surface.

A FRET-based DNA biosensor tracks OmpR-dependent acidification of Salmonella during macrophage infection.

In bacteria, one paradigm for signal transduction is the two-component regulatory system, consisting of a sensor kinase (usually a membrane protein) and a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system responds to osmotic stress and regulates expression of outer membrane proteins. In Salmonella, EnvZ/OmpR also controls expression of another two-component system SsrA/B, which is located on Salmonella Pathogenicity Island (SPI) 2. SPI-2 encodes a type III secretion system, which functions as a nanomachine to inject bacterial effector proteins into eukaryotic cells. During the intracellular phase of infection, Salmonella switches from assembling type III secretion system structural components to secreting effectors into the macrophage cytoplasm, enabling Salmonella to replicate in the phagocytic vacuole. Major questions remain regarding how bacteria survive the acidified vacuole and how acidification affects bacterial secretion. We previously reported that EnvZ sensed cytoplasmic signals rather than extracellular ones, as intracellular osmolytes altered the dynamics of a 17-amino-acid region flanking the phosphorylated histidine. We reasoned that the Salmonella cytoplasm might acidify in the macrophage vacuole to activate OmpR-dependent transcription of SPI-2 genes. To address these questions, we employed a DNA-based FRET biosensor (“I-switch”) to measure bacterial cytoplasmic pH and immunofluorescence to monitor effector secretion during infection. Surprisingly, we observed a rapid drop in bacterial cytoplasmic pH upon phagocytosis that was not predicted by current models. Cytoplasmic acidification was completely dependent on the OmpR response regulator, but did not require known OmpR-regulated genes such as ompC, ompF, or ssaC (SPI-2). Microarray analysis highlighted the cadC/BA operon, and additional experiments confirmed that it was repressed by OmpR. Acidification was blocked in the ompR null background in a Cad-dependent manner. Acid-dependent activation of OmpR stimulated type III secretion; blocking acidification resulted in a neutralized cytoplasm that was defective for SPI-2 secretion. Based upon these findings, we propose that Salmonella infection involves an acid-dependent secretion process in which the translocon SseB moves away from the bacterial cell surface as it associates with the vacuolar membrane, driving the secretion of SPI-2 effectors such as SseJ. New steps in the SPI-2 secretion process are proposed.

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. One structure of a GPCR bound to a G protein was solved, but the structure of a GPCR-arrestin complex has remained unknown despite its central role in GPCR biology. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. The structure reveals that arrestin binding induces large and unexpected conformational changes at both the extracellular and intracellular sides of rhodopsin. Arrestin also undergoes dramatic rearrangements from its inactive well-ordered β-sheet structure into a more flexible molten globule-type state, allowing a snake-like movement of the first 77 arrestin residues that shortens its central crest finger loop by seven residues to accommodate the concave surface of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling, reveals a new paradigm of signal transduction by a molten globule, and demonstrates the extraordinary power of X-ray lasers for advancing the frontiers of structural biology.

Phosphorylation of the Retinoic Acid Receptor Alpha Induces a Mechanical Allosteric Regulation and Changes in Internal Dynamics

Nuclear receptor proteins constitute a superfamily of proteins that function as ligand dependent transcription factors. They are implicated in the transcriptional cascades underlying many physiological phenomena, such as embryogenesis, cell growth and differentiation, and apoptosis, making them one of the major signal transduction paradigms in metazoans. Regulation of these receptors occurs through the binding of hormones, and in the case of the retinoic acid receptor (RAR), through the binding of retinoic acid (RA). In addition to this canonical scenario of RAR activity, recent discoveries have shown that RAR regulation also occurs as a result of phosphorylation. In fact, RA induces non-genomic effects, such as the activation of kinase signaling pathways, resulting in the phosphorylation of several targets including RARs themselves. In the case of RARα, phosphorylation of Ser369 located in loop L9–10 of the ligand-binding domain leads to an increase in the affinity for the protein cyclin H, which is part of the Cdk-activating kinase complex of the general transcription factor TFIIH. The cyclin H binding site in RARα is situated more than 40 Å from the phosphorylated serine. Using molecular dynamics simulations of the unphosphorylated and phosphorylated forms of the receptor RARα, we analyzed the structural implications of receptor phosphorylation, which led to the identification of a structural mechanism for the allosteric coupling between the two remote sites of interest. The results show that phosphorylation leads to a reorganization of a local salt bridge network, which induces changes in helix extension and orientation that affects the cyclin H binding site. This results in changes in conformation and flexibility of the latter. The high conservation of the residues implicated in this signal transduction suggests a mechanism that could be applied to other nuclear receptor proteins.

Higher-Order Assemblies in a New Paradigm of Signal Transduction

Recent studies have revealed that multiple intracellular signaling proteins may assemble into structured, yet sometimes infinite, higher-order signaling machines for transmission of receptor activation information to cellular responses. These studies advance our understanding of cell signaling and implicate new molecular mechanisms in proximity-driven enzyme activation, threshold behavior, signal amplification, reduction of biological noise, and temporal and spatial control of signal transduction.

Minireview: Nuclear Insulin and Insulin-like Growth Factor-1 Receptors: A Novel Paradigm in Signal Transduction

The specificity of the insulin receptor (InsR) and insulin-like growth factor-1 receptor (IGF1R) signaling pathways has been the focus of significant debate over the past few years. Recent evidence showing nuclear import and a direct transcriptional role for both InsR and IGF1R adds a new layer of complexity to this dialog. Hence, in addition to the classical roles associated with cell-surface receptors (eg, ligand binding, autophosphorylation of the tyrosine kinase domain, activation of insulin receptor substrate 1 (IRS-1) and additional substrates, protein-protein interactions with membrane and cytoplasm components), new data are consistent with nuclear (genomic) role(s) for both InsR and IGF1R. The present review provides a brief overview of the physical and functional similarities and differences between InsR and IGF1R and describes data from a number of laboratories providing evidence for a new layer of signaling regulation (ie, the ability of InsR and IGF1R to translocate to the cell nucleus and to elicit genomic activities usually associated with transcription factors). The ability of InsR and IGF1R to function as transcription factors, although poorly understood, constitutes a new paradigm in signal transduction. Although research on the role of nuclear InsR/IGF1R is still in its infancy, we believe that this rapidly developing area may have a major basic and translational impact on the fields of metabolism, diabetes, and cancer.

New Insights into S-nitrosylation in Multiple Sclerosis

S-nitrosylation is a biologically relevant post-translational protein modification with signaling consequence. In eukaryotes, a large number of proteins have been identified as S-nitrosylation targets. Derangement in protein S-nitrosylation has been implicated in the pathogenesis of a number of different disease entities including Multiple Sclerosis (MS). A growing body of evidence has shown that Nitric oxide (NO) plays a critical role in MS. NO and other reactive nitrogen species (RNS) are involved in neuroinflammation and neurodegeneration in MS. Signaling by RNS is carried out mainly by S-nitrosylation of critical cysteine residues in targeted proteins. In recent years, newer roles in MS have been attributed to RNS. These roles relate to S-nitrosylation of cysteines in proteins which has emerged as a potential new paradigm in signal transduction and regulation of protein function. In the present review we discuss the evidence for the diverse roles of S-nitrosylation in MS, including nitrosative stress-induced gene expression in MS, and S-nitrosylation of transcription factors in MS. In addition, S-nitrosylation can be therapeutically used in MS. Recent studies providing evidence for SNO-based therapy strategy in the treatment of MS will also be discussed. Undoubtedly, new exciting results will contribute to the expanding area of MS research.

Protein Implicated in Nonsyndromic Mental Retardation Regulates Protein Kinase A (PKA) Activity

Mutation of the coiled-coil and C2 domain-containing 1A (CC2D1A) gene, which encodes a C2 domain and DM14 domain-containing protein, has been linked to severe autosomal recessive nonsyndromic mental retardation. Using a mouse model that produces a truncated form of CC2D1A that lacks the C2 domain and three of the four DM14 domains, we show that CC2D1A is important for neuronal differentiation and brain development. CC2D1A mutant neurons are hypersensitive to stress and have a reduced capacity to form dendrites and synapses in culture. At the biochemical level, CC2D1A transduces signals to the cyclic adenosine 3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit to the nucleus is also defective in CC2D1A mutant cells. Consistently, phosphorylation of the PKA target cAMP-responsive element-binding protein, at serine 133, is nearly abolished in CC2D1A mutant cells. The defects in cAMP/PKA signaling were observed in fibroblast, macrophage, and neuronal primary cells derived from the CC2D1A KO mice. CC2D1A associates with the cAMP-PKA complex following forskolin treatment and accumulates in vesicles or on the plasma membrane in wild-type cells, suggesting that CC2D1A may recruit the PKA complex to the membrane to facilitate signal transduction. Together, our data show that CC2D1A is an important regulator of the cAMP/PKA signaling pathway, which may be the underlying cause for impaired mental function in nonsyndromic mental retardation patients with CC2D1A mutation.

Activation of Myeloid Cell-Specific Adhesion Class G Protein-Coupled Receptor EMR2 via Ligation-Induced Translocation and Interaction of Receptor Subunits in Lipid Raft Microdomains

The adhesion class G protein-coupled receptors (adhesion-GPCRs) play important roles in diverse biological processes ranging from immunoregulation to tissue polarity, angiogenesis, and brain development. These receptors are uniquely modified by self-catalytic cleavage at a highly conserved GPCR proteolysis site (GPS) dissecting the receptor into an extracellular subunit (α) and a seven-pass transmembrane subunit (β) with cellular adhesion and signaling functions, respectively. Using the myeloid cell-restricted EMR2 receptor as a paradigm, we exam the mechanistic relevance of the subunit interaction and demonstrate a critical role for GPS autoproteolysis in mediating receptor signaling and cell activation. Interestingly, two distinct receptor complexes are identified as a result of GPS proteolysis: one consisting of a noncovalent α-β heterodimer and the other comprising two completely independent receptor subunits which distribute differentially in membrane raft microdomains. Finally, we show that receptor ligation induces subunit translocation and colocalization within lipid rafts, leading to receptor signaling and inflammatory cytokine production by macrophages. Our present data resolve earlier conflicting results and provide a new mechanism of receptor signaling, as well as providing a paradigm for signal transduction within the adhesion-GPCR family.

S-Nitrosylation and S-Palmitoylation Reciprocally Regulate Synaptic Targeting of PSD-95

PSD-95, a principal scaffolding component of the postsynaptic density, is targeted to synapses by palmitoylation, where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA-mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.

Bistability in the EnvZ/OmpR Operon Controls Osmotic Signaling in E. coli

In bacteria, the paradigm for signal transduction is the two-component regulatory system. The first component is a sensor kinase and the second component is a response regulator (usually a DNA binding protein). The EnvZ/OmpR two-component system regulates expression of outer membrane proteins in response to osmotic stress. At low osmolality, the major porin is OmpF, at high osmolality, ompF is repressed and OmpC becomes the major porin. EnvZ is an inner membrane protein that transduces the osmotic signal, although the signal is presently unknown. EnvZ is autophosphorylated by ATP on a conserved histidine residue and then transfers the phosphoryl group to the response regulator OmpR on an aspartic acid residue. OmpR∼P binds to the regulatory regions of the porin genes to differentially control their expression. Although high salt or 20% sucrose have been used interchangeably as osmotic stimuli, it is apparent that they cause distinct morphological effects as well as differentially affecting EnvZ. Using an ompC-GFP transcriptional fusion, we examined transcription in single E. coli cells. Approximately 25% of the cells exhibited high fluorescence at high osmolality, but the remainder of the cells were less responsive or unresponsive. In order to understand the underlying basis for this bistability, we constructed photoactivatable chimeras to EnvZ and OmpR and used photoactivatable localization microscopy (PALM) to examine their abundance and localization at low and high osmolality. Surprisingly, EnvZ levels varied dramatically; some cells had low levels, but others had very high levels of EnvZ. Additional experiments are in progress to further characterize this bistability of EnvZ and will be discussed. Supported by NIH GM-058746 and the Mechanobiology Institute (MBI), National University of Singapore.

Phosphorylation-Dependent Regulation of PSF by GSK3 Controls CD45 Alternative Splicing

Signal-induced alternative splicing of the CD45 gene in human Tcells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon Tcell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting Tcells PSF is directly phosphorylated by GSK3, thus promoting interaction of PSF with TRAP150, which prevents PSF from binding CD45 pre-mRNA. Upon Tcell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a paradigm for signal transduction from the cell surface to the RNA processing machinery through the multifunctional protein PSF.

Imaging signal transduction during phagocytosis: phospholipids, surface charge, and electrostatic interactions

Together with the development of genetically encoded fluorescent probes, digital imaging has provided great impetus to the study of cell signaling by providing enhanced sensitivity and much-improved spatial and temporal resolution. We have used phagocytosis as a paradigm of signal transduction, taking advantage of the generous size of phagosomes and of their comparatively leisurely rate of formation. Aided by the design of specific probes, we demonstrated a highly localized and elegantly choreographed sequence of changes in the level of several phosphoinositides and were able to also monitor the fate of phosphatidylserine. The net changes in the content of these anionic phospholipids are accompanied by marked alterations in the surface charge of the membrane of nascent phagosomes. These, in turn, cause the relocation of proteins that associate with the membrane by electrostatic interactions. Our studies suggest that anionic lipids control protein targeting not only through stereospecific recognition by specialized domains but also by electrostatic association mediated by polycationic motifs. The "electrostatic switch" can be turned on or off by altering the charge of the protein ligand (e.g., by phosphorylation) or, alternatively, by modifying the lipid composition of the target membrane.

Systems level analysis of two-component signal transduction systems in Erwinia amylovora: Role in virulence, regulation of amylovoran biosynthesis and swarming motility

BackgroundTwo-component signal transduction systems (TCSTs), consisting of a histidine kinase (HK) and a response regulator (RR), represent a major paradigm for signal transduction in prokaryotes. TCSTs play critical roles in sensing and responding to environmental conditions, and in bacterial pathogenesis. Most TCSTs in Erwinia amylovora have either not been identified or have not yet been studied.ResultsWe used a systems approach to identify TCST and related signal transduction genes in the genome of E. amylovora. Comparative genomic analysis of TCSTs indicated that E. amylovora TCSTs were closely related to those of Erwinia tasmaniensis, a saprophytic enterobacterium isolated from apple flowers, and to other enterobacteria. Forty-six TCST genes in E. amylovora including 17 sensor kinases, three hybrid kinases, 20 DNA- or ligand-binding RRs, four RRs with enzymatic output domain (EAL-GGDEF proteins), and two kinases were characterized in this study. A systematic TCST gene-knockout experiment was conducted, generating a total of 59 single-, double-, and triple-mutants. Virulence assays revealed that five of these mutants were non-pathogenic on immature pear fruits. Results from phenotypic characterization and gene expression experiments indicated that several groups of TCST systems in E. amylovora control amylovoran biosynthesis, one of two major virulence factors in E. amylovora. Both negative and positive regulators of amylovoran biosynthesis were identified, indicating a complex network may control this important feature of pathogenesis. Positive (non-motile, EnvZ/OmpR), negative (hypermotile, GrrS/GrrA), and intermediate regulators for swarming motility in E. amylovora were also identified.ConclusionOur results demonstrated that TCSTs in E. amylovora played major roles in virulence on immature pear fruit and in regulating amylovoran biosynthesis and swarming motility. This suggested presence of regulatory networks governing expression of critical virulence genes in E. amylovora.