G protein threshold behavior in the human neutrophil oxidant response: measurement of G proteins available for signaling in responding and nonresponding subpopulations

Threshold behavior is an important aspect of signal transduction pathways that allows for responses to be turned on or off. Human neutrophil responses to N-formyl peptides, including oxidant production and release, exhibit threshold behavior with respect to the number of G proteins available for signaling; progressive treatment of neutrophils with pertussis toxin causes the conversion of responding cells to nonresponding cells. To quantify the threshold level of G proteins required for signaling of N-formyl peptide stimulated oxidant production in a neutrophil population, we used a plasma membrane associated G protein quantification assay in conjunction with a sorting flow cytometer and measured differences in the average number of G proteins available for signaling per cell in both the responding and the nonresponding subpopulations after pertussis toxin treatment. Although there appeared to be a threshold separating responding cells and nonresponding cells for a given sample, no discrete threshold was measured across multiple treatment conditions. A mathematical model of the early steps in signaling suggests that cell-to-cell variability in signal parameters, such as numbers of signal components and values of kinetic rate constants, obscures the measurement of a discrete threshold and leads to an apparent decrease in the threshold level of G proteins available for signaling as the total G proteins are decreased.     

Multisite phosphorylation and network dynamics of cyclin-dependent kinase signaling in the eukaryotic cell cycle

Multisite phosphorylation of regulatory proteins has been proposed to underlie ultrasensitive responses required to generate nontrivial dynamics in complex biological signaling networks. We used a random search strategy to analyze the role of multisite phosphorylation of key proteins regulating cyclin-dependent kinase (CDK) activity in a model of the eukaryotic cell cycle. We show that multisite phosphorylation of either CDK, CDC25, wee1, or CDK-activating kinase is sufficient to generate dynamical behaviors including bistability and limit cycles. Moreover, combining multiple feedback loops based on multisite phosphorylation do not destabilize the cell cycle network by inducing complex behavior, but rather increase the overall robustness of the network. In this model we find that bistability is the major dynamical behavior of the CDK signaling network, and that negative feedback converts bistability into limit cycle behavior. We also compare the dynamical behavior of several simplified models of CDK regulation to the fully detailed model. In summary, our findings suggest that multisite phosphorylation of proteins is a critical biological mechanism in generating the essential dynamics and ensuring robust behavior of the cell cycle.     

Influence of the extracellular domain size on the dynamic behavior of membrane proteins

The dynamic behavior of plasma membrane proteins mediates various cellular processes such as cellular motility, communication, and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interactions of the intracellular domain with cytosolic components such as cortical actin. Although initiation of different cellular signaling events at the plasma membrane has been attributed to the extracellular domain (ECD) properties recently, the impact of ECDs on the dynamic behavior of membrane proteins is rather unexplored. Here, we investigate how ECD properties influence protein dynamics in the lipid bilayer by reconstituting ECDs of different sizes or glycosylation in model membrane systems and analyzing ECD-driven protein sorting in lipid domains as well as protein mobility. Our data show that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconcile different mechanisms proposed for the initiation of cellular signaling by linking the ECD size of membrane proteins with their localization and diffusion dynamics in the plasma membrane.     

Regulators of G protein signaling & drugs of abuse

Drugs of abuse such as opioids and stimulants share a common dopaminergic reward pathway; however, in response to continual intermittent exposure to such drugs, there are neuronal alterations leading to changes in behavior. Regulators of G protein signaling (RGS) are proteins that negatively regulate G protein signaling and are expressed in brain areas important for the pharmacology of abused drugs. Moreover, the level of expression of several of these proteins is regulated by abused drugs. In this article, we discuss RGS proteins, their regulation by morphine and stimulants, and how altered levels of these proteins affect cell signaling to contribute to the pharmacology and behavioral consequence of abused drugs. Finally, we consider if RGS proteins represent viable targets for drug abuse medications.     

Modular engineering of cellular signaling proteins and networks

Living cells respond to their environment using networks of signaling molecules that act as sensors, information processors, and actuators. These signaling systems are highly modular at both the molecular and network scales, and much evidence suggests that evolution has harnessed this modularity to rewire and generate new physiological behaviors. Conversely, we are now finding that, following nature's example, signaling modules can be recombined to form synthetic tools for monitoring, interrogating, and controlling the behavior of cells. Here we highlight recent progress in the modular design of synthetic receptors, optogenetic switches, and phospho-regulated proteins and circuits, and discuss the expanding role of combinatorial design in the engineering of cellular signaling proteins and networks.     

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form

There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor zeta subunit. This domain exhibits specific dimerization that is distinct from non-specific aggregation behavior seen in many systems. In this work, we use diffusion and chemical shift mapping NMR data to show that the protein does not undergo a transition between disordered and ordered states upon dimerization. This finding opposes the generally accepted view on the behavior of intrinsically disordered proteins, provides evidence for the existence of specific dimerization interactions for intrinsically disordered protein species and opens a new line of research in this new and quickly developing field.     

Neuroblastoma Tyrosine Kinase Signaling Networks Involve FYN and LYN in Endosomes and Lipid Rafts

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.     

Brain signaling and behavioral responses induced by exposure to (56)Fe-particle radiation

Previous experiments have demonstrated that exposure to 56Fe-particle irradiation (1.5 Gy, 1 GeV) produced aging-like accelerations in neuronal and behavioral deficits. Astronauts on long-term space flights will be exposed to similar heavy-particle radiations that might have similar deleterious effects on neuronal signaling and cognitive behavior. Therefore, the present study evaluated whether radiation-induced spatial learning and memory behavioral deficits are associated with region-specific brain signaling deficits by measuring signaling molecules previously found to be essential for behavior [pre-synaptic vesicle proteins, synaptobrevin and synaptophysin, and protein kinases, calcium-dependent PRKCs (also known as PKCs) and PRKA (PRKA RIIbeta)]. The results demonstrated a significant radiation-induced increase in reference memory errors. The increases in reference memory errors were significantly negatively correlated with striatal synaptobrevin and frontal cortical synaptophysin expression. Both synaptophysin and synaptobrevin are synaptic vesicle proteins that are important in cognition. Striatal PRKA, a memory signaling molecule, was also significantly negatively correlated with reference memory errors. Overall, our findings suggest that radiation-induced pre-synaptic facilitation may contribute to some previously reported radiation-induced decrease in striatal dopamine release and for the disruption of the central dopaminergic system integrity and dopamine-mediated behavior.     

Synthetic biology with surgical precision: targeted reengineering of signaling proteins

The complexity of living systems exceeds everything else studied by natural sciences. Sophisticated networks of intimately intertwined signaling pathways coordinate cellular functions. Clear understanding how the integration of multiple inputs produces coherent behavior is one of the major challenges of cell biology. Integration via perfectly timed highly regulated protein-protein interactions and precise targeting of the "output" proteins to particular substrates is emerging as a common theme of signaling regulation. This often involves specialized scaffolding proteins, whose key function is to ensure that correct partners come together in an appropriate place at the right time. Defective or faulty signaling underlies many congenital and acquired human disorders. Several pioneering studies showed that ectopic expression of existing proteins or their elements can restore functions destroyed by mutations or normalize the signaling pushed out of balance by disease and/or current small molecule-based therapy. Several recent studies show that proteins with new functional modalities can be generated by mixing and matching existing domains, or via functional recalibration and fine-tuning of existing proteins by precisely targeted mutations. Using arrestins as an example, we describe how manipulation of individual functions yields signaling-biased proteins. Creative protein redesign generates novel tools valuable for unraveling the intricacies of cell biology. Engineered proteins with specific functional changes also have huge therapeutic potential in disorders associated with inherited or acquired signaling errors.     

Simulated Evolution of Signal Transduction Networks

Signal transduction is the process of routing information inside cells when receiving stimuli from their environment that modulate the behavior and function. In such biological processes, the receptors, after receiving the corresponding signals, activate a number of biomolecules which eventually transduce the signal to the nucleus. The main objective of our work is to develop a theoretical approach which will help to better understand the behavior of signal transduction networks due to changes in kinetic parameters and network topology. By using an evolutionary algorithm, we designed a mathematical model which performs basic signaling tasks similar to the signaling process of living cells. We use a simple dynamical model of signaling networks of interacting proteins and their complexes. We study the evolution of signaling networks described by mass-action kinetics. The fitness of the networks is determined by the number of signals detected out of a series of signals with varying strength. The mutations include changes in the reaction rate and network topology. We found that stronger interactions and addition of new nodes lead to improved evolved responses. The strength of the signal does not play any role in determining the response type. This model will help to understand the dynamic behavior of the proteins involved in signaling pathways. It will also help to understand the robustness of the kinetics of the output response upon changes in the rate of reactions and the topology of the network.     

D1 dopamine receptor signaling is modulated by the R7 RGS protein EAT-16 and the R7 binding protein RSBP-1 in Caenoerhabditis elegans motor neurons

Dopamine signaling modulates voluntary movement and reward-driven behaviors by acting through G protein-coupled receptors in striatal neurons, and defects in dopamine signaling underlie Parkinson's disease and drug addiction. Despite the importance of understanding how dopamine modifies the activity of striatal neurons to control basal ganglia output, the molecular mechanisms that control dopamine signaling remain largely unclear. Dopamine signaling also controls locomotion behavior in Caenorhabditis elegans. To better understand how dopamine acts in the brain we performed a large-scale dsRNA interference screen in C. elegans for genes required for endogenous dopamine signaling and identified six genes (eat-16, rsbp-1, unc-43, flp-1, grk-1, and cat-1) required for dopamine-mediated behavior. We then used a combination of mutant analysis and cell-specific transgenic rescue experiments to investigate the functional interaction between the proteins encoded by two of these genes, eat-16 and rsbp-1, within single cell types and to examine their role in the modulation of dopamine receptor signaling. We found that EAT-16 and RSBP-1 act together to modulate dopamine signaling and that while they are coexpressed with both D1-like and D2-like dopamine receptors, they do not modulate D2 receptor signaling. Instead, EAT-16 and RSBP-1 act together to selectively inhibit D1 dopamine receptor signaling in cholinergic motor neurons to modulate locomotion behavior.     

Does diacylglycerol serve as a signaling molecule in plants?

Diacylglycerol (DAG) is an important signaling phospholipid in animals, specifically binding to the C1 domain of proteins such as protein kinase C. In most plant species, however, DAG is present at low abundance, and no interacting proteins have yet been identified. As a result, it has been proposed that the signaling function of DAG has been discarded by plants during their evolution. In this mini-review, we summarize the accumulating experimental evidence which supports that notion that changes in DAG content in response to particular cues are a feature of plant cells. This behavior suggests that DAG does indeed act as a signaling molecule during plant development and in response to certain environmental stimuli.     

Dynamic control of TGF-beta signaling and its links to the cytoskeleton

Transforming growth factor beta (TGF-beta) regulates cellular behavior in embryonic and adult tissues. TGF-beta binding to serine/threonine kinase receptors on the plasma membrane activates Smad molecules and additional signaling proteins that coordinately regulate gene expression or cytoplasmic processes such as cytoskeletal dynamics. In turn, the activity and duration of the Smad pathway seems to be regulated by cytoskeletal components, which facilitate the shuttling process that segregates Smad proteins in the cytoplasm and nucleus. We discuss mechanisms and models that aim at explaining the coordination between several components of the signaling network downstream of the TGF-beta signal.     

Signaling interactions between the aerotaxis transducer Aer and heterologous chemoreceptors in Escherichia coli

Aer, a low-abundance signal transducer in Escherichia coli, mediates robust aerotactic behavior, possibly through interactions with methyl-accepting chemotaxis proteins (MCP). We obtained evidence for interactions between Aer and the high-abundance aspartate (Tar) and serine (Tsr) receptors. Aer molecules bearing a cysteine reporter diagnostic for trimer-of-dimer formation yielded cross-linking products upon treatment with a trifunctional maleimide reagent. Aer also formed mixed cross-linking products with a similarly marked Tar reporter. An Aer trimer contact mutation known to abolish trimer formation by MCPs eliminated Aer trimer and mixed trimer formation. Trimer contact alterations known to cause epistatic behavior in MCPs also produced epistatic properties in Aer. Amino acid replacements in the Tar trimer contact region suppressed an epistatic Aer signaling defect, consistent with compensatory conformational changes between directly interacting proteins. In cells lacking MCPs, Aer function required high-level expression, comparable to the aggregate number of receptors in a wild-type cell. Aer proteins with clockwise (CW)-biased signal output cannot function under these conditions but do so in the presence of MCPs, presumably through formation of mixed signaling teams. The Tar signaling domain was sufficient for functional rescue. Moreover, CW-biased lesions did not impair aerotactic signaling in a hybrid Aer-Tar transducer capable of adjusting its steady-state signal output via methylation-dependent sensory adaptation. Thus, MCPs most likely assist mutant Aer proteins to signal productively by forming collaborative signaling teams. Aer evidently evolved to operate collaboratively with high-abundance receptors but can also function without MCP assistance, provided that it can establish a suitable prestimulus swimming pattern.     

Cooperative Binding of MgATP and MgADP in the Trimeric PII Protein GlnK2 from Archaeoglobus fulgidus

P_II-like proteins, such as GlnK, found in a wide variety of organisms from prokaryotes to plants constitute a family of cytoplasmic signaling proteins that play a central regulatory role in the assimilation of nitrogen for biosyntheses. They specifically bind and are modulated by effector molecules such as adenosine triphosphate, adenosine diphosphate and 2-oxoglutarate. Their highly conserved, trimeric structure suggests that cooperativity in effector binding might be the basis for the ability to integrate and respond to a wide range of concentrations, but to date no direct quantification of this cooperative behavior has been presented. The hyperthermophilic archaeon Archaeoglobus fulgidus contains three GlnK proteins, functionally associated with ammonium transport proteins (Amt). We have characterized GlnK2 and its interaction with effectors by high-resolution X-ray crystallography and isothermal titration calorimetry. Binding of adenosine nucleotides resulted in distinct, cooperative behavior for ATP and ADP. While 2-oxoglutarate has been shown to interact with other GlnK proteins, GlnK2 was completely insensitive to this key indicator of a low level of intracellular nitrogen. These findings point to different regulation and modulation patterns and add to our understanding of the flexibility and versatility of the GlnK family of signaling proteins.     

Loss- and gain-of-function mutations in the F1-HAMP region of the Escherichia coli aerotaxis transducer Aer

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants.     

Rho and Rac take center stage

Many features of cell behavior are regulated by Rho family GTPases, but the most profound effects of these proteins are on the actin cytoskeleton and it was these that first drew attention to this family of signaling proteins. Focusing on Rho and Rac, we will discuss how their effectors regulate the actin cytoskeleton. We will describe how the activity of Rho proteins is regulated downstream from growth factor receptors and cell adhesion molecules by guanine nucleotide exchange factors and GTPase activating proteins. Additionally, we will discuss how there is signaling crosstalk between family members and how various bacterial pathogens have developed strategies to manipulate Rho protein activity so as to enhance their own survival.     

Structural analysis of bacterial chemotaxis proteins: components of a dynamic signaling system

Most motile bacteria are capable of directing their movement in response to chemical gradients, a behavior known as chemotaxis. The signal transduction system that mediates chemotaxis in enteric bacteria consists of a set of six cytoplasmic proteins that couple stimuli sensed by a family of transmembrane receptors to behavioral responses generated by the flagellar motors. Signal transduction occurs via a phosphotransfer pathway involving a histidine protein kinase, CheA, and a response regulator protein, CheY, that in its phosphorylated state, modulates the direction of flagellar rotation. Two auxiliary proteins, CheW and CheZ, and two receptor modification enzymes, methylesterase CheB and methyltransferase CheR, influence the flux of phosphoryl groups within this central pathway. This paper focuses on structural characteristics of the four signaling proteins (CheA, CheY, CheB, and CheR) for which NMR or x-ray crystal structures have been determined. The proteins are examined with respect to their signaling activities that involve reversible protein modifications and transient assembly of macromolecular complexes. A variety of data suggest conformational flexibility of these proteins, a feature consistent with their multiple roles in a dynamic signaling pathway.     

Small G protein signaling in neuronal plasticity and memory formation: the specific role of ras family proteins

Small G proteins are an extensive family of proteins that bind and hydrolyze GTP. They are ubiquitous inside cells, regulating a wide range of cellular processes. Recently, many studies have examined the role of small G proteins, particularly the Ras family of G proteins, in memory formation. Once thought to be primarily involved in the transduction of a variety of extracellular signals during development, it is now clear that Ras family proteins also play critical roles in molecular processing underlying neuronal and behavioral plasticity. We here review a number of recent studies that explore how the signaling of Ras family proteins contributes to memory formation. Understanding these signaling processes is of fundamental importance both from a basic scientific perspective, with the goal of providing mechanistic insights into a critical aspect of cognitive behavior, and from a clinical perspective, with the goal of providing effective therapies for a range of disorders involving cognitive impairments.