Chemical approaches for investigating phosphorylation in signal transduction networks

The power and scope of chemical synthesis offer considerable opportunities to broaden the lexicon of chemical tools that can be implemented for the study of complex biological systems. To investigate individual signaling proteins and pathways, chemical tools provide a powerful complement to existing genetic, chemical genetic and immunologic methods. In particular, understanding phosphorylation-mediated signaling in real time yields important information about the regulation of cellular function and insights into the origin of disease. Recent advances in the development of photolabile caged analogs of bioactive species and fluorescence-based sensors of protein kinase activities are useful for investigating protein phosphorylation and the roles of phosphoproteins. Photolabile caged analogs allow spatial and temporal control over the release of a compound, while fluorescence-based sensors allow the real-time visualization of kinase activity. Here, we discuss recent advances that have increased the specificity and availability of these tools.     

Chemical genetic strategies to delineate MAP kinase signaling pathways using protein-fragment complementation assays (PCA)

Signal transduction pathways mediated by MAP kinases are among the most studied. Direct analysis of MAP kinase pathways has been difficult because some details of MAP kinase signaling cannot be studied in vitro. Here, we describe a strategy for directly analyzing MAP kinase signaling pathways in living cells using protein-fragment complementation assays (PCA) based on intensely fluorescent proteins. The assays allow for spatial and temporal analysis of protein complexes including those that form upstream and downstream from MAPKs as well as complexes of MAPKs with regulator and effector proteins. We describe high-content assays, high-throughput quantitative microscopic methods to follow temporal changes in complex subcellular location and quantity. Spatial and temporal changes in response to perturbations (chemical, siRNA, and hormones) allow for delineation of MAPK signaling networks and a general and high-throughput approach to identify small molecules that act directly or indirectly on MAPK pathways.     

Rational design of genetically encoded fluorescence resonance energy transfer-based sensors of cellular Cdc42 signaling

The temporal and spatial control of Rho GTPase signaling pathways is a central issue in understanding the molecular mechanisms that generate complex cellular movements. The Rho protein Cdc42 induces a significant conformational change in its downstream effector, the Wiskott-Aldrich syndrome protein (WASP). On the basis of this conformational change, we have created a series of single-molecule sensors for both active Cdc42 and Cdc42 guanine nucleotide exchange factors (GEFs) that utilize fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. In vitro, the Cdc42 sensors produce up to 3.2-fold FRET emission ratio changes upon binding active Cdc42. The GEF sensors yield up to 1.7-fold changes in FRET upon exchange of GDP for GTP. The GEF-catalyzed rate of nucleotide exchange for the GEF sensor is indistinguishable from that of wild-type Cdc42, but GAP-catalyzed nucleotide hydrolysis is slowed approximately 16-fold. In vivo, both sensors faithfully report on Cdc42 and/or Cdc42-GEF activity. These results establish the successful creation of rationally designed and genetically encoded tools that can be used to image the activity of biologically and medically important molecules in living systems.     

Shining light on signaling and metabolic networks by genetically encoded biosensors

Fluorescent labels have revolutionized cell biology. Signaling intermediates and metabolites can be measured in real time with subcellular spatial resolution. Most of these sensors are based on fluorescent proteins, and many report fluorescence resonance energy transfer. Because the biosensors are genetically encoded, a toolbox for addressing cell biological questions at the systems level is now available. Fluorescent biosensors are able to determine the localization of proteins and their dynamics, to reveal the cellular and subcellular localization of the respective interactions and activities, and to provide complementary data on the steady state levels of ions, metabolites, and signaling intermediates with high temporal and spatial resolution. They represent the basis for cell-based high-throughput assays that are necessary for a systems perspective on plant cell function.     

Fluorescent biosensors of protein function

Fluorescent biosensors allow researchers to image and quantify protein activity and small molecule signals in living cells with high spatial and temporal resolution. Genetically encoded sensors are coded by a DNA sequence and hence constructed entirely out of amino acids. These biosensors typically utilize light-emitting proteins, such as derivatives of the green fluorescent protein (GFP), and have been developed for a wide range of small molecules and enzyme activities. Fluorescent biosensors can be genetically targeted to distinct locations within cells, such as organelles and membranes. This feature facilitates elucidation of how protein activities and cellular signals are modulated in different regions of the cell. Improvements in the dynamic range and robustness of sensors have enabled high throughput screening for molecules that act as agonists or antagonists of protein function.     

SLAM and CD31: signaling molecules involved in cytokine secretion during the development of innate and adaptive immune responses

Immune cells are modulated through the crosslinking of receptors named "immunoreceptors". Ligation of immunoreceptors by their ligands induces a tyrosine-phosphorylation signal that is essential for cell activation or inhibition. Physiologically, immunoreceptor triggering is not enough for cell activation, and stimulation of co-receptors is necessary for antigen-evoked cytokine production. Thus, signal transduction pathways mediated by proteins that regulate cytokine secretion are critical to achieve an effective immune response of the host, where the balance between positive and negative signaling allows effective immune responses, preventing tolerance and autoimmunity. This review deals with recent studies based on the role of the receptor signaling lymphocytic activation molecule (SLAM), a signaling protein that modulates cytokine secretion by immune cells, and the transmembrane glycoprotein CD31, which plays multiple roles in cellular signaling events by modulating the balance between inhibitory and stimulatory signals to immune cells. Recent studies have shed light on the ability of these molecules to transmit different signals that regulate the ability of innate and adaptive immune cells to synthesize stimulatory and inhibitory cytokines.     

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.     

A photoactivatable small-molecule inhibitor for light-controlled spatiotemporal regulation of Rho kinase in live embryos

To uncover the molecular mechanisms of embryonic development, the ideal loss-of-function strategy would be capable of targeting specific regions of the living embryo with both temporal and spatial precision. To this end, we have developed a novel pharmacological agent that can be light activated to achieve spatiotemporally limited inhibition of Rho kinase activity in vivo. A new photolabile caging group, 6-nitropiperonyloxymethyl (NPOM), was installed on a small-molecule inhibitor of Rho kinase, Rockout, to generate a 'caged Rockout' derivative. Complementary biochemical, cellular, molecular and morphogenetic assays in both mammalian cell culture and Xenopus laevis embryos validate that the inhibitory activity of the caged compound is dependent on exposure to light. Conveniently, this unique reagent retains many of the practical advantages of conventional small-molecule inhibitors, including delivery by simple diffusion in the growth medium and concentration-dependent tuneability, but can be locally activated by decaging with standard instrumentation. Application of this novel tool to the spatially heterogeneous problem of embryonic left-right asymmetry revealed a differential requirement for Rho signaling on the left and right sides of the primitive gut tube, yielding new insight into the molecular mechanisms that generate asymmetric organ morphology. As many aromatic/heterocyclic small-molecule inhibitors are amenable to installation of this caging group, our results indicate that photocaging pharmacological inhibitors might be a generalizable technique for engendering convenient loss-of-function reagents with great potential for wide application in developmental biology.     

Phosphoryltyrosyl mimetics in the design of peptide-based signal transduction inhibitors

The central roles played by protein-tyrosine kinase (PTK)-dependent signal transduction in normal cellular regulation and homeostasis have made inappropriate or aberrant functions of certain of these pathways contributing factors to a variety of diseases, including several cancers. For this reason, development of PTK signaling inhibitors has evolved into an important approach toward new therapeutics. Since in these pathways phosphotyrosyl (pTyr) residues provide unique and defining functions either by their creation under the catalysis of PTKs, their recognition and binding by protein modules such as SH2 and phosphotyrosyl binding (PTB) domains, or their destruction by protein-tyrosine phosphatases, pTyr mimetics provide useful general starting points for inhibitor design. Important considerations in the development of such pTyr mimetics include enzymatic stability (particularly toward PTPs), high affinity recognition by target pTyr binding proteins, and good cellular bioavailability. Although small molecule, nonpeptide inhibitors may be ultimate objectives of inhibitor development, peptides frequently serve as display platforms for pTyr mimetics, which afford useful and conceptually straightforward starting points in the development process. Reported herein is a limited overview of pTyr mimetic development as it relates to peptide-based agents. Of particular interest are recent findings that highlight potential limitations of peptides as display platforms for the identification of small molecule leads. One conclusion that results from this work is that while peptide-based approaches toward small molecule inhibitor design are often intellectually satisfying from a structure-based perspective, extrapolation of negative findings to small molecule, nonpeptide contexts should be undertaken with extreme caution.     

Role of systems biology and multi-omics analyses in delineating spatial interconnectivity and temporal dynamicity of ER stress mediated cellular responses

The endoplasmic reticulum (ER) is a membranous organelle involved in calcium storage, lipid biosynthesis, protein folding and processing. Many patho-physiological conditions and pharmacological agents are known to perturb normal ER function and can lead to ER stress, which severely compromise protein folding mechanism and hence poses high risk of proteotoxicity. Upon sensing ER stress, the different stress signaling pathways interconnect with each other and work together to preserve cellular homeostasis. ER stress response is a part of the integrative stress response (ISR) and might play an important role in the pathogenesis of chronic neurodegenerative diseases, where misfolded protein accumulation and cell death are common. The initiation, manifestation and progression of ER stress mediated unfolded protein response (UPR) is a complex procedure involving multiple proteins, pathways and cellular organelles. To understand the cause and consequences of such complex processes, implementation of an integrative holistic approach is required to identify novel players and regulators of ER stress. As multi-omics data-based systems analyses have shown potential to unravel the underneath molecular mechanism of complex biological systems, it is important to emphasize the utility of this approach in understanding the ER stress biology. In this review we first discuss the ER stress signaling pathways and regulatory players, along with their inter-connectivity. We next highlight the importance of systems and network biology approaches using multi-omics data in understanding ER stress mediated cellular responses. This report would help advance our current understanding of the multivariate spatial interconnectivity and temporal dynamicity of ER stress.     

Research advances on selective phosphatidylinositol 3 kinase δ (PI3Kδ) inhibitors

PI3Kδ in B cells mediates antigen receptor signaling and promote neutrophil chemotaxis. The activation of PI3Kδ can cause mast cell maturation and degranulation, myeloid cell dysfunction, and cytokine release. As a key signal molecule, PI3Kδ interacts with the lipid binding domain of a variety of cellular proteins as a secondary messenger, ultimately affecting a series of significant cellular pathways in disease pathology. Therefore, many research organizations and pharmaceutical companies have studied it to develop effectively selective PI3Kδ inhibitors as therapeutics. This review summarizes research advances in varying chemical classes of selective PI3Kδ inhibitors and the structure-activity relationship, and it mainly focuses on the propeller- versus flat-type class of inhibitors.     

Covalent Modification Cycles through the Spatial Prism

Covalent modification cycles are basic units and building blocks of posttranslational modification and cellular signal transduction. We systematically explore different spatial aspects of signal transduction in covalent modification cycles by starting with a basic temporal cycle as a reference and focusing on steady-state signal transduction. We consider, in turn, the effect of diffusion on spatial signal transduction, spatial analogs of ultrasensitive behavior, and the interplay between enzyme localization and substrate diffusion. Our analysis reveals the need to explicitly account for kinetics and diffusional transport (and localization) of enzymes, substrates, and complexes. It demonstrates a complex and subtle interplay between spatial heterogeneity, diffusion, and localization. Overall, examining the spatial dimension of covalent modification reveals that 1), there are important differences between spatial and temporal signal transduction even in this cycle; and 2), spatial aspects may play a substantial role in affecting and distorting information transfer in modules/networks that are usually studied in purely temporal terms. This has important implications for the systematic understanding of signaling in covalent modification cycles, pathways, and networks in multiple cellular contexts.     

Sodium nitroprusside affects the level of photosynthetic enzymes and glucose metabolism in Phaseolus aureus (mung bean)

Nitric oxide (NO) is an important signaling molecule in plants. The present study aims to investigate the downstream signaling pathways of NO in plants using a proteomic approach. Phaseolus aureus (mung bean) leaf was treated with sodium nitroprusside (SNP), which releases nitric oxide in the form of nitrosonium cation (NO+) upon light irradiation. Changes in protein expression profiles of the SNP treated mung bean leaf were analyzed by two-dimensional gel electrophoresis (2-DE). Comparison of 2-DE electropherograms revealed seven down-regulated and two up-regulated proteins after treatment with 0.5 mM SNP for 6 h. The identities of these proteins were analyzed by a combination of peptide mass fingerprinting and post-source decay using a matrix-assisted-laser-desorption-ionisation-time-of-flight (MALDI-TOF) mass spectrometer. Six out of these nine proteins found are involved in either photosynthesis or cellular metabolism. We have taken our investigation further by studying the effect of NO+ on glucose contents in mung bean leaves. Our results clearly demonstrated that NO+ rapidly and drastically decrease the amount of glucose in mung bean leaves. Moreover, four out of nine of these proteins are chloroplastic isoforms. These results suggested that chloroplasts might be one of the main sub-cellular targets of NO in plants.     

Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets

Cytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.     

Temporal/spatial expression of retinoid binding proteins and RAR isoforms in the postnatal lung

Endogenous retinoids have been implicated in alveologenesis in both the rat and the mouse, and exogenous retinoic acid (RA) can reverse or partially reverse experimental emphysema in adult rat and mouse models by an unknown mechanism. In this study, we examine the cellular and molecular biology of retinoid signaling during alveologenesis in the mouse. We describe the temporal and spatial expression of the retinoid binding proteins CRBP-I, CRBP-II, and CRABP-I using RT-PCR and immunohistochemistry. We identify the retinoic acid receptor isoforms RAR-alpha 1, RAR-beta 2, RAR-beta 4, and RAR-gamma 2 and describe their temporal and spatial expression using RT-PCR and in situ hybridization. We demonstrate that both retinoid binding proteins and RAR isoforms are temporally regulated and found within the alveolar septal regions during alveologenesis. These data support a role of dynamic endogenous RA signaling during alveolar formation.     

Covalent Modification Cycles through the Spatial Prism

Covalent modification cycles are basic units and building blocks of posttranslational modification and cellular signal transduction. We systematically explore different spatial aspects of signal transduction in covalent modification cycles by starting with a basic temporal cycle as a reference and focusing on steady-state signal transduction. We consider, in turn, the effect of diffusion on spatial signal transduction, spatial analogs of ultrasensitive behavior, and the interplay between enzyme localization and substrate diffusion. Our analysis reveals the need to explicitly account for kinetics and diffusional transport (and localization) of enzymes, substrates, and complexes. It demonstrates a complex and subtle interplay between spatial heterogeneity, diffusion, and localization. Overall, examining the spatial dimension of covalent modification reveals that 1), there are important differences between spatial and temporal signal transduction even in this cycle; and 2), spatial aspects may play a substantial role in affecting and distorting information transfer in modules/networks that are usually studied in purely temporal terms. This has important implications for the systematic understanding of signaling in covalent modification cycles, pathways, and networks in multiple cellular contexts.     

Complexity of calcium signaling in synaptic spines

Long-term potentiation and long-term depression are thought to be cellular mechanisms contributing to learning and memory. Although the physiological phenomena have been well characterized, little consensus of their underlying molecular mechanisms has emerged. One reason for this may be the under-appreciated complexity of the signaling pathways that can arise if key signaling molecules are discretely localized within the synapse. Recent findings suggest an unanticipated degree of structural organization at the synapse, and improved methods in cellular imaging of living tissue have provided much-needed information about the intracellular dynamics of Ca(2+), thought to be critical for both LTP and LTD. In this review, we briefly summarize some of these developments, and show that a more complete understanding of cellular signaling depends on the successful integration of traditional biochemistry and molecular biology with the spatial and temporal details of synaptic ultrastructure. Biophysically realistic computer simulations can have an important role in bridging these disciplines.     

Scaffold proteins in mammalian MAP kinase cascades

The mitogen-activated protein kinase (MAPK) signaling pathway, which is conserved from yeast to humans, is activated in response to a variety of extra- and intracellular stimuli, and plays key roles in multiple cellular processes, including proliferation, differentiation, and apoptosis. The MAPK pathway transmits its signal through the sequential phosphorylation of MAPK kinase kinase to MAPK kinase to MAPK. Specific and efficient activation of the MAPK cascades is crucial for proper cellular responses to stimuli. As shown in yeast, the mammalian MAPK signaling system may also employ scaffold proteins, in part, to organize the MAPK signaling components into functional MAPK modules, thereby enabling the efficient activation of specific MAPK pathways. This review article describes recent advances in the study of potential mammalian scaffold proteins that may help us understand the complex regulation, including the spatial and temporal control, of the mammalian MAPK signaling pathways.