Positional Information Generated by Spatially Distributed Signaling Cascades

The temporal and stationary behavior of protein modification cascades has been extensively studied, yet little is known about the spatial aspects of signal propagation. We have previously shown that the spatial separation of opposing enzymes, such as a kinase and a phosphatase, creates signaling activity gradients. Here we show under what conditions signals stall in the space or robustly propagate through spatially distributed signaling cascades. Robust signal propagation results in activity gradients with long plateaus, which abruptly decay at successive spatial locations. We derive an approximate analytical solution that relates the maximal amplitude and propagation length of each activation profile with the cascade level, protein diffusivity, and the ratio of the opposing enzyme activities. The control of the spatial signal propagation appears to be very different from the control of transient temporal responses for spatially homogenous cascades. For spatially distributed cascades where activating and deactivating enzymes operate far from saturation, the ratio of the opposing enzyme activities is shown to be a key parameter controlling signal propagation. The signaling gradients characteristic for robust signal propagation exemplify a pattern formation mechanism that generates precise spatial guidance for multiple cellular processes and conveys information about the cell size to the nucleus.     

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.     

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.     

An Algebraic Approach to Signaling Cascades with n Layers

Posttranslational modification of proteins is key in transmission of signals in cells. Many signaling pathways contain several layers of modification cycles that mediate and change the signal through the pathway. Here, we study a simple signaling cascade consisting of n layers of modification cycles such that the modified protein of one layer acts as modifier in the next layer. Assuming mass-action kinetics and taking the formation of intermediate complexes into account, we show that the steady states are solutions to a polynomial in one variable and in fact that there is exactly one steady state for any given total amounts of substrates and enzymes.     

Compartmentalization of ROS-mediated signal transduction

Localization of signaling molecules close to their targets is the central principle of cell signaling. The colocalization of multicomponent signaling complexes is realized through protein scaffolds that provide better specificity than undirected diffusion of the same components. It has been suggested that ROS-generating complexes follow the principle by specific intracellular localization of ROS production and the limitation of ROS diffusion distances. However, the lack of adequate methods did not allow direct detection of local ROS production to confirm the model of redox signaling compartmentalization. Nevertheless, evidence of local ROS production and the restriction of diffusion were provided by kinetic modeling and data on the subcellular localization of NADPH oxidase isoforms, their adapter proteins, and local restriction of ROS diffusion. Here we shall discuss the properties of antioxidant systems which prevent uncontrolled ROS diffusion from sites of generation to the adjacent subcellular compartments; the current data on the specific localization of NADPH oxidase activity and its influence on intracellular processes; and the recent evidence of the ROS diffusion restriction.     

Phosphorylation-mediated regulation of GEFs for RhoA

Spatio-temporal control of RhoA GTPase is critical for regulation of cell migration, attachment to extracellular matrix, and cell–cell adhesions. Activation of RhoA is mediated by guanine nucleotide exchange factors (GEFs), a diverse family of enzymes that are controlled by multiple signaling pathways regulating actin cytoskeleton and cell migration. GEFs can be regulated by different mechanisms. Growing evidence demonstrates that phosphorylation serves as one of the predominant signals controlling activity, interactions, and localization of RhoGEFs. It acts as a positive and a negative regulator, and allows for regulation of RhoGEFs by multiple signaling cascades. Although there are common trends in phosphorylation-mediated regulation of some RhoGEF homologs, the majority of GEFs utilize distinct mechanisms that are dictated by their unique structure and interaction networks. This diversity enables multiple signaling pathways to use different RhoGEFs for regulation of a single central—RhoA. Here, we review current examples of phosphorylation-mediated regulation of GEFs for RhoA and its role in cell migration, discuss mechanisms, and provide insights into potential future directions.     

Signaling Cascades Modulate the Speed of Signal Propagation through Space

Background

Cells are not mixed bags of signaling molecules. As a consequence, signals must travel from their origin to distal locations. Much is understood about the purely diffusive propagation of signals through space. Many signals, however, propagate via signaling cascades. Here, we show that, depending on their kinetics, cascades speed up or slow down the propagation of signals through space, relative to pure diffusion.

Methodology/Principal Findings

We modeled simple cascades operating under different limits of Michaelis-Menten kinetics using deterministic reaction-diffusion equations. Cascades operating far from enzyme saturation speed up signal propagation; the second mobile species moves more quickly than the first through space, on average. The enhanced speed is due to more efficient serial activation of a downstream signaling module (by the signaling molecule immediately upstream in the cascade) at points distal from the signaling origin, compared to locations closer to the source. Conversely, cascades operating under saturated kinetics, which exhibit zero-order ultrasensitivity, can slow down signals, ultimately localizing them to regions around the origin.

Conclusions/Significance

Signal speed modulation may be a fundamental function of cascades, affecting the ability of signals to penetrate within a cell, to cross-react with other signals, and to activate distant targets. In particular, enhanced speeds provide a way to increase signal penetration into a cell without needing to flood the cell with large numbers of active signaling molecules; conversely, diminished speeds in zero-order ultrasensitive cascades facilitate strong, but localized, signaling.     

Emerging themes of cAMP regulation of the pulmonary endothelial barrier

The presence of excess fluid in the interstitium and air spaces of the lung presents severe restrictions to gas exchange. The pulmonary endothelial barrier regulates the flux of fluid and plasma proteins from the vascular space into the underlying tissue. The integrity of this endothelial barrier is dynamically regulated by transitions in cAMP (3',5'-cyclic adenosine monophosphate), which are synthesized in discrete subcellular compartments. Cyclic AMP generated in the subplasma membrane compartment acts through PKA and Epac (exchange protein directly activated by cAMP) to tighten cell adhesions, strengthen cortical actin, reduce actomyosin contraction, and decrease permeability. Confining cAMP within the subplasma membrane space is critical to its barrier-protective properties. When cAMP escapes the near membrane compartment and gains access to the cytosolic compartment, or when soluble adenylyl cyclases generate cAMP within the cytosolic compartment, this second messenger activates established cytosolic cAMP signaling cascades to perturb the endothelial barrier through PKA-mediated disruption of microtubules. Thus the concept of cAMP compartmentalization in endothelial barrier regulation is gaining momentum and new possibilities are being unveiled for cytosolic cAMP signaling with the emergence of the bicarbonate-regulated mammalian soluble adenylyl cyclase (sAC or AC10).     

Spatial association of signaling proteins and F-actin effects on cluster assembly analyzed via photoactivation localization microscopy in T cells

Recognition of antigens by T cell receptors (TCRs) triggers cellular signaling cascades initiated by recruitment to the plasma membrane of numerous effector molecules to form signaling microclusters (MCs). Here we show that the method of high-resolution photoactivation localization microscopy (PALM) imaging can be used to analyze the spatial correlation between kinase ZAP70 and adaptor SLP76 MCs at the cell periphery and the effects of F-actin on MC assembly. We first determined the photophysical rate constants of Dronpa and tdEos fluorescence probes, which allowed us to optimize our dual-color PALM imaging method. We next analyzed the degrees of spatial association through determination of Mander's colocalization coefficients from PALM images, which revealed increasing spatial segregation of ZAP70 and SLP76 MCs at the cell periphery after initiation of signaling. We showed that this spatial segregation at the cell periphery occurred in parallel with the reduction of MC phosphorylation levels. Furthermore, we used Ripley's K function to analyze spatial randomness, and determined average radii of clusters as a function of activation time. The average radii of SLP76 and LAT MCs were found to decrease, whereas ZAP70 MC radii remained relatively constant. Finally, effects of F-actin depolymerization on MC morphology were studied by determining radial distributions of cluster circularity. Our data suggest that MC morphology is affected by actin polymerization. The quantitative analysis of sub-diffraction PALM images may provide a starting point for a molecular interpretation of cluster-cluster interactions and of the regulation of T cell signaling MCs by the cytoskeleton.     

Differential modification of Ras proteins by ubiquitination

Ras proteins are essential components of signal transduction pathways that control cell proliferation, differentiation, and survival. It is well recognized that the functional versatility of Ras proteins is accomplished through their differential compartmentalization, but the mechanisms that control their spatial segregation are not fully understood. Here we show that HRas is subject to ubiquitin conjugation, whereas KRas is refractory to this modification. The membrane-anchoring domain of HRas is necessary and sufficient to direct the mono- and diubiquitination of HRas. Ubiquitin attachment to HRas stabilizes its association with endosomes and modulates its ability to activate the Raf/MAPK signaling pathway. Therefore, differential ubiquitination of Ras proteins may control their location-specific signaling activities.     

Characterization of Golgi scaffold proteins and their roles in compartmentalizing cell signaling

Subcellular compartmentalization has become an important theme in cell signaling. In particular, the Golgi apparatus (GA) plays a prominent role in compartmentalizing signaling cascades that originate at the plasma membrane or other organelles. To precisely regulate this process, cells have evolved a unique class of organizer proteins, termed “scaffold proteins”. Sef, PAQR3, PAQR10 and PAQR11 are scaffold proteins that have recently been identified on the GA and are referred to as Golgi scaffolds. The major cell growth signaling pathways, such as Ras/MAPK, PI3K/AKT, insulin and VEGF (vascular endothelial growth factor), are tightly regulated spatially and temporally by these Golgi scaffolds to ensure a physiologically appropriate outcome. Here, we discuss the subcellular localization and characterization of the topology and functional domains of these Golgi scaffolds and summarize their roles in the compartmentalization of cell signaling. We also highlight the physiological and pathological roles of these Golgi scaffolds in tumorigenesis and developmental disorders.     

Spatial mutation of the T cell immunological synapse

The hardware for intracellular signaling networks consists of cascades of chemical reactions. It is becoming increasingly apparent that the large-scale spatial organization of molecules in these networks can lead to differential outcomes from otherwise chemically equivalent systems. This has amplified interest in controlled spatial organization as a regulator of cellular signal transduction. In response, a new category of experimentation is developing, in which the spatial positions of signaling molecules in living cells are directly manipulated through mechanical means. These methodologies complement conventional genetic and pharmacological approaches, both of which are chemical in nature, by perturbing the system through exclusively physical mechanisms.     

Tyrosine phosphorylation of HuR by JAK3 triggers dissociation and degradation of HuR target mRNAs

In response to stress conditions, many mammalian mRNAs accumulate in stress granules (SGs) together with numerous RNA-binding proteins that control mRNA turnover and translation. However, the signaling cascades that modulate the presence of ribonucleoprotein (RNP) complexes in SGs are poorly understood. Here, we investigated the localization of human antigen R (HuR), an mRNA-stabilizing RNA-binding protein, in SGs following exposure to the stress agent arsenite. Unexpectedly, the mobilization of HuR to SGs was prevented through the activation of Janus kinase 3 (JAK3) by the vitamin K3 analog menadione. JAK3 phosphorylated HuR at tyrosine 200, in turn inhibiting HuR localization in SGs, reducing HuR interaction with targets SIRT1 and VHL mRNAs, and accelerating target mRNA decay. Our findings indicate that HuR is tyrosine-phosphorylated by JAK3, and link this modification to HuR subcytoplasmic localization and to the fate of HuR target mRNAs.     

17-Beta-estradiol-mediated activation of extracellular-signal regulated kinase, phosphatidylinositol 3-kinase/protein kinase B-Akt and N-methyl-D-aspartate receptor phosphorylation in cortical synaptoneurosomes

In addition to its well-known activational mechanism, the steroid hormone 17-beta-estradiol (E2) has been shown to rapidly activate various signal transduction pathways that could participate in estrogen-mediated regulation of synaptic plasticity. Although the mechanisms underlying these effects are not clearly understood, it has been repeatedly suggested that they involve a plasma membrane receptor which has direct links to several intracellular signaling cascades. To further address the question of whether E2 acts directly at the synapse and through membrane-bound receptors, we studied the effects of E2 and of ligands of estrogen receptors on various signaling pathways in cortical synaptoneurosomes. Our results demonstrate that E2 elicits N-methyl-D-aspartate receptor phosphorylation and activates the extracellular signal-regulated kinase and the phosphatidylinositol 3-kinase/Akt signal transduction pathways in this cortical membrane preparation. Furthermore, we provide evidence for the presence of a membrane-bound estrogen receptor responsible for these effects in cortical synaptoneurosomes. Our study demonstrates that E2 directly acts at cortical synapses, and that synaptoneurosomes provide a useful system to investigate the mechanisms by which E2 regulates synaptic transmission and plasticity.     

Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation.     

NADPH氧化酶与ROS信号区域化

最近有关活性氧物质 (ROS)的研究取得了突飞猛进的进展,尤其是其作为第二信使介导了许多生理性与病理性细胞事件,包括细胞分化、过度生长、增殖及凋亡.为了避免ROS的毒性产生特异性的信号转导,ROS的产生与代谢必须被严格调控;其具体的调控机制一直是人们关注的焦点. 最近有关ROS区域化观点的提出解决了这一问题. NADPH是生成ROS的主要来源. 研究发现,NADPH氧化酶及其衍生的ROS存在于机体的多种组织内,且在细胞中呈区域化分布,对细胞内信号的精确调控具有至关重要的作用. NADPH一方面通过小窝/脂筏组装成功能型复合物,从而产生ROS区域化;另一方面,NADPH通过其不同亚细胞定位亚基与各种靶蛋白之间的相互作用,产生ROS特异性. 本文系统综述了NADPH衍生的ROS信号区域化,为进一步理解ROS信号在各种生理或病理过程的分子调控机制提供理论依据.     

Ultrasensitivity in multisite phosphorylation of membrane-anchored proteins

Cellular signaling is initially confined to the plasma membrane, where the cytoplasmic tails of surface receptors and other membrane-anchored proteins are phosphorylated in response to ligand binding. These proteins often contain multiple phosphorylation sites that are regulated by membrane-confined enzymes. Phosphorylation of these proteins is thought to be tightly regulated, because they initiate and regulate signaling cascades leading to cellular activation, yet how their phosphorylation is regulated is poorly understood. Ultrasensitive or switchlike responses in their phosphorylation state are not expected because the modifying enzymes are in excess. Here, we describe a novel mechanism of ultrasensitivity exhibited by multisite membrane-anchored proteins, but not cytosolic proteins, even when enzymes are in excess. The mechanism underlying this concentration-independent ultrasensitivity is the local saturation of a single enzyme by multiple sites on the substrate. Local saturation is a passive process arising from slow membrane diffusion, steric hindrances, and multiple sites, and therefore may be widely applicable. Critical to this ultrasensitivity is the brief enzymatic inactivation that follows substrate modification. Computations are presented using ordinary differential equations and stochastic spatial simulations. We propose a new role, to our knowledge, for multisite membrane-anchored proteins, discuss experiments that can be used to probe the model, and relate our findings to previous theoretical work.