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.     

Spatially distributed cell signalling

Emerging evidence indicates that complex spatial gradients and (micro)domains of signalling activities arise from distinct cellular localization of opposing enzymes, such as a kinase and phosphatase, in signal transduction cascades. Often, an interacting, active form of a target protein has a lower diffusivity than an inactive form, and this leads to spatial gradients of the protein abundance in the cytoplasm. A spatially distributed signalling cascade can create step-like activation profiles, which decay at successive distances from the cell surface, assigning digital positional information to different regions in the cell. Feedback and feedforward network motifs control activity patterns, allowing signalling networks to serve as cellular devices for spatial computations.     

Formation of Intracellular Concentration Landscapes by Multisite Protein Modification

Multiple cellular proteins are covalently modified (e.g., phosphorylated/dephosphorylated) at several sites, which leads to diverse signaling activities. Here, we consider a signaling cascade that is activated at the plasma membrane and composed of two-site protein modification cycles, and we focus on the radial profile of the concentration landscapes created by different protein forms in the cytoplasm. We show that under proper conditions, the concentrations of modified proteins generate a series of peaks that propagate into the cell interior. Proteins modified at both sites form activity gradients with long plateaus that abruptly decay at successive locations along the path from the membrane to the nucleus. We demonstrate under what conditions signals generated at the membrane stall in the vicinity of that membrane or propagate into the cell. We derive analytical approximations for the main characteristics of the protein concentration profiles and demonstrate what we believe to be a novel steady-state pattern formation mechanism capable of generating precise spatial guidance for diverse cellular processes.     

Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase

Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.     

Conformational changes in the GTPase modules of the signal reception particle and its receptor drive initiation of protein translocation

During cotranslational protein targeting, two guanosine triphosphatase (GTPase) in the signal recognition particle (SRP) and its receptor (SR) form a unique complex in which hydrolyses of both guanosine triphosphates (GTP) are activated in a shared active site. It was thought that GTP hydrolysis drives the recycling of SRP and SR, but is not crucial for protein targeting. Here, we examined the translocation efficiency of mutant GTPases that block the interaction between SRP and SR at specific stages. Surprisingly, mutants that allow SRP-SR complex assembly but block GTPase activation severely compromise protein translocation. These mutations map to the highly conserved insertion box domain loops that rearrange upon complex formation to form multiple catalytic interactions with the two GTPs. Thus, although GTP hydrolysis is not required, the molecular rearrangements that lead to GTPase activation are essential for protein targeting. Most importantly, our results show that an elaborate rearrangement within the SRP-SR GTPase complex is required to drive the unloading and initiate translocation of cargo proteins.     

Structure of the small G protein Rap2 in a non-catalytic complex with GTP

We report a novel crystal form of the small G protein Rap2A in complex with GTP which has no GTPase activity in the crystal. The asymmetric unit contains two complexes which show that a conserved switch I residue, Tyr 32, contributes an extra hydrogen bond to the gamma-phosphate of GTP as compared to related structures with GTP analogs. Since GTP is not hydrolyzed in the crystal, this interaction is unlikely to contribute to the intrinsic GTPase activity. The comparison of other G protein structures to the Rap2-GTP complex suggests that an equivalent interaction is likely to exist in their GTP form, whether unbound or bound to an effector. This interaction has to be released to allow the GAP-activated GTPase, and presumably the intrinsic GTPase activity as well. We also discuss the definition of the flexible regions and their hinges in the light of this structure and the expanding database of G protein structures. We propose that the switch I and switch II undergo either partial or complete disorder-to-order transitions according to their cellular status, thus defining a complex energy landscape comprising more than two conformational states. We observe in addition that the region connecting the switch I and switch II is flexible in Rap2 and other G proteins. This region may be important for protein-protein interactions and possibly behave as a conformational lever arm, as characterized for Arf. Taken together, these observations suggest that the structural mechanisms of small G proteins are significantly driven by entropy-based free energy changes.     

Role of SRP RNA in the GTPase cycles of Ffh and FtsY.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.     

Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins

The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.     

Structure-based prediction of DNA target sites by regulatory proteins

Regulatory proteins play a critical role in controlling complex spatial and temporal patterns of gene expression in higher organism, by recognizing multiple DNA sequences and regulating multiple target genes. Increasing amounts of structural data on the protein-DNA complex provides clues for the mechanism of target recognition by regulatory proteins. The analyses of the propensities of base-amino acid interactions observed in those structural data show that there is no one-to-one correspondence in the interaction, but clear preferences exist. On the other hand, the analysis of spatial distribution of amino acids around bases shows that even those amino acids with strong base preference such as Arg with G are distributed in a wide space around bases. Thus, amino acids with many different geometries can form a similar type of interaction with bases. The redundancy and structural flexibility in the interaction suggest that there are no simple rules in the sequence recognition, and its prediction is not straightforward. However, the spatial distributions of amino acids around bases indicate a possibility that the structural data can be used to derive empirical interaction potentials between amino acids and bases. Such information extracted from structural databases has been successfully used to predict amino acid sequences that fold into particular protein structures. We surmised that the structures of protein-DNA complexes could be used to predict DNA target sites for regulatory proteins, because determining DNA sequences that bind to a particular protein structure should be similar to finding amino acid sequences that fold into a particular structure. Here we demonstrate that the structural data can be used to predict DNA target sequences for regulatory proteins. Pairwise potentials that determine the interaction between bases and amino acids were empirically derived from the structural data. These potentials were then used to examine the compatibility between DNA sequences and the protein-DNA complex structure in a combinatorial "threading" procedure. We applied this strategy to the structures of protein-DNA complexes to predict DNA binding sites recognized by regulatory proteins. To test the applicability of this method in target-site prediction, we examined the effects of cognate and noncognate binding, cooperative binding, and DNA deformation on the binding specificity, and predicted binding sites in real promoters and compared with experimental data. These results show that target binding sites for several regulatory proteins are successfully predicted, and our data suggest that this method can serve as a powerful tool for predicting multiple target sites and target genes for regulatory proteins.     

Different import pathways through the mitochondrial intermembrane space for inner membrane proteins.

Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.     

A multiscale whole-cell theory for mechanosensitive migration on viscoelastic substrates

Increasing experimental evidence validates that both the elastic stiffness and viscosity of the extracellular matrix regulate mesenchymal cell behavior, such as the rational switch between durotaxis (cell migration to stiffer regions), anti-durotaxis (migration to softer regions), and adurotaxis (stiffness-insensitive migration). To reveal the mechanisms underlying the crossover between these motility regimes, we have developed a multiscale chemomechanical whole-cell theory for mesenchymal migration. Our framework couples the subcellular focal adhesion dynamics at the cell-substrate interface with the cellular cytoskeletal mechanics and the chemical signaling pathways involving Rho GTPase proteins. Upon polarization by the Rho GTPase gradients, our simulated cell migrates by concerted peripheral protrusions and contractions, a hallmark of the mesenchymal mode. The resulting cell dynamics quantitatively reproduces the experimental migration speed as a function of the uniform substrate stiffness and explains the influence of viscosity on the migration efficiency. In the presence of stiffness gradients and absence of chemical polarization, our simulated cell can exhibit durotaxis, anti-durotaxis, and adurotaxis respectively with increasing substrate stiffness or viscosity. The cell moves toward an optimally stiff region from softer regions during durotaxis and from stiffer regions during anti-durotaxis. We show that cell polarization through steep Rho GTPase gradients can reverse the migration direction dictated by the mechanical cues. Overall, our theory demonstrates that opposing durotactic behaviors emerge via the interplay between intracellular signaling and cell-medium mechanical interactions in agreement with experiments, thereby elucidating complex mechanosensing at the single-cell level.     

Sec14 homology domain targets p50RhoGAP to endosomes and provides a link between Rab and Rho GTPases

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.     

Mechanisms of NOS2 regulation by Rho GTPase signaling in airway epithelial cells

The aberrant dysregulation of the inducible form of nitric oxide synthase (NOS2) is thought to play a role in many inflammatory disorders including cystic fibrosis (CF). The complex regulation of NOS2 expression is the subject of intense investigation, and one intriguing regulatory pathway known to influence NOS2 expression is the Rho GTPase cascade. We examined NOS2 regulation in response to inflammatory cytokines in a human alveolar epithelial cell line treated with inhibitors of different upstream and downstream components of the Rho GTPase pathway to better define potential signaling mechanisms. Statin-mediated 3-hydroxy-3-methylglutaryl-CoA reductase inhibition increased cytokine-dependent activation of the NOS2 promoter, reversible by the addition of geranylgeranyl pyrphosphate. However, inhibition of Rho-associated kinase (ROCK) with Y-27632 resulted in a decrease in NOS2 promoter activity, yet an increase in NOS2 mRNA and protein levels. Our results suggest that prenylation events influence NOS2 promoter activity independently of the Rho GTPase pathway and that Rho GTPase signaling mediated through ROCK suppresses NOS2 production downstream of promoter function at the message and protein level.     

Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase.

Termination of translation in eukaryotes is governed by two polypeptide chain release factors, eRF1 and eRF3 on the ribosome. eRF1 promotes stop-codon-dependent hydrolysis of peptidyl-tRNA, and eRF3 interacts with eRF1 and stimulates eRF1 activity in the presence of GTP. Here, we have demonstrated that eRF3 is a GTP-binding protein endowed with a negligible, if any, intrinsic GTPase activity that is profoundly stimulated by the joint action of eRF1 and the ribosome. Separately, neither eRF1 nor the ribosome display this effect. Thus, eRF3 functions as a GTPase in the quaternary complex with ribosome, eRF1, and GTP. From the in vitro uncoupling of the peptidyl-tRNA and GTP hydrolyses achieved in this work, we conclude that in ribosomes both hydrolytic reactions are mediated by the formation of the ternary eRF1-eRF3-GTP complex. eRF1 and the ribosome form a composite GTPase-activating protein (GAP) as described for other G proteins. A dual role for the revealed GTPase complex is proposed: in " GTP state," it controls the positioning of eRF1 toward stop codon and peptidyl-tRNA, whereas in "GDP state," it promotes release of eRFs from the ribosome. The initiation, elongation, and termination steps of protein synthesis seem to be similar with respect to GTPase cycles.     

Predictive Spatiotemporal Manipulation of Signaling Perturbations Using Optogenetics

Recently developed optogenetic methods promise to revolutionize cell biology by allowing signaling perturbations to be controlled in space and time with light. However, a quantitative analysis of the relationship between a custom-defined illumination pattern and the resulting signaling perturbation is lacking. Here, we characterize the biophysical processes governing the localized recruitment of the Cryptochrome CRY2 to its membrane-anchored CIBN partner. We develop a quantitative framework and present simple procedures that enable predictive manipulation of protein distributions on the plasma membrane with a spatial resolution of 5 μm. We show that protein gradients of desired levels can be established in a few tens of seconds and then steadily maintained. These protein gradients can be entirely relocalized in a few minutes. We apply our approach to the control of the Cdc42 Rho GTPase activity. By inducing strong localized signaling perturbation, we are able to monitor the initiation of cell polarity and migration with a remarkable reproducibility despite cell-to-cell variability.     

How regulators of G protein signaling achieve selective regulation

The regulators of G protein signaling (RGS) are a family of cellular proteins that play an essential regulatory role in G protein-mediated signal transduction. There are multiple RGS subfamilies consisting of over 20 different RGS proteins. They are basically the guanosine triphosphatase (GTPase)-accelerating proteins that specifically interact with G protein alpha subunits. RGS proteins display remarkable selectivity and specificity in their regulation of receptors, ion channels, and other G protein-mediated physiological events. The molecular and cellular mechanisms underlying such selectivity are complex and cooperate at many different levels. Recent research data have provided strong evidence that the spatiotemporal-specific expression of RGS proteins and their target components, as well as the specific protein-protein recognition and interaction through their characteristic structural domains and functional motifs, are determinants for RGS selectivity and specificity. Other molecular mechanisms, such as alternative splicing and scaffold proteins, also significantly contribute to RGS selectivity. To pursue a thorough understanding of the mechanisms of RGS selective regulation will be of great significance for the advancement of our knowledge of molecular and cellular signal transduction.     

MAP kinase cascade signaling and endocytic trafficking: a marriage of convenience?

A hallmark of many signaling pathways is the spatial separation of activation and deactivation of signaling proteins. Quantitative analysis demonstrates that the spatial separation of a membrane-bound kinase and a cytosolic phosphatase potentially results in precipitous gradients of target phosphoproteins. Hypothetically, such gradients in the mitogen-activated protein kinase (MAPK) cascade would result in a strong attenuation of the phosphorylation signal towards the nucleus. When effective signal transduction is hampered by slow protein diffusion and rapid dephosphorylation, phosphoprotein trafficking within endocytic vesicles might be an efficient way to propagate the signals. Additional mechanisms facilitating information transfer could involve the assembly of MAP kinases on a scaffolding protein and active transport of signaling complexes by molecular motors. The proposed mechanism explains recent observations that MAPK activation can be strongly suppressed by various inhibitors of endocytosis.     

Geographic variation in the relationship between large-scale environmental determinants and bat species richness

Several studies have already shown the close relationship between geographic gradients of biodiversity and distinct environmental determinants such as energy, environmental heterogeneity and seasonality. Nevertheless, whether and how such relationships vary around the globe remains poorly understood. Here we used spatial models to answer whether the bat species richness-environment relationship on a global scale are constant across geographic space. We also partitioned the contribution of the different environmental determinants on bat species richness at different regions of the globe. We found that the relationship between bat species richness and environment is not constant across geographic space and that the shared contributions of environmental determinants are more important than their unique contributions. We conclude that understanding geographic gradients of biodiversity and its environmental determinants, particularly for bats, is more complex than previously thought because the relationship between species richness and environment varies considerably across geographic space.     

GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling

Pleiotropic G proteins are essential for the action of hormones and neurotransmitters and are activated by stimulation of G protein-coupled receptors (GPCR), which initiates heterotrimer dissociation of the G protein, exchange of GDP for GTP on its Galpha subunit and activation of effector proteins. Regulator of G protein signaling (RGS) proteins regulate this cascade and can be recruited to the membrane upon GPCR activation. Direct functional interaction between RGS and GPCR has been hypothesized. We show that recruitment of GAIP (RGS19) by the dopamine D2 receptor (D2R), a GPCR, required the scaffold protein GIPC (GAIP-interacting protein, C terminus) and that all three were coexpressed in neurons and neuroendocrine cells. Dynamic translocation of GAIP to the plasma membrane and coassembly in a protein complex in which GIPC was a required component was dictated by D2R activation and physical interactions. In addition, two different D2R-mediated responses were regulated by the GTPase activity of GAIP at the level of the G protein coupling in a GIPC-dependent manner. Since GIPC exclusively interacted with GAIP and selectively with subsets of GPCR, this mechanism may serve to sort GPCR signaling in cells that usually express a large repertoire of GPCRs, G proteins, and RGS.     

Characterization of a GTPase-activating protein for the Ras-related Ral protein

We have demonstrated the presence of a GTPase-activating protein (GAP) for the Ras-related Ral A protein in the cytosolic fraction of brain and testis. This protein, designated Ral-GAP, was distinguished from Ras-GAP by its behavior in two chromatography systems and by the fact that the two GAP proteins did not stimulate the GTPase activity of each others target GTP binding proteins. The lack of effect of Ral-GAP on Ras GTPase activity also distinguished it from the product of the neurofibromatosis gene NF-1. Ral-GAP also differed from Rho-GAP and Rap-GAP by virtue of its elution from a gel filtration column with proteins of Mr greater than 10(6). This was likely an overestimate of the protein's molecular mass, however, since it sedimented in sucrose gradients between standard proteins of 150 and 443 kDa. Ral-GAP failed to promote the GTPase activity of mutant Ral proteins containing amino acid substitutions that in Ras lead to GAP-insensitive proteins.