Actin’ up: Herpesvirus Interactions with Rho GTPase Signaling

Herpesviruses constitute a very large and diverse family of DNA viruses, which can generally be subdivided in alpha-, beta- and gammaherpesvirus subfamilies. Increasing evidence indicates that many herpesviruses interact with cytoskeleton-regulating Rho GTPase signaling pathways during different phases of their replication cycle. Because of the large differences between herpesvirus subfamilies, the molecular mechanisms and specific consequences of individual herpesvirus interactions with Rho GTPase signaling may differ. However, some evolutionary distinct but similar general effects on Rho GTPase signaling and the cytoskeleton have also been reported. Examples of these include Rho GTPase-mediated nuclear translocation of virus during entry in a host cell and Rho GTPase-mediated viral cell-to-cell spread during later stages of infection. The current review gives an overview of both general and individual interactions of herpesviruses with Rho GTPase signaling

Genetic Interactions Between The Guanine Nucleotide Exchange Factor Gefmeso And Gtpase Signaling Components In The Drosophila Wing Reveal Microenvironment Dependent Variation Within Gtpase Signaling N

The Ras superfamily of GTPases are important regulators of morphogenesis involved in control of cytoskeletal dynamics, intracellular trafficking, apical-basal polarity and cell migration. Mis-regulation of GTPase signaling interferes with development and is linked to pathogenesis. Traditionally, GTPase signaling has been depicted as a series of independent linear pathways. However, recently it has become apparent that multiple GTPases can interact to regulate a single cellular process, functioning in poorly understood networks of cross talk between pathways during development. Jim Fristrom (unpublished data) identified a mutation (18-5) that interacts with components of the GTPases Rho1, Rala, and Cdc42 signaling in multiple developmental contexts. Genetic analysis, physical mapping studies, and sequencing of the mutant allele have indicated that the gene was an allele of GEFmeso (CG30115), which encodes guanine nucleotide exchange factor. To show that 18-5 is an allele of GEFmeso, I demonstrated that a GEFmeso transgene could functionally rescue developmental defects associated with the 18-5 mutation. I also investigated cross talk and network variation in signaling interactions between GEFmeso and other GTPase signaling components in the Drosophila wing. My data provide evidence for microenvironment-dependent variation in GTPase signaling networks in specific domains of the wing, and reveal intercellular variation in GTPase signaling within an otherwise uniform epithelium

Dual positive and negative regulation of GPCR signaling by GTP hydrolysis

G protein-coupled receptors (GPCRs) regulate a variety of intracellular pathways through their ability to promote the binding of GTP to heterotrimeric G proteins. Regulator of G protein signaling (RGS) proteins increase the intrinsic GTPase activity of G-subunits and are widely regarded as negative regulators of G protein signaling. Using yeast we demonstrate that GTP hydrolysis is not only required for desensitization, but is essential for achieving a high maximal (saturated level) response. Thus RGS-mediated GTP hydrolysis acts as both a negative (low stimulation) and positive (high stimulation) regulator of signaling. To account for this we generated a new kinetic model of the G protein cycle where GGTP enters an inactive GTP-bound state following effector activation. Furthermore, in vivo and in silico experimentation demonstrates that maximum signaling output first increases and then decreases with RGS concentration. This unimodal, non-monotone dependence on RGS concentration is novel. Analysis of the kinetic model has revealed a dynamic network motif that shows precisely how inclusion of the inactive GTP-bound state for the G produces this unimodal relationship

Characterization of ROP GTPase-activated Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI_A)

Plants have to respond and adapt to a variety of continuously changing environmental factors in order to establish an appropriate developmental strategy to ensure survival. There are ample data showing that protein phosphorylation/dephosphorylation plays a central role in cellular signal transduction in all organisms (Herrmann et al. 2006; Stone and Walker 1995). Interestingly, plants have a similar system as mammals to detect and transfer signals across the cell membrane into the nucleus where adaptations could be initiated. For the detection and transfer of an external signal, mammalian systems have receptor protein kinases. The proteins contain a single hydrophobic transmembrane domain, an extracellular domain and protein kinase domain. The majority of animal receptor kinases are phosphorylated on tyrosine residues within the kinase domain (receptor tyrosin kinases or RTKs; Ullrich and Schlessinger 1990), but a few were discovered which are phosphorylated on serine and threonine residues (Lin et al. 1992). In plants, two different types of transmembrane receptor kinases are known, including receptor-like serine/threonine (Ser/Thr) kinases (receptor-like kinases RLKs; Shiu and Bleecker 2001, 2003; Shiu et al. 2004; Walker 1994), structurally similar to mammalian RTKs, and receptor histidine (His) kinases (Grefen and Harter 2004; Mizuno 2005; Urao et al. 2000). Since the first RLK-encoding gene family was found in Zea mays (Walker and Zhang 1990), thousands of RLK genes have been identified from many different plant species. The Arabidopsis genome contains more than 600 members, representing nearly 2.5% of the annotated protein-coding genes; and more than 1000 members were annotated in the rice genome (Shiu et al. 2004). Approximately 25% of the Arabidopsis RLKs contain only a kinase domain with no apparent signal sequence or transmembrane region and thus were collectively named as receptor like cytoplasmic kinases (RLCKs). Arabidopsis RLCKs can be subdivided into 12 classes with 193 protein coding genes all together. Concerning the function of plant RLCKs, at the present only few members have been characterized and it is very likely that they play major role in the perception and 93 transmission of external signals perceived by RLKs (Zhou et al. 1995; Murase et al. 2004). Recently, our group as well as a group in Germany reported a direct interaction of plant ROP GTPases with receptor-like cytoplasmic kinases (RLCK class VI) from Arabidopsis (Molendijk et al. 2008) and alfalfa (Dorjgotov et al. 2009). Moreover, we provide evidences that kinases belonging to the RLCK Class VI family of Medicago truncatula and Arabidopsis thaliana can be specifically activated by GTP-bound ROP GTPases in vitro further supporting the view that plant Rho (ROP) G-proteins may directly regulate downstream kinase signaling. A further kinase designated as cysteine-rich receptor kinase (NCRK) belonging to a distinct kinase family has also been shown to interact with ROPs (Molendijk et al. 2008). None of these plant specific ROP-interacting kinases has any characteristic domain or motif that could be correlated with their ability to bind ROP GTPases. Plant specific ROP GTPases are versatile molecular switches in many processes during plant growth, development and responses to the environment and thus a possible implication of RLCKs in these ROP-dependent signal transduction pathways is in discussion. Our general aim was to characterize the members of the Arabidopsis thaliana RLCK Class VI family of protein kinases. Despite of their potential significance in ROP GTPase mediated signaling, hardly any functional information was available until now about the fourteen Arabidopsis RLCK Class VI members. Sequence comparison and phylogenetic analysis revealed that gene duplication played a significant role in the formation of this kinase family and allowed the separation of the 14 RCLK VI kinases into two groups with seven members each (A1 to A7 and B1 to B7). The proteins are highly homologous to each other, especially in the kinase domain, but are divergent from the related kinase families. It was established that, several members have an N-terminal UspA (“universal stress protein”) domain (group B members) or an N-terminal serine-rich region (group A members). In order to formulate a possible biological role of AtRLCK_VI kinases, real-time quantitative reverse transcription-polymerase reaction (qRT-PCR) was used to determine relative transcript levels in the various organs (root, rosette leaves, cauline leaves, 94 inflorescence stem, flower buds, open flowers, siliques. exponentially dividing cultured cells) of the Arabidopsis plant as well as under a series of abiotic stress/hormone (osmotic, sugar, salt stress, oxidative stress, cold and hormone treatment) treatments in seedlings. AtRLCK VI genes exhibited diverse expression patterns in the various plant organs as well as in response to stress/hormone treatments..

Reciprocal regulation of PKA and rac signaling

Activated G protein-coupled receptors (GPCRs) and receptor tyrosine kinases relay extracellular signals through spatial and temporal controlled kinase and GTPase entities. These enzymes are coordinated by multifunctional scaffolding proteins for precise intracellular signal processing. The cAMP-dependent protein kinase A (PKA) is the prime example for compartmentalized signal transmission downstream of distinct GPCRs. A-kinase anchoring proteins tether PKA to specific intracellular sites to ensure precision and directionality of PKA phosphorylation events. Here, we show that the Rho-GTPase Rac contains A-kinase anchoring protein properties and forms a dynamic cellular protein complex with PKA. The formation of this transient core complex depends on binary interactions with PKA subunits, cAMP levels and cellular GTP-loading accounting for bidirectional consequences on PKA and Rac downstream signaling. We show that GTP-Rac stabilizes the inactive PKA holoenzyme. However, β-adrenergic receptor-mediated activation of GTP-Rac–bound PKA routes signals to the Raf-Mek-Erk cascade, which is critically implicated in cell proliferation. We describe a further mechanism of how cAMP enhances nuclear Erk1/2 signaling: It emanates from transphosphorylation of p21-activated kinases in their evolutionary conserved kinase-activation loop through GTP-Rac compartmentalized PKA activities. Sole transphosphorylation of p21-activated kinases is not sufficient to activate Erk1/2. It requires complex formation of both kinases with GTP-Rac1 to unleash cAMP-PKA–boosted activation of Raf-Mek-Erk. Consequently GTP-Rac functions as a dual kinase-tuning scaffold that favors the PKA holoenzyme and contributes to potentiate Erk1/2 signaling. Our findings offer additional mechanistic insights how β-adrenergic receptor-controlled PKA activities enhance GTP-Rac–mediated activation of nuclear Erk1/2 signaling

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity

Systems Analysis Implicates WAVE2 Complex in the Pathogenesis of Developmental Left-Sided Obstructive Heart Defects.

Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho) physiological conditions of stress

A RAC/CDC-42–Independent GIT/PIX/PAK Signaling Pathway Mediates Cell Migration in C. elegans

P21 activated kinase (PAK), PAK interacting exchange factor (PIX), and G protein coupled receptor kinase interactor (GIT) compose a highly conserved signaling module controlling cell migrations, immune system signaling, and the formation of the mammalian nervous system. Traditionally, this signaling module is thought to facilitate the function of RAC and CDC-42 GTPases by allowing for the recruitment of a GTPase effector (PAK), a GTPase activator (PIX), and a scaffolding protein (GIT) as a regulated signaling unit to specific subcellular locations. Instead, we report here that this signaling module functions independently of RAC/CDC-42 GTPases in vivo to control the cell shape and migration of the distal tip cells (DTCs) during morphogenesis of the Caenorhabditis elegans gonad. In addition, this RAC/CDC-42–independent PAK pathway functions in parallel to a classical GTPase/PAK pathway to control the guidance aspect of DTC migration. Among the C. elegans PAKs, only PAK-1 functions in the GIT/PIX/PAK pathway independently of RAC/CDC42 GTPases, while both PAK-1 and MAX-2 are redundantly utilized in the GTPase/PAK pathway. Both RAC/CDC42–dependent and –independent PAK pathways function with the integrin receptors, suggesting that signaling through integrins can control the morphology, movement, and guidance of DTC through discrete pathways. Collectively, our results define a new signaling capacity for the GIT/PIX/PAK module that is likely to be conserved in vertebrates and demonstrate that PAK family members, which are redundantly utilized as GTPase effectors, can act non-redundantly in pathways independent of these GTPases