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Investigating cell migration in 3D settings has revealed that specific extracellular matrix environments require differential activities of the Rho GTPases for efficient migration. However, it is largely unknown how the activities of specific Rho GTPases are modulated to direct cell migration in response to different extracellular matrix cues. We have recently reported that extracellular matrix-dependent regulation of a specific Rho GEF is a fundamental mechanism governing cell migration in different microenvironments, providing a direct mechanism for extracellular matrix-specific regulation of Rho GTPase activity directing cell motility. We discovered that the Rho GEF βPix has a unique function during cell migration in fibrillar collagen environments by restraining RhoA signaling through a conserved signaling axis involving Cdc42 and the Rho GAP srGAP1. In this Commentary, we expand upon this new pathway and discuss potential mechanotransductive and therapeutic applications. Additionally, we speculate on a generalized role for Rho GEFs and GAPs in providing localized, context-dependent responses to the cellular microenvironment during cell migration and other cellular processes. [ABSTRACT FROM PUBLISHER]

Rho GTPases are involved in the acquisition of all the hallmarks of cancer, which comprise 6 biological capabilities acquired during the development of human tumors. The hallmarks include proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis programs, as defined by Hanahan and Weinberg.1 Controlling these hallmarks are genome instability and inflammation. Emerging hallmarks are reprogramming of energy metabolism and evading immune destruction. To give a different view to the readers, we will not be focusing on invasion, metastasis, or cytoskeletal remodeling, but we will review here how Rho GTPases contribute to other hallmarks of cancer with a special emphasis on malignant transformation. [ABSTRACT FROM AUTHOR]

DBS/MCF2Lhas been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration. [ABSTRACT FROM PUBLISHER]

Cell polarity, the asymmetric distribution of proteins and lipids, is essential for a variety of cellular functions. One mechanism orchestrating cell polarity is polarized vesicle trafficking; whereby cargo loaded secretory vesicles are specifically transported to predetermined areas of the cell. The evolutionarily conserved exocyst complex and its small GTPase regulators play crucial roles in spatiotemporal control of polarized vesicle trafficking. In studies on neuronal membrane remodeling and synaptic plasticity, conserved mechanisms of exocyst regulation and cargo recycling during polarized vesicle trafficking are beginning to emerge as well. Recently, our lab demonstrated that RhoGTPase-binding proteins in both yeast (Bem3) and mammals (Ocrl1) are also required for the efficient traffic of secretory vesicles to sites of polarized growth and signaling. Together with our studies, we highlight the evolutionary conservation of the basic elements essential for polarized vesicle traffic across different cellular functions and model systems. In conclusion, we emphasize that studies on RhoGTPase-binding proteins in these processes should be included in the next level of investigation, for a more complete understanding of their hitherto unknown roles in polarized membrane traffic and exocyst regulation. [ABSTRACT FROM PUBLISHER]

Rho-GTPases belong to the Ras superfamily and are crucial signal transducing proteins downstream of many receptors. In general, the Rho-GTPases function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states. The activated GTP bound Rho-GTPases interact with a broad spectrum of effectors to regulate a plethora of biological pathways including cytoskeletal dynamics, motility, cytokinesis, cell growth, apoptosis, transcriptional activity and nuclear signaling. Recently, gene targeting in mice allowed the selective inactivation of different Rho-GTPases and has advanced our understanding of the physiological role of these proteins, particularly in the immune system. Particularly, these proteins are key signaling molecules in T lymphocytes, which are generated in the thymus and are major players in the immune system. The scope of this review is to discuss recent data obtained in Rho-GTPases deficient mice by focusing on the role-played by Rho-GTPases in T-lymphocyte development, migration, activation and differentiation. [ABSTRACT FROM PUBLISHER]

The establishment and maintenance of epithelial cell-cell junctions is crucially important to regulate adhesion, apico-basal polarity and motility of epithelial cells, and ultimately controls the architecture and physiology of epithelial organs. Junctions are supported, shaped and regulated by cytoskeletal filaments, whose dynamic organization and contractility are finely tuned by GTPases of the Rho family, primarily RhoA, Rac1 and Cdc42. Recent research has identified new molecular mechanisms underlying the cross-talk between these GTPases and epithelial junctions. Here we briefly summarize the current knowledge about the organization, molecular evolution and cytoskeletal anchoring of cell-cell junctions, and we comment on the most recent advances in the characterization of the interactions between Rho GTPases and junctional proteins, and their consequences with regards to junction assembly and regulation of cell behavior in vertebrate model systems. The concept of “zonular signalosome” is proposed, which highlights the close functional relationship between proteins of zonular junctions (zonulae occludentesandadhaerentes) and the control of cytoskeletal organization and signaling through Rho GTPases, transcription factors, and their effectors. [ABSTRACT FROM PUBLISHER]

The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture. [ABSTRACT FROM PUBLISHER]

Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease. [ABSTRACT FROM PUBLISHER]

Cell adhesion and migration are regulated through the concerted action of cytoskeletal dynamics and adhesion proteins, the activity of which is governed by RhoGTPases. Specific RhoGTPase signaling requires spatio-temporal activation and coordination of subsequent protein-protein and protein-lipid interactions. The nature, location and duration of these interactions are dependent on polarized extracellular triggers, such as cell-cell contact, and intracellular modifying events, such as phosphorylation. RhoA, RhoB, and RhoC are highly homologous GTPases that, however, succeed in generating specific intracellular responses. Here, we discuss the key features that contribute to this specificity. These not only include the well-studied switch regions, the conformation of which is nucleotide-dependent, but also additional regions and seemingly small differences in primary sequence that also contribute to specific interactions. These differences translate into differential surface charge distribution, local exposure of amino acid side-chains and isoform-specific post-translational modifications. The available evidence supports the notion that multiple regions in RhoA/B/C cooperate to provide specificity in binding to regulators and effectors. These specific interactions are highly regulated in time and space. We therefore subsequently discuss current approaches means to visualize and analyze localized GTPase activation using biosensors that allow imaging of isoform-specific, localized regulation. [ABSTRACT FROM PUBLISHER]

Many bacterial pathogens manipulate the host cell cytoskeleton during infection. Such cytoskeletal modulation can occur at several points of contact between the pathogen and the host, and involves extracellular receptors, intracellular signal transduction and cytoskeletal proteins themselves. The field of bacterial pathogenesis has progressed dramatically over the past decade, such that structural knowledge is both timely and essential for a full appreciation of the biology at the pathogen–host interface. Several recent examples involving bacterial proteins that target actin, Rho family GTPases and extracellular receptors have contributed to a structural understanding of eukaryotic cytoskeletal modulation by pathogens. [Copyright &y& Elsevier]

Living cells rival computers in their ability to process external information and make complex behavioral decisions. Many of these decisions are made by networks of interacting signaling proteins. Ongoing structural, biochemical and cell-based studies have begun to reveal several common principles by which protein components are used to specifically transmit and process information. Recent engineering studies demonstrate that these relatively simple principles can be used to rewire signaling behavior in a process that mimics the evolution of new phenotypic responses. [Copyright &y& Elsevier]

The general organization and function of the endomembrane system is highly conserved in eukaryotic cells. In addition, increasing numbers of studies demonstrate that normal plant growth and development are dependent on specialized tissue and subcellular-specific components of the plant membrane trafficking machinery. New approaches, including chemical genomics and proteomics, will likely accelerate our understanding of the diverse functions of the plant endomembrane system. [Copyright &y& Elsevier]

Plant small GTPases belonging to the Rop, Arf, and Rab families are regulators of vesicle trafficking. Rop GTPases regulate actin dynamics and modulate H2O2 production in polar cell growth and pathogen defence. A candidate Rop GDP to Rop GTP exchange factor (RopGEF) SPIKE1 is involved in the morphogenesis of leaf epidermal cells. The ArfGEF GNOM regulates the endosomal recycling of the PIN proteins, which are involved in polar auxin transport. Intracellular localisation of small GTPases and functional studies using dominant mutant versions of Arf and Rab GTPases are defining novel plant-specific membrane compartments, especially those that participate in endosomal vesicle trafficking. [Copyright &y& Elsevier]

The ROP family of small GTPases has emerged as a versatile and pivotal regulator in plant signal transduction. Recent studies have implicated ROP signaling in diverse processes ranging from cytokseletal organization to hormone and stress responses. Acting as a switch early in signaling cascades, ROPs are also capable of orchestrating several downstream pathways to amplify a specific signal. [Copyright &y& Elsevier]

Ephrins are cell-surface-tethered ligands for Eph receptors, the largest family of receptor tyrosine kinases. During development, the Eph/ephrin cell communication system appears to influence cell behavior such as attraction/repulsion, adhesion/de-adhesion and migration, thereby influencing cell fate, morphogenesis and organogenesis. During adulthood, the Eph/ephrin system continues to play roles in tissue plasticity, for example in shaping dendritic spines during neuronal plasticity. Mechanistically, Eph–ephrin repulsive behavior appears to require ligand–receptor internalization and signaling to Rho GTPases. [Copyright &y& Elsevier]

Immunoglobulin-like nectins contribute to the formation of a variety of cell–cell junctions, acting cooperatively with, or independently of, cadherins. In addition, nectins heterophilically trans-interact with nectin-like molecules (Necls), which are involved in cell adhesion, migration, and proliferation, and assist or modify their functions. On the other hand, nectins and Necls serve as viral receptors and are associated with human diseases (including cancer) when mutated or upregulated. [Copyright &y& Elsevier]

The small GTPase Rab9 is an essential regulator of vesicular transport from the late endosome to the trans-Golgi network, as monitored by the redirection of the mannose-6-phosphate receptors. The crystal structure of Rab9 complexed to GDP, Mg2+, and Sr2+ reveals a unique dimer formed by an intermolecular β-sheet that buries the switch I regions. Surface area and shape complementarity calculations suggest that Rab9 dimers can form an inactive, membrane-bound pool of Rab9 · GDP that is independent of GDI. Mg2+-bound Rab9 represents an inactive state, but Sr2+-bound Rab9 · GDP displays activated switch region conformations, mimicking those of the GTP state. A hydrophobic tetrad is formed resembling an effector-discriminating epitope found only in GTP-bound Rab proteins. [Copyright &y& Elsevier]

Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes. Bacterial Obg is essential for cellular growth, sporulation, and differentiation. Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A˚ resolution, in the nucleotide-free state. It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain. The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively. These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis. On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold). A comparison of the T. thermophilus and B. subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain. Notably, the N-terminal domain is rotated drastically, by almost 180°, around the G domain axis. In the T. thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule. These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state. [Copyright &y& Elsevier]

Specific protein–protein interactions play crucial roles in the regulation of any biological process. Recently, a new protein–protein interaction domain termed PB1 (Phox and Bem1) was identified, which is conserved throughout evolution and present in diverse proteins functioning in signal transduction, cell polarity and survival. Here, we investigated the structure and molecular interactions of the PB1 heterodimer complex composed of the PB1 domains of the yeast proteins Bem1 and Cdc24. A structural model of the Cdc24 PB1 was built by homology modeling and molecular dynamics simulations, and experimentally validated by 15N nuclear Overhauser effect spectroscopy (NOESY)–heteronuclear single quantum coherence (HSQC) analysis. Residues at the interface of the complex for both proteins were identified by NMR titration experiments. A model of the heterodimer was obtained by docking of the two PB1 domains with HADDOCK, which applies ambiguous interaction restraints on residues at the interface to drive the docking procedure. The refined model was validated by site-directed mutagenesis on both Bem1 and Cdc24. Finally, the docking was repeated from the newly published NMR structure of Cdc24, allowing us to assess the performance of homology-based docking. Our results provide insight into the molecular structure of the Bem1–Cdc24 PB1-mediated heterodimer, which allowed identification of critical residues at the binding interface. [Copyright &y& Elsevier]

Insulin-like growth factor-I (IGF-I) is an important anabolic growth factor in the maintenance of articular cartilage phenotypic expression. Chondrocyte morphology is also tightly linked to phenotype. The small G-protein Cdc42 plays a key role in regulation of cell morphology and phenotypic expression in several cell types and, we show here, in articular chondrocytes. The purpose of these studies was to investigate possible links between the intracellular signaling pathways of IGF-I and Cdc42 in articular chondrocytes. Treatment of chondrocytes with IGF-I resulted in a rapid and sustained decrease in the activation state (decreased GTP-bound) of Cdc42. Nucleotide exchange and hydrolysis experiments suggest that the decreased activation occurs through increased hydrolysis. Transient expression of dominant-negative Cdc42(T17N) allowed for enhanced expression of normal chondrocyte phenotype as determined by increased mRNA expression of collagen type II (Coll II) with decreased matrix metalloproteinase-3 (MMP-3) expression. The results of these studies suggest a novel link between IGF-I and Cdc42 signaling pathways. Further, an additional mechanism for the regulation of chondrocyte phenotype is defined through the IGF-I induced down-regulation of Cdc42 activation. [Copyright &y& Elsevier]

HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF4−) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF4−, in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions. [Copyright &y& Elsevier]

Small GTPases of the Rho family including Rac, Rho and Cdc42 regulate different cellular processes like reorganization of the actin cytoskeleton by acting as molecular switches. The three distinct mammalian Rac proteins share very high sequence identity but how their specificity is achieved is hitherto unknown. Here we show that Rac1 and Rac3 are very closely related concerning their biochemical properties, such as effector interaction, nucleotide binding and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis support the idea that altered dynamics of Rac2 at the switch I region may be responsible for different biochemical properties. These results provide insight into the individual functionalities of the Rac isoforms. [Copyright &y& Elsevier]

The regulation of gene expression at the translational level not only allows for rapid changes in specific protein levels but also provides an opportunity to alter codon specificity. For the incorporation of selenocysteine (Sec) into protein, the UGA codon is transformed from one that signals translation termination to one specific for Sec. This review provides a look at Sec incorporation from the perspective of the individual steps involved in protein synthesis: initiation, elongation and termination. The roles of the factors known to be required for Sec incorporation are considered in the context of each step in translation including structural modeling of the differences between the standard elongation factor eEF1A and the Sec-specific counterpart, eEFSec. [Copyright &y& Elsevier]

The GTPases of the Rho family are molecular switches that play an important role in a wide range of cellular processes and are increasingly implicated in tumourigenesis. Unlike what was found for the Ras oncogenes in tumours, hardly any activating mutations have been found in the genes encoding Rho proteins. In the past, we have identified Tiam1 (T-lymphoma invasion and metastasis) as a specific activator for the Rho-like GTPase Rac. In vivo, Tiam1 deficiency protects against Ras-induced skin carcinogenesis, underscoring the consequences of deregulated signalling for the onset and progression of tumours. Thus, an important level of regulation of signalling via the Rho-like GTPases comes from the specific control of their activators. In this paper, we review what is known on the specific regulation of Tiam1 signalling towards Rac. [Copyright &y& Elsevier]

Ras GTPases are binary switches, cycling between an inactive GDP-bound form and an active GTP-bound form at the membrane. They transduce signals into the cytoplasm via effector pathways that regulate cell growth, differentiation and apoptosis. Ras activation is enhanced by guanine nucleotide exchange factors (GEFs); deactivation is accelerated by GTPase-activating proteins (GAPs). Recently, new roles for Ca2+ and diacylglycerol (DAG) in the control of Ras cycling have emerged with the discovery of a series of novel GEFs and GAPs. These regulators of Ras cycling are likely to play a key role in the information processing of Ca2+ and DAG signals. [Copyright &y& Elsevier]

The Ras homology (Rho) guanine nucleotide exchange factor p115-RhoGEF couples the α13 heterotrimeric guanine nucleotide binding protein (G protein) subunit to Rho GTPase. α13 binds to a regulator of G protein signaling (RGS) domain in p115-RhoGEF, but the mechanism of α13 activation of p115-RhoGEF is poorly understood. In this report, we demonstrate in cell-based assays that the acidic-rich N-terminus, adjacent to the RGS domain, is required for binding to activated α13, and refine the importance of this region by showing that mutation of glutamic acids 27 and 29 in full-length p115-RhoGEF is sufficient to prevent interaction with activated α13. However, α13-interacting deficient N-terminal mutants of p115-RhoGEF retain α13-dependent plasma membrane recruitment. Overall, these findings demonstrate a critical role for the N-terminal extension of p115-RhoGEF in mediating binding to α13 and dissociate two activities of p115-RhoGEF: binding to activated α13 and translocation to the PM in response to activated α13. [Copyright &y& Elsevier]

Pak interacting exchange factor (βPix) is a recently cloned protein that contains a multidomain with many potential binding sites and is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. These domains of βPix appear to play a critical role in the regulation of the cytoskeletal organization. The overexpression of βPix enhances the activation of p38, which is thought to be an important downstream effector of the Rho GTPase family (Rac, Cdc42), which are involved in increased membrane ruffling and cell motility. This increase of cell mobility is an important feature of cancer invasion. We examined the expression of βPix-a in human breast cancer tissues and adjacent normal tissues obtained from 39 breast cancer patients. Immunoblot analysis and RT-PCR revealed that βPix-a expression was significantly increased in 37 of the 39 breast cancer tissues (94.9%) versus normal breast tissues. Immunohistochemical analysis showed that breast cancer tissues have consistently stronger immunoreactivity to βPix-a antibodies than normal tissues. βPix-a overexpression was inversely associated with extensive intraductal component (P<0.001). In conclusion, βPix-a expression was found to be higher in human breast cancer tissues than in normal breast tissues, which implies a role for βPix-a in human breast tumorigenesis. We suggest that βPix-a may be a useful marker of malignant disease in the breast. [Copyright &y& Elsevier]

Matrix metalloproteinase-9 (MMP-9) is a collagenolytic enzyme secreted by cancer cells and involved in invasiveness and metastasis. Its secretion from human fibrosarcoma HT 1080 cells is markedly enhanced by phorbol 12-myristate 13-acetate (PMA) and abolished by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation. These results support a role for ARF in PMA-stimulated MMP-9 secretion. Overexpression of arfaptin 1, a 39 kDa ARF-binding protein that inhibits in vitro activation of cholera toxin ADP-ribosyltransferase and phospholipase D (PLD) by ARF, inhibited PMA-stimulated MMP-9 and PLD activation. These data are in agreement with previous results demonstrating a significant role for PLD in regulating MMP-9 secretion. [Copyright &y& Elsevier]

Internalisation of the human pathogen Yersinia pseudotuberculosis via interaction of bacterial invasin with host β1 integrins depends on the actin cytoskeleton and involves Src family kinases, focal adhesion kinase, p130Crk-associated substrate, proline-rich tyrosine kinase 2, Rac, Arp 2/3 complex and WASP family members. We show here that Rho GTPases are regulated by the microtubule system during bacterial uptake. Interfering with microtubule organisation using nocodazole or paclitaxel suppressed uptake by HeLa cells. The nocodazole effect on microtubule depolymerisation was partially inhibited through overexpression of Rac, Cdc42, RhoG or RhoA and completely prevented by expression of Vav2. This suggests that microtubules influence Rho GTPases during invasin-mediated phagocytosis and in the absence of functional microtubules Vav2 can mimic their effect on one, or more, of the Rho family GTPases. Lastly, overexpression of p50 dynamitin partially inhibited bacterial uptake and this effect was also blocked by co-expression of Vav2, thus further implicating this guanine nucleotide exchange factor in activating Rho GTPases for internalisation during loss of microtubule function. [Copyright &y& Elsevier]

Plexins are receptors for the repulsive axon guidance molecules semaphorins. Previously, we have shown that plexin-B1 binds activated Rac, but that clustering of plexin-B1 causes Rho activation, resulting in stress fiber formation. Using the yeast two-hybrid system, we found that the C-terminus of B plexins interacted directly with Rho-specific exchange factors, via their PDZ domain. Mutation of the carboxy-terminal amino acids of plexin-B1 or coexpression of a dominant negative PDZ-RhoGEF abrogated the ability of plexin-B1 to cause stress fiber formation. Our results demonstrate a role for PDZ-RhoGEF in B plexin-mediated activation of Rho/Rho kinase signaling, implicated in the regulation of axon guidance and cell migration. [Copyright &y& Elsevier]

The hair cells (HCs) are the most vulnerable elements in the cochlea and damage to them is the most common cause of sensorineural hearing loss. Understanding the intracellular events that lead to the death of HCs is a key to developing protective strategies. Recently, it has been shown that the c-Jun-N-terminal kinase (JNK) pathway is activated in HCs in response to aminoglycosides (J. Neurosci. 20 (2000) 43). We have studied the upstream events leading to JNK activation in aminoglycoside toxicity in vitro. The small GTPases Rac and Cdc42 are well known upstream activators of JNK in other cell types. Clostridium difficile toxin B monoglucosylates all members of the Rho GTPase subfamily (Rho, Rac and Cdc42 isoforms) and inhibits GTP binding by steric interference (Nature 341 (1989) 209). Organ of Corti explants from p5 rat basal turns were maintained in tissue culture and treated with C. difficile toxin B for 12 h. They were then treated with toxin B plus gentamicin for 72 h. Significantly less HC death was observed compared to with gentamicin alone. Toxin B alone had no effect on HCs at the highest concentration used. Using antibodies against phospho-c-Jun, we observed background immunoreactivity in control explants, strong staining of outer hair cell nuclei in gentamicin treated explants, and weaker immunostaining in explants treated with gentamicin and C. difficile toxin B. We conclude that Rho family small GTPases play a role in aminoglycoside toxicity signaling as upstream activators of the JNK signaling pathway. [Copyright &y& Elsevier]

The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p-related sequences. We demonstrate that the Yip1p-related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C-terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family. [Copyright &y& Elsevier]

The functionality and efficacy of Rho GTPase signaling is pivotal for a plethora of biological processes. Due to the integral nature of these molecules, the dysregulation of their activities can result in diverse aberrant phenotypes. Dysregulation can, as will be described below, be based on an altered signaling strength on the level of a specific regulator or that of the respective GTPase itself. Alternatively, effector pathways emanating from a specific Rho GTPase may be under- or overactivated. In this review, we address the role of the Rho-type GTPases as a subfamily of the Ras-superfamily of small GTP-binding proteins in the development of various disease phenotypes. The steadily growing list of genetic alterations that specifically impinge on proper Rho GTPase function corresponds to pathological categories such as cancer progression, mental disabilities and a group of quite diverse and unrelated disorders. We will provide an overview of disease-rendering mutations in genes that have been positively correlated with Rho GTPase signaling and will discuss the cellular and molecular mechanisms that may be affected by them. [Copyright &y& Elsevier]

The role of Vav in the transcriptional regulation of the human interferon-γ (IFN-γ) promoter was investigated. Overexpression of Vav in Jurkat-TAg cells enhanced T cell receptor (TCR)-induced activation of a luciferase (Luc) reporter gene construct driven by cis-regulatory element of the IFN-γ gene (−346 to +7). Electrophoresis mobility shift and Luc reporter assays demonstrated that the DNA-binding and transcriptional activity of the proximal AP-1-dependent NFAT site (positions −172 to −138), the AP-1/Ying-Yang 1 (YY1)-binding site (−209 to −184), and a consensus AP-1-binding site were upregulated by Vav. Vav enhanced TCR-induced activation of c-Jun N-terminal kinase (JNK) and its upstream regulator, Rho family GTPases. Finally, coexpression of a dominant-negative Rac1 mutant suppressed Vav-mediated upregulation of the transcriptional and DNA-binding activity of the proximal NFAT/AP-1 site and the AP-1/YY1 site, as well as the complete IFN-γ promoter activity. Vav activates the IFN-γ promoter via upregulation of AP-1-binding through a Rac1/JNK pathway. [Copyright &y& Elsevier]

Multidomain guanine nucleotide (GDP/GTP) exchange factor (GEF) proteins coordinate diverse inputs that signal the actin cytoskeleton. Mammals have two such proteins (Kalirin, Trio), while Drosophila has one, which plays essential roles within and outside the nervous system. For Kalirin, numerous isoforms containing different combinations of functional domains are generated through alternative splicing and use of alternative transcriptional start sites. These different isoforms potentially allow a wide variety of proteins to interact with Kalirin, thereby affecting the activity of the functional domains. Humans, like rats, express a large set of Kalirin isoform mRNAs, and we identified a novel Kalirin isoform, containing only the second GEF domain. Kalirin isoforms are predominantly expressed in the brain, while Trio is expressed in a wider variety of tissues. Alternative splicing and transcription of Kalirin are differentially regulated during development in rats and humans, resulting in expression of isoforms of Kalirin containing different functional domains at different times and locations. The prevalence of Kalirin in the cortex throughout life suggests roles in axonal development and the mature brain. [Copyright &y& Elsevier]