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G Protein Signaling Modulator-3 (GPSM3) is a leukocyte-specific regulator of G protein-coupled receptors (GPCRs), which binds inactivated Gαi·GDP subunits and precludes their reassociation with Gβγ subunits. GPSM3 deficiency protects mice from inflammatory arthritis and, in humans, GPSM3 single-nucleotide polymorphisms (SNPs) are inversely associated with the risk of rheumatoid arthritis development; recently, these polymorphisms were linked to one particular SNP (rs204989) that decreases GPSM3 transcript abundance. However, the precise role of GPSM3 in leukocyte biology is unknown. Here, we show that GPSM3 is induced in the human promyelocytic leukemia NB4 cell line following retinoic acid treatment, which differentiates this cell line into a model of neutrophil physiology (NB4*). Reducing GPSM3 expression in NB4* cells, akin to the effect ascribed to the rs204989 C>T transition, disrupts cellular migration toward leukotriene B4 (LTB4) and (to a lesser extent) interleukin-8 (a.k.a. IL-8 or CXCL8), but not migration toward formylated peptides (fMLP). As the chemoattractants LTB4 and CXCL8 are involved in recruitment of neutrophils to the arthritic joint, our results suggest that the arthritis-protective GPSM3 SNP rs204989 may act to decrease neutrophil chemoattractant responsiveness., Competing Interests: The authors declare no conflict of interest.

Respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung infections have critical consequences on mortality and morbidity in humans. The aims of the present study were to examine the mechanisms by which CXCL12 affects MUC1 transcription and airway inflammation, which depend on activator of G-protein signaling (AGS) 3 and to identify specific molecules that suppress CXCL12-induced airway inflammation by acting on G-protein-coupled receptors. Herein, AGS3 suppresses CXCL12-mediated upregulation of MUC1 and TNFα by regulating Gαi. We found that the G-protein regulatory (GPR) motif peptide in AGS3 binds to Gαi and downregulates MUC1 expression; in contrast, this motif upregulates TNFα expression. Mutated GPR Q34A peptide increased the expression of MUC1 and TGFβ but decreased the expression of TNFα and IL-6. Moreover, CXCR4-induced dendritic extensions in 2D and 3D matrix cultures were inhibited by the GPR Q34A peptide compared with a wild-type GPR peptide. The GPR Q34A peptide also inhibited CXCL12-induced morphological changes and inflammatory cell infiltration in the mouse lung, and production of inflammatory cytokines in bronchoalveolar lavage (BAL) fluid and the lungs. Our data indicate that the GPR motif of AGS3 is critical for regulating MUC1/Muc1 expression and cytokine production in the inflammatory microenvironment.

Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. Here, we show that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. We show that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins(-) mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. Our data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells.

Dental pulp stem cells (DPSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulating differentiation of DPSCs may be a useful tool for regenerative medicine and cell-based therapy in oral diseases. Multisignaling pathways are involved in osteogenic differentiation of DPSCs. Recent studies show that cAMP/PKA/CREB signaling could stimulate the expression of genes such as bone morphogenic proteins 2 (BMP2), inhibitor of DNA binding 2 (ID2), bone sialoprotein, osteocalcin, and type XXIV collagen, which have been implicated in osteogenesis and bone formation. Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, plays an important role in regulating the phosphorylation of cyclic AMP response element-binding protein (p-CREB). However, the involvement of AGS3 in osteogenic differentiation of DPSCs has not been explored. Our data indicated that increased expression of AGS3 would inhibit osteogenic differentiation of DPSCs exposed to inflammatory cytokine tumor necrosis factor α (TNF-α) via cAMP/PKA/CREB signaling. The negative role of AGS3 in osteogenic differentiation was further confirmed by knocking down and over expression of AGS3. Our findings may provide clinical implications for osteoporosis., (Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.

Rab proteins constitute the largest family within the Ras superfamily of small GTPases (>60 in mammals) and are essential regulators of transport between intracellular organelles. Key to this activity is their targeting to specific compartments within the cell. However, although great strides have been made over the last 25 years in assigning functions to individual Rabs and identifying their downstream effectors, the mechanism(s) regulating their targeting to specific subcellular membranes remains less well understood. In the present paper, we review the evidence supporting the proposed mechanisms of Rab targeting and highlight insights into this process provided by studies of Rab27a.

Despite over two decades of research, the mechanism of Rab targeting to specific intracellular membranes is still not completely understood. Present evidence suggests that the original hypothesis that the message for targeting resides solely in the hypervariable C-terminus is incorrect, and a second mechanism involving a GDF [GDI (guanine-nucleotide-dissociation inhibitor) displacement factor] to disrupt stable Rab-GDI complexes has only been shown to apply in one case, despite the need for targeting over 60 human Rab proteins. Evidence for the involvement of Rab-effector interactions has only been presented for a few cases or in a very specific context. There is mounting evidence that GEFs (guanine-nucleotide-exchange factors) are essential for membrane targeting, although contributions from additional factors are likely to be of importance, at least in specific cases.

Rab GTPases are master regulators of membrane traffic. By binding to distinct sets of effector proteins, Rabs catalyse the formation of function-specifying membrane microdomains. They are delivered to membranes by a protein named GDI (guanine-nucleotide-dissociation inhibitor) and are stabilized there after nucleotide exchange by effector binding. In the present mini-review, I discuss what we know about how Rab GTPases are delivered to the correct membrane-bound compartments and how Rab GTPase cascades order Rabs within the secretory and endocytic pathways. Finally, I describe how Rab cascades may establish the distinct compartments of the Golgi complex to permit ordered processing, sorting and secretion of secretory cargoes.

Half of patients with muscle-invasive bladder cancer develop metastatic disease, and this is responsible for most of the deaths from this cancer. Low expression of RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI) is associated with metastatic disease in patients with muscle-invasive bladder cancer. Moreover, a reduction in metastasis is observed upon reexpression of RhoGDI2 in xenograft models of metastatic cancer. Here, we show that RhoGDI2 suppresses lung metastasis in mouse models by reducing the expression of isoforms V1 and V3 of the proteoglycan versican (VCAN; also known as chondroitin sulfate proteoglycan 2 [CSPG2]). In addition, we found that high versican levels portended poor prognosis in patients with bladder cancer. The functional importance of tumor expression of versican in promoting metastasis was established in in vitro and in vivo studies in mice that implicated a role for the chemokine CCL2 (also known as MCP1) and macrophages. Further analysis indicated that RhoGDI2 suppressed metastasis by altering inflammation in the tumor microenvironment. In summary, we demonstrate what we believe to be a new mechanism of metastasis suppression that works by reducing host responses that promote metastatic colonization of the lung. Therapeutic targeting of these interactions may provide a novel adjuvant strategy for delaying the appearance of clinical metastasis in patients.

Since metastasis is one of the most important prognostic factors in colorectal cancer, development of new methods to diagnose and prevent metastasis is highly desirable. However, the molecular mechanisms leading to the metastatic phenotype have not been well elucidated. In this study, a proteomics-based search was carried out for metastasis-related proteins in colorectal cancer by analyzing the differential expression of proteins in primary versus metastasis focus-derived colorectal tumor cells. Protein expression profiles were determined using a tissue microarray (TMA), and the results identified Rho GDP-dissociation inhibitor alpha (Rho GDI) as a metastasis-related protein in colon and prostate cancer patients. Consequently, Rho GDI may be useful as a diagnostic biomarker and/or a therapeutic to prevent colon and prostate cancer metastasis.

Date hub proteins have 1 or 2 interaction interfaces but many interaction partners. This raises the question of whether all partner proteins compete for the interaction interface of the hub or if the cell carefully regulates aspects of this process? Here, we have used real-time rendering of protein interaction networks to analyse the interactions of all the 1 or 2 interface hubs of Saccharomyces cerevisiae during the cell cycle. By integrating previously determined structural and gene expression data, and visually hiding the nodes (proteins) and their edges (interactions) during their troughs of expression, we predict when interactions of hubs and their partners are likely to exist. This revealed that 20 out of all 36 one- or two- interface hubs in the yeast interactome fell within two main groups. The first was dynamic hubs with static partners, which can be considered as 'competitive hubs'. Their interaction partners will compete for the interaction interface of the hub and the success of any interaction will be dictated by the kinetics of interaction (abundance and affinity) and subcellular localisation. The second was static hubs with dynamic partners, which we term 'non-competitive hubs'. Regulatory mechanisms are finely tuned to lessen the presence and/or effects of competition between the interaction partners of the hub. It is possible that these regulatory processes may also be used by the cell for the regulation of other, non-cell cycle processes.

Cellular signaling pathways exhibit complex response profiles with features such as thresholds and steep activation (i.e., ultrasensitivity). In a reconstituted mitotic spindle orientation pathway, activation of Drosophila Pins (LGN in mammals) by Gαi is ultrasensitive (apparent Hill coefficient of 3.1), such that Pins recruitment of the microtubule binding protein Mud (NuMA) occurs over a very narrow Gαi concentration range. Ultrasensitivity is required for Pins function in neuroblasts as a nonultrasensitive Pins mutant fails to robustly couple spindle position to cell polarity. Pins contains three Gαi binding GoLoco domains (GLs); Gαi binding to GL3 activates Pins, whereas GLs 1 and 2 shape the response profile. Although cooperative binding is one mechanism for generating ultrasensitivity, we find GLs 1 and 2 act as "decoys" that compete against activation at GL3. Many signaling proteins contain multiple protein interaction domains, and the decoy mechanism may be a common method for generating ultrasensitivity in regulatory pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Proteins containing the G protein regulator (GPR) domain bind the major neural G protein Gα(o) in vitro. However, the biological functions of GPR proteins in neurons remain undefined, and based on the in vitro activities of GPR proteins it is unclear whether these proteins activate or inhibit G protein signaling in vivo. We found that the conserved GPR domain protein AGS-3 activates Gα(o) signaling in vivo to allow Caenorhabditis elegans to alter several behaviors after food deprivation, apparently so that the animals can more effectively seek food. AGS-3 undergoes a progressive change in its biochemical fractionation upon food deprivation, suggesting that effects of food deprivation are mediated by modifying this protein. We analyzed one C. elegans food-regulated behavior in depth; AGS-3 activates Gα(o) in the ASH chemosensory neurons to allow food-deprived animals to delay response to the aversive stimulus octanol. Genetic epistasis experiments show the following: (1) AGS-3 and the guanine nucleotide exchange factor RIC-8 act in ASH in a mutually dependent fashion to activate Gα(o); (2) this activation requires interaction of the GPR domains of AGS-3 with Gα(o); and (3) Gα(o)-GTP is ultimately the signaling molecule that acts in ASH to delay octanol response. These results identify a biological role for AGS-3 in response to food deprivation and indicate the mechanism for its activation of Gα(o) signaling in vivo.

Ly-GDI, Rho GTPase dissociation inhibitor beta, was found to be expressed parallel to the GM3 level in mouse B16 cells whose GM3 contents were modified by B4galt6 sense, B4galt6 antisense cDNA, or St3galt5 siRNA transfection. Ly-GDI expression was increased on GM3 addition to these cells and decreased with D-PDMP treatment, a glucosylceramide synthesis inhibitor. Suppression of GM3 or Ly-GDI by RNAi was concomitantly associated with an increase in anchorage-independent growth in soft agar. These results clearly indicate that GM3 suppresses anchorage-independent growth through Ly-GDI. GM3 signals regulating Ly-GDI expression was inhibited by LY294002, siRNA against Akt1 and Akt2 and rapamycin, showing that GM3 signals are transduced via the PI3K/Akt/mTOR pathway. Either siRNA towards Rictor or Raptor suppressed Ly-GDI expression. The Raptor siRNA suppressed the effects of GM3 on Ly-GDI expression and Akt phosphorylation at Thr(308) , suggesting GM3 signals to be transduced to mTOR-Raptor and Akt-Thr(308) , leading to Ly-GDI stimulation. siRNA targeting Pdpk1 reduced Akt phosphorylation at Thr(308) and rendered the cells insensitive to GM3 stimulation, indicating that Akt-Thr(308) plays a critical role in the pathway. The components aligned in this pathway showed similar effects on anchorage-independent growth as GM3 and Ly-GDI. Taken together, GM3 signals are transduced in B16 cells through PI3K, Pdpk1, Akt(Thr308) and the mTOR/Raptor pathway, leading to enhanced expression of Ly-GDI mRNA, which in turn suppresses anchorage-independent growth in melanoma B16 cells., (© 2011 Japanese Cancer Association.)

RhoGDIs (Rho GDP-dissociation inhibitors) are the natural inhibitors of Rho GTPases. They interfere with Rho protein function by either blocking upstream activation or association with downstream signalling molecules. RhoGDIs can also extract membrane-bound Rho GTPases to form soluble cytosolic complexes. We have shown previously that purified yeast RhoGDI Rdi1p, can inhibit vacuole membrane fusion in vitro. In the present paper we functionally dissect Rdi1p to discover its mode of regulating membrane fusion. Overexpression of Rdi1p in vivo profoundly affected cell morphology including increased actin patches in mother cells indicative of polarity defects, delayed ALP (alkaline phosphatase) sorting and the presence of highly fragmented vacuoles indicative of membrane fusion defects. These defects were not caused by the loss of typical transport and fusion proteins, but rather were linked to the reduction of membrane localization and activation of Cdc42p and Rho1p. Subcellular fractionation showed that Rdi1p is predominantly a cytosolic monomer, free of bound Rho GTPases. Overexpression of endogenous Rdi1p, or the addition of exogenous Rdi1p, generated stable cytosolic complexes. Rdi1p structure-function analysis showed that membrane association via the C-terminal β-sheet domain was required for the functional inhibition of membrane fusion. Furthermore, Rdi1p inhibited membrane fusion through the binding of Rho GTPases independent from its extraction activity.

Ovarian cancer is a gynecological malignancy with the highest mortality. Chemoresistance is an important subject for the treatment of ovarian cancer, because obtaining significant drug resistance to the first line chemotherapy, paclitaxel, causes major therapeutic obstacles. It is essential to improve the survival rate of ovarian cancer patients by mining the biomarkers indicating the drug resistance and prognosis, and by further understanding underlying mechanisms of drug resistance. In the present study, we established paclitaxel-resistant subline (SKpac) from human epithelial ovarian cancer cell line, SKOV3, and performed comparative analysis of whole proteomes between paclitaxel-resistant SKpac sublines and paclitaxel-sensitive parental SKOV3 cells to identify differentially expressed proteins and useful biomarkers indicating chemoresistance. Proteins related to chemoresistant process were identified by two-dimensional gel electrophoresis (2DE) with mass spectrometry (MALDI-TOF and LC-MS/MS). Eighteen spots were differentially expressed and were identified in SKpac chemoresistant cells compared to SKOV3. The expressions of ALDH 1A1, annexin A1, hnRNP A2, and GDI 2 proteins were validated by Western blot, which was consistent with proteomic analysis. Among the selected proteins, downregulation of hnRNP A2 and GDI 2 was found to be the most significant finding in SKpac cells and chemoresistant ovarian cancer tissues. Our results suggest that hnRNP A2 and GDI 2 may represent potential biomarkers of the paclitaxel-resistant ovarian cancers for tailored cancer therapy.

Background/aims: The acquisition of resistance to 5-FU is one of the most prominent obstacles to successful chemotherapy, and the mechanisms underlying the resistance are not fully understood. The aim of this study is to identify novel mediators of 5-FU resistance in colon cancer cells., Methodology: LoVo colon cancer cells were induced to 5-FU resistance in vitro. The global protein profiles between LoVo and its 5-FU resistant derivative cell line LoVo/5-FU were analyzed by two dimensional gel electrophoresis-based comparative proteomics. The identified proteins expression was confirmed by Western blot analysis. The cytotoxicity of 5-FU was measured in LoVo/5-FU after knockdown of RhoGDI2 (one of the identified protien)., Results: Three differentially expressed proteins were identified. RhoGDI2 and CapG were upregulated, whereas proapoptotic protein Maspin was down-regulated in LoVo/5-FU and validated by Western blotting. Furthermore, knockdown of RhoGDI2 expression by transfection with the RhoGDI2-specific siRNA significantly reduced the resistance to 5-FU in LoVo/5-FU (p < 0.05)., Conclusions: These novel data suggest that these differentially expressed proteins may contribute to the development of 5-FU resistance in colon cancer cells.

Rho GDP dissociation inhibitors (RhoGDIs) are important regulators of the GTP hydrolase activity and biological functions of Rho GTPases. RhoGDI2 has been shown to be a metastasis suppressor in bladder cancer and several other cancers. However, the underlying mechanism, effector targets, and the cognate biological functions of RhoGDI2 are not fully understood. To investigate the possible role of RhoGDI2 in lung cancer tumorigenesis and metastasis, the expression pattern of RhoGDI2 in various lung cancer tissue samples and lung cancer-derived cell lines were profiled at both mRNA and protein levels. Furthermore, possible interplay between PI3K/Akt/mTOR pathway activation/inhibition and RhoGDI2 signalling is examined in lung cancer-related cell lines. Our results suggest that RhoGDI2 is likely to be involved in lung tumor malignancy and metastasis.

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil-containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras-induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.

At steady state, most Rho GTPases are bound in the cytosol to Rho guanine nucleotide dissociation inhibitors (RhoGDIs). RhoGDIs have generally been considered to hold Rho proteins passively in an inactive state within the cytoplasm. Here we describe an evolutionarily conserved mechanism by which RhoGDI1 controls the homeostasis of Rho proteins in eukaryotic cells. We found that depletion of RhoGDI1 promotes misfolding and degradation of the cytosolic geranylgeranylated pool of Rho GTPases while activating the remaining membrane-bound fraction. Because RhoGDI1 levels are limiting, and Rho proteins compete for binding to RhoGDI1, overexpression of an exogenous Rho GTPase displaces endogenous Rho proteins bound to RhoGDI1, inducing their degradation and inactivation. These results raise important questions about the conclusions drawn from studies that manipulate Rho protein levels. In many cases the response observed may arise not simply from the overexpression itself but from additional effects on the levels and activity of other Rho GTPases as a result of competition for binding to RhoGDI1; this may require a re-evaluation of previously published studies that rely exclusively on these techniques.

The prion protein (PrP) is a glycosylphosphatidylinositol-anchored membrane glycoprotein that plays a vital role in prion diseases, a class of fatal neurodegenerative disorders of humans and animals. Approximately 20% of human prion diseases display autosomal dominant inheritance and are linked to mutations in the PrP gene on chromosome 20. PrP mutations are thought to favor the conformational conversion of PrP into a misfolded isoform that causes disease by an unknown mechanism. The PrP mutation D178N/Met-129 is linked to fatal familial insomnia, which causes severe sleep abnormalities and autonomic dysfunction. We showed by immunoelectron microscopy that this mutant PrP accumulates abnormally in the endoplasmic reticulum and Golgi of transfected neuroblastoma N2a cells. To investigate the impact of intracellular PrP accumulation on cellular homeostasis, we did a two-dimensional gel-based differential proteomics analysis. We used wide range immobilized pH gradient strips, pH 4-7 and 6-11, to analyze a large number of proteins. We found changes in proteins involved in energy metabolism, redox regulation, and vesicular transport. Rab GDP dissociation inhibitor alpha (GDI) was one of the proteins that changed most. GDI regulates vesicular protein trafficking by acting on the activity of several Rab proteins. We found a specific reduction in the level of functional Rab11 in mutant PrP-expressing cells associated with impaired post-Golgi trafficking. Our data are consistent with a model by which mutant PrP induces overexpression of GDI, activating a cytotoxic feedback loop that leads to protein accumulation in the secretory pathway.

Cdc42 cycling through GTP/GDP states is critical for its function in the second/granule mobilization phase of insulin granule exocytosis in pancreatic islet beta cells, although the identities of the Cdc42 cycling proteins involved remain incomplete. Using a tandem affinity purification-based mass spectrometry screen for Cdc42 cycling factors in beta cells, RhoGDI was identified. RNA interference-mediated depletion of RhoGDI from isolated islets selectively amplified the second phase of insulin release, consistent with the role of RhoGDI as a Cdc42 cycling factor. Replenishment of RhoGDI to RNA interference-depleted cells normalized secretion, confirming the action of RhoGDI to be that of a negative regulator of Cdc42 activation. Given that RhoGDI also regulates Rac1 activation in beta cells, and that Rac1 activation occurs in a Cdc42-dependent manner, the question as to how the beta cell utilized RhoGDI for differential Cdc42 and Rac1 cycling was explored. Co-immunoprecipitation was used to determine that RhoGDI-Cdc42 complexes dissociated upon stimulation of beta cells with glucose for 3 min, correlating with the timing of glucose-induced Cdc42 activation and the onset of RhoGDI tyrosine phosphorylation. Glucose-induced disruption of RhoGDI-Rac1 complexes occurred subsequent to this, coincident with Rac1 activation, which followed the onset of RhoGDI serine phosphorylation. RhoGDI-Cdc42 complex dissociation was blocked by mutation of RhoGDI residue Tyr-156, whereas RhoGDI-Rac1 dissociation was blocked by RhoGDI mutations Y156F and S101A/S174A. Finally, expression of a triple Y156F/S101A/S174A-RhoGDI mutant specifically inhibited only the second/granule mobilization phase of glucose-stimulated insulin secretion, overall supporting the integration of RhoGDI into the activation cycling mechanism of glucose-responsive small GTPases.

Importance of the Field: Rho GDP dissociation inhibitor 2 (RhoGDI2) has been identified as a regulator of Rho GTPases that play important roles in the development of numerous aspects of the malignant phenotype, including cell cycle progression, resistance to apoptotic stimuli, neovascularization, tumor cell motility, invasiveness, and metastasis. Although RhoGDI2 has been known to be expressed only in hematopoietic tissues, recent studies suggest that this protein is also aberrantly expressed in several human cancers and contributes to aggressive phenotypes, such as invasion and metastasis. Hence, RhoGDI2 appears to be a target of interest for therapeutic manipulation., Areas Covered in This Review: Here, we summarize the role of RhoGDI2 in human cancers, specifically metastasis-related processes, and discuss its potential as a therapeutic target., What the Reader Will Gain: RhoGDI2 modulates the invasiveness and metastatic ability of cancer cells through regulation of Rac1 activity., Take Home Message: RhoGDI2 may be a useful marker for tumor progression in human cancers, and interruption of the RhoGDI2-mediated cancer cell invasion and metastasis by an interfacial inhibitor may be a powerful therapeutic approach to cancer.

Despite the recent advances in the diagnosis of bladder cancer, recurrence after surgical intervention for muscle invasive disease is still problematic as nearly half of the patients harbor occult distant metastases and this, in turn, is associated with poor 5-year survival rate. We have recently identified Rho family GDP dissociation inhibitor 2 (RhoGDI2) protein as functional metastasis suppressor and a prognostic marker in patients after cystectomy. In identifying the mechanisms underlying metastasis suppression by RhoGDI2, we found this protein to be associated with the c-Src kinase in human tumors, where the expression of both is diminished as a function of stage. Interestingly, c-Src bound to and phosphorylated RhoGDI2 resulting in enhanced metastasis suppressive potency. In this review, we will discuss the established roles of c-Src and RhoGDI2 in bladder cancer and speculate on their therapeutic relevance.

Insulin stimulates GLUT4 (glucose transporter 4) translocation in adipocytes and muscles. An emerging picture is that Rab10 could bridge the gap between the insulin signalling cascade and GLUT4 translocation in adipocytes. In the present study, two potential effectors of Rab10, GDI (guanine-nucleotide-dissociation inhibitor)-1 and GDI-2, are characterized in respect to their roles in insulin-stimulated GLUT4 translocation. It is shown that both GDI-1 and GDI-2 exhibit similar distribution to GLUT4 and Rab10 at the TGN (trans-Golgi network) and periphery structures. Meanwhile, GDI-1 clearly interacts with Rab10 with higher affinity, as shown by both immunoprecipitation and in vivo FRET (fluorescence resonance energy transfer). In addition, the participation of GDIs in GLUT4 translocation is illustrated when overexpression of either GDI inhibits insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Taken together, we propose that GDI-1 is preferentially involved in insulin-stimulated GLUT4 translocation through facilitating Rab10 recycling.

Farnesol, a precursor in the isoprenoid/sterol pathway, was recently identified as a quorum-sensing molecule produced by the fungal pathogen Candida albicans. Farnesol is involved in the inhibition of germination and biofilm formation by C. albicans and can be cytotoxic at certain concentrations. In addition, we have shown that farnesol can trigger apoptosis in mammalian cells via the classical apoptotic pathways. In order to elucidate the mechanism behind farnesol cytotoxicity in C. albicans, the response to farnesol was investigated, using proteomic analysis. Global protein expression profiles demonstrated significant changes in protein expression resulting from farnesol exposure. Among the downregulated proteins were those involved in metabolism, glycolysis, protein synthesis, and mitochondrial electron transport and the respiratory chain, whereas proteins involved in folding, protection against environmental and oxidative stress, actin cytoskeleton reorganization, and apoptosis were upregulated. Cellular changes that accompany apoptosis (regulated cell death) were further analyzed using fluorescent microscopy and gene expression analysis. The results indicated reactive oxygen species accumulation, mitochondrial degradation, and positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) in the farnesol-exposed cells concurrent with increased expression of antioxidant-encoding and drug response genes. More importantly, the results demonstrated farnesol-induced upregulation of the caspase gene MCA1 and the intracellular presence of activated caspases. In conclusion, this study demonstrated that farnesol promotes apoptosis in C. albicans through caspase activation, implying an important physiological role for farnesol in the fungal cell life cycle with important implications for adaptation and survival.

Rho-GDP dissociation inhibitors (Rho-GDI) are repressors of Rho-type monomeric GTPases that control fundamental cellular processes, such as cytoskeletal arrangement, vesicle trafficking, and polarized growth. We identified and altered the expression of the gene encoding a Rho-GDI homolog in the human fungal pathogen Cryptococcus neoformans and investigated its impact on pathogenicity in animal models of cryptococcosis. Consistent with its predicted function to inhibit and sequester Rho-type GTPases, overexpression of RDI1 results in cytosolic localization of Cdc42. Likely as a result of this finding, RDI1-overexpressing strains exhibited altered morphology compared to that of the wild type, with apparent defects in maintaining proper cell polarity and cytokinesis. RDI1 deletion resulted in increased vacuole size in tissue culture medium and aberrant cell morphology at neutral pH. Maintenance of normal cell morphology is vital for C. neoformans pathogenicity. Accordingly, the rdi1Delta mutant strain also showed reduced intracellular survival in macrophages and severe attenuation of virulence in two murine models of cryptococcosis. This reduction in virulence of the rdi1Delta mutant occurs in the absence of major growth defects in rich medium and with classical virulence-associated phenotypes.

The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3beta homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.

Invasion and metastasis are the critical steps in cancer progression that lead to death from this disease. Intense investigation into the underlying mechanisms of metastasis has revealed a complex set of signaling pathways that regulate the process. Since the mid-1980s, it has been demonstrated that the Rho family of proteins plays a major role in these pathways. Proteins that regulate Rho, including guanine nucleotide exchange factors, GTPase-activating proteins, and Rho GDP dissociation inhibitors (RhoGDIs), have also been shown to contribute to cancer progression. Among this group of Rho-regulating proteins is RhoGDI2 (RhoGDIbeta/LyGDI/GDID4/RabGDIbeta). Our laboratory initially identified RhoGDI2 as a metastasis suppressor due to its differential expression between metastatically capable and poorly metastatic bladder cancer cell lines. Over the subsequent years, in vivo and in vitro systems have been used to model steps in the metastatic cascade and to test how the expression of RhoGDI2 affected those processes. This chapter describes several of the more significant methods used to investigate the role of RhoGDI2 in bladder cancer invasion and metastasis. These methods include an in vitro assay for invasion using bladder organ cultures, lung metastasis assays in immunocompromised murine hosts, polymerase chain reaction-based quantification of metastatic burden, and derivation of increasingly metastatic cell lines.

Objective: Rho GTPase may be involved in human cancer invasion via the augmentation of cell motility and adhesion. We report on two point mutations of the D4-guanine diphosphate (GDP)-dissociation inhibitor (GDI) gene, one of the Rho-GDIs, which were found in a human leukemic cell line, Reh, and the mutated D4-GDI functions as an accelerator of leukemic cell invasion., Material and Methods: We investigated the altered activity of GDP dissociation by mutated (mt) D4-GDI and the functions of this mt and wild-type (wt) D4-GDI in invasion. The mice inoculated with wt or mt D4-GDI vector-transfected Raji cells were observed and examined pathologically. Adhesiveness and cell motility of wt or mt D4-GDI vector-transfected Raji cells were examined. Finally, it was examined whether Rho activation was changed by mutation of D4-GDI under the condition of Rho-GDI knockdown., Results: Two point mutations of the D4-GDI gene were found in Reh cells. The region of mutations is conserved among members of the Rho-GDI family at the amino acid level. D4-GDI with two mutations (V68L and V69A) functioned in a dominant negative manner in the inhibition of GDP dissociation from Rho. Severe combined immune-deficient mice inoculated with Raji cells developed hemiparalysis. The Raji cells were present in bone marrow and peripheral blood, and hepatic invasion was observed in 20% of the mice. Mice inoculated with wt D4-GDI vector-transfected Raji cells (wt D4) showed later paralysis and none developed hepatic invasion. Mice inoculated with mt D4-GDI-transfected Raji cells (mt D4) showed a 5-day reduction in the time to paraplegia and death. In addition, hepatic invasion was evident in 80% of mice transplanted with mt D4 cells. There were no differences in growth rates and amounts of guanine triphosphate (GTP)-bound Rho, cdc42, or Rac among all clones, however, GTP-bound Rho in mt D4 clone with short hairpin RNA (shRNA) vector for Rho-GDI knockdown was increased compared with wt D4 clone with shRNA vector for Rho-GDI knockdown. The mt D4 cells showed an augmentation of adhesiveness and cell motility. On the other hand, wt D4 cells showed a decreased ability of cell motility., Conclusion: These results suggest the mutated D4-GDI functions as a dominant negative molecule against the wt D4-GDI and accelerates invasion via regulation of cytoskeletal machinery.

Lipid droplets play a critical role in a variety of metabolic diseases. Numerous proteomic studies have provided detailed information about the protein composition of the droplet, which has revealed that they are functional organelles involved in many cellular processes, including lipid storage and metabolism, membrane traffic, and signal transduction. Thus, the droplet proteome indicates that lipid accumulation is only one of a constellation of organellar functions critical for normal lipid metabolism in the cell. As a result of this new understanding, we suggested the name adiposome for this organelle. The trafficking ability of the adiposome is likely to be very important for lipid uptake, retention, and distribution, as well as membrane biogenesis and lipid signaling. We have taken advantage of the ease of purifying lipid-filled adiposomes to develop a cell-free system for studying adiposome-mediated traffic. Using this approach, we have determined that the interaction between adiposomes and endosomes is dependent on Rab GTPases but is blocked by ATPase. These methods also allowed us to identify multiple proteins that dynamically associate with adiposomes in a nucleotide-dependent manner. An adiposome-endosome interaction in vitro occurs in the absence of cytosolic factors, which simplifies the assay dramatically. This assay will enable researchers to dissect the molecular mechanisms of interaction between these two organelles. This chapter provides a detailed account of the methods developed.

Estrogen receptor alpha (ER alpha) is a ligand-activated transcription factor that regulates expression of estrogen-responsive genes. Upon binding of the ligand-occupied ER alpha to estrogen response elements (EREs) in DNA, the receptor interacts with a variety of coregulatory proteins to modulate transcription of target genes. We have isolated and identified a number of proteins associated with the DNA-bound ER alpha. One of these proteins, Rho guanosine diphosphate (GDP) dissociation inhibitor alpha (RhoGDI alpha), is a negative regulator of the Rho family of GTP-binding proteins. In this study, we demonstrate that endogenously expressed RhoGDI alpha is present in the nucleus as well as the cytoplasm of MCF-7 breast cancer cells, and that RhoGDI alpha binds directly to ER alpha, alters the ER alpha-ERE interaction, and influences the ability of ER alpha to regulate transcription of a heterologous estrogen-responsive reporter plasmid in transient transfection assays as well as endogenous, estrogen-responsive genes in MCF-7 cells. Our studies suggest that, in addition to the activity of RhoGDI alpha in the cytoplasm, it also influences ER alpha signaling in the nucleus.

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1(-/-) mice. We observed that basal endothelial permeability in lungs of RhoGDI-1(-/-) mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1(-/-) lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1-mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

Although the molecular function of sigma receptors has not been fully defined and the natural ligand(s) is still not known, there is increasing evidence that these receptors and their ligands might play a significant role in cancer biology. 4-(N-benzylpiperidin-4-yl)-4-iodobenzamide (4-IBP), a selective sigma1 agonist, has been used to investigate whether this compound is able to modify: 1) in vitro the migration and proliferation of human cancer cells; 2) in vitro the sensitivity of human glioblastoma cells to cytotoxic drugs; and 3) in vivo in orthotopic glioblastoma and non-small cell lung carcinoma (NSCLC) models the survival of mice co-administered cytotoxic agents. 4-IBP has revealed weak antiproliferative effects on human U373-MG glioblastoma and C32 melanoma cells but induced marked concentration-dependent decreases in the growth of human A549 NSCLC and PC3 prostate cancer cells. The compound was also significantly antimigratory in all four cancer cell lines. This may result, at least in U373-MG cells, from modifications to the actin cytoskeleton. 4-IBP modified the sensitivity of U373-MG cells in vitro to proapoptotic lomustin and proautophagic temozolomide, and markedly decreased the expression of two proteins involved in drug resistance: glucosylceramide synthase and Rho guanine nucleotide dissociation inhibitor. In vivo, 4-IBP increased the antitumor effects of temozolomide and irinotecan in immunodeficient mice that were orthotopically grafted with invasive cancer cells.

Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudotuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.

Rho family small GTP-binding proteins, including Rho, Rac, and Cdc42, are key determinants of cell movement and actin-dependent cytoskeletal morphogenesis. Rho GDP-dissociation inhibitor (GDI) alpha and Rho GDIbeta (or D4/Ly-GDI), closely related regulators for Rho proteins, are both expressed in hemopoietic cell lineages. Nevertheless, the functional contributions of Rho GDIs remain poorly understood in vivo. In this study, we report that combined disruption of both the Rho GDIalpha and Rho GDIbeta genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers. Furthermore, these mice showed lower CD3 expression and impaired CD3-mediated proliferation of T cells. While B cells showed slightly enhanced chemotactic migration in response to CXCL12, peripheral T cells showed markedly reduced chemotactic migration in response to CCL21 and CCL19 associated with decreased receptor levels of CCR7. Overall, Rho protein levels were reduced in the bone marrow, spleen, and thymus but sustained activation of the residual part of RhoA, Rac1, and Cdc42 was detected mainly in the bone marrow and spleen. Rho GDIalpha and Rho GDIbeta thus play synergistic roles in lymphocyte migration and development by modulating activation cycle of the Rho proteins in a lymphoid organ-specific manner.

During the last years, post-translational modification of peripheral membrane proteins with hydrophobic side groups has been attributed to a couple of additional functions than just simple anchoring into lipid bilayers. In particular isoprenylation and N- and S-acylation did quicken interest in terms of specific recognition elements for protein-protein interactions and as hydrophobic switches that allow for temporal regulated association with distinct target structures. Furthermore new insights into the heterogeneity of natural membranes have connected the physical properties of e.g. farnesyl or palmitoyl side chains with a preference for such sub-compartments as lipid rafts or caveolae. In this review the impact of the two frequently realized modifications by isoprenylation and S-acylation on the process of cellular signal transduction is exemplified with proteins of the Ras and Rab family of small GTP-binding proteins.

The Rho GDP dissociation inhibitors (RhoGDIs) are a major class of regulators of Rho GTPases and play essential roles in normal cell growth and malignant transformation. Although RhoGDIs are known to inhibit Rho activities, recent studies indicate that RhoGDIs can also act as positive regulators through their ability to target Rho GTPases to specific subcellular membranes or to protect the GTPases from degradation by caspases. RhoGDIs are aberrantly expressed in human tumors and this may contribute to Rho-induced cancer progression. This review will discuss the dual roles of RhoGDIs in the regulation of Rho GTPases, highlighting a possible role in regulating tumorigenicity. In addition, the potential for targeting RhoGDIs for anticancer therapy will be discussed.

Rho guanine nucleotide dissociation inhibitors (RhoGDIs) regulate the activity of Rho family GTPases. RhoGDIbeta (LyGDI/GDID4/RhoGDI2) has two caspase cleavage sites after Asp19 and Asp55. The resulting cleavage products, DeltaN(1-19)RhoGDIbeta and DeltaN(1-55)RhoGDIbeta, are expressed in cells under conditions that activate caspases. DeltaN(1-19)RhoGDIbeta, which can inhibit GDP dissociation, is implicated in the process of apoptosis, whereas the physiological roles for DeltaN(1-55)RhoGDIbeta, which lacks the ability to inhibit GDP dissociation, are largely unknown. To explore the roles of DeltaN(1-55)RhoGDIbeta, we examined the phenotypes of v-src-transformed metastatic fibroblasts transfected with plasmids for expressing DeltaN(1-55)RhoGDIbeta. Although the expression of DeltaN(1-55)RhoGDIbeta had no effect on the rate of growth in vitro, it suppressed experimental metastasis and decreased the rate of growth in vivo. In addition, DeltaN(1-55)RhoGDIbeta-expressing cells had enhanced adhesion to fibronectin, laminin, and collagens but reduced retention in the lung after intravenous injection. Also, the expression of DeltaN(1-55)RhoGDIbeta promoted anoikis without affecting the levels of activated Rac1 or Cdc42. Furthermore, DeltaN(1-55)RhoGDIbeta did not affect the expression or phosphorylation of focal adhesion kinase, p44/p42 mitogen-activated protein kinases, or Akt1 before or after induction of anoikis. Thus, DeltaN(1-55)RhoGDIbeta appears to promote anoikis by undefined mechanisms, thereby suppressing metastasis in v-src-transformed fibroblasts.

Cortical cell polarity controls mitotic spindle orientation in many cell types. In this issue of Cell, it is turned around and shown that the transfer of polarity information between the cortex and the spindle is not just one way. In Drosophila neuroblasts, the spindle also has polarizing activity on the cortex.

The Ypt/Rab family of small G-proteins is important in regulating vesicular transport. Rabs hydrolyze GTP very slowly on their own and require GTPase-activating proteins (GAPs). Here we report the identification and characterization of OsGAP1, a Rab-specific rice GAP. OsGAP1 strongly stimulated OsRab8a and OsRab11, which are homologs of the mammalian Rab8 and Rab11 proteins that are essential for Golgi to plasma membrane (PM) and trans-Golgi network (TGN) to PM trafficking, respectively. Substitution of two invariant arginines within the catalytic domain of Oryza sativa GTPase-activating protein 1 (OsGAP1) with alanines significantly inhibited its GAP activity. In vivo targeting experiments revealed that OsGAP1 localizes to the TGN or pre-vacuolar compartment (PVC). A yeast expression system demonstrated that wild-type OsGAP1 facilitates O. sativa dissociation inhibitor 3 (OsGDI3)-catalyzed OsRab11 recycling at an early stage, but the OsGAP1(R385A) and (R450A) mutants do not. Thus, GTP hydrolysis is essential for Rab recycling. Moreover, expression of the OsGAP1 mutants in Arabidopsis protoplasts inhibited the trafficking of some cargo proteins, including the PM-localizing H+-ATPase-green fluorescent protein (GFP) and Ca2+-ATPase8-GFP and the central vacuole-localizing Arabidopsis aleurain-like protein (AALP)-GFP. The OsGAP1 mutants caused these proteins to accumulate at the Golgi apparatus. Surprisingly, OsRab11 overproduction relieved the inhibitory effect of the OsGAP1 mutants on vesicular trafficking. OsRab8a had no such effect. Thus, the OsGAP1 mutants may inhibit TGN to PM or central vacuole trafficking because they induce the sequestration of endogenous Rab11. We propose that OsGAP1 facilitates vesicular trafficking from the TGN to the PM or central vacuole by both stimulating the GTPase activity of OsRab11 and increasing the recycling of inactive OsRab11.

Inhibition of the type 5 phosphodiesterase and inhibition of Rho kinase are both effective in reducing pulmonary hypertension (PH). Here we investigate whether Rho kinase inhibition is involved in the beneficial effect of the type 5 phosphodiesterase inhibitor sildenafil on PH. Chronic hypoxia-induced PH in rats is associated with an increase in RhoA activity in pulmonary artery that was maximal after 2 days (10.7+/-0.9-fold increase, n=6, P<0.001). The activity of Rho kinase assessed by measuring the level of myosin phosphatase target subunit 1 (MYPT1) phosphorylation was also increased (5.7+/-0.8-fold over control, n=8). Chronic fasudil (30 mg kg(-1) day(-1); 14 days) and sildenafil (25 mg kg(-1) day(-1); 14 days) treatments reduced PH and pulmonary cardiovascular remodelling, and inhibited the MYPT1 phosphorylation in pulmonary artery from hypoxic rats by 82.3+/-3% (n=4) and by 76.6+/-2% (n=4), respectively. The inhibitory effect of sildenafil (10 microM) on MYPT1 phosphorylation was demonstrated by the loss of actin stress fibres in vascular smooth muscle cells. However, in vitro kinase assays indicated that sildenafil had no direct inhibitory action on Rho kinase activity. Sildenafil treatment induced increased RhoA phosphorylation and association to its cytosolic inhibitory protein, guanine dissociation inhibitor (GDI) in pulmonary artery.We propose that sildenafil inhibits RhoA/Rho kinase-dependent functions in pulmonary artery through enhanced RhoA phosphorylation and cytosolic sequestration by GDI. The inhibition of intracellular events downstream of RhoA thus participates in the beneficial effect of sildenafil on PH.

Extant studies have implicated the Rho subfamily of guanosine triphosphate-binding proteins (G-proteins; e.g., Rac1) in physiological insulin secretion from isolated beta-cells. However, very little is known with regard to potential regulation by G-protein regulatory factors (e.g., the guanosine diphosphate-dissociation inhibitor [GDI]) of insulin secretion from the islet beta-cell. To this end, using Triton X-114 phase partition, co-immunoprecipitation, and sucrose density gradient centrifugation approaches, we report coexistence of GDI with Rac1 in insulin-secreting beta-cells (INS cells). Overexpression of wild-type GDI significantly inhibited glucose-induced, but not KCl- or mastoparan-induced, insulin secretion from INS cells. Furthermore, glucose-stimulated insulin secretion (GSIS) was significantly increased in INS cells in which expression of GDI was inhibited via the small interfering RNA-mediated knockdown approach. Together, these data appear to suggest an inhibitory role for GDI in the glucose metabolic signaling cascade, which may be relevant for GSIS.

Asymmetric division of Drosophila neuroblasts (NBs) and the Caenorhabditis elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. We show here that the Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pl cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13Fsingle and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Gai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry.

RhoGDI (Rho GDP-dissociation inhibitor) was identified as a down-regulator of Rho family GTPases typified by its ability to prevent nucleotide exchange and membrane association. Structural studies on GTPase-RhoGDI complexes, in combination with biochemical and cell biological results, have provided insight as to how RhoGDI exerts its effects on nucleotide binding, the membrane association-dissociation cycling of the GTPase and how these activities are controlled. Despite the initial negative roles attributed to RhoGDI, recent evidence has come to suggest that it may also act as a positive regulator necessary for the correct targeting and regulation of Rho activities by conferring cues for spatial restriction, guidance and availability to effectors. These potential functions are discussed in the context of RhoGDI-associated multimolecular complexes, the newly emerged shuttling capability and the importance of the particular membrane microenvironment that represents the site of action for GTPases. All these results point to a wider role for RhoGDI than initially perceived, making it a binding partner that can tightly control Rho GTPases, but which also allows them to reach their full spectrum of activities.

Rho GDP dissociation inhibitor (RhoGDI) plays an essential role in control of a variety of cellular functions through interactions with Rho family GTPases, including Rac1, Cdc42, and RhoA. RhoGDI is frequently overexpressed in human tumors and chemo-resistant cancer cell lines, raising the possibility that RhoGDI might play a role in the development of drug resistance in cancer cells. We found that overexpression of RhoGDI increased resistance of cancer cells (MDA-MB-231 human breast cancer cells and JLP-119 lymphoma cells) to the induction of apoptosis by two chemotherapeutic agents: etoposide and doxorubicin. Conversely, silencing of RhoGDI expression by DNA vector-mediated RNA interference (small interfering RNA) sensitized MDA-MB-231 cells to drug-induced apoptosis. Resistance to apoptosis was restored by reintroduction of RhoGDI protein expression. The mechanism for the anti-apoptotic activity of RhoGDI may derive from its ability to inhibit caspase-mediated cleavage of Rac1 GTPase, which is required for maximal apoptosis to occur in response to cytotoxic drugs. Taken together, the data show that RhoGDI is an anti-apoptotic molecule that mediates cellular resistance to these chemotherapy agents.

The GDP dissociation inhibitors (GDIs) are pivotal regulators of Rho GTPase function. GDIs control the access of Rho GTPases to regulatory guanine nucleotide exchange factors and GTPase-activating proteins, to effector targets and to membranes where such effectors reside. We discuss here our current understanding of how Rho GTPase-GDI complexes are regulated by various proteins, lipids and enzymes that exert GDI displacement activity. We propose that phosphorylation mediated by diverse kinases might provide a means of controlling and coordinating Rho GTPase activation.

Through tropomyosin-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

The small GTPase Rab5 is one of the key regulators of early endocytic traffic and, like other GTPases, cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle is controlled by a guanine nucleotide dissociation inhibitor (GDI), which functions as a Rab vehicle in the cytosol. GDI extracts from membranes the inactive GDP-bound form of the Rab. Then, the cytosolic GDI:Rab complex is delivered to the appropriate target membrane, where the Rab protein is reloaded, presumably via a GDI displacement factor (Pfeffer and Aivazian, 2004). We previously reported that the formation of the GDI:Rab5 complex is stimulated by the mitogen-activated protein kinase p38 (Cavalli et al., 2001). Mol. Cell7, 421-432.]. Selective activation of p38 MAPK increases endocytic rates in vivo, presumably allowing more efficient internalization of cell surface components for repair, storage, or degradation. These observations emphasize the possibility that external stimuli contribute to the regulation of membrane traffic. Here, we describe how to monitor the ability of GDI to extract Rab5 from early endosomal membranes in vitro and the role of p38 MAPK in this process. In addition, we detail how to investigate the possible role of p38 MAPK in the regulation of endocytosis in vivo.