Identification of a novel Rac1-interacting protein involved in membrane ruffling.

The Rac GTP binding proteins are implicated in actin cytoskeleton-membrane interaction in mammalian cells. In fibroblast cells, Rac has been shown to mediate growth factor-induced polymerization of actin to form membrane ruffles and lamellipodia. We report here the isolation of a noval Rac1-interacting protein, POR1. POR1 binds directly to Rac1, and the interaction of POR1 with Rac1 is GTP dependent. A mutation in the Rac1 effector binding loop shown to abolish membrane ruffling also abolishes interaction with POR1. Truncated versions of POR1 inhibit the induction of membrane ruffling by an activated mutant of Rac1, V12Rac1, in quiescent rat embryonic fibroblast REF52 cells. Furthermore, POR1 synergizes with an activated mutant of Ras, V12Ras, in the induction of membrane ruffling. These results suggest a potential role for POR1 in Rac1-mediated signaling pathways.     

cAMP induced Rac 1-mediated cytoskeletal reorganization in microvascular endothelium

It is well established that cAMP stabilizes endothelial barrier functions, in part by regulation of VE-cadherin via EPAC/Rap 1. The aim of the present study was to investigate whether cAMP activates Rac 1 in microvascular endothelium. In human dermal microvascular endothelial cells (HDMEC), treatment with forskolin/rolipram (F/R) to increase cAMP by as well as the Epac/Rap 1-stimulating cAMP analogue 8-pCPT-2'-O-methyl-cAMP (O-Me-cAMP) stabilized endothelial barrier properties as revealed by raised transendothelial electrical resistance (TER). Under these conditions, immunostaining of VE-cadherin and claudin 5 were increased and linearized. This was paralleled by activation of Rac 1 by 153 +/- 16% (F/R) or 281 +/- 65% (O-Me-cAMP) whereas activity of Rho A was unchanged. F/R and O-Me-cAMP increased the peripheral actin belt and recruited the Rac 1 effector cortactin to cell junctions, similar to direct activation of Rac 1 by CNF-1. Thrombin was used to further test the physiologic relevance of cAMP-mediated Rac 1 activation. Thrombin-induced drop of TER was paralleled by intercellular gap formation, inactivation of Rac 1 and activation of Rho A at 5 and 15 min whereas baseline conditions where re-established following 60 min. Both, F/R and O-Me-cAMP completely blocked the thrombin-induced barrier breakdown. F/R completely abolished thrombin-induced Rac 1 inactivation and Rho A activation whereas O-Me-cAMP only partially blocked Rac 1 inactivation. Taken together, these results indicate that Rac 1 activation likely contributes to the barrier-stabilizing effects of cAMP in microvascular endothelium and that these effects may in part be mediated by Epac/Rap 1.     

Use of a novel coinfection system reveals a role for Rac1, H-Ras, and CrkII phosphorylation in Helicobacter pylori-induced host cell actin cytoskeletal rearrangements

The Helicobacter pylori CagA protein induces profound morphological changes in the host cytoskeleton and cell scattering, but the signalling involved is poorly understood. Pseudomonas aeruginosa also affects host actin cytoskeleton in a variety of ways by injecting the ExoS and ExoT toxins which encode N-terminal GTPase activating protein and C-terminal ADP-ribosyltransferase (ADPRT) activities. In this study we developed a novel coinfection assay to gain new insights into CagA effector protein functions. We found that P. aeruginosa injecting either ExoT or ExoS efficiently prevented the H. pylori-induced scattering phenotype. Both the Rho-GAP and the ADPRT domains of ExoS were needed to block the H. pylori-induced actin cytoskeletal rearrangements, whereas either domain of ExoT was sufficient for this activity. This strategy revealed common pathways subverted by different pathogens, and aided in the definition of signalling cascades that control the CagA-mediated cell scattering and elongation. We identified Crk adapter proteins, Rac1 and H-Ras, but not RhoA or Cdc42, which are the ExoS and/or ExoT targets, as crucial components of the CagA-induced phenotype. In addition, we show that ADP-ribosylation of CrkII by ExoT blocks phosphorylation of CrkII at Y-221, which is also important for the CagA-induced signalling.     

Expression of ARF6 mutants in neuroendocrine cells suggests a role for ARF6 in synaptic vesicle biogenesis.

ARF6 regulates membrane trafficking between the plasma membrane and endosomes. We investigated the role of ARF6 in synaptic vesicle biogenesis as this process occurs both at the plasma membrane and at endosomes. We used a synaptic vesicle marker protein, p-selectin-horseradish peroxidase (HRP), to follow the effects of ARF6 expression on synaptic vesicle biogenesis in PC12 neuroendocrine cells. Expression of a constitutively active ARF6 mutant increased, while expression of a nucleotide-free ARF6 mutant decreased, p-selectin-HRP levels in the synaptic vesicle peak. These results provide the first direct evidence for a role for ARF6 in synaptic vesicle biogenesis.     

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and cofilin activity. The TGFβ-like activity of the vitreous may participate in this effect. Actin polymerization causes the cytoskeletal rearrangements that lead to the plasticity of vitreous-transformed RPE cells in PVR.     

c-Cbl facilitates cytoskeletal effects in v-Abl transformed fibroblast through Rac1- and Rap1-mediated signaling

c-Cbl functions as a multifunctional adaptor and an E3 ubiquitin ligase. Several studies have shown that c-Cbl is involved in cytoskeleton-mediated events, but the molecular mechanisms linking c-Cbl to cytoskeletal rearrangements remain to be elucidated. Our previous results indicated that c-Cbl facilitates spreading and migration of v-Abl-transformed NIH 3T3 fibroblasts and suggested that small GTPases play important roles in the cytoskeletal effects of c-Cbl in this system. To elucidate the individual contributions of small GTPases to these effects, we assessed the roles of endogenous Rac1, RhoA and Rap1 in the c-Cbl-dependent spreading and migration of v-Abl-transformed fibroblasts overexpressing c-Cbl, using RNAi. Furthermore, since it has been shown that Rap1 can act as an upstream regulator of Rac1 in inducing cell spreading, we analyzed the interplay between Rap1 and Rac1 in the signaling pathways connecting c-Cbl to the cytoskeletal events. Our results indicate that Rac1 is essential for cell migration and spreading, whereas activation of RhoA exerts a negative effect. We have also shown that Rap1 is essential for cell spreading, although not for migration in our experimental system. Furthermore, we provide evidence that Rap1 is located upstream of Rac1 in one of the signaling pathways that regulate c-Cbl-facilitated cell spreading. Overall, our findings are consistent with the model describing the connection of c-Cbl to the cytoskeletal rearrangements via two pathways, one of which is mediated by PI3K and Rac1, and the other, by CrkL/C3G, Rap1 and Rac1.     

Establishing epithelial glandular polarity: interlinked roles for ARF6, Rac1, and the matrix microenvironment

Epithelial cysts comprise the structural units of the glandular epithelium. Although glandular inversion in epithelial tumors is thought to be a potential mechanism for the establishment of metastatic disease, little is known about the morphogenic cues and signaling pathways that govern glandular polarity and organization. Using organotypic cultures of Madin-Darby canine kidney cells in reconstituted basement membrane, we show that cellular depletion of the small GTP-binding protein ARF6 promotes the formation of inverted cysts, wherein the apical cell membrane faces the cyst exterior, and the basal domain faces the central lumen, while individual cell polarity is maintained. These cysts are also defective in interactions with laminin at the cyst–matrix interface. This inversion of glandular orientation is accompanied by Rac1 inactivation during early cystogenesis, and temporal activation of Rac1 is sufficient to recover the normal cyst phenotype. In an unnatural collagen I microenvironment, ARF6-depleted, inverted epithelial cysts exhibit some loss of cell polarity, a marked increase in Rho activation and Rac1 inactivation, and striking rearrangement of the surrounding collagen I matrix. These studies demonstrate the importance of ARF6 as a critical determinant of glandular orientation and the matrix environment in dictating structural organization of epithelial cysts.     

Cytokine expression and AIF-1-mediated activation of Rac2 in vascular smooth muscle cells: a role for Rac2 in VSMC activation

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein involved in vascular smooth muscle cell (VSMC) migration and proliferation. The objective of this study is to characterize AIF-1 functional protein interactions that may regulate VSMC activation. Through use of a bacterial two-hybrid screen, we identified a molecular interaction between AIF-1 and the small GTPase, Rac2, which was verified by pull-down and colocalization experiments. This was unexpected in that Rac2 expression had been considered to be restricted to hematopoietic cells. The Rac2/AIF-1 interaction is functional, in that a loss-of-function, point-mutated AIF-1 does not interact with Rac2; Rac2 colocalizes with AIF-1 in the cytoplasm of VSMC and cotranslocates to lamellopodia upon platelet-derived growth factor stimulation; and AIF-1 expression in VSMC leads to Rac2 activation. Because Rac2 function in VSMC had not been described, we focused on characterization of its function in these cells. Rac2 protein expression in VSMC is inducible by inflammatory cytokines, and Rac2 activation in VSMC is also responsive to inflammatory cytokines. Rac2 expression and activation patterns differ from the ubiquitously expressed Rac1. We hypothesized that Rac2 participates in VSMC activation. Retroviral overexpression of Rac2 in primary VSMC leads to increased migration, activation of the NADPH oxidation cascade, and increased activation of the Rac2 effector protein Pak1 and its proximal effectors, ERK1/2, and p38 (P < 0.05 for all). The major points of this study indicate a functional interaction between AIF-1 and Rac2 in VSMC leading to Rac2 activation and a potential function for Rac2 in inflammation-driven VSMC response to injury. allograft inflammatory factor-1; signal transduction     

An essential role for p120-catenin in Src- and Rac1-mediated anchorage-independent cell growth

p120-catenin regulates epithelial cadherin stability and has been suggested to function as a tumor suppressor. In this study, we used anchorage-independent growth (AIG), a classical in vitro tumorigenicity assay, to examine the role of p120 in a different context, namely oncogene-mediated tumorigenesis. Surprisingly, p120 ablation by short hairpin RNA completely blocked AIG induced by both Rac1 and Src. This role for p120 was traced to its activity in suppression of the RhoA–ROCK pathway, which appears to be essential for AIG. Remarkably, the AIG block associated with p120 ablation was completely reversed by inhibition of the downstream RhoA effector ROCK. Harvey-Ras (H-Ras)–induced AIG was also dependent on suppression of the ROCK cascade but was p120 independent because its action on the pathway occurred downstream of p120. The data suggest that p120 modulates oncogenic signaling pathways important for AIG. Although H-Ras bypasses p120, a unifying theme for all three oncogenes is the requirement to suppress ROCK, which may act as a gatekeeper for the transition to anchorage independence.     

Antigen-stimulated activation of phospholipase D1b by Rac1, ARF6, and PKCalpha in RBL-2H3 cells

Phospholipase D (PLD) activity can be detected in response to many agonists in most cell types; however, the pathway from receptor occupation to enzyme activation remains unclear. In vitro PLD1b activity is phosphatidylinositol 4,5-bisphosphate dependent via an N-terminal PH domain and is stimulated by Rho, ARF, and PKC family proteins, combinations of which cooperatively increase this activity. Here we provide the first evidence for the in vivo regulation of PLD1b at the molecular level. Antigen stimulation of RBL-2H3 cells induces the colocalization of PLD1b with Rac1, ARF6, and PKCalpha at the plasma membrane in actin-rich structures, simultaneously with cooperatively increasing PLD activity. Activation is both specific and direct because dominant negative mutants of Rac1 and ARF6 inhibit stimulated PLD activity, and surface plasmon resonance reveals that the regulatory proteins bind directly and independently to PLD1b. This also indicates that PLD1b can concurrently interact with a member from each regulator family. Our results show that in contrast to PLD1b's translocation to the plasma membrane, PLD activation is phosphatidylinositol 3-kinase dependent. Therefore, because inactive, dominant negative GTPases do not activate PLD1b, we propose that activation results from phosphatidylinositol 3-kinase-dependent stimulation of Rac1, ARF6, and PKCalpha.     

Role of MLK3-mediated activation of p70 S6 kinase in Rac1 transformation.

The signaling pathways that mediate the transforming activity of the Rac1 GTPase remain to be determined. In the present study, we used effector domain mutants of the constitutively activated Rac(61L) mutant that display differential transforming activities and differential activation of downstream effector pathways to investigate the contribution of p70 S6 kinase (p70(S6K)) to Rac1 transformation and to decipher the signaling pathways leading from Rac1 to p70(S6K). First, we found that Rac1 transforming activity could be dissociated from Rac1 activation of p70(S6K). A weakly transforming Rac1 mutant retained the ability to activate p70(S6K), whereas some potently transforming effector mutants were impaired in their ability to activate p70(S6K). These data suggest that p70(S6K) is not necessary to promote full Rac1 transforming activity. We also found a strong correlation between the ability of the Rac(61L) effector mutants to activate p70(S6K) and their ability to activate the JNK mitogen-activated protein kinase. We found that the MLK3 serine/threonine kinase activated JNK and p70(S6K), whereas activation of p70(S6K) by Rac(61L) was significantly inhibited by dominant-negative MLK3. Additionally, the ability of the Rac(61L) effector mutants to activate MLK3 correlated well with their ability to activate p70(S6K) and JNK. Taken together, these results provide evidence that Rac1 coordinately activates p70(S6K) and JNK via MLK3 activation. Finally, we found that co-expression of wild type, but not kinase-dead, MLK3 significantly inhibited Rac1 transforming activity. These results suggest that MLK3 may be a negative regulator of the growth-promoting and transforming properties of Rac1.     

Gamma-BAR, a novel AP-1-interacting protein involved in post-Golgi trafficking

A novel peripheral membrane protein (2c18) that interacts directly with the gamma 'ear' domain of the adaptor protein complex 1 (AP-1) in vitro and in vivo is described. Ultrastructural analysis demonstrates a colocalization of 2c18 and gamma1-adaptin at the trans-Golgi network (TGN) and on vesicular profiles. Overexpression of 2c18 increases the fraction of membrane-bound gamma1-adaptin and inhibits its release from membranes in response to brefeldin A. Knockdown of 2c18 reduces the steady-state levels of gamma1-adaptin on membranes. Overexpression or downregulation of 2c18 leads to an increased secretion of the lysosomal hydrolase cathepsin D, which is sorted by the mannose-6-phosphate receptor at the TGN, which itself involves AP-1 function for trafficking between the TGN and endosomes. This suggests that the direct interaction of 2c18 and gamma1-adaptin is crucial for membrane association and thus the function of the AP-1 complex in living cells. We propose to name this protein gamma-BAR.     

The role of VASP in regulation of cAMP- and Rac 1-mediated endothelial barrier stabilization

Regulation of actin dynamics is critical for endothelial barrier functions. We provide evidence that the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) is required for endothelial barrier maintenance. Baseline permeability was significantly increased in VASP-deficient (VASP(-/-)) microvascular myocardial endothelial cells (MyEnd) in the absence of discernible alterations of immunostaining for adherens and tight junctions. We tested whether VASP is involved in the endothelium-stabilizing effects of cAMP or Rac 1. Forskolin and rolipram (F/R) to increase cAMP and cytotoxic necrotizing factor 1 (CNF-1) to activate Rac 1 were equally efficient to stabilize barrier functions in VASP(-/-) and wild-type (wt) cells. In wt cells, VASP was phosphorylated in response to F/R but did not localize to intercellular junctions. In contrast, CNF-1 and expression of constitutively active Rac 1 induced translocation of VASP to cell borders in wt cells, where it colocalized with active Rac 1. In VASP(-/-) cells, Rac 1 activity was reduced to 0.4 of wt levels in controls and increased approximately 20-fold in response to CNF-1 compared with 7-fold activation in wt cells. Moreover, inactivation of Rac 1 by lethal toxin led to a greater increase of permeability compared with wt cells. All these data suggest that VASP is involved in the regulation of Rac 1 activity. Taking these findings together, our study indicates that VASP at least in part stabilizes endothelial barrier functions by control of Rho-family GTPases.     

ARF6-dependent interaction of the TWIK1 K+ channel with EFA6, a GDP/GTP exchange factor for ARF6

TWIK1 belongs to a family of K(+) channels involved in neuronal excitability and cell volume regulation. Its tissue distribution suggests a role in epithelial potassium transport. Here we show that TWIK1 is expressed in a subapical compartment in renal proximal tubules and in polarized MDCK cells. In nonpolarized cells, this compartment corresponds to pericentriolar recycling endosomes. We identified EFA6, an exchange factor for the small G protein ADP-ribosylation factor 6 (ARF6), as a protein binding to TWIK1. EFA6 interacts with TWIK1 only when it is bound to ARF6. Because ARF6 modulates endocytosis at the apical surface of epithelial cells, the ARF6/EFA6/TWIK1 association is probably important for channel internalization and recycling.     

Rac1 function is required for Src-induced transformation. Evidence of a role for Tiam1 and Vav2 in Rac activation by Src

The proto-oncogene c-Src has been implicated in the development and progression of a number of human cancers including those of colon and breast. Accumulating evidence indicates that activated alleles of Src may induce cell transformation through Ras-ERK-dependent and -independent pathways. Here we show that Rac1 activity is strongly elevated in Src-transformed cells and that this small G protein is a critical component of the pathway connecting oncogenic Src with cell transformation. We further show that Vav2 and the ubiquitously expressed Rac1 guanine nucleotide exchange factor Tiam1 are phosphorylated in tyrosine residues in cells transfected with active and oncogenic Src. Moreover, phosphorylation of Tiam1 in cells treated with pervanadate, a potent inhibitor of tyrosine phosphatases, was partially inhibited by the Src inhibitor SU6656. Using truncated mutants of Tiam1, we demonstrate that multiple sites can be tyrosine-phosphorylated by Src. Furthermore, Tiam1 cooperated with Src to induce activation of Rac1 in vivo and the formation of membrane ruffles. Similarly, activation of JNK and the c-jun promoter by Src were also potently increased by Tiam1. Together, these results suggest that Vav2 and Tiam1 may act as downstream effectors of Src, thereby regulating Rac1-dependent pathways that participate in Src-induced cell transformation.     

GEFT, A Rho family guanine nucleotide exchange factor, regulates lens differentiation through a Rac1-mediated mechanism

The Rho-family of small GTPase specific guanine nucleotide exchange factor, GEFT, is expressed at high levels in adult human excitable tissues including the brain, heart, and skeletal muscle. Previously, we demonstrated that GEFT is specifically expressed in the adult mouse hippocampus and cerebellum, and that overexpression of this protein can result in neurite and dendrite remodeling. This finding prompted us to explore the expression of GEFT in other tissues, which share common developmental ancestry to the nervous system, specifically the ocular system. Using immunohistochemical analysis specific for GEFT protein expression, we observed the highest ocular expression of GEFT occurring in the neuroblastic layer and differentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lens epithelium, and throughout the retina. Exogenous expression of GEFT in N/N1003A rabbit lens epithelial cells induced lens fiber differentiation as reflected by cell elongation and lentoid formation, as well as a strong increase in β-crystallin and filensin expression. Moreover, transfection of lens epithelial cells with GEFT resulted in a Rac-1 mediated up-regulation of αA-, αB-, βB-, γC-, or γF-crystallin promoter activities that is in part dependent on the nuclear localization of Rac1. Furthermore, pharmacological inhibition of Rac1 blocked GEFT-induced N/N1003A lens fiber differentiation and βB-crystallin expression in ex vivo mouse lens explants. These results demonstrate for the first time a role for GEFT in lens cell differentiation and mouse eye development. Moreover, GEFT regulation of lens differentiation and eye development occurs through a Rac1-dependent mechanism.     

The Kar3-interacting protein Cik1p plays a critical role in passage through meiosis I in Saccharomyces cerevisiae.

Meiosis I in Saccharomyces cerevisiae is dependent upon the motor protein Kar3. Absence of Kar3p in meiosis results in an arrest in prophase I. Cik1p and Vik1p are kinesin-associated proteins known to modulate the function of Kar3p in the microtubule-dependent processes of karyogamy and mitosis. Experiments were performed to determine whether Cik1p and Vik1p are also important for the function of Kar3p during meiosis. The meiotic phenotypes of a cik1 mutant were found to be similar to those of kar3 mutants. Cells without Cik1p exhibit a meiotic defect in homologous recombination and synaptonemal complex formation. Most cik1 mutant cells, like kar3 mutants, arrest in meiotic prophase; however, in cik1 mutants this arrest is less severe. These data are consistent with the model that Cik1p is necessary for some, but not all, of the roles of Kar3p in meiosis I. vik1 mutants sporulate at wild-type levels, but have reduced spore viability. This loss in viability is partially attributable to vegetative chromosome loss in vik1 diploids. Cellular localization experiments reveal that Kar3p, Cik1p, and Vik1p are present throughout meiosis and are consistent with Cik1p and Vik1p having different meiotic roles.     

A genomewide screen for suppressors of Alu-mediated rearrangements reveals a role for PIF1

Alu-mediated rearrangement of tumor suppressor genes occurs frequently during carcinogenesis. In breast cancer, this mechanism contributes to loss of the wild-type BRCA1 allele in inherited disease and to loss of heterozygosity in sporadic cancer. To identify genes required for suppression of Alu-mediated recombination we performed a genomewide screen of a collection of 4672 yeast gene deletion mutants using a direct repeat recombination assay. The primary screen and subsequent analysis identified 12 candidate genes including TSA, ELG1, and RRM3, which are known to play a significant role in maintaining genomic stability. Genetic analysis of the corresponding human homologs was performed in sporadic breast tumors and in inherited BRCA1-associated carcinomas. Sequencing of these genes in high risk breast cancer families revealed a potential role for the helicase PIF1 in cancer predisposition. PIF1 variant L319P was identified in three breast cancer families; importantly, this variant, which is predicted to be functionally damaging, was not identified in a large series of controls nor has it been reported in either dbSNP or the 1000 Genomes Project. In Schizosaccharomyces pombe, Pfh1 is required to maintain both mitochondrial and nuclear genomic integrity. Functional studies in yeast of human PIF1 L319P revealed that this variant cannot complement the essential functions of Pfh1 in either the nucleus or mitochondria. Our results provide a global view of nonessential genes involved in suppressing Alu-mediated recombination and implicate variation in PIF1 in breast cancer predisposition.