Rac-specific Guanine Nucleotide Exchange Factor Research Articles

The cooperative action of bacterial fibronectin-binding proteins and secreted proteins promote maximal Campylobacter jejuni invasion of host cells by stimulating membrane ruffling

This study was performed to elucidate the host cell scaffolding and signalling molecules that Campylobacter jejuni utilizes to invade epithelial cells. We hypothesized that the C. jejuni fibronectin-binding proteins and secreted proteins are required for cell signalling and maximal invasion of host cells. C. jejuni binding to host cells via the CadF and FlpA fibronectin-binding proteins activated the epidermal growth factor (EGF) pathway, as evidenced by inhibitor studies and immunoprecipitation coupled with immunoblot analysis using antibodies reactive against total and active EGF receptor. Inhibitor studies revealed maximal C. jejuni host cell invasion was dependent upon PI3-Kinase, c-Src and focal adhesion kinase (FAK), all of which are known to participate in cytoskeletal rearrangements. Knockdown of endogenous Dock180, which is a Rac1-specific guanine nucleotide exchange factor, using siRNA revealed that C. jejuni invasion was significantly reduced compared with cells treated with scrambled siRNA. We further demonstrated that the C. jejuni Cia proteins are, in part, responsible for Rho GTPase Rac1 recruitment and activation, as judged by immunofluorescence microscopy and Rac1 activation. Based on these data, we present a model that illustrates that C. jejuni utilizes a coordinated mechanism involving both adhesins and secreted proteins to promote membrane ruffling and host cell invasion.

CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1

Son of sevenless 1 (SOS1) is a dual guanine nucleotide exchange factor (GEF) that activates the guanosine triphosphatases Rac1 and Ras, which mediate signaling initiated by peptide growth factors. In this paper, we show that CIIA is a new binding partner of SOS1. CIIA promoted the SOS1-Rac1 interaction and inhibited the SOS1-Ras interaction. Furthermore, CIIA promoted the formation of an SOS1-EPS8 complex and SOS1-mediated Rac1 activation, whereas it inhibited SOS1-mediated activation of Ras. Transforming growth factor β (TGF-β) up-regulated the expression of CIIA and thereby promoted the association between CIIA and SOS1 in A549 human lung adenocarcinoma cells. Depletion of CIIA in these cells by ribonucleic acid interference inhibited the TGF-β-induced interaction between SOS1 and EPS8, activation of Rac1, and cell migration. Together, these results suggest that CIIA mediates the TGF-β-induced activation of SOS1-Rac1 signaling and cell migration in A549 cells. They further show that CIIA functions as a molecular switch for the GEF activity of SOS1, directing this activity toward Rac1.

MTORC1‐ and mTORC2‐interacting proteins keep their multifunctional partners focused

The mammalian target of rapamycin, best known as mTOR, is a phylogenetically conserved serine/threonine kinase that controls life-defining cellular processes such as growth, metabolism, survival, and migration under the influence of multiple interacting proteins. Historically, the cellular activities blocked by rapamycin in mammalian cells were considered the only events controlled by mTOR. However, this paradigm changed with the discovery of two signaling complexes differentially sensitive to rapamycin, whose catalytic component is mTOR. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to growth factors and nutrients via the phosphorylation of p70S6K and 4EBP1; while the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of AKT, respectively. Although mTORC2 kinase activity is not inhibited by rapamycin, hours of incubation with this antibiotic can impede the assembly of this signaling complex. The direct mechanism by which mTORC2 leads to cell migration depends on its interaction with P-Rex1, a Rac-specific guanine nucleotide exchange factor, while additional indirect pathways involve the intervention of PKC or AKT, multifunctional ubiquitous serine/threonine kinases that activate effectors of cell migration upon being phosphorylated by mTORC2 in response to chemotactic signals. These mTORC2 effectors are altered in metastatic cancer. Numerous clinical trials are testing mTOR inhibitors as potential antineoplasic drugs. Here, we briefly review the actions of mTOR with emphasis on the controlling role of mTORC1 and mTORC2-interacting proteins and highlight the mechanisms linked to cell migration.

P-Rex1 is required for efficient melanoblast migration and melanoma metastasis

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

Cla4 kinase triggers destruction of the Rac1-GEF Cdc24 during polarized growth inUstilago maydis

Dimorphic switching from budding to filamentous growth is a characteristic feature of many pathogenic fungi. In the fungal model organism Ustilago maydis polarized growth is induced by the multiallelic b mating type locus and requires the Rho family GTPase Rac1. Here we show that mating type-induced polarized growth involves negative feedback regulation of the Rac1-specific guanine nucleotide exchange factor (GEF) Cdc24. Although Cdc24 is essential for polarized growth, its concentration is drastically diminished during filament formation. Cdc24 is part of a protein complex that also contains the scaffold protein Bem1 and the PAK kinase Cla4. Activation of Rac1 results in Cla4-dependent degradation of the Rac1-GEF Cdc24, thus creating a regulatory negative feedback loop. We generated mutants of Cdc24 that are resistant to Cla4-dependent destruction. Expression of stable Cdc24 variants interfered with filament formation, indicating that negative feedback regulation of Cdc24 is critical for the establishment of polarized growth.

Expression of T‐cell lymphoma invasion and metastasis 2 (TIAM2) promotes proliferation and invasion of liver cancer

The T-cell lymphoma invasion and metastasis 2 (TIAM2) gene is the homolog of human TIAM1, a Rac-specific guanine nucleotide exchange factor that plays important roles in neuron development and human malignancies. Although the role of TIAM1 is well characterized, the physiological and pathological functions of TIAM2 remain unknown. In our study, human cDNA and protein panels were evaluated for endogenous expression of TIAM2. Four hepatocellular carcinoma (HCC) cell lines and 91 HCC samples were used to demonstrate expression of TIAM2S (the short form of TIAM2) in cancer cells. In addition, HepG2 cells stably expressing TIAM2S were used for tumorigenic assays in both cellular and mouse models. We demonstrate that endogenous TIAM2S was induced in several human cancers including HCC. TIAM2S expression was undetectable in normal human liver but was induced in all HCC cell lines and in 86% (78/91) of HCC biopsies. TIAM2S expression was positively associated with TIAM1 expression, hepatitis B virus (HBV) infection and metastatic phenotype. Expression of recombinant TIAM2S in HepG2 cells promoted growth and invasiveness. In vivo study using a xenografted mouse model demonstrated that induced endogenous expression of TIAM2S converted non-invasive human HCC cells into highly aggressive vascular tumors. Further examination revealed that TIAM2S expression resulted in up-regulation of N-cadherin and vimentin, and in redistribution of E-cadherin. These findings show, for the first time, that human TIAM2S is involved in HCC pathogenesis, and that increased expression of TIAM2S promotes epithelial-to-mesenchymal transition and results in proliferation and invasion in liver cancer cells.

Abstract 3852: Identification and functional characterization of T-cell lymphoma invasion and metastasis 2 (TIAM2) in neuronal cells

Abstract T-Cell Lymphoma Invasion and Metastasis 2 (TIAM2) gene was identified as the homolog for human TIAM1 gene, a Rac-specific guanine nucleotide exchange factor (GEF), and classified as a member of TIAM GEF subfamily. Although functions of TIAM family proteins have been extensively studied, the characteristics of human TIAM2 remain largely unknown. In this study we systematically investigate the cellular function of TIAM2. Two mRNA isoforms of TIAM2, TIAM2S and TIAM2L, were detected and showed distinct expression patterns in various human tissues. Although both mRNAs are highly expressed in many human tissues, no TIAM2L protein can be detected in any of the examined human tissues and cell lines. In addition, TIAM2S protein only expressed in neuron of restricted regions in the human brain. Overexpression of TIAM2S-GFP fusion protein revealed a cytosolic distribution in both HeLa and NIH3T3 cells. Using LC/MS/MS and enzymatic analysis, TIAM2S was shown to be an O-linked glycoprotein. As transiently transfected TIAM2S-fusion protein neither translocate onto the cell membrane, nor exhibit GEF activity to RhoGTPases in vivo, these data suggested human TIAM2S is not a classical GEF and its functional role in normal brain may differ from other TIAM members. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3852. doi:10.1158/1538-7445.AM2011-3852

A Minimal Rac Activation Domain in the Unconventional Guanine Nucleotide Exchange Factor Dock180

Guanine nucleotide exchange factors (GEFs) activate Rho GTPases by catalyzing the exchange of bound GDP for GTP, thereby resulting in downstream effector recognition. Two metazoan families of GEFs have been described: Dbl-GEF family members that share conserved Dbl homology (DH) and Pleckstrin homology (PH) domains and the more recently described Dock180 family members that share little sequence homology with the Dbl family and are characterized by conserved Dock homology regions 1 and 2 (DHR-1 and -2, respectively). While extensive characterization of the Dbl family has been performed, less is known about how Dock180 family members act as GEFs, with only a single X-ray structure having recently been reported for the Dock9-Cdc42 complex. To learn more about the mechanisms used by the founding member of the family, Dock180, to act as a Rac-specific GEF, we set out to identify and characterize its limit functional GEF domain. A C-terminal portion of the DHR-2 domain, composed of approximately 300 residues (designated as Dock180(DHR-2c)), is shown to be necessary and sufficient for robust Rac-specific GEF activity both in vitro and in vivo. We further show that Dock180(DHR-2c) binds to Rac in a manner distinct from that of Rac-GEFs of the Dbl family. Specifically, Ala(27) and Trp(56) of Rac appear to provide a bipartite binding site for the specific recognition of Dock180(DHR-2c), whereas for Dbl family Rac-GEFs, Trp(56) of Rac is the sole primary determinant of GEF specificity. On the basis of our findings, we are able to define the core of Dock180 responsible for its Rac-GEF activity as well as highlight key recognition sites that distinguish different Dock180 family members and determine their corresponding GTPase specificities.

P190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.

EphA8-ephrinA5 signaling and clathrin-mediated endocytosis is regulated by Tiam-1, a Rac-specific guanine nucleotide exchange factor.

Recent studies indicate that endocytosis of Eph-ephrin complexes may be one of the mechanisms by which a high affinity cell-cell adhesion is converted to a repulsive interaction. In this study, we show that EphA8 undergoes clathrin-mediated endocytosis upon treatment with ephrin-A5, and that EphA8 is associated tightly with Tiam-1, a Rac-specific guanine nucleotide exchange factor. Analysis of EphA8 deletion mutants revealed that a juxtamembrane region in EphA8 is critically involved in endocytosis of EphA8-ephrinA5 complexes. An EphA8 mutant lacking this juxtamembrane portion was defective for endocytosis with ephrinA5, and also displayed a weak association with Tiam-1. Expression of an endocytosis-defective version of EphA8 resulted in a low level of Rac activity in response to ephrin-A5 stimulation. More importantly, down-regulation of Tiam-1 resulted in inefficient endocytosis of EphA8-ephrinA5 complexes. These results suggest that Tiam-1 plays a role in clathrin-dependent endocytosis of EphA8-ephrinA5 complexes.

CADM1 Interacts with Tiam1 and Promotes Invasive Phenotype of Human T-cell Leukemia Virus Type I-transformed Cells and Adult T-cell Leukemia Cells

CADM1 encodes a multifunctional immunoglobulin-like cell adhesion molecule whose cytoplasmic domain contains a type II PSD95/Dlg/ZO-1 (PDZ)-binding motif (BM) for associating with other intracellular proteins. Although CADM1 lacks expression in T lymphocytes of healthy individuals, it is overexpressed in adult T-cell leukemia-lymphoma (ATL) cells. It has been suggested that the expression of CADM1 protein promotes infiltration of leukemic cells into various organs and tissues, which is one of the frequent clinical manifestations of ATL. Amino acid sequence alignment revealed that Tiam1 (T-lymphoma invasion and metastasis 1), a Rac-specific guanine nucleotide exchange factor, has a type II PDZ domain similar to those of membrane-associated guanylate kinase homologs (MAGUKs) that are known to bind to the PDZ-BM of CADM1. In this study, we demonstrated that the cytoplasmic domain of CADM1 directly interacted with the PDZ domain of Tiam1 and induced formation of lamellipodia through Rac activation in HTLV-I-transformed cell lines as well as ATL cell lines. Our results indicate that Tiam1 integrates signals from CADM1 to regulate the actin cytoskeleton through Rac activation, which may lead to tissue infiltration of leukemic cells in ATL patients.

Characterization of P-Rex1 for its role in fMet-Leu-Phe-induced superoxide production in reconstituted COS phox cells

P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gβγ subunits and by PtdIns (3,4,5)P 3. Recent studies indicate that P-Rex1 plays an important role in signaling downstream of neutrophil chemoattractant receptors. Here we report that heterologous expression of P-Rex1, but not Vav1, reconstitutes formyl peptide receptor 1 (FPR1)-mediated NADPH oxidase activation in the transgenic COS phox cells expressing gp91 phox, p22 phox, p67 phox and p47 phox. A successful reconstitution requires the expression of a full-length P-Rex1 with intact DH and PH domains, and is accompanied by P-Rex1 membrane localization as well as Rac1 activation. P-Rex1-dependent superoxide generation in the reconstituted COS phox cells was further enhanced by expression of the novel PKC isoform PKCδ and by overexpression of Akt. Heterologous expression of P-Rex1 in COS phox cells potentiated fMet-Leu-Phe-induced Akt phosphorylation, whereas expression of a constitutively active form of Akt enhanced Rac1 activation. In contrast, a dominant negative Akt mutant reduced the fMet-Leu-Phe stimulated superoxide generation as well as Rac1 activation. These results demonstrate that in COS phox cells, P-Rex1 is a critical component for FPR1-mediated signaling leading to NADPH oxidase activation, and there is a crosstalk between the P-Rex1-Rac pathway and Akt in superoxide generation.

A Modified Tandem Affinity Purification Technique Identifies That 14-3-3 Proteins Interact with Tiam1, an Interaction Which Controls Tiam1 Stability

The Rac-specific GEF (guanine-nucleotide exchange factor) Tiam1 has important functions in multiple cellular processes including proliferation, apoptosis and adherens junction maintenance. Here we describe a modified tandem affinity purification (TAP) technique that we have applied to specifically enrich Tiam1-containing protein complexes from mammalian cells. Using this technique in conjunction with LC-MS/MS mass spectrometry, we have identified additional Tiam1-interacting proteins not seen with the standard technique, and have identified multiple 14-3-3 family members as Tiam1 interactors. We confirm the Tiam1/14-3-3 protein interaction by GST-pulldown and coimmunoprecipitation experiments, show that it is phosphorylation-dependent, and that they colocalize in cells. The interaction is largely dependent on the N-terminal region of Tiam1; within this region, there are four putative phospho-serine-containing 14-3-3 binding motifs, and we confirm that two of them (Ser172 and Ser231) are phosphorylated in cells using mass spectrometry. Moreover, we show that phosphorylation at three of these motifs (containing Ser60, Ser172 and Ser231) is required for the binding of 14-3-3 proteins to this region of Tiam1. We show that phosphorylation of these sites does not affect Tiam1 activity; significantly however, we demonstrate that phosphorylation of the Ser60-containing motif is required for the degradation of Tiam1. Thus, we have established and proven methodology that allows the identification of additional protein-protein interactions in mammalian cells, resulting in the discovery of a novel mechanism of regulating Tiam1 stability.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the β-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

Regulation of Cross-linked Actin Network (CLAN) Formation in Human Trabecular Meshwork (HTM) Cells by Convergence of Distinct β1 and β3 Integrin Pathways

To determine the beta1/beta3 integrin-mediated pathways that regulate cross-linked actin network (CLAN) formation in human trabecular meshwork (HTM) cells. CLANs form in glaucomatous and steroid-treated TM cells, which may contribute to reducing outflow facility through the TM. Expression of CD47 (an alphavbeta3 integrin coreceptor/thrombospondin-1 receptor) and integrins alphavbeta3 and beta1 was assessed by FACS. CLANs were induced by plating cells on fibronectin (a beta1 integrin ligand) in the absence or presence of the beta3 integrin-activating mAb AP-5 and were identified by phalloidin labeling. The role of Src kinases, PI-3 kinase (PI-3K), Rac1, and CD47 was determined by incubating cells with the inhibitors PP2 and EPA (Src kinases), LY294002 (PI-3K), or NSC23766 (Rac1). Tiam1 and Trio siRNAs and dominant-negative Tiam1 were used to determine which Rac1-specific guanine nucleotide exchange factor was involved. The role of CD47 was determined using the thrombospondin-1-derived agonist peptide 4N1K and the CD47 function blocking antibody B6H12.2. HTM cells expressed CD47 and integrins alphavbeta3 and beta1. beta3 Integrin or CD47 activation significantly increased CLAN formation over beta1 integrin-induced levels, whereas anti-CD47 mAb B6H12.2 inhibited this increase. PP2, NSC23766, and Trio siRNA decreased beta3-induced CLAN formation by 72%, 45%, and 67%, respectively, whereas LY294002 and dominant negative Tiam1 had no effect. LY294002 decreased beta1 integrin-mediated CLAN formation by 42%, and PP2 completely blocked it. Distinct beta1 and alphavbeta3 integrin signaling pathways converge to enhance CLAN formation. beta1-Mediated CLAN formation was PI-3K dependent, whereas beta3-mediated CLAN formation was CD47 and Rac1/Trio dependent and might have been regulated by thrombospondin-1. Both integrin pathways were Src dependent.

Maximizing tumor cell invasion

The Rho family of proteins, com-prised of Rho, Rac, and Cdc42 is impli-cated actomyosin contractility andmicrotubule arrangement (Ridley, 2001;Scott, 2002). RhoA, B and C activateRho kinase (ROCK), which causes theformation of actin stress fibers by rein-forcing actomysin contractile force. Racactivation, on the other hand, results inlamellipodia formation (Ridley, 2001).Tumor cell migration is highly depen-dent on cellular actin cytoskeletondynamics. In order for cancer cells tometastasize, the primary tumor mustinvade and migrate through tissues soas to reside in a secondary location.The invasion process can either be pro-tease-dependent or -independent. Todate, cancer cells exhibit two forms ofcellular movement: mesenchymal thatinvolves elongated cells and amoeboidthat involves rounded blebbing cells.The mesenchymal mode of invasionengages the extracellular matrixremodeling by proteases, whereas theamoeboid movement is proteases-inde-pendent; instead, it relies on extracellu-lar matrix remodeling by force (Sahaiand Marshall, 2003). Ameoboid move-ment is a result of ROCK activation asit requires high actomyosin contractibil-ity. Although aberrant small Rho-GTPase activation has been linked totumor cell metastasis, little is knownabout how tumor cells switch frommesenchymal to amoeboid modes ofmovement or vice versa.Sanz-Moreno et al. (2008) unveiledthe factors that control the differentmodes of cell mobility through a sys-tematic guanine nucleotide exchangefactors (GEFs) siRNA screening. Thestudy aimed to identify GEFs that regu-late cell morphology and movement inorder to understand the interchangebetween two different modes of move-ment (mesenchymal and amoeboidmovement) in melanoma cells. Fromthe screen, DOCK3 was discovered tocontrol elongated movement. DOCK3 isa Rac specific GEF that, together withNEDD9, regulates Rac and WAVE2 toinitiate mesenchymal movement, thusinhibiting amoeboid movement. Con-versely, amoeboid movement requiresRhoA activation of ROCK and ARHGAP,which in turn inactivates mesenchymalmovement.The siRNA based screen was firsttested in A375M2 cell line, a low meta-static melanoma cells that are in amoe-boid and mesenchymal transition. Fromthe 83 GEFs that were targeted, thesuppression of DOCK3 expressionresulted in the exhibition of an ameo-boid morphology, and the inhibition ofthe transition from the amoeboid tomesenchymal mode. In this study,Sanz-Moreno et al. also found that inhi-bition of ROCK activity resulted in aconversion of the round cells to elon-gated cells, although it did not affectthe speed of cancer cell migration.DOCK3’s effect is specific, as otherDBL families do not cause cell elonga-tion. In addition, knock-down of DOCK3or expression of an internal deletionmutant, DELDOCK3, suppressed theconversion of the rounded cells to elon-gated cells. Similarly, Rac1 inhibition bysiRNA or by inhibitor also abolished theelongated morphology. Taken together

Mechanical stretch decreases migration of alveolar epithelial cells through mechanisms involving Rac1 and Tiam1

Mechanical ventilation can overdistend the lungs or generate shear forces in them during repetitive opening/closing, contributing to lung injury and inflammation in patients with acute respiratory distress syndrome (ARDS). Repair of the injured lung epithelium is important for restoring normal barrier and lung function. In the current study, we investigated the effects of cyclic mechanical strain (CS), constant distention strain (CD), and simulated positive end-expiratory pressure (PEEP) on activation of Rac1 and wound closure of rat primary alveolar type 2 (AT2) cells. Cyclic stretch inhibited the migration of wounded AT2 cells in a dose-dependent manner with no inhibition occurring with 5% CS, but significant inhibition with 10% and 15% CS. PEEP conditions were investigated by stretching AT2 cells to 15% maximum strain (at a frequency of 10 cycles/min) with relaxation to 10% strain. AT2 cells were also exposed to 20% CD. All three types of mechanical strain inhibited wound closure of AT2 cells compared with static controls. Since lamellipodial extensions in migrating cells at the wound edge were significantly smaller in stretched cells, we measured Rac1 activity and found it to be decreased in stretched cells. We also demonstrate that Tiam1, a Rac1-specific guanine nucleotide exchange factor, was expressed mainly in the cytosol of AT2 cells exposed to mechanical strain compared with membrane localization in static cells. Downregulation of Tiam1 with 100 microM NSC-23766 inhibited activation of Rac1 and migration of AT2 cells, suggesting its involvement in repair mechanisms of AT2 cells subjected to mechanical strain.

Cross talk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids

We previously reported that the barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cells (ECs) delineate the role of Rac- and Cdc42-dependent mechanisms and described the involvement of the focal adhesion (FA) protein paxillin in enhancement of the EC barrier upon OxPAPC challenge. This study examined a potential role of paxillin in the feedback mechanism of Rac regulation by FAs in OxPAPC-stimulated ECs. Our results demonstrate that OxPAPC induced Rac-dependent, Rho-independent peripheral accumulation of paxillin-containing FAs and time-dependent paxillin phosphorylation. Molecular inhibition of Rac decreased association of paxillin with the Rac-specific guanine nucleotide exchange factor beta-PIX. Molecular inhibition of paxillin also attenuated OxPAPC-induced enhancement of adherens junctions critical for the EC barrier-protective response, accumulation of vascular endothelial cadherin in the membrane fractions, and decreased activation of Rac and its effector p21-activated kinase (PAK1). Expression of paxillin with a mutated PAK1-dependent phosphorylation site (S273A) attenuated OxPAPC-induced PAK1 activation and the EC barrier-protective response. These results suggest that PAK1-specific paxillin phosphorylation at Ser(273) is critically involved in the positive-feedback regulation of the Rac-PAK1 pathway and may contribute to sustained enhancement of the EC barrier caused by oxidized phospholipids.

Specific Recognition of Rac2 and Cdc42 by DOCK2 and DOCK9 Guanine Nucleotide Exchange Factors

Recognition of cognate Rho GTPases by guanine-nucleotide exchange factors (GEF) is fundamental to Rho GTPase signaling specificity. Two main GEF families use either the Dbl homology (DH) or the DOCK homology region 2 (DHR-2) catalytic domain. How DHR-2-containing GEFs distinguish between the GTPases Rac and Cdc42 is not known. To determine how these GEFs specifically recognize the two Rho GTPases, we studied the amino acid sequences in Rac2 and Cdc42 that are crucial for activation by DOCK2, a Rac-specific GEF, and DOCK9, a distantly related Cdc42-specific GEF. Two elements in the N-terminal regions of Rac2 and Cdc42 were found to be essential for specific interactions with DOCK2 and DOCK9. One element consists of divergent amino acid residues in the switch 1 regions of the GTPases. Significantly, these residues were also found to be important for GTPase recognition by Rac-specific DOCK180, DOCK3, and DOCK4 GEFs. These findings were unexpected because the same residues were shown previously to interact with GTPase effectors rather than GEFs. The other element comprises divergent residues in the beta3 strand that are known to mediate specific recognition by DH domain containing GEFs. Remarkably, Rac2-to-Cdc42 substitutions of four of these residues were sufficient for Rac2 to be specifically activated by DOCK9. Thus, DOCK2 and DOCK9 specifically recognize Rac2 and Cdc42 through their switch 1 as well as beta2-beta3 regions and the mode of recognition via switch 1 appears to be conserved among diverse Rac-specific DHR-2 GEFs.

Rho-Kinase Phosphorylates PAR-3 and Disrupts PAR Complex Formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.