G2Cdb::Gene report

Gene id
G00001481
Gene symbol
RAC2 (HGNC)
Species
Homo sapiens
Description
ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)
Orthologue
G00000232 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000030487 (Vega human gene)
Gene
ENSG00000128340 (Ensembl human gene)
5880 (Entrez Gene)
545 (G2Cdb plasticity & disease)
RAC2 (GeneCards)
Literature
602049 (OMIM)
Marker Symbol
HGNC:9802 (HGNC)
Protein Sequence
P15153 (UniProt)

Synonyms (1)

  • EN-7

Literature (64)

Pubmed - other

  • Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells.

    Niggli V, Schlicht D and Affentranger S

    Department of Pathology, University of Bern, Murtenstr. 31, CH-3010 Bern, Switzerland. verena.niggli@pathology.unibe.ch

    Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis.

    Biochemical and biophysical research communications 2009;386;4;688-92

  • Human neutrophils coordinate chemotaxis by differential activation of Rac1 and Rac2.

    Zhang H, Sun C, Glogauer M and Bokoch GM

    Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, CA 92037-1092, USA.

    Rac1 and Rac2, members of the small Rho GTPase family, play essential roles in coordinating directional migration and superoxide production during neutrophil responses to chemoattractants. Although earlier studies in Rac1 and Rac2 knockout mice have demonstrated unique roles for each Rac isoform in chemotaxis and NADPH oxidase activation, it is still unclear how human neutrophils use Rac1 and Rac2 to achieve their immunological responses to foreign agent stimulation. In the current study, we used TAT dominant-negative Rac1-T17N and Rac2-T17N fusion proteins to acutely alter the activity of Rac1 and Rac2 individually in human neutrophils. We demonstrate distinct activation kinetics and different roles for Rac1 and Rac2 in response to low vs high concentrations of fMLP. These observations were verified using neutrophils from mice in which Rac1 or Rac2 was genetically absent. Based on these results, we propose a model to explain how human neutrophils kill invading microbes while limiting oxidative damage to the adjacent surrounding healthy tissue through the differential activation of Rac1 and Rac2 in response to different concentrations of chemoattractant.

    Funded by: NIGMS NIH HHS: GM39434, R01 GM039434-21

    Journal of immunology (Baltimore, Md. : 1950) 2009;183;4;2718-28

  • Lead exposure, polymorphisms in genes related to oxidative stress, and risk of adult brain tumors.

    Bhatti P, Stewart PA, Hutchinson A, Rothman N, Linet MS, Inskip PD and Rajaraman P

    Department of Health ad Human Services, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland, USA. pbhatti@fhcrc.org

    There is some evidence that oxidative stress plays a role in lead-induced toxicity. Mechanisms for dealing with oxidative stress may be of particular relevance in the brain given the high rate of oxygen metabolism. Using a hospital-based case-control study, we investigated the role of oxidative stress in the potential carcinogenicity of lead through examination of effect modification of the association between occupational lead exposure and brain tumors by single nucleotide polymorphisms in genes with functions related to oxidative stress. The study included 362 patients with glioma (176 of which had glioblastoma multiforme), 134 patients with meningioma, and 494 controls. Lead exposure was estimated by expert review of detailed job history data for each participant. We evaluated effect modification with 142 single nucleotide polymorphisms using likelihood ratio tests that compared nested unconditional logistic regression models that did and did not include a cross-product term for cumulative lead exposure and genotype. When the analyses were restricted to cases with glioblastoma multiforme, RAC2 rs2239774 and two highly correlated GPX1 polymorphisms (rs1050450 and rs18006688) were found to significantly modify the association with lead exposure (P <or= 0.05) after adjustment for multiple comparisons. Furthermore, the same GPX1 polymorphisms and XDH rs7574920 were found to significantly modify the association between cumulative lead exposure and meningioma. Although the results of this study provide some evidence that lead may cause glioblastoma multiforme and meningioma through mechanisms related to oxidative damage, the results must be confirmed in other populations.

    Funded by: Intramural NIH HHS: NIH0011946573, Z01 CP010135-12; NCI NIH HHS: N01-CO-12400, N01CO12400

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2009;18;6;1841-8

  • Genetic polymorphisms in nitric oxide synthase genes modify the relationship between vegetable and fruit intake and risk of non-Hodgkin lymphoma.

    Han X, Zheng T, Lan Q, Zhang Y, Kilfoy BA, Qin Q, Rothman N, Zahm SH, Holford TR, Leaderer B and Zhang Y

    Yale University School of Public Health, New Haven, CT 06520-8034, USA.

    Oxidative damage caused by reactive oxygen species and other free radicals is involved in carcinogenesis. It has been suggested that high vegetable and fruit intake may reduce the risk of non-Hodgkin lymphoma (NHL) as vegetables and fruit are rich in antioxidants. The aim of this study is to evaluate the interaction of vegetable and fruit intake with genetic polymorphisms in oxidative stress pathway genes and NHL risk. This hypothesis was investigated in a population-based case-control study of NHL and NHL histologic subtypes in women from Connecticut, including 513 histologically confirmed incident cases and 591 randomly selected controls. Gene-vegetable/fruit joint effects were estimated using unconditional logistic regression model. The false discovery rate method was applied to adjust for multiple comparisons. Significant interactions with vegetable and fruit intake were mainly found for genetic polymorphisms on nitric oxide synthase (NOS) genes among those with diffuse large B-cell lymphoma and follicular lymphoma. Two single nucleotide polymorphisms in the NOS1 gene were found to significantly modify the association between total vegetable and fruit intake and risk of NHL overall, as well as the risk of follicular lymphoma. When vegetables, bean vegetables, cruciferous vegetables, green leafy vegetables, red vegetables, yellow/orange vegetables, fruit, and citrus fruits were examined separately, strong interaction effects were narrowed to vegetable intake among patients with diffuse large B-cell lymphoma. Our results suggest that genetic polymorphisms in oxidative stress pathway genes, especially in the NOS genes, modify the association between vegetable and fruit intake and risk of NHL.

    Funded by: FIC NIH HHS: 1D43TW007864-01, D43 TW007864; Intramural NIH HHS: Z01 CP010123-12; NCI NIH HHS: R01 CA062006; NCRR NIH HHS: UL1 RR024139

    Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 2009;18;5;1429-38

  • OAG induces an additional PKC-, PI3K-, and Rac2-mediated signaling pathway up-regulating NOX2 activity, independently of Ca2+ entry.

    Bréchard S, Salsmann A and Tschirhart EJ

    Life Sciences Research Unit, University of Luxembourg, Avenue de la Faïencerie, L-1511 Luxembourg.

    The requirement of calcium ion (Ca(2)(+)) entry for neutrophil NADPH oxidase (NOX2) regulation is clearly established. However, its role in the signaling pathway leading to NOX2 activation is still elusive. 1-oleoyl-2-acetyl-sn-glycerol (OAG) causes an increase in NOX2 activity and has been shown to directly modulate Ca(2)(+) channels unrelated to the well-known store-operated Ca(2)(+) entry (SOCE) mechanism. In our study, we have investigated the potential role of OAG in Ca(2)(+) influx-mediated NOX2 activity in neutrophil-like-differentiated HL-60 cells to further characterize second signals involved in the regulation of NOX2. OAG inhibited fMLF- and thapsigargin-induced Ca(2)(+) entry, a phenomenon that was not restored by protein kinase C (PKC) or PI3K blockade. Addition of OAG resulted in a rapid decrease of maximal intracellular Ca(2)(+) concentration induced by thapsigargin. Both results suggest that OAG has an inhibitory effect, independent of PI3K and PKC, on the regulation of SOCE. In contrast to SOCE inhibition, OAG-induced NOX2 activation was mediated by PKC and PI3K. Our data establish that both kinases exert their effects through the regulation of Rac2 activity. In addition, OAG potentiated the effect of fMLF on the activation of NOX2 and led to a discernible activity of NOX2 upon thapsigargin stimulation. In conclusion, our results demonstrate that an additional PKC- and/or PI3K-dependent signal may act in synergy with Ca(2)(+) influx to trigger NOX2 activation.

    Journal of leukocyte biology 2009;85;4;638-47

  • Rho GTPase-mediated pathways in mature CD4+ T cells.

    Pernis AB

    Department of Medicine, Columbia University, 630 West 168th Street, New York, NY 10032, USA. abp1@columbia.edu

    Effective immune responses require the appropriate activation and differentiation of peripheral CD4(+) T cells. These processes need to be followed by the timely elimination of the responding T cells in order to restore T cell homeostasis. Defects in the appropriate regulation of T cell activation, expansion, and survival underlie the pathogenesis of many autoimmune disorders including SLE. The molecular machinery employed by T cells to properly control these processes and prevent the onset of autoimmunity has not been fully elucidated. Rho GTPases (which include the Rac, Cdc42, and Rho subfamilies) are molecular switches that control a wide range of cellular processes. Their fundamental role in biology is due to their ability to regulate both cytoskeletal dynamics and a large number of signal transduction pathways. Activation of Rho GTPases is now recognized as a key event in the coordination of immune responses and, particularly, in the activation of T cells. In this review, we will first provide an overview of the role of Rho GTPase-mediated pathways in mature CD4(+) T cells and then we will discuss recent studies, which suggest that deregulation of these pathways may play a role in the pathogenesis of SLE.

    Funded by: NHLBI NIH HHS: R01 HL-62215

    Autoimmunity reviews 2009;8;3;199-203

  • Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling.

    Mitchell T, Lo A, Logan MR, Lacy P and Eitzen G

    Dept. of Cell Biology, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7.

    The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

    Funded by: Canadian Institutes of Health Research: 43913

    American journal of physiology. Cell physiology 2008;295;5;C1354-65

  • Parallel phosphatidylinositol 3-kinase (PI3K)-dependent and Src-dependent pathways lead to CXCL8-mediated Rac2 activation and chemotaxis.

    Sai J, Raman D, Liu Y, Wikswo J and Richmond A

    Department of Cancer Biology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.

    The requirement for phosphatidylinositol 3-kinase (PI3K) in the establishment of cell polarity and motility in a number of cell types has recently come into question. In this study, we demonstrate that inhibition of PI3K by wortmannin in neutrophil-like differentiated HL60 cells expressing CXCR2 resulted in reduced cell motility but normal chemotaxis in response to a gradient of CXCL8. However, wortmannin inhibition of PI3K did impair the ability of cells to re-orient their polarity and respond quickly to a change in the direction of the CXCL8 gradient. We hypothesized that Src-regulated ELMO-Dock2-Rac2 activation mediates chemotaxis in the absence of PI3K activity. Inhibition of Src with the small molecule inhibitor, PP2, or inhibition of Dock2 by shRNA knockdown confirmed the functional role of Src and Dock2 in regulating chemotaxis when PI3K was inhibited. Moreover, neutrophils isolated from bone marrow of hck(-/-)fgr(-/-)lyn(-/-) mice exhibited much more severe inhibition of chemotaxis when PI3K was blocked with wortmannin as compared with neutrophils isolated from bone marrow of wild-type mice. Thus, PI3K and Src-ELMO-Dock2 pathways work in parallel to activate Rac2 and modulate chemotaxis in response to a CXCL8 gradient in neutrophils.

    Funded by: NCI NIH HHS: CA34590, CA68485, R01 CA034590, U54CA113007

    The Journal of biological chemistry 2008;283;39;26538-47

  • Neither replication nor simulation supports a role for the axon guidance pathway in the genetics of Parkinson's disease.

    Li Y, Rowland C, Xiromerisiou G, Lagier RJ, Schrodi SJ, Dradiotis E, Ross D, Bui N, Catanese J, Aggelakis K, Grupe A and Hadjigeorgiou G

    Celera, Alameda, California, United States of America. yonghong.li@celera.com

    Susceptibility to sporadic Parkinson's disease (PD) is thought to be influenced by both genetic and environmental factors and their interaction with each other. Statistical models including multiple variants in axon guidance pathway genes have recently been purported to be capable of predicting PD risk, survival free of the disease and age at disease onset; however the specific models have not undergone independent validation. Here we tested the best proposed risk panel of 23 single nucleotide polymorphisms (SNPs) in two PD sample sets, with a total of 525 cases and 518 controls. By single marker analysis, only one marker was significantly associated with PD risk in one of our sample sets (rs6692804: P = 0.03). Multi-marker analysis using the reported model found a mild association in one sample set (two sided P = 0.049, odds ratio for each score change = 1.07) but no significance in the other (two sided P = 0.98, odds ratio = 1), a stark contrast to the reported strong association with PD risk (P = 4.64x10(-38), odds ratio as high as 90.8). Following a procedure similar to that used to build the reported model, simulated multi-marker models containing SNPs from randomly chosen genes in a genome wide PD dataset produced P-values that were highly significant and indistinguishable from similar models where disease status was permuted (3.13x10(-23) to 4.90x10(-64)), demonstrating the potential for overfitting in the model building process. Together, these results challenge the robustness of the reported panel of genetic markers to predict PD risk in particular and a role of the axon guidance pathway in PD genetics in general.

    PloS one 2008;3;7;e2707

  • Mutational analysis of Rac2 in gliomas.

    Idbaih A, Paris S, Boisselier B, Marie Y, Sanson M, Thillet J, Hoang-Xuan K and Delattre JY

    Journal of neuro-oncology 2008;87;3;365-6

  • Specific recognition of Rac2 and Cdc42 by DOCK2 and DOCK9 guanine nucleotide exchange factors.

    Kwofie MA and Skowronski J

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

    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.

    Funded by: NIAID NIH HHS: AI-42561

    The Journal of biological chemistry 2008;283;6;3088-96

  • Cutting edge: Rac GTPases sensitize activated T cells to die via Fas.

    Ramaswamy M, Dumont C, Cruz AC, Muppidi JR, Gomez TS, Billadeau DD, Tybulewicz VL and Siegel RM

    Immunoregulation Unit, Autoimmunity Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

    In activated CD4(+) T cells, TCR restimulation triggers apoptosis that depends on interactions between the death receptor Fas and its ligand, FasL. This process, termed restimulation-induced cell death (RICD), is a mechanism of peripheral immune tolerance. TCR signaling sensitizes activated T cells to Fas-mediated apoptosis, but what pathways mediate this process are not known. In this study we identify the Rho GTPases Rac1 and Rac2 as essential components in restimulation-induced cell death. RNA interference-mediated knockdown of Rac GTPases greatly reduced Fas-dependent, TCR-induced apoptosis. The ability of Rac1 to sensitize T cells to Fas-induced apoptosis correlated with Rac-mediated cytoskeletal reorganization, dephosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal linker proteins, and the translocation of Fas to lipid raft microdomains. In primary activated CD4(+) T cells, Rac1 and Rac2 were independently required for maximal TCR-induced apoptosis. Activating Rac signaling may be a novel way to sensitize chronically stimulated lymphocytes to Fas-induced apoptosis, an important goal in the treatment of autoimmune diseases.

    Funded by: Intramural NIH HHS; Medical Research Council: MC_U117527252

    Journal of immunology (Baltimore, Md. : 1950) 2007;179;10;6384-8

  • Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils.

    El Bekay R, Alba G, Reyes ME, Chacón P, Vega A, Martín-Nieto J, Jiménez J, Ramos E, Oliván J, Pintado E and Sobrino F

    Departamento de Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, Avda. Sánchez Pizjuán 4, E-41009 Sevilla, Spain.

    Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.

    Journal of molecular endocrinology 2007;39;5;351-63

  • Signaling requirements for translocation of P-Rex1, a key Rac2 exchange factor involved in chemoattractant-stimulated human neutrophil function.

    Zhao T, Nalbant P, Hoshino M, Dong X, Wu D and Bokoch GM

    Department of Immunology, IMM14, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

    PI 3,4,5-trisphosphate [PI(3,4,5)P3; PIP3]-dependent Rac exchanger 1 (P-Rex1) is a Rac-specific guanine nucleotide exchange factor abundant in neutrophils and myeloid cells. As a selective catalyst for Rac2 activation, P-Rex1 serves as an important regulator of human neutrophil NADPH oxidase activity and chemotaxis in response to a variety of extracellular stimuli. The exchange activity of P-Rex1 is synergistically activated by the binding of PIP3 and betagamma subunits of heterotrimeric G proteins in vitro, suggesting that the association of P-Rex1 with membranes is a prerequisite for cellular activation. However, the spatial regulation of endogenous P-Rex1 has not been well defined, particularly in human neutrophils activated through G protein-coupled receptors. Upon stimulation of neutrophil chemoattractant receptors, we observed that P-Rex1 translocated from cytoplasm to the leading edge of polarized cells in a G protein betagamma subunit- and PIP3-dependent manner, where it colocalized with F-actin and its substrate, Rac2. Redistribution of P-Rex1 to the leading edge was also dependent on tyrosine kinase activity and was modulated by cell adhesion. Furthermore, we observed that activation of cAMP-dependent protein kinase A (PKA), which phosphorylates and inactivates P-Rex1, inhibited its translocation. Our data indicate that endogenous P-Rex1 translocates to areas of Rac2 and cytoskeletal activation at the leading edge in response to chemoattractant stimuli in human neutrophils and that this translocation can be negatively modulated by activation of PKA and by cell adhesion.

    Funded by: NHLBI NIH HHS: HL48008, HL70694, HL80706, R01 HL080706, R01 HL080706-10, R01 HL080706-11, R01 HL080706-12; NIGMS NIH HHS: GM39434, R01 GM054597

    Journal of leukocyte biology 2007;81;4;1127-36

  • Characterization of IQGAP1-containing complexes in NK-like cells: evidence for Rac 2 and RACK1 association during homotypic adhesion.

    Meng X, Krokhin O, Cheng K, Ens W and Wilkins JA

    Manitoba Centre for Proteomics and Systems Biology, University of Manitoba, 799 JBRC, 715 McDermot Avenue, Winnipeg, MB, Canada R3E 3P4.

    IQGAP1 is a scaffolding protein that binds to a diverse array of signaling and structural molecules that are often associated with cell polarization and adhesion. Through interaction with its target proteins, IQGAP1 participates in multiple cellular functions, including Ca2+-calmodulin signaling, definition of cytoskeletal architecture, regulation of Cdc42 and Rac1 dependent cytoskeletal changes, and control of E-cadherin mediated intercellular adhesion. These analysis have been largely restricted to cells of epithelial and fibroblast origin. The present studies were initiated to examine the role of IQGAP1 in cellular interactions involving the lymphoid cells. A mass spectrometric based analysis of IQGAP1 containing complexes isolated from the human NK-like cell line, YTS, identified several known and new potential IQGAP1 interaction partners including receptor of activated C kinase 1 (RACK1) and the small GTPase, Rac2. Immunofluorescence analysis of YTS cells indicated that a minor component of IQGAP1 was localized at the cell membrane with the remainder diffusely distributed through out the cytoplasm. However, at sites of cellular contact, there was a marked accumulation of IQGAP1. Staining for RACK1 and Rac2 revealed that both of these proteins accumulated these contact sites. Antibody-based studies suggested that a subset of RACK1 was associated in an IQGAP1-containing complex, which prevented recognition of RACK1 by monoclonal antibody. These results suggest that RACK1, Rac2, and IQGAP1 are components of complexes involved in NK cell homotypic adhesion.

    Journal of proteome research 2007;6;2;744-50

  • Rac1 and Rac2 regulate macrophage morphology but are not essential for migration.

    Wheeler AP, Wells CM, Smith SD, Vega FM, Henderson RB, Tybulewicz VL and Ridley AJ

    Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, 91 Riding House Street, London, W1W 7BS, UK.

    Rac GTPases are believed to contribute to migration in leukocytes by transducing signals from cell surface receptors to the actin and microtubule cytoskeletons. Mammals have three closely related Rac isoforms, Rac1, Rac2 and Rac3, and it is widely assumed that cell migration requires the activity of these Rac GTPases. We have previously shown that Rac1-null mouse macrophages have altered cell shape and reduced membrane ruffling but normal migration speed. Here we investigate the behaviour of macrophages lacking Rac2 (Rac2(-/-)) or Rac1 and Rac2 (Rac1/2(-/-)). Rac2(-/-) macrophages have reduced F-actin levels and lack podosomes, which are integrin-based adhesion sites, and their migration speed is similar to or slightly slower than wild-type macrophages, depending on the substrate. Unexpectedly, Rac1/2(-/-) macrophages, which do not express Rac1, Rac2 or Rac3, migrate at a similar speed to wild-type macrophages on a variety of substrates and perform chemotaxis normally, although their morphology and mode of migration is altered. However, Rac1(-/-) and Rac1/2(-/-) but not Rac2(-/-) macrophages are impaired in their ability to invade through Matrigel. Together, these data show that Rac1 and Rac2 have distinct roles in regulating cell morphology, migration and invasion, but are not essential for macrophage migration or chemotaxis.

    Funded by: Medical Research Council: MC_U117527252

    Journal of cell science 2006;119;Pt 13;2749-57

  • Nox1-dependent reactive oxygen generation is regulated by Rac1.

    Cheng G, Diebold BA, Hughes Y and Lambeth JD

    Department of Pathology and Laboratory Medicine, Emory University Medical School, 615 Michael Street, Atlanta, GA 30322, USA.

    Rac1 has been implicated in the generation of reactive oxygen species (ROS) in several cell types, but the enzymatic origin of the ROS has not been proven. The present studies demonstrate that Nox1, a homolog of the phagocyte NADPH-oxidase component gp91(phox), is activated by Rac1. When Nox1 is co-expressed along with its regulatory subunits NOXO1 and NOXA1, significant ROS generation is seen. Herein, co-expression of constitutively active Rac1(G12V), but not wild-type Rac1, resulted in marked further stimulation of activity. Decreased Rac1 expression using small interfering RNA reduced Nox1-dependent ROS. CDC42(G12V) failed to increase activity, and small interfering RNA directed against CDC42 failed to decrease activity, pointing to specificity for Rac. TPR domain mutants of NOXA1 that interfere with Rac1 binding were ineffective in supporting Nox1-dependent ROS generation. Immunoprecipitation experiments demonstrated a complex containing Rac1(G12V), NOXO1, NOXA1, and Nox1. CDC42(G12V) could not substitute for Rac1(G12V) in such a complex. Nox1 formed a complex with Rac1(G12V) that was independent of NOXA1 and NOXO1, consistent with direct binding of Rac1(G12V) to Nox1. Rac1(G12V) interaction with NOXA1 was enhanced by Nox1 and NOXO1, suggesting cooperative binding. A model is presented comparing activation by regulatory subunits of Nox1 versus gp91(phox) (Nox2) in which Rac1 activation provides a major trigger that acutely activates Nox1-dependent ROS generation.

    Funded by: NCI NIH HHS: CA 105116, CA 84138

    The Journal of biological chemistry 2006;281;26;17718-26

  • HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway.

    Toschi E, Bacigalupo I, Strippoli R, Chiozzini C, Cereseto A, Falchi M, Nappi F, Sgadari C, Barillari G, Mainiero F and Ensoli B

    AIDS National Center, Istituto Superiore di Sanità, 00161 Rome, Italy.

    Tat, the transactivator of HIV-1 gene expression, is released by acutely HIV-1-infected T-cells and promotes adhesion, migration, and growth of inflammatory cytokine-activated endothelial and Kaposi's sarcoma cells. It has been previously demonstrated that these effects of Tat are due to its ability to bind through its arginine-glycine-aspartic (RGD) region to the alpha5beta1 and alphavbeta3 integrins. However, the signaling pathways linking Tat to the regulation of cellular functions are incompletely understood. Here, we report that Tat ligation on human endothelial cells results in the activation of the small GTPases Ras and Rac and the mitogen-activated protein kinase ERK, specifically through its RGD region. In addition, we demonstrated that Tat activation of Ras, but not of Rac, induces ERK phosphorylation. We also found that the receptor proximal events accompanying Tat-induced Ras activation are mediated by tyrosine phosphorylation of Shc and recruitment of Grb2. Moreover, Tat enabled endothelial cells to progress through the G1 phase in response to bFGF, and the process is linked to ERK activation. Taken together, these data provide novel evidence about the ability of Tat to activate the Ras-ERK cascade which may be relevant for endothelial cell proliferation and for Kaposi's sarcoma progression.

    Molecular biology of the cell 2006;17;4;1985-94

  • Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression.

    Coiras M, Camafeita E, Ureña T, López JA, Caballero F, Fernández B, López-Huertas MR, Pérez-Olmeda M and Alcamí J

    AIDS Immunopathology Unit, National Centre of Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

    The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.

    Proteomics 2006;6 Suppl 1;S63-73

  • Rac2 concentrations in umbilical cord neutrophils.

    Meade VM, Barese CN, Kim C, Njinimbam CG, Marchal CC, Ingram DA, Clapp DW, Dinauer MC and Yoder MC

    Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

    Background: Human newborn infants display a variety of immunodeficiencies of immaturity, including diminished neutrophil adhesion, chemotaxis, and migration. Rac2, a guanosine triphosphate-binding protein, is an essential regulator of human neutrophil migration and chemotaxis. Since human subjects and mice deficient in Rac2 display deficiencies in neutrophil functions similar to newborn infants, we postulated that newborn neutrophils may be deficient in Rac2.

    Objectives: The aim of the study was to measure Rac1 and Rac2 concentrations in neutrophils from umbilical cord blood.

    Methods: Neutrophils from cord and adult blood were isolated, total cell lysates extracted, and Rac protein concentrations determined using Western blot analysis.

    Results: Rac2 concentrations were significantly lower in the neutrophil protein lysates isolated from cord blood compared to adult blood despite similar levels of Rac1.

    Conclusions: Diminished Rac2 expression in cord blood neutrophils may contribute to the defects observed in cord blood neutrophil function.

    Funded by: NICHD NIH HHS: T32 HD04299

    Biology of the neonate 2006;90;3;156-9

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • p190 Rho-GTPase activating protein associates with plexins and it is required for semaphorin signalling.

    Barberis D, Casazza A, Sordella R, Corso S, Artigiani S, Settleman J, Comoglio PM and Tamagnone L

    Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, Candiolo, Torino 10060, Italy.

    Plexins are transmembrane receptors for semaphorins, guiding cell migration and axon extension. Plexin activation leads to the disassembly of integrin-based focal adhesive structures and to actin cytoskeleton remodelling and inhibition of cell migration; however, the underlying molecular mechanisms are unclear. We consistently observe a transient decrease of cellular RhoA-GTP levels upon plexin activation in adherent cells. One of the main effectors of RhoA downregulation is p190, a ubiquitously expressed GTPase activating protein (GAP). We show that, in p190-deficient fibroblasts, the typical functional activities mediated by plexins (such as cell collapse and inhibition of integrin-based adhesion) are blocked or greatly impaired. Notably, the functional response can be rescued in these cells by re-expressing exogenous p190, but not a mutant form specifically lacking RhoGAP activity. We furthermore demonstrate that semaphorin function is blocked in epithelial cells, primary endothelial cells and neuroblasts upon treatment with small interfering RNAs that knockdown p190 expression. Finally, we show that p190 transiently associates with plexins, and its RhoGAP activity is increased in response to semaphorin stimulation. We conclude that p190-RhoGAP is crucially involved in semaphorin signalling to the actin cytoskeleton, via interaction with plexins.

    Journal of cell science 2005;118;Pt 20;4689-700

  • Rac2 expression and mutation in human brain tumors.

    Hwang SL, Lieu AS, Chang JH, Cheng TS, Cheng CY, Lee KS, Lin CL, Howng SL and Hong YR

    Division of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.

    Background: Rac1 and Rac2 are interchangeable in NADPH oxidase activation. Rac1 plays an important role in regulating nuclear signalling and in the activation of transcriptional factors that regulate gene expression and cell growth. Our previous study observed mutation in effector region of Rac1 gene in brain tumors. Little is known about the expression and mutation of Rac2 in human brain tumors.

    Method: We examined the expression of Rac2 by using reverse transcriptase-polymerase chain reaction (RT-PCR) and northern blot analysis and the mutation of Rac2 gene by using DNA sequence analysis.

    Findings: The decreased expression of Rac2 was found in 15 cases (57.7%) including 8 of 10 astrocytomas, 2 of 8 meningiomas, and 5 of 8 pituitary adenomas. Two of 13 cases with decreased expression of Rac2 had gene mutation. Only two of 26 cases had Rac2 overexpression in which no Rac2 gene mutation was found. Four of 8 cases with normal Rac2 expression had Rac2 gene mutation. The site of Rac2 gene mutation had no hot spots and was not concentrated in the effector region.

    Conclusions: Our results showed a low frequency of mutation and no hot spots of mutation in Rac2 gene in brain tumors, suggesting a decreased possibility of Rac2 in the brain tumorigenesis. The role of high frequency of decreased Rac2 expression in brain tumors, particularly in malignant astrocytomas, needs further investigations to be elucidated.

    Acta neurochirurgica 2005;147;5;551-4; discussion 554

  • High-throughput mapping of a dynamic signaling network in mammalian cells.

    Barrios-Rodiles M, Brown KR, Ozdamar B, Bose R, Liu Z, Donovan RS, Shinjo F, Liu Y, Dembowy J, Taylor IW, Luga V, Przulj N, Robinson M, Suzuki H, Hayashizaki Y, Jurisica I and Wrana JL

    Program in Molecular Biology and Cancer, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada, M5G 1X5.

    Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.

    Funded by: NIGMS NIH HHS: P50 GM-62413

    Science (New York, N.Y.) 2005;307;5715;1621-5

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • DEF6, a novel PH-DH-like domain protein, is an upstream activator of the Rho GTPases Rac1, Cdc42, and RhoA.

    Mavrakis KJ, McKinlay KJ, Jones P and Sablitzky F

    Institute of Genetics, Queen's Medical Centre, School of Biology, The University of Nottingham, Nottingham NG7 2UH, UK.

    In this paper, we describe the characterization of DEF6, a novel PH-DH-like protein related to SWAP-70 that functions as an upstream activator of Rho GTPases. In NIH 3T3 cells, stimulation of the PI 3-kinase signaling pathway with either H2O2 or platelet-derived growth factor (PDGF) resulted in the translocation of an overexpressed DEF6-GFP fusion protein to the cell membrane and induced the formation of filopodia and lamellipodia. In contrast to full-length DEF6, expression of the DH-like (DHL) domain as a GFP fusion protein potently induced actin polymerization, including stress fiber formation in COS-7 cells, in the absence of PI 3-kinase signaling, indicating that it was constitutively active. The GTP-loading of Cdc42 was strongly enhanced in NIH 3T3 cells expressing the DH domain while filopodia formation, membrane ruffling, and stress fiber formation could be inhibited by the co-expression of the DH domain with dominant negative mutants of either N17Rac1, N17Cdc42, or N19RhoA, respectively. This indicated that DEF6 acts upstream of the Rho GTPases resulting in the activation of the Cdc42, Rac1, and RhoA signaling pathways. In vitro, DEF6 specifically interacted with Rac1, Rac2, Cdc42, and RhoA, suggesting a direct role for DEF6 in the activation of Rho GTPases. The ability of DEF6 to both stimulate actin polymerization and bind to filamentous actin suggests a role for DEF6 in regulating cell shape, polarity, and movement.

    Experimental cell research 2004;294;2;335-44

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis.

    Janardhan A, Swigut T, Hill B, Myers MP and Skowronski J

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

    The infectious cycle of primate lentiviruses is intimately linked to interactions between cells of the immune system. Nef, a potent virulence factor, alters cellular environments to increase lentiviral replication in the host, yet the mechanisms underlying these effects have remained elusive. Since Nef likely functions as an adaptor protein, we exploited a proteomic approach to directly identify molecules that Nef targets to subvert the signaling machinery in T cells. We purified to near homogeneity a major Nef-associated protein complex from T cells and identified by mass spectroscopy its subunits as DOCK2-ELMO1, a key activator of Rac in antigen- and chemokine-initiated signaling pathways, and Rac. We show that Nef activates Rac in T cell lines and in primary T cells following infection with HIV-1 in the absence of antigenic stimuli. Nef activates Rac by binding the DOCK2-ELMO1 complex, and this interaction is linked to the abilities of Nef to inhibit chemotaxis and promote T cell activation. Our data indicate that Nef targets a critical switch that regulates Rac GTPases downstream of chemokine- and antigen-initiated signaling pathways. This interaction enables Nef to influence multiple aspects of T cell function and thus provides an important mechanism by which Nef impacts pathogenesis by primate lentiviruses.

    Funded by: NIAID NIH HHS: AI-42561, R01 AI042561

    PLoS biology 2004;2;1;E6

  • A genome annotation-driven approach to cloning the human ORFeome.

    Collins JE, Wright CL, Edwards CA, Davis MP, Grinham JA, Cole CG, Goward ME, Aguado B, Mallya M, Mokrab Y, Huckle EJ, Beare DM and Dunham I

    The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

    We have developed a systematic approach to generating cDNA clones containing full-length open reading frames (ORFs), exploiting knowledge of gene structure from genomic sequence. Each ORF was amplified by PCR from a pool of primary cDNAs, cloned and confirmed by sequencing. We obtained clones representing 70% of genes on human chromosome 22, whereas searching available cDNA clone collections found at best 48% from a single collection and 60% for all collections combined.

    Genome biology 2004;5;10;R84

  • Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex.

    Zhao X, Carnevale KA and Cathcart MK

    Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.

    Funded by: NHLBI NIH HHS: HL51068, HL61971

    The Journal of biological chemistry 2003;278;42;40788-92

  • Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils.

    Forsberg M, Druid P, Zheng L, Stendahl O and Särndahl E

    Department of Cell Biology, Faculty of Health Sciences, Linköping University, Sweden.

    Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcgammaR)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcgammaR and CR3- and FcgammaR-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or FcgammaRs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.

    Journal of leukocyte biology 2003;74;4;611-9

  • Direct interaction of Rnd1 with Plexin-B1 regulates PDZ-RhoGEF-mediated Rho activation by Plexin-B1 and induces cell contraction in COS-7 cells.

    Oinuma I, Katoh H, Harada A and Negishi M

    Laboratory of Molecular Neurobiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.

    Plexins are receptors for the axon guidance molecule semaphorins, and several lines of evidence suggest that Rho family small GTPases are implicated in the downstream signaling of Plexins. Recent studies have demonstrated that Plexin-B1 activates RhoA and induces growth cone collapse through Rho-specific guanine nucleotide exchange factor PDZ-RhoGEF. Here we show that Rnd1, a member of Rho family GTPases, directly interacted with the cytoplasmic domain of Plexin-B1. In COS-7 cells, coexpression of Rnd1 and Plexin-B1 induced cell contraction in response to semaphorin 4D (Sema4D), a ligand for Plexin-B1, whereas expression of Plexin-B1 alone or coexpression of Rnd1 and a Rnd1 interaction-defective mutant of Plexin-B1 did not. The Sema4D-induced contraction in Plexin-B1/Rnd1-expressing COS-7 cells was suppressed by dominant negative RhoA, a Rho-associated kinase inhibitor, a dominant negative form of PDZ-RhoGEF, or deletion of the carboxyl-terminal PDZ-RhoGEF-binding region of Plexin-B1, indicating that the PDZ-RhoGEF/RhoA/Rho-associated kinase pathway is involved in this morphological effect. We also found that Rnd1 promoted the interaction between Plexin-B1 and PDZ-RhoGEF and thereby dramatically potentiated the Plexin-B1-mediated RhoA activation. We propose that Rnd1 plays an important role in the regulation of Plexin-B1 signaling, leading to Rho activation during axon guidance and cell migration.

    The Journal of biological chemistry 2003;278;28;25671-7

  • Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases.

    Takeya R, Ueno N, Kami K, Taura M, Kohjima M, Izaki T, Nunoi H and Sumimoto H

    Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

    The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

    The Journal of biological chemistry 2003;278;27;25234-46

  • Down-regulation of Rac activity during beta 2 integrin-mediated adhesion of human neutrophils.

    Dib K, Melander F, Axelsson L, Dagher MC, Aspenström P and Andersson T

    Department of Laboratory Medicine, Lund University, Malmö University Hospital, Entrance 78, SE-205 02 Malmö, Sweden. Karim.Dib@exppat.mas.lu.se

    In human neutrophils, beta2 integrin engagement mediated a decrease in GTP-bound Rac1 and Rac2. Pretreatment of neutrophils with LY294002 or PP1 (inhibiting phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases, respectively) partly reversed the beta2 integrin-induced down-regulation of Rac activities. In contrast, beta2 integrins induced stimulation of Cdc42 that was independent of Src family members. The PI 3-kinase dependence of the beta2 integrin-mediated decrease in GTP-bound Rac could be explained by an enhanced Rac-GAP activity, since this activity was blocked by LY204002, whereas PP1 only had a minor effect. The fact that only Rac1 but not Rac2 (the dominating Rac) redistributed to the detergent-insoluble fraction and that it was independent of GTP loading excludes the possibility that down-regulation of Rac activities was due to depletion of GTP-bound Rac from the detergent-soluble fraction. The beta2 integrin-triggered relocalization of Rac1 to the cytoskeleton was enabled by a PI 3-kinase-induced dissociation of Rac1 from LyGDI. The dissociations of Rac1 and Rac2 from LyGDI also explained the PI 3-kinase-dependent translocations of Rac GTPases to the plasma membrane. However, these accumulations of Rac in the membrane, as well as that of p47phox and p67phox, were also regulated by Src tyrosine kinases. Inasmuch as Rac GTPases are part of the NADPH oxidase and the respiratory burst is elicited in neutrophils adherent by beta2 integrins, our results indicate that activation of the NADPH oxidase does not depend on the levels of Rac-GTP but instead requires a beta2 integrin-induced targeting of the Rac GTPases as well as p47phox and p67phox to the plasma membrane.

    The Journal of biological chemistry 2003;278;26;24181-8

  • Ephrin stimulation modulates T cell chemotaxis.

    Sharfe N, Freywald A, Toro A, Dadi H and Roifman C

    Division of Immunology and Allergy, Department of Pediatrics, Infection, Immunity, Injury and Repair Program, Research Institute, The Hospital for Sick Children and the University of Toronto, Toronto, Canada.

    Eph receptor tyrosine kinases and their ligands, the ephrins, are known to play an important role in regulating cell migration and targeting in neuronal and endothelial cells. Recently, it has been shown that lymphoid cells also express Eph receptors, raising the possibility that Eph receptors may similarly regulate lymphocyte migration. Chemotaxis in response to soluble chemokine factors is an essential facet of T cell biology. We demonstrate here that T cell chemotaxis in response to both the stromal cell-derived factor (SDF)-1alpha and macrophage inflammatory protein 3beta chemokines is modulated by costimulation with ephrins. Both ephrin-A and ephrin-B ligands were found to modify the chemotactic responses of a T cell line and primary T cells. Ephrin-A1, in particular, strongly inhibited chemotaxis. In accordance with the tyrosine kinase activity of EphA receptors, ephrin-A1 stimulation induced rapid intracellular tyrosine phosphorylation in T cells. Although strongly inhibiting chemotaxis, ephrin-A1 costimulus did not affect many of the signaling events downstream of the SDF-1alpha receptor CXCR4, including calcium flux and activation of MAPK. Rather, ephrin-A1 altered the balance of small G protein activity in T cells. Ephrin-A1 stimulation prevented SDF-1alpha-induced activation of cdc42, while simultaneously inducing rho activation. Ultimately, ephrin-A1 was found to inhibit chemokine-induced actin polymerization, thereby blocking migration. Ubiquitous ephrin expression in vivo creates enormous potential for T cells to encounter these ligands, suggesting that Eph receptors and ephrins may be important regulators of T cell migration.

    European journal of immunology 2002;32;12;3745-55

  • DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription.

    Nishihara H, Maeda M, Tsuda M, Makino Y, Sawa H, Nagashima K and Tanaka S

    Laboratory of Molecular and Cellular Pathology, Hokkaido University School of Medicine, N 15, W7, Kita-ku, Sapporo 060-8638, Japan.

    DOCK2, a CDM family protein exclusively found in hematopoietic cells, has been shown to play a role in lymphocyte migration by the regulation of actin cytoskeleton. Although DOCK2 has been shown to induce the activation of Rac1, the regulatory mechanism of Rac2, which is a hematopoietic cell-specific small GTPase, is still unknown. In this study, we examined the role of DOCK2 in the activation of Rac2 in hematopoietic cells. DOCK2 was found to associate with the zeta subunit of the CD3 complex of T cell receptors in Jurkat cells and to activate forced expressed Rac2 in 293T cells. In addition, the stable expression of DOCK2 in Jurkat cells exhibited the elevated activity of endogenous Rac2. Furthermore, the transcriptional activity of interleukin-2 (IL-2) was enhanced in DOCK2-expressing Jurkat cells and the dominant negative form of Rac2 suppressed its elevated IL-2 promoter activity. These results suggest that DOCK2 mediates TCR-dependent activation of Rac2, leading to the regulation of IL-2 promoter activity in T cells.

    Biochemical and biophysical research communications 2002;296;3;716-20

  • Interaction between p21-activated protein kinase and Rac during differentiation of HL-60 human promyelocytic leukemia cell induced by all-trans-retinoic acid.

    Nisimoto Y and Ogawa H

    Department of Biochemistry, Aichi Medical University, School of Medicine, Nagakute, Aichi, Japan. nisiio@amugw.aichi-med-u.ac.jp

    Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O(2)(-) concentration upon incubation with all-trans-retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA- induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K(d) value of 6.7 nm.

    European journal of biochemistry 2002;269;10;2622-9

  • Plexin signaling via off-track and rho family GTPases.

    Whitford KL and Ghosh A

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

    Two papers in this issue of Neuron examine new aspects of Semaphorin signaling via Plexin receptors. Winberg et al. present evidence that the transmembrane protein Off-track (OTK) interacts biochemically and genetically with Plexin A and is important for Sema 1a repulsive signaling. Hu et al. examine the coupling of Plexin B to Rac and RhoA and propose that Plexin B signaling involves inhibition of Rac function by direct sequestration and simultaneous activation of RhoA.

    Neuron 2001;32;1;1-3

  • Crystal structure of the Rac1-RhoGDI complex involved in nadph oxidase activation.

    Grizot S, Fauré J, Fieschi F, Vignais PV, Dagher MC and Pebay-Peyroula E

    Institut de Biologie Structurale, CEA-CNRS-UJF, UMR 5075, 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France.

    A heterodimer of prenylated Rac1 and Rho GDP dissociation inhibitor was purified and found to be competent in NADPH oxidase activation. Small angle neutron scattering experiments confirmed a 1:1 stoichiometry. The crystal structure of the Rac1-RhoGDI complex was determined at 2.7 A resolution. In this complex in which Rac1 is bound to GDP, the switch I region of Rac1 is in the GDP conformation whereas the switch II region resembles that of a GTP-bound GTPase. Two types of interaction between RhoGTPases and RhoGDI were investigated. The lipid-protein interaction between the geranylgeranyl moiety of Rac1 and RhoGDI resulted in numerous structural changes in the core of RhoGDI. The interactions between Rac1 and RhoGDI occur through hydrogen bonds which involve a number of residues of Rac1, namely, Tyr64(Rac), Arg66(Rac), His103(Rac), and His104(Rac), conserved within the Rho family and localized in the switch II region or in its close neighborhood. Moreover, in the switch II region of Rac1, hydrophobic interactions involving Leu67(Rac) and Leu70(Rac) contribute to the stability of the Rac1-RhoGDI complex. Inhibition of the GDP-GTP exchange in Rac1 upon binding to RhoGDI partly results from interaction of Thr35(Rac) with Asp45(GDI). In the Rac1-RhoGDI complex, the accessibility of the effector loops of Rac1 probably accounts for the ability of the Rac1-RhoGDI complex to activate the NADPH oxidase.

    Biochemistry 2001;40;34;10007-13

  • The small GTPase Rac interacts with ubiquitination complex proteins Cullin-1 and CDC23.

    Senadheera D, Haataja L, Groffen J and Heisterkamp N

    Division of Hematology/Oncology, Section of Molecular Carcinogenesis, Childrens Hospital of Los Angeles Research Institute, Los Angeles, CA 90027, USA.

    Racs are involved in the regulation of important cellular processes including mitogenesis. We found that the E3 ubiquitination ligase subunit Cullin-1 interacts with constitutively active Rac3 but not with wild-type Rac3 in yeast. In mammalian cell lysates, Cullin-1 bound to V12Rac3, effector domain mutants V12L37Rac3 and V12H40Rac3, and insert domain deletion mutant V12Rac3DeltaIns(124-135). Cullin-1 also formed a clearly detectable complex with other activated Rac3-related proteins including Rac1, Rac2, Cdc42 and RhoA but not with the distantly related small GTPase Rap1. Since the proteasome is involved in cell cycle control through the programmed degradation of cell cycle proteins, the possible regulation of Rac levels during the cell cycle was examined. However, Rac was expressed at constant levels throughout the cell cycle, and a specific proteasome inhibitor had no effect on Rac protein levels. These combined results indicate that the binding of activated Rac to Cullin-1 does not affect Rac protein levels, nor does it mediate the regulation of mitogenesis by Rac. However, Rac-Cullin-1 interactions may serve to regulate other E3 ligase functions such as subcellular localization. Indeed, activated Rac3 and Cullin-1 co-localized to the perinuclear region of the cell. We also detected complex formation between Rac and the APC component CDC23. These results indicate that Rac may regulate specific proteolytic processes through directed subcellular localization of SCF or APC complexes.

    Funded by: NCI NIH HHS: CA47456, CA90321; NEI NIH HHS: EY03040

    International journal of molecular medicine 2001;8;2;127-33

  • Specific association of nitric oxide synthase-2 with Rac isoforms in activated murine macrophages.

    Kuncewicz T, Balakrishnan P, Snuggs MB and Kone BC

    Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas 77030, USA.

    Nitric oxide synthase-2 (NOS2) is responsible for high-output nitric oxide production important in renal inflammation and injury. Using a yeast two-hybrid assay, we identified Rac2, a Rho GTPase member, as a NOS2-interacting protein. NOS2 and Rac2 proteins coimmunoprecipitated from activated RAW 264.7 macrophages. The two proteins colocalized in an intracellular compartment of these cells. Glutathione-S-transferase (GST) pull-down assays revealed that both Rac1 and Rac2 associated with GST-NOS2 and that the NOS2 oxygenase domain was necessary and sufficient for the interaction. [(35)S]methionine-labeled NOS2 interacted directly with GST-Rac2 in the absence of GTP, calmodulin, or NOS2 substrates or cofactors. Stable overexpression of Rac2 in RAW 264.7 cells augmented LPS-induced nitrite generation (~60%) and NOS2 activity (~45%) without measurably affecting NOS2 protein abundance and led to a redistribution of NOS2 to a high-speed Triton X-100-insoluble fraction. We conclude that Rac1 and Rac2 physically interact with NOS2 in activated macrophages and that the interaction with Rac2 correlates with a posttranslational stimulation of NOS2 activity and likely its spatial redistribution within the cell.

    Funded by: NIDDK NIH HHS: DK-50745; NIGMS NIH HHS: GM-20529

    American journal of physiology. Renal physiology 2001;281;2;F326-36

  • The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways.

    Tarricone C, Xiao B, Justin N, Walker PA, Rittinger K, Gamblin SJ and Smerdon SJ

    Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 IAA, UK.

    Small G proteins are GTP-dependent molecular switches that regulate numerous cellular functions. They can be classified into homologous subfamilies that are broadly associated with specific biological processes. Cross-talk between small G-protein families has an important role in signalling, but the mechanism by which it occurs is poorly understood. The coordinated action of Arf and Rho family GTPases is required to regulate many cellular processes including lipid signalling, cell motility and Golgi function. Arfaptin is a ubiquitously expressed protein implicated in mediating cross-talk between Rac (a member of the Rho family) and Arf small GTPases. Here we show that Arfaptin binds specifically to GTP-bound Arf1 and Arf6, but binds to Rac.GTP and Rac.GDP with similar affinities. The X-ray structure of Arfaptin reveals an elongated, crescent-shaped dimer of three-helix coiled-coils. Structures of Arfaptin with Rac bound to either GDP or the slowly hydrolysable analogue GMPPNP show that the switch regions adopt similar conformations in both complexes. Our data highlight fundamental differences between the molecular mechanisms of Rho and Ras family signalling, and suggest a model of Arfaptin-mediated synergy between the Arf and Rho family signalling pathways.

    Funded by: Medical Research Council: MC_U117565398

    Nature 2001;411;6834;215-9

  • Interactions between Rho GTPases and Rho GDP dissociation inhibitor (Rho-GDI).

    Fauré J and Dagher MC

    Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (BBSI), UMR CEA/CNRS/UJF 5092, CEA Grenoble, 17, rue des Martyrs, 38054 cedex 9, Grenoble, France.

    The Rho-GDP dissociation inhibitor (Rho-GDI) was used as bait in a two-hybrid screen of a human leucocyte cDNA library. Most of the isolated cDNAs encoded GTPases of the Rho subfamily: RhoA, B, C, Rac1, 2, CDC42 and RhoG. The newly discovered RhoH interacted very poorly with Rho-GDI. Another protein partner shared a homology with RhoA that points to Asp67(RhoA)-Arg68(RhoA)-Leu69(RhoA) as critical for interaction with Rho-GDI. A second screen with RhoA as bait led to the isolation of GDI only. In order to investigate the relative role of protein-protein and protein-lipid interactions between Rho GTPases and Rho-GDI, CAAX box mutants of RhoA were produced. They were found to interact with Rho-GDI as efficiently as wild type RhoA, indicating that protein-protein interactions alone lead to strong binding of the two proteins. The C-terminal polybasic region of RhoA was also shown to be a site of protein-protein interaction with Rho-GDI.

    Biochimie 2001;83;5;409-14

  • Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase.

    Tamura M, Kai T, Tsunawaki S, Lambeth JD and Kameda K

    Department of Applied Chemistry, Ehime University, Matsuyama, Ehime, 790-8577, Japan. miketamu@en3.ehime-u.ac.jp

    The cell-free activation of human neutrophil NADPH oxidase is enhanced by actin, and actin filaments formed during activation are suggested to stabilize the oxidase. In an attempt to elucidate the mechanism, we examined the protein-protein interactions between actin and cytosolic components of the oxidase. Far-Western blotting using recombinant phox proteins showed that both alpha- and beta-actin interacted with p47(phox) and rac1, and weakly with rac2. A deletion mutant of p47(phox) proved that its C-terminal region was essential for the interaction. The dissociation constant (K(d)) for interaction between actin and p47(phox) was estimated to be 0.45 microM by surface plasmon resonance, and that between actin and rac1 or rac2 was 1.7 or 4.6 microM, respectively. Far-Western blotting using cytosol as a target showed an interaction between actin and endogenous p47(phox) and rac proteins. These results suggest that actin can directly interact with p47(phox) and possibly with rac in the cells.

    Biochemical and biophysical research communications 2000;276;3;1186-90

  • Cloning and functional analysis of cDNAs with open reading frames for 300 previously undefined genes expressed in CD34+ hematopoietic stem/progenitor cells.

    Zhang QH, Ye M, Wu XY, Ren SX, Zhao M, Zhao CJ, Fu G, Shen Y, Fan HY, Lu G, Zhong M, Xu XR, Han ZG, Zhang JW, Tao J, Huang QH, Zhou J, Hu GX, Gu J, Chen SJ and Chen Z

    Shanghai Institute of Hematology (SIH), Rui Jin Hospital affiliated with Shanghai Second Medical University, Shanghai 200025, China.

    Three hundred cDNAs containing putatively entire open reading frames (ORFs) for previously undefined genes were obtained from CD34+ hematopoietic stem/progenitor cells (HSPCs), based on EST cataloging, clone sequencing, in silico cloning, and rapid amplification of cDNA ends (RACE). The cDNA sizes ranged from 360 to 3496 bp and their ORFs coded for peptides of 58-752 amino acids. Public database search indicated that 225 cDNAs exhibited sequence similarities to genes identified across a variety of species. Homology analysis led to the recognition of 50 basic structural motifs/domains among these cDNAs. Genomic exon-intron organization could be established in 243 genes by integration of cDNA data with genome sequence information. Interestingly, a new gene named as HSPC070 on 3p was found to share a sequence of 105bp in 3' UTR with RAF gene in reversed transcription orientation. Chromosomal localizations were obtained using electronic mapping for 192 genes and with radiation hybrid (RH) for 38 genes. Macroarray technique was applied to screen the gene expression patterns in five hematopoietic cell lines (NB4, HL60, U937, K562, and Jurkat) and a number of genes with differential expression were found. The resource work has provided a wide range of information useful not only for expression genomics and annotation of genomic DNA sequence, but also for further research on the function of genes involved in hematopoietic development and differentiation.

    Genome research 2000;10;10;1546-60

  • Structure of the TPR domain of p67phox in complex with Rac.GTP.

    Lapouge K, Smith SJ, Walker PA, Gamblin SJ, Smerdon SJ and Rittinger K

    Division of Protein Structure, National Institute for Medical Research, London, United Kingdom.

    p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.

    Funded by: Medical Research Council: MC_U117565398

    Molecular cell 2000;6;4;899-907

  • Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

    Williams DA, Tao W, Yang F, Kim C, Gu Y, Mansfield P, Levine JE, Petryniak B, Derrow CW, Harris C, Jia B, Zheng Y, Ambruso DR, Lowe JB, Atkinson SJ, Dinauer MC and Boxer L

    Department of Pediatrics, Wells Center for Pediatric Research, Howard Hughes Medical Institute, Indiana University School of Medicine, Indianapolis, IN 46202-5225, USA. dwilliam@iupui.edu

    Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

    Funded by: NCI NIH HHS: 1P01CA71932; NHLBI NIH HHS: R01 HL-45635; NIAID NIH HHS: R01 AI-20065; ...

    Blood 2000;96;5;1646-54

  • Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.

    Ambruso DR, Knall C, Abell AN, Panepinto J, Kurkchubasche A, Thurman G, Gonzalez-Aller C, Hiester A, deBoer M, Harbeck RJ, Oyer R, Johnson GL and Roos D

    Bonfils Blood Center, Denver, CO 80220, USA. daniel.ambruso@UCHSC.edu

    A 5-week-old male infant presented with severe bacterial infections and poor wound healing, suggesting a neutrophil defect. Neutrophils from this patient exhibited decreased chemotaxis, polarization, azurophilic granule secretion, and superoxide anion (O(2)(-)) production but had normal expression and up-regulation of CD11b. Rac2, which constitutes >96% of the Rac in neutrophils, is a member of the Rho family of GTPases that regulates the actin cytoskeleton and O(2)(-) production. Western blot analysis of lysates from patient neutrophils demonstrated decreased levels of Rac2 protein. Addition of recombinant Rac to extracts of the patient neutrophils reconstituted O(2)(-) production in an in vitro assay system. Molecular analysis identified a point mutation in one allele of the Rac2 gene resulting in the substitution of Asp57 by an Asn (Rac2(D57N)). Asp57 is invariant in all defined GTP-binding proteins. Rac2(D57N) binds GDP but not GTP and inhibits oxidase activation and O(2)(-) production in vitro. These data represent the description of an inhibitory mutation in a member of the Rho family of GTPases associated with a human immunodeficiency syndrome.

    Funded by: NIGMS NIH HHS: GM30324, R01 GM030324, R37 GM030324

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;9;4654-9

  • Activation of the small GTPases, rac and cdc42, after ligation of the platelet PAR-1 receptor.

    Azim AC, Barkalow K, Chou J and Hartwig JH

    Division of Hematology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

    Stimulation of platelet PAR-1 receptors results in the rapid (10 to 30 seconds) and extensive (30% to 40% of total) guanosine triphosphate (GTP) charging of endogenous platelet rac, previously identified as a possible key intermediate in the signal pathway between PAR-1 and actin filament barbed-end uncapping, leading to actin assembly. During PAR-1-mediated platelet activation, rac distributes from the cell interior to the cell periphery, and this reorganization is resistant to the inhibition of PI-3-kinase activity. Rac, in resting or activated platelets, is Triton X-100 soluble, suggesting that it does not form tight complexes with actin cytoskeletal proteins, though its retention in octyl-glucoside-treated platelets and ultrastructural observations of activated platelets implies that rac binds to plasma membranes, where it can interact with phosphoinositide kinases implicated in actin assembly reactions. PAR-1 stimulation also rapidly and extensively activates cdc42, though, in contrast to rac, some cdc42 associates with the actin cytoskeleton in resting platelets, and the bound fraction increases during stimulation. The differences in subcellular distribution and previous evidence showing quantitatively divergent effects of rac and cdc42 on actin nucleation in permeabilized platelets indicate different signaling roles for these GTPases.

    Funded by: NHLBI NIH HHS: HL56252, HL56949

    Blood 2000;95;3;959-64

  • The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI.

    Scheffzek K, Stephan I, Jensen ON, Illenberger D and Gierschik P

    Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany. klaus@mpimf-heidelberg.mpg.de

    Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2, which are abundant in leukocytes, representing the cytosolic, resting pool of Rho species to be activated by extracellular signals. The N-terminal domain of LyGDI (LyN), which has been reported to be flexible in isolated RhoGDIs, becomes ordered upon complex formation and contributes more than 60% to the interface area. The structure is consistent with the C-terminus of Rac2 binding to a hydrophobic cavity previously proposed as isoprenyl binding site. An inner segment of LyN forms a helical hairpin that contacts mainly the switch regions of Rac2. The architecture of the complex interface suggests a mechanism for the inhibition of guanine nucleotide dissociation that is based on the stabilization of the magnesium (Mg2+) ion in the nucleotide binding pocket.

    Nature structural biology 2000;7;2;122-6

  • The DNA sequence of human chromosome 22.

    Dunham I, Shimizu N, Roe BA, Chissoe S, Hunt AR, Collins JE, Bruskiewich R, Beare DM, Clamp M, Smink LJ, Ainscough R, Almeida JP, Babbage A, Bagguley C, Bailey J, Barlow K, Bates KN, Beasley O, Bird CP, Blakey S, Bridgeman AM, Buck D, Burgess J, Burrill WD, O'Brien KP et al.

    Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK. id1@sanger.ac.uk

    Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

    Nature 1999;402;6761;489-95

  • Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.

    Nishihara H, Kobayashi S, Hashimoto Y, Ohba F, Mochizuki N, Kurata T, Nagashima K and Matsuda M

    Department of Pathology, Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku-ku, Tokyo, Japan.

    Human DOCK180, which was originally identified as a major protein bound to the Crk oncogene product, is an archetype of the CDM family of proteins, including Ced-5 of Caenorhabditis elegans and Mbc of Drosophila melanogaster. After DOCK180, at least three putative human proteins that manifest high amino acid sequence similarity to DOCK180 have been registered in the GenBank/EMBL database. We have designated one of them, KIAA0209, as DOCK2 and characterize here. DOCK2 mRNA was expressed mostly in peripheral blood cells, followed by slight expression in the spleen and thymus, whereas DOCK180 was expressed in all tissues tested except in peripheral blood cells. Immunostaining of human cadaver tissues revealed that the expression of DOCK2 was limited to the lymphocytes and macrophages of various organs. DOCK2 bound to and activated Rac1, as did DOCK180; however, DOCK2 did not bind to CrkII, which transduces signals at focal adhesions. Thus, DOCK180 and DOCK2 are regulators of Rac and function in adherent and non-adherent cells, respectively.

    Biochimica et biophysica acta 1999;1452;2;179-87

  • Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation.

    Zhang B and Zheng Y

    Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163, USA.

    The Rho family GTPases are tightly regulated between the active GTP-bound state and the inactive GDP-bound state in a variety of signal transduction processes. Here the Rho family members Cdc42, Rac2, and RhoA were found to form reversible homodimers in both the GTP- and the GDP-bound states. The homophilic interaction of Cdc42 and Rac2, but not RhoA, in the GTP-bound state, caused a significant stimulation of the intrinsic GTPase activity, i.e. the activated form of Cdc42 and Rac2 acts as GTPase-activating proteins toward Cdc42-GTP or Rac2-GTP. The dimerization of the GTPases appeared to be mediated by the carboxyl-terminal polybasic domain, and the specific GTPase-activating effects of Cdc42 and Rac2 were also attributed to the structural determinant(s) in the same region of the molecules. Moreover, similar to the case of Cdc42 and Cdc42GAP interaction, Cdc42-GDP interacted with tetrafluoroaluminate and Cdc42-GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) to form a transition state complex of the GTPase-activating reaction in which the carboxyl-terminal determinant(s) of the GTPgammaS-bound Cdc42 plays a critical role. These results provide a rationale for the fast rate of intrinsic GTP hydrolysis by Cdc42 and Rac and suggest that dimerization may play a role in the negative regulation of specific Rho family GTPases mediated by the carboxyl-terminal polybasic domain.

    Funded by: NIGMS NIH HHS: GM53943

    The Journal of biological chemistry 1998;273;40;25728-33

  • Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).

    Ahmed S, Prigmore E, Govind S, Veryard C, Kozma R, Wientjes FB, Segal AW and Lim L

    Department of Neurochemistry, Institute of Neurology, 1 Wakefield Street, London WC1N 1PJ, Great Britain.

    Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67(phox), but not with the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling. Deletion of p67(phox) C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by approximately 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase.

    Funded by: Wellcome Trust

    The Journal of biological chemistry 1998;273;25;15693-701

  • Phagocyte NADPH oxidase p67-phox possesses a novel carboxylterminal binding site for the GTPases Rac2 and Cdc42.

    Faris SL, Rinckel LA, Huang J, Hong YR and Kleinberg ME

    Baltimore Veterans Affairs Medical Center and the Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, 21201, USA.

    Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection. GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase. Binding was independent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox.

    Funded by: NIAID NIH HHS: AI32220, AI40077

    Biochemical and biophysical research communications 1998;247;2;271-6

  • Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum.

    Dai Q, Choy E, Chiu V, Romano J, Slivka SR, Steitz SA, Michaelis S and Philips MR

    Departments of Medicine and Cell Biology, New York University School of Medicine, New York, New York 10016, USA.

    Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pcCMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pcCMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pcCMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.

    Funded by: NIAID NIH HHS: AI36224, R01 AI036224; NIGMS NIH HHS: GM41223, GM55279

    The Journal of biological chemistry 1998;273;24;15030-4

  • Differential properties of D4/LyGDI versus RhoGDI: phosphorylation and rho GTPase selectivity.

    Gorvel JP, Chang TC, Boretto J, Azuma T and Chavrier P

    Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, France.

    RhoA/B/C and CDC42/Rac, which form two subgroups of the rho guanosine triphosphatase (GTPase) family, regulate various aspects of actin cytoskeleton organisation. In cytosol, guanosine diphosphate (GDP) dissociation inhibitor (GDI) interacts with and maintains rho GTPases in their inactive GDP-bound form. RhoGDI is a ubiquitously expressed GDI, whereas D4/LyGDI is hematopoietic cell-specific and 10-fold less potent than RhoGDI in binding to and regulating rho GTPases. We have combined microanalytical liquid chromatography with the use of specific antibodies in order to separate D4/LyGDI and RhoDGI-complexes from the cytosol of U937 cells and to demonstrate that the two GDIs associate with different rho protein partners. RhoGDI can form a complex with CDC42Hs, RhoA, Rac1 and Rac2, while none of these GTPases was found to interact with D4/LyGDI. In addition, we found that stimulation of U937 cells with phorbol ester leads to phosphorylation of D4/LyGDI. Our results suggest that LyGDI forms complexes with specific rho GTPases expressed in hematopoietic cells where it may regulate specific pathways.

    FEBS letters 1998;422;2;269-73

  • Structure and chromosomal assignment to 22q12 and 17qter of the ras-related Rac2 and Rac3 human genes.

    Courjal F, Chuchana P, Theillet C and Fort P

    IGM-UMR5535, CNRS, route de Mende, Montpellier cedex 5, 34293, France.

    Members of the Rho/Rac/Cdc42Hs family of GTPases have been shown to participate in many aspects of the signaling of cell growth and differentiation. Although the biochemical properties of these GTPases have been extensively studied, very little is known about the structure of the corresponding genes. To gain insight on the evolution of the Rho family, we were interested in studying the genomic structure of several members. We report here the structure and the localization to 22q12 of the human Rac2 gene, as well as the localization to 17qter of Rac3, a new member closely related to Rac1 and Rac2. Unlike the structure of its closest relative ARH-G gene, which contains a single intron, Rac2 is made of at least 7 exons, spanning over 18 kb of DNA. Comparison of gene structure and exonic borders suggests that the emergence of the whole superfamily took place early during evolution.

    Genomics 1997;44;2;242-6

  • Regulation of the human neutrophil NADPH oxidase by rho-related G-proteins.

    Kwong CH, Malech HL, Rotrosen D and Leto TL

    Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.

    Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).

    Biochemistry 1993;32;21;5711-7

  • Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

    Mizuno T, Kaibuchi K, Ando S, Musha T, Hiraoka K, Takaishi K, Asada M, Nunoi H, Matsuda I and Takai Y

    Department of Biochemistry, Kobe University School of Medicine, Japan.

    The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.

    The Journal of biological chemistry 1992;267;15;10215-8

  • Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA.

    Kinsella BT, Erdman RA and Maltese WA

    Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.

    Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.

    Funded by: NCI NIH HHS: CA-34569

    The Journal of biological chemistry 1991;266;15;9786-94

  • A hemopoietic specific gene encoding a small GTP binding protein is overexpressed during T cell activation.

    Reibel L, Dorseuil O, Stancou R, Bertoglio J and Gacon G

    Institut Cochin de Génétique Moléculaire, INSERM U. 257, Paris, France.

    We have isolated, from a human B cell line cDNA library, a cDNA (Gx) encoding a small G protein identical to rac 2, a member of the ras superfamily. Gx/rac 2 gene is expressed as a unique mRNA of 1,7 Kb in peripheral blood lymphocytes, in purified B and T cells, in thymus as well as in several B and T cell lines. It is not expressed in many other tissues analysed including liver, brain, lung, heart and kidney. Upon in vitro stimulation with phytohemagglutinin A, peripheral blood lymphocytes show a clear increase of the Gx/rac 2 mRNA after 6 hours; a 30-50 fold accumulation is reached at 24 hours and persists thereafter. Purified T lymphocytes exhibit a similar increase in Gx/rac 2 mRNA expression upon mitogenic stimulation. Therefore, the expression of the Gx/rac 2 gene appears to be restricted to cells of the hemopoietic lineage and to be strongly stimulated during T cell activation. Gx/rac 2 protein must fulfill a specific role in activated T cells that could provide a new model for studying the function of small G proteins.

    Biochemical and biophysical research communications 1991;175;2;451-8

  • rac, a novel ras-related family of proteins that are botulinum toxin substrates.

    Didsbury J, Weber RF, Bokoch GM, Evans T and Snyderman R

    Department of Pharmacological Sciences, Genentech, Incorporated, South San Francisco, California 94080.

    A new family of ras-related proteins, designated rac (ras-related C3 botulinum toxin substrate) has been identified. rac1 and rac2 cDNA clones were isolated from a differentiated HL-60 library and encode proteins that are 92% homologous and share 58% and 26-30% amino acid homology with human rhos and ras, respectively. Nucleotide sequence analysis predicts both rac1 and rac2 proteins to contain 192 amino acids with molecular masses of 21,450 and 21,429 daltons, respectively. rac1 and rac2 possess four of the five conserved functional domains in ras associated with binding and hydrolysis of guanine nucleotides. They also contain the COOH-terminal consensus sequence Cys-X-X-X-COOH which localizes ras to the inner plasma membrane and the residues Gly12 and Ala59, at which sites mutations elicit transforming potential to ras. The rac transcripts, particularly rac2, display relative myeloid tissue selectivity. Both rac1 transcripts (2.4 and 1.1 kilobases (kb] increase when HL-60 cells differentiate to neutrophil-like morphology. In contrast, differentiation of U937 cells to monocyte-like morphology causes no change in the 2.4-kb mRNA and a decrease in the 1.1-kb mRNA species. rac2 mRNA (1.45 kb) increases 7-9-fold and 3-fold upon differentiation of HL-60 and U937 cells, respectively. Neither rac mRNAs are present in a Jurkat T cell line, and unlike rac1, rac2 mRNA is absent in human brain and liver tissue. Transfection experiments permitted the demonstration that rac1 and rac2 are substrates for ADP-ribosylation by the C3 component of botulinum toxin. The data suggest that racs are plasma membrane-associated GTP-binding proteins which could regulate secretory processes, particularly in myeloid cells.

    Funded by: NIDCR NIH HHS: 5R01-DE-03738-16; NIGMS NIH HHS: GM39434

    The Journal of biological chemistry 1989;264;28;16378-82

Gene lists (2)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

Cookies Policy | Terms and Conditions. This site is hosted by Edinburgh University and the Genes to Cognition Programme.