Journal articles on the topic 'Rac1 protein'
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1
Dumontier, M., P. Hocht, U. Mintert, and J. Faix. "Rac1 GTPases control filopodia formation, cell motility, endocytosis, cytokinesis and development in Dictyostelium." Journal of Cell Science 113, no. 12 (June 15, 2000): 2253–65. http://dx.doi.org/10.1242/jcs.113.12.2253.
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The function of the highly homologous Rac1A, Rac1B, and Rac1C GTPases of the Dictyostelium Rac1 group was investigated. All three GTPases bound with an equal capacity to the IQGAP-related protein DGAP1, with a preference for the activated GTP-bound form. Strong overexpression of wild-type Rac1 GTPases N-terminally tagged with green fluorescent protein (GFP), predominantly induced the formation of numerous long filopodia. Remarkably, expression of the constitutively-activated GTPases resulted in dominant-negative phenotypes: these Rac1-V12 mutants completely lacked filopodia but formed numerous crown shaped structures resembling macropinosomes. Moreover, these mutants were severely impaired in cell motility, colony growth, phagocytosis, pinocytosis, cytokinesis and development. Transformants expressing constitutively-inactivated Rac1-N17 proteins were similar to wild-type cells, but displayed abundant and short filopodia and exhibited a moderate defect in cytokinesis. Taken together, our results indicate that the three GTPases play an identical role in signaling pathways and are key regulators of cellular activities that depend on the re-organization of the actin cytoskeleton in Dictyostelium.
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Seiz, Julia R., Johannes Klinke, Laura Scharlibbe, Dirk Lohfink, Marisa Heipel, Hendrik Ungefroren, Klaudia Giehl, and Andre Menke. "Different signaling and functionality of Rac1 and Rac1b in the progression of lung adenocarcinoma." Biological Chemistry 401, no. 4 (March 26, 2020): 517–31. http://dx.doi.org/10.1515/hsz-2019-0329.
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AbstractRac1 is a ubiquitously expressed Rho GTPase and an important regulator of the actin cytoskeleton. Its splice variant Rac1b exhibits a 19-amino acid (aa) in-frame insertion and is predominantly active. Both proteins were described in tumorigenesis or metastasis. We investigated the contribution of Rac1 and Rac1b to tumor progression of human non-small-cell lung adenocarcinoma (NSCLA). Rac1 protein was present in 8/8 NSCLA cell lines analyzed, whereas Rac1b was expressed in only 6/8. In wound-healing assays, enhanced green fluorescence protein (EGFP)-Rac1 slightly decreased cell migration, whereas proliferation was increased in both, Rac1- and Rac1b-expressing cells. In the in vivo chorioallantoic invasion model, EGFP-Rac1-expressing cells formed more invasive tumors compared to EGFP-Rac1b. This increased invasiveness correlated with enhanced phosphorylation of p38α, AKT and glycogen synthase kinase 3β (GSK3β), and activation of serum response- and Smad-dependent gene promoters by Rac1. In contrast, Rac1b solely activated the mitogen-activated protein kinase (MAPK) JNK2, together with TCF/LEF1- and nuclear factor kappa B (NFκB)-responsive gene reporters. Rac1b, as Rac1, phosphorylated p38α, AKT and GSK3β. Knockdown of the splicing factor epithelial splicing regulatory protein 1 (ESRP1), which mediates out-splicing of exon 3b from Rac1 pre-messenger RNA, resulted in increased Rac1b messenger RNA (mRNA) and suppression of the epithelial-mesenchymal transition (EMT)-associated transcription factor ZEB1. Our data demonstrate different signaling and functional activities of Rac1 and Rac1b and an important role for Rac1 in lung cancer metastasis.
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Nagata, Koh-ichi, Yukio Okano, and Yoshinori Nozawa. "Differential Expression of Low Mr GTP-binding Proteins in Human Megakaryoblastic Leukemia Cell Line, MEG-01, and their Possible Involvement in the Differentiation Process." Thrombosis and Haemostasis 77, no. 02 (1997): 368–75. http://dx.doi.org/10.1055/s-0038-1655970.
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SummaryThe expression of various low Mr GTP-binding proteins at various states of differentiation of a human megakaryoblastic leukemia cell line, MEG-01, was analyzed using thermocycle amplification of mRNA and immunoblotting. MEG-01 cells were found to express mRNAs of rap1A, rap1B, rap2B, ralA, rhoA, rac1, rac2, CDC42Hs, rab1, rab3B, rab6, ram and ran, but not rab4, and the proteins of Rap 1, Rap2, RhoA, Rac1, Rac2, Rab3B, Rab4, Rab6 and Rab8 were expressed. Differentiation of MEG-01 cells induced by 100 nM 12-O-tetradecanoylphorbol-13-acetate revealed the considerable increases in mRNA expression of rap1B, rab3B, rabA, ram and ran whereas the levels of rap2B, rhoA and rac1 decreased. During the differentiation process, significant changes in protein levels of Rap1, RhoA, Rac1, Rac2, Rab3B, Rab4 and Rab6 were observed among three subcellular (cytosol, Triton X-100-soluble membrane and -insoluble cytoskeleton) fractions. The present investigation may be useful for the study of the megakaryocyte differentiation.
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Hoppe, Adam D., and Joel A. Swanson. "Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during Phagocytosis." Molecular Biology of the Cell 15, no. 8 (August 2004): 3509–19. http://dx.doi.org/10.1091/mbc.e03-11-0847.
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The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcγ receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.
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Kalfa, Theodosia A., Suvarnamala Pushkaran, Narla Mohandas, John H. Hartwig, Velia M. Fowler, James F. Johnson, Clinton H. Joiner, David A. Williams, and Yi Zheng. "Rac GTPases regulate the morphology and deformability of the erythrocyte cytoskeleton." Blood 108, no. 12 (December 1, 2006): 3637–45. http://dx.doi.org/10.1182/blood-2006-03-005942.
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Abstract Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase–induced deletion of Rac1 on a Rac2 null genetic background. The Rac1–/–;Rac2–/– mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1–/–;Rac2–/– RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1–/–;Rac2–/– erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1–/–;Rac2–/– erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.
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Thomas, Emily K., Jose A. Cancelas, Heedon Chae, Adrienne D. Cox, Patricia J. Keller, Danilo Perrotti, Paolo Neviani, et al. "Rac GTPases Are Potential Therapeutic Targets in p210-BCR-ABL-Induced Myeloproliferative Disease (MPD)." Blood 110, no. 11 (November 16, 2007): 465. http://dx.doi.org/10.1182/blood.v110.11.465.465.
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Abstract The p210-BCR-ABL fusion protein is a constitutively active tyrosine kinase that is necessary and sufficient for the development of chronic myelogenous leukemia (CML). ABL-kinase inhibitors such as imatinib mesylate (Gleevec, STI571) potently block BCR-ABL activation, but the continued presence of leukemic stem cells and the emergence of imatinib-resistant BCR-ABL mutants suggest that ABL kinase inhibitors alone cannot completely eradicate disease. Rac GTPases have been implicated in BCR-ABL-mediated proliferation in cell lines and regulate many of the same signaling pathways as BCR-ABL, suggesting that these proteins could be additional therapeutic targets in CML. We have found that Rac1, Rac2, and, to a lesser extent, Rac3 were hyperactivated in CD34+ cells purified from the peripheral blood of two CML patients. To better study the role of Rac in BCR-ABL disease development, murine hematopoietic stem cells (HSC) genetically deficient in Rac1 and/or Rac2 were transduced with a retroviral vector expressing p210-BCR-ABL. Wild type (WT) and Rac1−/− mice experienced similar disease progression [median survival 23 ± 6 days (n=30) and 22 ± 4 days (n=8), respectively], Rac2−/− mice exhibited significantly attenuated development of BCR-ABL-mediated MPD [median survival 43 ± 27 days (n=18); p<0.001], and Rac1−/−;Rac2−/− animals showed markedly prolonged survival [median survival 92 ± 34 days (n=19); p<0.001]. p210-BCR-ABL WT, Rac1−/−, and Rac2−/− mice had elevated circulating myeloblasts 30 days post-transplant, while Rac1−/−;Rac2−/− mice had normal peripheral blood morphology. Attenuation of disease in Rac2- and Rac1/Rac2-deficient animals correlated with severely diminished activation of BCR-ABL-induced signaling pathways, including p44/42 and p38 ERK, JNK, CrkL, and Akt. The leukemogenesis impairment induced by Rac deficiency did not appear to be due to loss of p210-BCR-ABL vector integration, as clonal analysis of leukemic bone marrow from mice in each genotype by LAM-PCR showed similar, oligoclonal reconstitution of p210-BCR-ABL expressing cells. Interestingly, bone marrow cells obtained from Rac1/Rac2-deficient animals that developed late leukemia showed marked hyperactivation of Rac3 and initiated disease in recipients with a latency of three weeks, suggesting that leukemia-initiating cells were able to engraft, in spite of Rac1/Rac2 deficiency. Treatment of BCR-ABL-expressing murine HSC with NSC23766, a rationally-designed Rac-specific small molecule antagonist, potently inhibited cell proliferation in vitro and increased the survival of leukemic animals treated in vivo, compared to PBS control-treated animals (p<0.05). NSC23766 also inhibited the growth of an imatinib-resistant p210-BCR-ABL-T315I-expressing Ba/F3 leukemic cell line by 90%, compared to <5% by imatinib alone, blocked the growth of primary human chronic phase Rac-hyperactivated CML blast colonies by 80% in vitro, and inhibited survival of these cells in NOD-SCID mice. These results suggest that individual Rac proteins play both unique and combinatorial roles in stem cell transformation and may represent unique targets for therapy of BCR-ABL-persistent and imatinib-resistant CML.
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Chuang, T. H., X. Xu, L. A. Quilliam, and G. M. Bokoch. "SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism." Biochemical Journal 303, no. 3 (November 1, 1994): 761–67. http://dx.doi.org/10.1042/bj3030761.
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The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5′-[gamma-thio]-triphosphate GTP[gamma S] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[gamma S] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[gamma S]-binding during incubations at 30 degrees C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[gamma S] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.
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Engers, R., S. Ziegler, M. Mueller, A. Walter, R. Willers, and H. E. Gabbert. "Prognostic relevance of increased Rac GTPase expression in prostate carcinomas." Endocrine-Related Cancer 14, no. 2 (June 2007): 245–56. http://dx.doi.org/10.1677/erc-06-0036.
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Rac proteins of the Rho-like GTPase family, including the ubiquitous Rac1, the hematopoiesis-specific Rac2, and the least-characterized Rac3 play a major role in oncogenic transformation, tumor invasion and metastasis. However, the prognostic relevance of Rac expression in human tumors has not been investigated yet. In the present study, Rac protein expression was analyzed in benign secretory epithelium, high-grade prostatic intraepithelium neoplasia (HG-PIN), and prostate carcinomas of 60 R0-resected radical prostatectomy specimens by semiquantitative immunohistochemistry. Thus, Rac proteins were significantly strongly expressed in HG-PIN (P < 0.001) and prostate carcinomas (P < 0.001) when compared with benign secretory epithelium. Accordingly, all tumor tissues analyzed by isoform-specific real-time PCR (n = 7) exhibited significantly higher RNA expression levels of Rac (i.e. sum of Rac1 and Rac3 expression levels) than the respective benign counterparts (P = 0.018) and this appeared to result mainly from increased expression of the Rac3 isoform as verified by immunoblotting. Univariate analyses showed statistically significant associations of increased Rac protein expression in prostate cancer (P = 0.045), preoperative prostate-specific antigen levels (P = 0.044), pT stage (P = 0.002), and Gleason score (P = 0.001) with decreased disease-free survival (DFS). This prognostic effect of increased protein expression of Rac remained significant even in a multivariate analysis including all these four factors (relative risk = 3.22, 95% confidence interval = 1.04–10.00; P = 0.043). In conclusion, our data suggest that increased Rac protein expression in prostate cancer relative to the corresponding benign secretory epithelium is an independent predictor of decreased DFS and appears to result mainly from increased expression of the Rac3 isoform.
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Kuncewicz, Teresa, Priya Balakrishnan, Mark B. Snuggs, and Bruce C. Kone. "Specific association of nitric oxide synthase-2 with Rac isoforms in activated murine macrophages." American Journal of Physiology-Renal Physiology 281, no. 2 (August 1, 2001): F326—F336. http://dx.doi.org/10.1152/ajprenal.2001.281.2.f326.
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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. [35S]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.
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Michaelson, David, Joseph Silletti, Gretchen Murphy, Peter D'Eustachio, Mark Rush, and Mark R. Philips. "Differential Localization of Rho Gtpases in Live Cells." Journal of Cell Biology 152, no. 1 (January 8, 2001): 111–26. http://dx.doi.org/10.1083/jcb.152.1.111.
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Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.
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RAMASWAMY, MADHU, CELINE DUMONT, JAGAN R. MUPPIDI, VICTOR L. TYBULEWICZ, and RICHARD M. SIEGEL. "Rac GTPases are required for restimulation-induced CD4+ T Cell Death (RICD) (87.20)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S131. http://dx.doi.org/10.4049/jimmunol.178.supp.87.20.
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Abstract In activated CD4 T cells, TCR restimulation or antigen leads to FasL mRNA synthesis, expression and eventually apoptosis through cis or trans FasL-Fas interactions, in the process known as restimulation-induced cell death (RICD). Apoptosis requires both FasL synthesis and secretion and a protein-synthesis independent ‘competency to die’ signal that sensitizes TCR-stimulated cells to apoptosis induced by FasL. Recent work in our lab correlated competency signals with translocation of Fas into specialized membrane micro-domains (lipid rafts) in TCR restimulated T cells. However, what signaling pathways underlie the TCR-induced ‘competancy to die’ signal is not known. We have now identified the small Rho GTPase Rac as a modulator of TCR signals that mediate competency. SiRNA knockdown of Rac1 in jurkats and human CD4 T cells, results in specific abrogation of TCR induced ‘competency to die’ via Fas. Transfection of constitutively active Rac1 rescues TCR mediated Fas apoptosis in Vav-1 deficient Jurkat cells, which are devoid of competency signals. Absence of Rac1 does not affect a number of other parameters of early T cell signaling, however, we observe reduction in dephoshorylation of the Ezrin-Radixin-Moesin (ERM) proteins. CD2Cre Rac1 flox/flox mouse T cells as well as Rac2 −/− T cells have deficient Restimulation Induced cell death (RICD), which is more severe in T cells lacking both Rac1 and Rac2 (Rac1−/+/ Rac2 −/−). Further, in Jurkat and mouse T cells, Rac1 deficiency inhibits FasL upregulation upon TCR stimulation. Therefore, Rho GTPase proteins are critically involved in two distinct aspects of restimulation-induced T cell apoptosis and may be a novel target for immunomodulatory therapy.
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Ma, Zhong, Keena S. Thomas, Donna J. Webb, Radim Moravec, Ana Maria Salicioni, Wendy M. Mars, and Steven L. Gonias. "Regulation of Rac1 activation by the low density lipoprotein receptor–related protein." Journal of Cell Biology 159, no. 6 (December 23, 2002): 1061–70. http://dx.doi.org/10.1083/jcb.200207070.
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The low density lipoprotein receptor–related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1–deficient MEFs demonstrated increased Rac1 activation compared with LRP-1–expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR−/− MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK− cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR−/− MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal–regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.
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Shutes, Adam, Cercina Onesto, Virginie Picard, Bertrand Leblond, Fabien Schweighoffer, and Channing J. Der. "Specificity and Mechanism of Action of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases." Journal of Biological Chemistry 282, no. 49 (October 11, 2007): 35666–78. http://dx.doi.org/10.1074/jbc.m703571200.
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There is now considerable experimental evidence that aberrant activation of Rho family small GTPases promotes the uncontrolled proliferation, invasion, and metastatic properties of human cancer cells. Therefore, there is considerable interest in the development of small molecule inhibitors of Rho GTPase function. However, to date, most efforts have focused on inhibitors that indirectly block Rho GTPase function, by targeting either enzymes involved in post-translational processing or downstream protein kinase effectors. We recently determined that the EHT 1864 small molecule can inhibit Rac function in vivo. In this study, we evaluated the biological and biochemical specificities and biochemical mechanism of action of EHT 1864. We determined that EHT 1864 specifically inhibited Rac1-dependent platelet-derived growth factor-induced lamellipodia formation. Furthermore, our biochemical analyses with recombinant Rac proteins found that EHT 1864 possesses high affinity binding to Rac1, as well as the related Rac1b, Rac2, and Rac3 isoforms, and this association promoted the loss of bound nucleotide, inhibiting both guanine nucleotide association and Tiam1 Rac guanine nucleotide exchange factor-stimulated exchange factor activity in vitro. EHT 1864 therefore places Rac in an inert and inactive state, preventing its engagement with downstream effectors. Finally, we evaluated the ability of EHT 1864 to block Rac-dependent growth transformation, and we determined that EHT 1864 potently blocked transformation caused by constitutively activated Rac1, as well as Rac-dependent transformation caused by Tiam1 or Ras. Taken together, our results suggest that EHT 1864 selectively inhibits Rac downstream signaling and transformation by a novel mechanism involving guanine nucleotide displacement.
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Zohn, Irene E., Marc Symons, Magdalena Chrzanowska-Wodnicka, John K. Westwick, and Channing J. Der. "Mas Oncogene Signaling and Transformation Require the Small GTP-Binding Protein Rac." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1225–35. http://dx.doi.org/10.1128/mcb.18.3.1225.
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ABSTRACT The Mas oncogene encodes a novel G-protein-coupled receptor that was identified originally as a transforming protein when overexpressed in NIH 3T3 cells. The mechanism and signaling pathways that mediate Mas transformation have not been determined. We observed that the foci of transformed NIH 3T3 cells caused by Mas were similar to those caused by activated Rho and Rac proteins. Therefore, we determined if Mas signaling and transformation are mediated through activation of a specific Rho family protein. First, we observed that, like activated Rac1, Mas cooperated with activated Raf and caused synergistic transformation of NIH 3T3 cells. Second, both Mas- and Rac1-transformed NIH 3T3 cells retained actin stress fibers and showed enhanced membrane ruffling. Third, like Rac, Mas induced lamellipodium formation in porcine aortic endothelial cells. Fourth, Mas and Rac1 strongly activated the JNK and p38, but not ERK, mitogen-activated protein kinases. Fifth, Mas and Rac1 stimulated transcription from common DNA promoter elements: NF-κB, serum response factor (SRF), Jun/ATF-2, and the cyclin D1 promoter. Finally, Mas transformation and some of Mas signaling (SRF and cyclin D1 but not NF-κB activation) were blocked by dominant negative Rac1. Taken together, these observations suggest that Mas transformation is mediated in part by activation of Rac-dependent signaling pathways. Thus, Rho family proteins are common mediators of transformation by a diverse variety of oncogene proteins that include Ras, Dbl family, and G-protein-coupled oncogene proteins.
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Yang, Shigao, Alfred T. Harding, Catherine Sweeney, David Miao, Gregory Swan, Connie Zhou, Zhaozhao Jiang, et al. "Control of antiviral innate immune response by protein geranylgeranylation." Science Advances 5, no. 5 (May 2019): eaav7999. http://dx.doi.org/10.1126/sciadv.aav7999.
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The mitochondrial antiviral signaling protein (MAVS) orchestrates host antiviral innate immune response to RNA virus infection. However, how MAVS signaling is controlled to eradicate virus while preventing self-destructive inflammation remains obscure. Here, we show that protein geranylgeranylation, a posttranslational lipid modification of proteins, limits MAVS-mediated immune signaling by targeting Rho family small guanosine triphosphatase Rac1 into the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) at the mitochondria-ER junction. Protein geranylgeranylation and subsequent palmitoylation promote Rac1 translocation into MAMs upon viral infection. MAM-localized Rac1 limits MAVS’ interaction with E3 ligase Trim31 and hence inhibits MAVS ubiquitination, aggregation, and activation. Rac1 also facilitates the recruitment of caspase-8 and cFLIPL to the MAVS signalosome and the subsequent cleavage of Ripk1 that terminates MAVS signaling. Consistently, mice with myeloid deficiency of protein geranylgeranylation showed improved survival upon influenza A virus infection. Our work revealed a critical role of protein geranylgeranylation in regulating antiviral innate immune response.
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KITAMURA, Yukari, Tadahiro KITAMURA, Hiroshi SAKAUE, Tetsuo MAEDA, Hikaru UENO, Shoko NISHIO, Shigeo OHNO, et al. "Interaction of Nck-associated protein 1 with activated GTP-binding protein Rac." Biochemical Journal 322, no. 3 (March 15, 1997): 873–78. http://dx.doi.org/10.1042/bj3220873.
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Bacterially expressed glutathione S-transferase fusion proteins containing Rac1 were used to identify binding proteins of this Rho family GTPase present in a bovine brain extract. Five proteins of 85, 110, 125, 140 and 170 kDa were detected, all of which were associated exclusively with guanosine 5´-[γ-thio]triphosphate-bound Rac1, not with GDP-bound Rac1. The 85 and 110 kDa proteins were identified as the regulatory and catalytic subunits respectively of phosphatidylinositol 3-kinase. Several lines of evidence suggested that the 125 kDa protein is identical with Nck-associated protein 1 (Nap1). The mobilities of the 125 kDa protein and Nap1 on SDS/PAGE were indistinguishable, and the 125 kDa protein was depleted from brain extract by preincubation with the Src homology 3 domain of Nck to which Nap1 binds. Furthermore, antibodies to Nap1 reacted with the 125 kDa protein. Nap1 was co-immunoprecipitated with a constitutively active form of Rac expressed in Chinese hamster ovary cells. The observation that complex formation between activated Rac and PAK, but not that between Rac and Nap1, could be reproduced in vitro with recombinant proteins indicates that the interaction of Nap1 with Rac is indirect. The 140 kDa Rac-binding protein is a potential candidate for a link that connects Nap1 to Rac. The multimolecular complex comprising Rac, Nap1 and probably the 140 kDa protein might mediate some of the biological effects transmitted by the multipotent GTPase.
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Gauthier-Rouvière, Cécile, Emmanuel Vignal, Mayya Mériane, Pierre Roux, Philippe Montcourier, and Philippe Fort. "RhoG GTPase Controls a Pathway That Independently Activates Rac1 and Cdc42Hs." Molecular Biology of the Cell 9, no. 6 (June 1998): 1379–94. http://dx.doi.org/10.1091/mbc.9.6.1379.
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RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.
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Choi, Ki Young, Min Sup Lee, Young Jun Cho, Myong Ho Jeong, Seung Jin Han, and Seung Hwan Hong. "p104 Binds to Rac1 and Reduces Its Activity during Myotube Differentiation of C2C12 Cell." Scientific World Journal 2014 (2014): 1–12. http://dx.doi.org/10.1155/2014/592450.
Full textAbstract:
The p104 protein inhibits cellular proliferation when overexpressed in NIH3T3 cells and has been shown to associate with p85α, Grb2, and PLCγ1. In order to isolate other proteins that interact with p104, yeast two-hybrid screening was performed. Rac1 was identified as a binding partner of p104 and the interaction between p104 and Rac1 was confirmed by immunoprecipitation. Using a glutathione S-transferase (GST) pull-down assay with various p104 fragments, the 814–848 amino acid residue at the carboxyl-terminal region of p104 was identified as the key component to interact with Rac1. The CrkII which is involved in the Rac1-mediated cellular response was also found to interact with p104 protein. NIH3T3 cells which overexpressed p104 showed a decrease of Rac1 activity. However, neither the proline-rich domain mutant, which is unable to interact with CrkII, nor the carboxy-terminal deletion mutant could attenuate Rac1 activity. During the differentiation of myoblasts, the amount of p104 protein as well as transcript level was increased. The overexpression of p104 enhanced myotube differentiation, whereas siRNA of p104 reversed this process. In this process, more Rac1 and CrkII were bound to increased p104. Based on these results, we conclude that p104 is involved in muscle cell differentiation by modulating the Rac1 activity.
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Heyworth, P. G., U. G. Knaus, J. Settleman, J. T. Curnutte, and G. M. Bokoch. "Regulation of NADPH oxidase activity by Rac GTPase activating protein(s)." Molecular Biology of the Cell 4, no. 11 (November 1993): 1217–23. http://dx.doi.org/10.1091/mbc.4.11.1217.
Full textAbstract:
Activation of the NADPH oxidase of phagocytic cells requires the action of Rac2 or Rac1, members of the Ras superfamily of GTP-binding proteins. Rac proteins are active when in the GTP-bound form and can be regulated by a variety of proteins that modulate the exchange of GDP for GTP and/or GTP hydrolysis. The p190 Rac GTPase Activating Protein (GAP) inhibits human neutrophil NADPH oxidase activity in a cell-free assay system with a K1 of approximately 100 nM. Inhibition by p190 was prevented by GTP gamma S, a nonhydrolyzable analogue of GTP. Similar inhibition was seen with a second protein exhibiting Rac GAP activity, CDC42Hs GAP. The effect of p190 on superoxide (O2-) formation was reversed by the addition of a constitutively GTP-bound Rac2 mutant or Rac1-GTP gamma S but not by RhoA-GTP gamma S. Addition of p190 to an activated oxidase produced no inhibitory effect, suggesting either that p190 no longer has access to Rac in the assembled oxidase or that Rac-GTP is not required for activity once O2- generation has been initiated. These data confirm the role of Rac in NADPH oxidase regulation and support the view that it is the GTP form of Rac that is necessary for oxidase activation. Finally, they raise the possibility that NADPH oxidase may be regulated by the action of GAPs for Rac proteins.
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Buscemi, Nina, Chris Murray, Amanda Doherty-Kirby, Gilles Lajoie, Mark A. Sussman, and Jennifer E. Van Eyk. "Myocardial subproteomic analysis of a constitutively active Rac1-expressing transgenic mouse with lethal myocardial hypertrophy." American Journal of Physiology-Heart and Circulatory Physiology 289, no. 6 (December 2005): H2325—H2333. http://dx.doi.org/10.1152/ajpheart.01041.2004.
Full textAbstract:
A two-dimensional gel electrophoresis (2-DE)-based proteomic approach was used to study a transgenic mouse model of acerbated dilated cardiomyopathy in which the small monomeric GTPase, Rac1, was constitutively expressed exclusively in the myocardium. A subfractionation procedure allowed for the focused analysis of both cytoplasmic and myofilament protein-enriched extracts of ventricular tissue from Rac1 transgenic and age-matched nontransgenic (NTG) mice. The majority of these mice displayed severe hypertrophy (heart-to-body weight ratios >2-fold greater in the Rac1 mice) and died from overt heart failure between days 14 and 17. Comparative 2-DE analysis (pH 3–10, 12% SDS-PAGE) derived from Rac1 ( n = 4) and NTG ( n = 4) groups revealed differences in mean protein spot intensities. Twelve proteins from the cytoplasmic protein-enriched extract met our criteria for robustness and spot resolution and were identified. These proteins represent a broad distribution of cellular functions with only some previously implicated in myocardial hypertrophy. The myofilament subproteome displayed no change in posttranslational modification, but further analysis by one-dimensional Western blot showed increased quantities of myofilament proteins in the Rac1 mouse ventricles. Additionally, three proteins with different functionality that were altered in the cytoplasmic protein-enriched subproteome, tubulin β-chain, manganese superoxide dismutase, and malate dehydrogenase, were analyzed at days 7, 9, and 11 to assess their role in the development of the dilated cardiomyopathic phenotype. The quantity of all three proteins peaked at day 9, suggesting an early response in cardiac hypertrophic failure.
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Hamill, Kevin J., Susan B. Hopkinson, Philip DeBiase, and Jonathan C. R. Jones. "BPAG1e Maintains Keratinocyte Polarity through β4 Integrin–mediated Modulation of Rac 1 and Cofilin Activities." Molecular Biology of the Cell 20, no. 12 (June 15, 2009): 2954–62. http://dx.doi.org/10.1091/mbc.e09-01-0051.
Full textAbstract:
α6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.
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Aranda, Juan F., Natalia Reglero-Real, Leonor Kremer, Beatriz Marcos-Ramiro, Ana Ruiz-Sáenz, María Calvo, Carlos Enrich, Isabel Correas, Jaime Millán, and Miguel A. Alonso. "MYADM regulates Rac1 targeting to ordered membranes required for cell spreading and migration." Molecular Biology of the Cell 22, no. 8 (April 15, 2011): 1252–62. http://dx.doi.org/10.1091/mbc.e10-11-0910.
Full textAbstract:
Membrane organization into condensed domains or rafts provides molecular platforms for selective recruitment of proteins. Cell migration is a general process that requires spatiotemporal targeting of Rac1 to membrane rafts. The protein machinery responsible for making rafts competent to recruit Rac1 remains elusive. Some members of the MAL family of proteins are involved in specialized processes dependent on this type of membrane. Because condensed membrane domains are a general feature of the plasma membrane of all mammalian cells, we hypothesized that MAL family members with ubiquitous expression and plasma membrane distribution could be involved in the organization of membranes for cell migration. We show that myeloid-associated differentiation marker (MYADM), a protein with unique features within the MAL family, colocalizes with Rac1 in membrane protrusions at the cell surface and distributes in condensed membranes. MYADM knockdown (KD) cells had altered membrane condensation and showed deficient incorporation of Rac1 to membrane raft fractions and, similar to Rac1 KD cells, exhibited reduced cell spreading and migration. Results of rescue-of-function experiments by expression of MYADM or active Rac1L61 in cells knocked down for Rac1 or MYADM, respectively, are consistent with the idea that MYADM and Rac1 act on parallel pathways that lead to similar functional outcomes.
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Wang, Xiaohui, Dongbin Liu, Fangzhen Wei, Yue Li, Xuefeng Wang, Linjie Li, Guan Wang, Shuli Zhang, and Lei Zhang. "Stress-Sensitive Protein Rac1 and Its Involvement in Neurodevelopmental Disorders." Neural Plasticity 2020 (November 24, 2020): 1–11. http://dx.doi.org/10.1155/2020/8894372.
Full textAbstract:
Ras-related C3 botulinum toxin substrate 1 (Rac1) is a small GTPase that is well known for its sensitivity to the environmental stress of a cell or an organism. It senses the external signals which are transmitted from membrane-bound receptors and induces downstream signaling cascades to exert its physiological functions. Rac1 is an important regulator of a variety of cellular processes, such as cytoskeletal organization, generation of oxidative products, and gene expression. In particular, Rac1 has a significant influence on certain brain functions like neuronal migration, synaptic plasticity, and memory formation via regulation of actin dynamics in neurons. Abnormal Rac1 expression and activity have been observed in multiple neurological diseases. Here, we review recent findings to delineate the role of Rac1 signaling in neurodevelopmental disorders associated with abnormal spine morphology, synaptogenesis, and synaptic plasticity. Moreover, certain novel inhibitors of Rac1 and related pathways are discussed as potential avenues toward future treatment for these diseases.
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Faix, J., C. Clougherty, A. Konzok, U. Mintert, J. Murphy, R. Albrecht, B. Muhlbauer, and J. Kuhlmann. "The IQGAP-related protein DGAP1 interacts with Rac and is involved in the modulation of the F-actin cytoskeleton and control of cell motility." Journal of Cell Science 111, no. 20 (October 15, 1998): 3059–71. http://dx.doi.org/10.1242/jcs.111.20.3059.
Full textAbstract:
DGAP1 of Dictyostelium discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP-related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated GTP-bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organised in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility.
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Kalfa, Theodosia A., Suvarnamala Pushkaran, James F. Johnson, Qian Wei, David A. Williams, and Yi Zheng. "Erythrocyte Cytoskeletal Defects Induced in Mice by Deletion of Rac GTPases." Blood 104, no. 11 (November 16, 2004): 1573. http://dx.doi.org/10.1182/blood.v104.11.1573.1573.
Full textAbstract:
Abstract The small Rho GTPases Rac1 and Rac2 have been implicated in regulating actin structures in a variety of cells, including hematopoietic stem cells and leucocytes. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. We explored the possible role of Rac1 and Rac2 signaling molecules in the dynamic assembly of actin in the red blood cells (RBC), and thus in the regulation of morphology and function of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases have been shown to have overlapping as well as distinct roles in actin organization, cell survival, and proliferation in hematopoietic stem cells (Gu et al. Science, 2003); we focused our study on the erythrocyte phenotype of Rac2−/− and Rac1−/−;Rac2−/− mice. Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background. Deletion of Rac1 after treatment with PolyI:PolyC to induce Cre recombinase was confirmed in bone marrow cells using DNA PCR and in erythrocytes by immunoblot. Since the erythrocytes consist a population of variable age, the optimal time of the maximum Rac1 deletion in erythrocytes was determined to be three to five weeks post induction. During this period, Rac1 protein in erythrocytes was decreased by 50–80% as determined by immunoblot densitometry. Rac2−/− and wild-type mice were subjected to the same treatment to control for any effects of PolyI:PolyC independent of the Rac1 deletion. Blood samples were obtained weekly after the completion of induction and the hematologic phenotype was studied by evaluation of complete blood counts, RBC indices, and reticulocyte counts. Erythrocyte morphology was examined on Wright-Giemsa smears of peripheral blood. Intact erythrocytes and erythrocyte ghosts were stained for actin with rhodamine-phalloidin and studied by confocal microscopy. The Rac2−/− mice appeared to have a rather mild erythrocyte phenotype with no significant anemia or reticulocytosis, although they did demonstrate a mild poikilocytosis and anisocytosis at baseline. The Rac1−/−;Rac2−/− mice developed a microcytic anemia with a hemoglobin drop of up to 30% in comparison to the baseline and to the wild-type hemoglobin values, with the nadir noted at three weeks post induction. The percentage of reticulocytes increased up to threefold in comparison to the control group. The mean corpuscular volume decreased up to 20% from the baseline in the Rac1−/−;Rac2−/− mice, and remained decreased up to six weeks post induction with an elevated red blood cell distribution width. Significant anisocytosis and poikilocytosis were observed with fragmented erythrocytes in the peripheral blood smear. Filamentous actin in the RBC cytoskeleton stained with rhodamine-phalloidin appeared to have a uniform distribution in intact and ghost erythrocytes under confocal microscopy. However, Rac1−/−;Rac2−/− erythrocytes demonstrated punctuate lesions on the cell surface while Rac1−/−;Rac2−/− erythrocyte ghosts appeared to collapse into irregular shapes. These data suggest that deficiency of Rac1 and Rac2 GTPases in mice cause a microcytic hemolytic anemia with poikilocytosis and red cell fragmentation indicating a possible dynamic regulation of the erythrocyte cytoskeleton organization by these signaling molecules.
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Zang, Li, Quan Hong, Guoqing Yang, Weijun Gu, Anping Wang, Jingtao Dou, Yiming Mu, Di Wu, and Zhaohui Lyu. "MACROD1/LRP16 Enhances LPS-Stimulated Inflammatory Responses by Up-Regulating a Rac1-Dependent Pathway in Adipocytes." Cellular Physiology and Biochemistry 51, no. 6 (2018): 2591–603. http://dx.doi.org/10.1159/000495931.
Full textAbstract:
Background/Aims: Chronic inflammation contributes to the development of type 2 diabetes mellitus by targeting the insulin receptor substrate protein-1 (IRS-1) signaling pathway. Previous studies showed that Leukemia related protein 16 (LRP16) reduced insulin stimulated glucose uptake in adipocytes by impairing the IRS-1 signaling pathway. We explored the mechanism by which LRP16 promotes the inflammatory response. Methods: We screened LRP16 induced proteins in the lipopolysaccharide (LPS)-stimulated inflammatory response using liquid chromatography-mass spectrometry (LC-MS) and analyzed the potential biological functions of these proteins using online bioinformatics tools. mRNA expression and protein expression of target genes were measured by real time PCR and Western blot, respectively. Results: A total of 390 differentially expressed proteins were identified. The mitogen-activated protein kinase (MAPK) signaling pathway was the primary activated pathway in LRP16-expressing cells. Overexpression of LRP16 activated ERK1/2 and Rac1, which are two key players related to the MAPK signaling pathway. Furthermore, knock down of endogenous LRP16 by RNA interference (RNAi) reduced Rac1 expression, ERK activation, and inflammatory cytokine expression in human adipocytes stimulated by LPS. The stimulatory effect of LRP16 was diminished by suppressing Rac1 expression and treating the cells with the ERK specific inhibitor, PD98059. Conclusion: These findings revealed the functions of LRP16 in promoting the inflammatory response through activating the Rac1-MAPK1/ERK pathway in human adipocytes.
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Weiß, Lukas, Lana Gaelings, Tina Reiner, Julia Mergner, Bernhard Kuster, Attila Fehér, Götz Hensel, et al. "Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination." PLOS ONE 17, no. 3 (March 25, 2022): e0258924. http://dx.doi.org/10.1371/journal.pone.0258924.
Full textAbstract:
Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.
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Eiden, Caroline, and Hendrik Ungefroren. "The Ratio of RAC1B to RAC1 Expression in Breast Cancer Cell Lines as a Determinant of Epithelial/Mesenchymal Differentiation and Migratory Potential." Cells 10, no. 2 (February 8, 2021): 351. http://dx.doi.org/10.3390/cells10020351.
Full textAbstract:
Breast cancer (BC) is a heterogenous disease encompassing tumors with different histomorphological phenotypes and transcriptionally defined subtypes. However, the non-mutational/epigenetic alterations that are associated with or causally involved in phenotype diversity or conversion remain to be elucidated. Data from the pancreatic cancer model have shown that the small GTPase RAC1 and its alternatively spliced isoform, RAC1B, antagonistically control epithelial–mesenchymal transition and cell motility induced by transforming growth factor β. Using a battery of established BC cell lines with either a well-differentiated epithelial or poorly differentiated mesenchymal phenotype, we observed subtype-specific protein expression of RAC1B and RAC1. While epithelial BC lines were RAC1Bhigh and RAC1low, mesenchymal lines exhibited the reverse expression pattern. High RAC1B and/or low RAC1 abundance also correlated closely with a poor invasion potential, and vice versa, as revealed by measuring random cell migration (chemokinesis), the preferred mode of cellular movement in cells that have undergone mesenchymal transdifferentiation. We propose that a high RAC1B:RAC1 ratio in BC cells is predictive of an epithelial phenotype, while low RAC1B along with high RAC1 is a distinguishing feature of the mesenchymal state. The combined quantitative assessment of RAC1B and RAC1 in tumor biopsies of BC patients may represent a novel diagnostic tool for probing molecular subtype and eventually predict malignant potential of breast tumors.
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Yang, Xiaoxu, Yongjin Sun, Xu Li, and Wenzhi Zhang. "Rac1 regulates nucleus pulposus cell degeneration by activating the Wnt/β-catenin signaling pathway and promotes the progression of intervertebral disc degeneration." American Journal of Physiology-Cell Physiology 322, no. 3 (March 1, 2022): C496—C507. http://dx.doi.org/10.1152/ajpcell.00355.2021.
Full textAbstract:
Nucleus pulposus cell (NPC) dysfunction is considered as an important event related to intervertebral disc degeneration (IVDD). In the present study, tandem mass spectrometry (TMT) was used to detect total protein expression of nucleus pulposus (NP) in patients with IVDD and healthy controls. Bioinformatic analysis was performed to identify differentially expressed proteins that may be involved in the degeneration of NP. The results show that Rac1 may be a key protein involved in the degeneration of NP via Wnt/β-catenin pathway activation. We investigated the influence of Rac1 on IVDD degeneration and associated mechanisms. Rac1 expression increased in interleukin (IL)-1β-stimulated human NPCs, consistent with the results of TMT. The Rac1 inhibitor NSC23766 alleviated the degeneration of NPCs in vitro. Furthermore, Rac1 activated Wnt/β-catenin signaling, and the inhibition of this pathway significantly ameliorated the Rac1-mediated degenerative phenotype. NSC23766 exerted protective effects on IVDD in a puncture rat model. Taken together, these data suggest that Rac1 inhibition can delay NPC degeneration, probably through the regulation of the Wnt/β-catenin pathway. This study has the potential to advance understanding of the mechanism of occurrence of degenerative NP tissues and to provide novel strategies for slowing IVDD progression.
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Chan, Diane, Allison Citro, Joanna M. Cordy, Grace C. Shen, and Benjamin Wolozin. "Rac1 Protein Rescues Neurite Retraction Caused by G2019S Leucine-rich Repeat Kinase 2 (LRRK2)." Journal of Biological Chemistry 286, no. 18 (March 16, 2011): 16140–49. http://dx.doi.org/10.1074/jbc.m111.234005.
Full textAbstract:
Mutations in leucine-rich repeat kinase 2 (LRRK2) are currently the most common genetic cause of familial late-onset Parkinson disease, which is clinically indistinguishable from idiopathic disease. The most common pathological mutation in LRRK2, G2019S LRRK2, is known to cause neurite retraction. However, molecular mechanisms underlying regulation of neurite length by LRRK2 are unknown. Here, we demonstrate a novel interaction between LRRK2 and the Rho GTPase, Rac1, which plays a critical role in actin cytoskeleton remodeling necessary for the maintenance of neurite morphology. LRRK2 binds strongly to endogenous or expressed Rac1, while showing weak binding to Cdc42 and no binding to RhoA. Co-expression with LRRK2 increases Rac1 activity, as shown by increased binding to the p21-activated kinase, which modulates actin cytoskeletal dynamics. LRRK2 constructs carrying mutations that inactivate the kinase or GTPase activities do not activate Rac1. Interestingly, LRRK2 does not increase levels of membrane-bound Rac1 but dramatically changes the cellular localization of Rac1, causing polarization, which is augmented further when LRRK2 is co-expressed with constitutively active Rac1. Four different disease-related mutations in LRRK2 altered binding to Rac1, with the G2019S and R1441C LRRK2 mutations attenuating Rac1 binding and the Y1699C and I2020T LRRK2 mutations increasing binding. Co-expressing Rac1 in SH-SY5Y cells rescues the G2019S mutant phenotype of neurite retraction. We hypothesize that pathological mutations in LRRK2 attenuates activation of Rac1, causing disassembly of actin filaments, leading to neurite retraction. The interactions between LRRK2 and Rho GTPases provide a novel pathway through which LRRK2 might modulate cellular dynamics and contribute to the pathophysiology of Parkinson disease.
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Azim, Anser C., Hongmei Cao, Xiaopei Gao, Myungsoo Joo, Asrar B. Malik, Richard B. van Breemen, Ruxana T. Sadikot, GyeYoung Park, and John W. Christman. "Regulation of cyclooxygenase-2 expression by small GTPase Rac2 in bone marrow macrophages." American Journal of Physiology-Lung Cellular and Molecular Physiology 293, no. 3 (September 2007): L668—L673. http://dx.doi.org/10.1152/ajplung.00043.2007.
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Cyclooxygenase 2 (COX-2) is induced by microbial products, proinflammatory cytokines, growth factors, and oncogenes. The Rho family includes RhoA, Rac1, Rac2, Rac3, and Cdc42 and is involved in regulation of the actin cytoskeleton organization, cell growth, vesicular cell trafficking, and transcriptional regulation. Rac2 binds to NADPH oxidase protein complex, and Rac2 null neutrophils are known to have poor phagocytic activity. We examined whether Rac2, the predominant small GTPase in hematopoietic cells, influences COX-2 expression in bone marrow-derived macrophages (BMDM). We showed that BMDM from Rac2−/−null mice have reduced COX-2 expression in response to treatment with endotoxin. Despite a compensatory increase in Rac1, BMDM from Rac2−/−null mice have less biologically active GTP-bound Rac in response to LPS stimulation. Signaling molecules (downstream of Rac2 and Toll-like receptor 4) such as p42/44, p38, and pAKT were also affected in BMDM from Rac2−/−null mouse macrophages. We also observed that BMDM from Rac2−/−null failed to degrade IκBα significantly and had less immunoreactive PU.1. We show that both NF-κB pathway and PU.1 are involved in normal macrophage function and play a role in macrophage COX-2 expression. In summary, these data indicate that Rac2 regulates COX-2 expression in BMDM.
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Clerk, Angela, Fong H. Pham, Stephen J. Fuller, Erik Sahai, Klaus Aktories, Richard Marais, Chris Marshall, and Peter H. Sugden. "Regulation of Mitogen-Activated Protein Kinases in Cardiac Myocytes through the Small G Protein Rac1." Molecular and Cellular Biology 21, no. 4 (February 15, 2001): 1173–84. http://dx.doi.org/10.1128/mcb.21.4.1173-1184.2001.
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ABSTRACT Small guanine nucleotide-binding proteins of the Ras and Rho (Rac, Cdc42, and Rho) families have been implicated in cardiac myocyte hypertrophy, and this may involve the extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK), and/or p38 mitogen-activated protein kinase (MAPK) cascades. In other systems, Rac and Cdc42 have been particularly implicated in the activation of JNKs and p38-MAPKs. We examined the activation of Rho family small G proteins and the regulation of MAPKs through Rac1 in cardiac myocytes. Endothelin 1 and phenylephrine (both hypertrophic agonists) induced rapid activation of endogenous Rac1, and endothelin 1 also promoted significant activation of RhoA. Toxin B (which inactivates Rho family proteins) attenuated the activation of JNKs by hyperosmotic shock or endothelin 1 but had no effect on p38-MAPK activation. Toxin B also inhibited the activation of the ERK cascade by these stimuli. In transfection experiments, dominant-negative N17Rac1 inhibited activation of ERK by endothelin 1, whereas activated V12Rac1 cooperated with c-Raf to activate ERK. Rac1 may stimulate the ERK cascade either by promoting the phosphorylation of c-Raf or by increasing MEK1 and/or -2 association with c-Raf to facilitate MEK1 and/or -2 activation. In cardiac myocytes, toxin B attenuated c-Raf(Ser-338) phosphorylation (50 to 70% inhibition), but this had no effect on c-Raf activity. However, toxin B decreased both the association of MEK1 and/or -2 with c-Raf and c-Raf-associated ERK-activating activity. V12Rac1 cooperated with c-Raf to increase expression of atrial natriuretic factor (ANF), whereas N17Rac1 inhibited endothelin 1-stimulated ANF expression, indicating that the synergy between Rac1 and c-Raf is potentially physiologically important. We conclude that activation of Rac1 by hypertrophic stimuli contributes to the hypertrophic response by modulating the ERK and/or possibly the JNK (but not the p38-MAPK) cascades.
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Brill, S., S. Li, C. W. Lyman, D. M. Church, J. J. Wasmuth, L. Weissbach, A. Bernards, and A. J. Snijders. "The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases." Molecular and Cellular Biology 16, no. 9 (September 1996): 4869–78. http://dx.doi.org/10.1128/mcb.16.9.4869.
Full textAbstract:
We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.
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Hope, Hannah, Stéphanie Bogliolo, Robert A. Arkowitz, and Martine Bassilana. "Activation of Rac1 by the Guanine Nucleotide Exchange Factor Dck1 Is Required for Invasive Filamentous Growth in the Pathogen Candida albicans." Molecular Biology of the Cell 19, no. 9 (September 2008): 3638–51. http://dx.doi.org/10.1091/mbc.e07-12-1272.
Full textAbstract:
Rho G proteins and their regulators are critical for cytoskeleton organization and cell morphology in all eukaryotes. In the opportunistic pathogen Candida albicans, the Rho G proteins Cdc42 and Rac1 are required for the switch from budding to filamentous growth in response to different stimuli. We show that Dck1, a protein with homology to the Ced-5, Dock180, myoblast city family of guanine nucleotide exchange factors, is necessary for filamentous growth in solid media, similar to Rac1. Our results indicate that Dck1 and Rac1 do not function in the same pathway as the transcription factor Czf1, which is also required for embedded filamentous growth. The conserved catalytic region of Dck1 is required for such filamentous growth, and in vitro this region directly binds a Rac1 mutant, which mimics the nucleotide-free state. In vivo overexpression of a constitutively active Rac1 mutant, but not wild-type Rac1, in a dck1 deletion mutant restores filamentous growth. These results indicate that the Dock180 guanine nucleotide exchange factor homologue, Dck1 activates Rac1 during invasive filamentous growth. We conclude that specific exchange factors, together with the G proteins they activate, are required for morphological changes in response to different stimuli.
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Vartiainen, Maria, Pauli J. Ojala, Petri Auvinen, Johan Peränen, and Pekka Lappalainen. "Mouse A6/Twinfilin Is an Actin Monomer-Binding Protein That Localizes to the Regions of Rapid Actin Dynamics." Molecular and Cellular Biology 20, no. 5 (March 1, 2000): 1772–83. http://dx.doi.org/10.1128/mcb.20.5.1772-1783.2000.
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ABSTRACT In our database searches, we have identified mammalian homologues of yeast actin-binding protein, twinfilin. Previous studies suggested that these mammalian proteins were tyrosine kinases, and therefore they were named A6 protein tyrosine kinase. In contrast to these earlier studies, we did not find any tyrosine kinase activity in our recombinant protein. However, biochemical analysis showed that mouse A6/twinfilin forms a complex with actin monomer and prevents actin filament assembly in vitro. A6/twinfilin mRNA is expressed in most adult tissues but not in skeletal muscle and spleen. In mouse cells, A6/twinfilin protein is concentrated to the areas at the cell cortex which overlap with G-actin-rich actin structures. A6/twinfilin also colocalizes with the activated forms of small GTPases Rac1 and Cdc42 to membrane ruffles and to cell-cell contacts, respectively. Furthermore, expression of the activated Rac1(V12) in NIH 3T3 cells leads to an increased A6/twinfilin localization to nucleus and cell cortex, whereas a dominant negative form of Rac1(V12,N17) induces A6/twinfilin localization to cytoplasm. Taken together, these studies show that mouse A6/twinfilin is an actin monomer-binding protein whose localization to cortical G-actin-rich structures may be regulated by the small GTPase Rac1.
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Khosravi-Far, R., P. A. Solski, G. J. Clark, M. S. Kinch, and C. J. Der. "Activation of Rac1, RhoA, and mitogen-activated protein kinases is required for Ras transformation." Molecular and Cellular Biology 15, no. 11 (November 1995): 6443–53. http://dx.doi.org/10.1128/mcb.15.11.6443.
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Although substantial evidence supports a critical role for the activation of Raf-1 and mitogen-activated protein kinases (MAPKs) in oncogenic Ras-mediated transformation, recent evidence suggests that Ras may activate a second signaling pathway which involves the Ras-related proteins Rac1 and RhoA. Consequently, we used three complementary approaches to determine the contribution of Rac1 and RhoA function to oncogenic Ras-mediated transformation. First, whereas constitutively activated mutants of Rac1 and RhoA showed very weak transforming activity when transfected alone, their coexpression with a weakly transforming Raf-1 mutant caused a greater than 35-fold enhancement of transforming activity. Second, we observed that coexpression of dominant negative mutants of Rac1 and RhoA reduced oncogenic Ras transforming activity. Third, activated Rac1 and RhoA further enhanced oncogenic Ras-triggered morphologic transformation, as well as growth in soft agar and cell motility. Finally, we also observed that kinase-deficient MAPKs inhibited Ras transformation. Taken together, these data support the possibility that oncogenic Ras activation of Rac1 and RhoA, coupled with activation of the Raf/MAPK pathway, is required to trigger the full morphogenic and mitogenic consequences of oncogenic Ras transformation.
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Liu, Kathleen D., Anirban Datta, Wei Yu, Paul R. Brakeman, Tzuu-Shuh Jou, Michael A. Matthay, and Keith E. Mostov. "Rac1 is required for reorientation of polarity and lumen formation through a PI 3-kinase-dependent pathway." American Journal of Physiology-Renal Physiology 293, no. 5 (November 2007): F1633—F1640. http://dx.doi.org/10.1152/ajprenal.00053.2007.
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Epithelial cells are characterized by the ability to form sheets of cells that surround fluid-filled lumens. Cells in these sheets exhibit a characteristic subcellular polarity, with an apical pole that faces the lumen and a basolateral pole that is in contact with other cells and the extracellular matrix (ECM). To investigate the signaling events required for polarization and lumen formation, we have taken advantage of the ability of Madin-Darby canine kidney (MDCK) cells to dynamically remodel their polarity in response to changes in ECM cues. When MDCK cells are grown in suspension culture, they form multicellular “inside-out” cysts with apical proteins found on the peripheral surface and basolateral markers on the interior surface. When these inside-out cysts are embedded in ECM, they rapidly reorient their polarity: apical proteins become localized to the inside surface, and basolateral proteins are found on the surface that contacts ECM. Here we have characterized the signaling requirements for these early molecular reorientation events. Specifically, expression of a dominant-negative form of Rac1 (DN-Rac1) blocks the reorientation of polarity. Phosphoinositide 3′-kinase is required for apical membrane protein remodeling from the initial apical membrane surface. Cells expressing DN-Rac1 fail to detectably activate the PI 3-kinase/protein kinase B pathway. Last, we found that atypical protein kinase C (aPKC) is also required for reorientation of polarity, since an inhibitor of atypical PKC blocks reorientation. This effect cannot be overcome by constitutively active Rac1, demonstrating that both Rac1 and atypical PKC are required for reorientation of cellular polarity.
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Abdrabou, Abdalla, Daniel Brandwein, Changyu Liu, and Zhixiang Wang. "Rac1 S71 Mediates the Interaction between Rac1 and 14-3-3 Proteins." Cells 8, no. 9 (August 30, 2019): 1006. http://dx.doi.org/10.3390/cells8091006.
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Both 14-3-3 proteins (14-3-3s) and Rho proteins regulate cytoskeleton remodeling and cell migration, which suggests a possible interaction between the signaling pathways regulated by these two groups of proteins. Indeed, more and more emerging evidence indicates the mutual regulation of these two signaling pathways. However, all of the data regarding the interaction between Rac1 signaling pathways and 14-3-3 signaling pathways are through either the upstream regulators or downstream substrates. It is not clear if Rac1 could interact with 14-3-3s directly. It is interesting to notice that the Rac1 sequence 68RPLSYP73 is likely a 14-3-3 protein binding motif following the phosphorylation of S71 by Akt. Thus, we hypothesize that Rac1 directly interacts with 14-3-3s. We tested this hypothesis in this research. By using mutagenesis, co-immunoprecipitation (co-IP), Rac1 activity assay, immunoblotting, and indirect immunofluorescence, we demonstrate that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners, but the phosphorylation-dependent interaction is much stronger. Epidermal growth factor (EGF) strongly stimulates the phosphorylation of Rac1 S71 and the interaction between 14-3-3s and Rac1. Mutating S71 to A completely abolishes both phosphorylation-dependent and -independent interactions between 14-3-3s and Rac1. The interaction between 14-3-3s and Rac1 mostly serve to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -σ, and -θ showed interactions with Rac1 in both Cos-7 and HEK 293 cells. 14-3-3γ also binds to Rac1 in HEK 293 cells, but not in Cos-7 cells. We conclude that 14-3-3s interact with Rac1. This interaction is mediated by Rac1 S71 in both phosphorylation-dependent and -independent manners. The interaction between 14-3-3 and Rac1 mostly serves to regulate the activity and subcellular localization of Rac1. Among the seven 14-3-3 isoforms, 14-3-3η, -γ, -σ, and -θ interact with Rac1.
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Tyasi, Thobela Louis, Xue Sun, Xuesong Shan, Simushi Liswaniso, Ignatius Musenge Chimbaka, Ning Qin, and Rifu Xu. "Effects of RAC1 on Proliferation of Hen Ovarian Prehierarchical Follicle Granulosa Cells." Animals 10, no. 9 (September 6, 2020): 1589. http://dx.doi.org/10.3390/ani10091589.
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RAC1 belongs to the small G protein Rho subfamily and is implicated in regulating gene expression, cell proliferation and differentiation in mammals and humans; nevertheless, the function of RAC1 in growth and development of hen ovarian follicles is still unclear. This study sought to understand the biological effects of RAC1 on granulosa cell (GC) proliferation and differentiation of hen ovarian prehierarchical follicles. Firstly, our results showed expression levels of RAC1 mRNA in the follicles with diameters of 7.0–8.0 mm, 6.0–6.9 mm and 1.0–3.9 mm were greater than other follicles (p < 0.05). The RAC1 protein was mainly expressed in oocyte and its around GCs and stromal tissues of the prehierarchical follicles by immunohistochemistry. Further investigation revealed the RAC1 gene remarkably enhanced the mRNA and protein expression levels of FSHR (a marker of follicle selection), CCND2 (a marker of cell-cycle progression and GC differentiation), PCNA (a marker of GC proliferation), StAR and CYP11A1 (markers of GC differentiation and steroidogenesis) (p < 0.05). Furthermore, our data demonstrated siRNA interference of RAC1 significantly reduced GC proliferation (p < 0.05), while RAC1 gene overexpression enhanced GC proliferation in vitro (p < 0.05). Collectively, this study provided new evidence that the biological effects of RAC1 on GC proliferation, differentiation and steroidogenesis of chicken ovary follicles.
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Zavarella, Salvatore, Mitsutoshi Nakada, Shawn Belverud, Salvatore J. Coniglio, Amanda Chan, Mark A. Mittler, Steven J. Schneider, and Marc Symons. "Role of Rac1-regulated signaling in medulloblastoma invasion." Journal of Neurosurgery: Pediatrics 4, no. 2 (August 2009): 97–104. http://dx.doi.org/10.3171/2009.4.peds08322.
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Object Medulloblastomas are the most common malignant brain tumors in children. These tumors are highly invasive, and patients harboring these lesions are frequently diagnosed with distant spread. In this study, the authors investigated the role of Rac1, a member of the Rho family of small guanosine triphosphatases, in medulloblastoma invasion. Methods Three established medulloblastoma cell lines were used: DAOY, UW-228, and ONS-76. Specific depletion of Rac1 protein was accomplished by transient transfection of small interfering RNA. Cell invasion through extracellular matrix (Matrigel) was quantified using a transwell migration assay. Mitogen activated protein kinase activation was determined using phospho-MAP kinase–specific antibodies, and inhibition of MAP kinase pathways was achieved by specific small molecule inhibitors. Localization of Rac1 and its expression levels were determined by immunohistochemical analysis using a Rac1-specific antibody, and Rac1 activation was qualitatively assessed by Rac1 plasma membrane association. Results Small interfering RNA–mediated depletion of Rac1 strongly inhibited medulloblastoma cell invasion. Although depletion of Rac1 inhibited the proliferation of UW-228 cells, and of ONS-76 cells to a lesser extent, it stimulated the proliferation of DAOY cells. Depletion of Rac1 also inhibited the activation of the ERK and JNK MAP kinase pathways, and inhibition of either pathway diminished invasion and proliferation. Immunohistochemical analysis demonstrated that the Rac1 protein was overexpressed in all medulloblastoma tumors examined, and indicated that Rac1 was hyperactive in 6 of 25 tumors. Conclusions The authors' data show that Rac1 is necessary for the invasive behavior of medulloblastoma cells in vitro, and plays a variable role in medulloblastoma cell proliferation. In addition, these results indicate that Rac1 stimulates medulloblastoma invasion by activating the ERK and JNK pathways. The authors suggest that Rac1 and signaling elements controlled by this guanosine triphosphatase may serve as novel targets for therapeutic intervention in malignant medulloblastomas.
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Vallim, Marcelo A., Connie B. Nichols, Larissa Fernandes, Kari L. Cramer, and J. Andrew Alspaugh. "A Rac Homolog Functions Downstream of Ras1 To Control Hyphal Differentiation and High-Temperature Growth in the Pathogenic Fungus Cryptococcus neoformans." Eukaryotic Cell 4, no. 6 (June 2005): 1066–78. http://dx.doi.org/10.1128/ec.4.6.1066-1078.2005.
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ABSTRACT The Cryptococcus neoformans Ras1 protein serves as a central regulator for several signaling pathways. Ras1 controls the induction of the mating pheromone response cascade as well as a distinct signaling pathway that allows this pathogenic fungus to grow at human physiological temperature. To characterize elements of the Ras1-dependent high-temperature growth pathway, we performed a multicopy suppressor screen, identifying genes whose overexpression allows the ras1 mutant to grow at 37°C. Using this genetic technique, we identified a C. neoformans gene encoding a Rac homolog that suppresses multiple ras1 mutant phenotypes. Deletion of the RAC1 gene does not affect high-temperature growth. However, a rac1 mutant strain demonstrates a profound defect in haploid filamentation as well as attenuated mating. In a yeast two-hybrid assay, Rac1 physically interacts with the PAK kinase Ste20, which similarly regulates hyphal formation in this fungus. Similar to Rac1, overexpression of the STE20α gene also restores high-temperature growth to the ras1 mutant. These results support a model in which the small G protein Rac1 acts downstream of Ras proteins and coordinately with Ste20 to control high-temperature growth and cellular differentiation in this human fungal pathogen.
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Chatterjee, Moumita, Linda Sequeira, Mashariki Jenkins-Kabaila, Cara W. Dubyk, Surabhi Pathak, and Kenneth L. van Golen. "Individual Rac GTPases Mediate Aspects of Prostate Cancer Cell and Bone Marrow Endothelial Cell Interactions." Journal of Signal Transduction 2011 (June 27, 2011): 1–13. http://dx.doi.org/10.1155/2011/541851.
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The Rho GTPases organize the actin cytoskeleton and are involved in cancer metastasis. Previously, we demonstrated that RhoC GTPase was required for PC-3 prostate cancer cell invasion. Targeted down-regulation of RhoC led to sustained activation of Rac1 GTPase and morphological, molecular and phenotypic changes reminiscent of epithelial to mesenchymal transition. We also reported that Rac1 is required for PC-3 cell diapedesis across a bone marrow endothelial cell layer. In the current study, we queried whether Rac3 and RhoG GTPases also have a role in prostate tumor cell diapedesis. Using specific siRNAs we demonstrate roles for each protein in PC-3 and C4-2 cell adhesion and diapedesis. We have shown that the chemokine CCL2 induces tumor cell diapedesis via Rac1 activation. Here we find that RhoG partially contributes to CCL2-induced tumor cell diapedesis. We also find that Rac1 GTPase mediates tight binding of prostate cancer cells to bone marrow endothelial cells and promotes retraction of endothelial cells required for tumor cell diapedesis. Finally, Rac1 leads to β1 integrin activation, suggesting a mechanism that Rac1 can mediate tight binding with endothelial cells. Together, our data suggest that Rac1 GTPase is key mediator of prostate cancer cell-bone marrow endothelial cell interactions.
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Khanday, Firdous A., Lakshmi Santhanam, Kenji Kasuno, Tohru Yamamori, Asma Naqvi, Jeremy DeRicco, Artem Bugayenko, et al. "Sos-mediated activation of rac1 by p66shc." Journal of Cell Biology 172, no. 6 (March 6, 2006): 817–22. http://dx.doi.org/10.1083/jcb.200506001.
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The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH2-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.
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Wang, Bo, Fiona G. Wylie, Rohan D. Teasdale, and Jennifer L. Stow. "Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells." American Journal of Physiology-Cell Physiology 288, no. 6 (June 2005): C1411—C1419. http://dx.doi.org/10.1152/ajpcell.00533.2004.
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E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120 ctn interacts with E-cadherin, because p120 ctn localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.
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DiPaolo, Brian C., Nurit Davidovich, Marcelo G. Kazanietz, and Susan S. Margulies. "Rac1 pathway mediates stretch response in pulmonary alveolar epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 305, no. 2 (July 15, 2013): L141—L153. http://dx.doi.org/10.1152/ajplung.00298.2012.
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Alveolar epithelial cells (AECs) maintain the pulmonary blood-gas barrier integrity with gasketlike intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We have previously shown that AEC monolayers stretched cyclically and equibiaxially undergo rapid magnitude- and frequency-dependent actin cytoskeletal remodeling to form perijunctional actin rings (PJARs). In this work, we show that even 10 min of stretch induced increases in the phosphorylation of Akt and LIM kinase (LIMK) and decreases in cofilin phosphorylation, suggesting that the Rac1/Akt pathway is involved in these stretch-mediated changes. We confirmed that Rac1 inhibitors wortmannin or EHT-1864 decrease stretch-stimulated Akt and LIMK phosphorylation and that Rac1 agonists PIP3 or PDGF increase phosphorylation of these proteins in unstretched cells. We also confirmed that Rac1 pathway inhibition during stretch modulated stretch-induced changes in occludin content and monolayer permeability, actin remodeling and PJAR formation, and cell death. As further validation, overexpression of Rac GTPase-activating protein β2-chimerin also preserved monolayer barrier properties in stretched monolayers. In summary, our data suggest that constitutive activity of Rac1, which is necessary for stretch-induced activation of the Rac1 downstream proteins, mediates stretch-induced increases in permeability and PJAR formation.
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Kwon, Taegun, Do Yoon Kwon, Jaesun Chun, Jae Hong Kim, and Sang Sun Kang. "Akt Protein Kinase Inhibits Rac1-GTP Binding through Phosphorylation at Serine 71 of Rac1." Journal of Biological Chemistry 275, no. 1 (January 7, 2000): 423–28. http://dx.doi.org/10.1074/jbc.275.1.423.
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Yakubchyk, Yury, Hanan Abramovici, Jean-Christian Maillet, Elias Daher, Christopher Obagi, Robin J. Parks, Matthew K. Topham, and Stephen H. Gee. "Regulation of Neurite Outgrowth in N1E-115 Cells through PDZ-Mediated Recruitment of Diacylglycerol Kinase ζ." Molecular and Cellular Biology 25, no. 16 (August 15, 2005): 7289–302. http://dx.doi.org/10.1128/mcb.25.16.7289-7302.2005.
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ABSTRACT Syntrophins are scaffold proteins that regulate the subcellular localization of diacylglycerol kinase ζ (DGK-ζ), an enzyme that phosphorylates the lipid second-messenger diacylglycerol to yield phosphatidic acid. DGK-ζ and syntrophins are abundantly expressed in neurons of the developing and adult brain, but their function is unclear. Here, we show that they are present in cell bodies, neurites, and growth cones of cultured cortical neurons and differentiated N1E-115 neuroblastoma cells. Overexpression of DGK-ζ in N1E-115 cells induced neurite formation in the presence of serum, which normally prevents neurite outgrowth. This effect was independent of DGK-ζ kinase activity but dependent on a functional C-terminal PDZ-binding motif, which specifically interacts with syntrophin PDZ domains. DGK-ζ mutants with a blocked C terminus acted as dominant-negative inhibitors of outgrowth from serum-deprived N1E-115 cells and cortical neurons. Several lines of evidence suggest DGK-ζ promotes neurite outgrowth through association with the GTPase Rac1. DGK-ζ colocalized with Rac1 in neuronal processes and DGK-ζ-induced outgrowth was inhibited by dominant-negative Rac1. Moreover, DGK-ζ directly interacts with Rac1 through a binding site located within its C1 domains. Together with syntrophin, these proteins form a tertiary complex in N1E-115 cells. A DGK-ζ mutant that mimics phosphorylation of the MARCKS domain was unable to bind an activated Rac1 mutant (Rac1V12) and phorbol myristate acetate-induced protein kinase C activation inhibited the interaction of DGK-ζ with Rac1V12, suggesting protein kinase C-mediated phosphorylation of the MARCKS domain negatively regulates DGK-ζ binding to active Rac1. Collectively, these findings suggest DGK-ζ, syntrophin, and Rac1 form a regulated signaling complex that controls polarized outgrowth in neuronal cells.
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Silva, Guillermo B., and Jeffrey L. Garvin. "Rac1 mediates NaCl-induced superoxide generation in the thick ascending limb." American Journal of Physiology-Renal Physiology 298, no. 2 (February 2010): F421—F425. http://dx.doi.org/10.1152/ajprenal.00472.2009.
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Superoxide (O2−) produced by NADPH oxidase regulates Na absorption and renal hemodynamics. Increased NaCl in the thick ascending limb (TAL) stimulates O2− generation. However, we do not know whether physiological changes in NaCl concentration augment O2− generation, nor do we know the mediator(s) involved. In other cells, Rac1, a regulatory subunit of NADPH oxidase, is activated by elevated NaCl. We hypothesized that increasing luminal NaCl within the physiological range activates Rac1 and NADPH oxidase and, thereby, increases O2− production. We increased NaCl from 10 to 57 mM in medullary TAL suspensions and used lucigenin to measure O2− generation and Western blot to measure Rac1 activity. Increasing NaCl stimulated O2− generation from 1.41 ± 0.16 to 2.71 ± 0.30 nmol O2−·min−1·mg protein−1 ( n = 6, P < 0.05). This increase was blocked by the Na-K-2Cl cotransporter inhibitor furosemide and the NADPH oxidase inhibitor apocynin. To examine the role of Rac1 in NaCl-induced O2− production, we measured Rac1 translocation by Western blot. When we added NaCl, Rac1 in the particulate fraction increased from 6.8 ± 0.8 to 11.7 ± 2.4% of total Rac1 ( n = 7, P < 0.05). Then we measured O2− generation in the presence and absence of the Rac1 inhibitor. In the absence of the Rac1 inhibitor, NaCl increased O2− generation from 1.07 ± 0.24 to 2.02 ± 0.49 nmol O2−·min−1·mg protein−1, and this increase was completely blocked by the inhibitor. Similarly, in vivo treatment of TALs with adenovirus expressing dominant-negative Rac1 decreased NaCl-induced O2− generation by 60% compared with control (0.33 ± 0.04 vs. 0.81 ± 0.17 nmol O2−·min−1·mg protein−1, n = 6, P < 0.05). We concluded that physiological increases in NaCl stimulate TAL O2− generation by activating Rac1.
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Arrizabalaga, Onetsine, Hadriano M. Lacerda, Ana M. Zubiaga, and José L. Zugaza. "Rac1 Protein Regulates Glycogen Phosphorylase Activation and Controls Interleukin (IL)-2-dependent T Cell Proliferation." Journal of Biological Chemistry 287, no. 15 (February 15, 2012): 11878–90. http://dx.doi.org/10.1074/jbc.m111.297804.
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Small GTPases of the Rho family have been implicated in important cellular processes such as cell migration and adhesion, protein secretion, and/or gene transcription. In the lymphoid system, these GTPases participate in the signaling cascades that are activated after engagement of antigen receptors. However, little is known about the role that Rho GTPases play in IL-2-mediated responses. Here, we show that IL-2 induces Rac1 activation in Kit 225 T cells. We identified by mass spectrometry the muscle isoform of glycogen phosphorylase (PYGM) as a novel Rac1 effector molecule in IL-2-stimulated cells. The interaction between the active form of Rac1 (Rac1-GTP) and PYGM was established directly through a domain comprising amino acids 191–270 of PYGM that exhibits significant homology with the Rac binding domain of PAK1. The integrity of this region was crucial for PYGM activation. Importantly, IL-2-dependent cellular proliferation was inhibited upon blocking both the activation of Rac1 and the activity of PYGM. These results reveal a new role for Rac1 in cell signaling, showing that this GTPase triggers T cell proliferation upon IL-2 stimulation by associating with PYGM and modulating its enzymatic activity.
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Modha, Rakhee, Louise J. Campbell, Daniel Nietlispach, Heeran R. Buhecha, Darerca Owen, and Helen R. Mott. "The Rac1 Polybasic Region Is Required for Interaction with Its Effector PRK1." Journal of Biological Chemistry 283, no. 3 (November 15, 2007): 1492–500. http://dx.doi.org/10.1074/jbc.m706760200.
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Protein kinase C-related kinase 1 (PRK1 or PKN) is involved in regulation of the intermediate filaments of the actin cytoskeleton, as well as having effects on processes as diverse as mitotic timing and apoptosis. It is activated by interacting with the Rho family small G proteins and arachidonic acid or by caspase cleavage. We have previously shown that the HR1b of PRK1 binds exclusively to Rac1, whereas the HR1a domain binds to both Rac1 and RhoA. Here, we have determined the solution structure of the HR1b-Rac complex. We show that HR1b binds to the C-terminal end of the effector loop and switch 2 of Rac1. Comparison with the HR1a-RhoA structure shows that this part of the Rac1-HR1b interaction is homologous to one of the contact sites that HR1a makes with RhoA. The Rac1 used in this study included the C-terminal polybasic region, which is frequently omitted from structural studies, as well as the core G domain. The Rac1 C-terminal region reverses in direction to interact with residues in switch 2, and the polybasic region itself interacts with residues in HR1b. The interactions with HR1b do not prevent the polybasic region being available to contact the negatively charged membrane phospholipids, which is considered to be its primary role. This is the first structural demonstration that the C terminus of a G protein forms a novel recognition element for effector binding.