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• W2052796543 abstract "The Rho family of small GTPases controls a wide range of cellular processes in eukaryotic cells, such as normal cell growth, proliferation, differentiation, gene regulation, actin cytoskeletal organization, cell fate determination, and neurite outgrowth. The activation of Rho-GTPases requires the exchange of GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors. We demonstrate that a newly identified guanine nucleotide exchange factor, GEFT, is widely expressed in the brain and highly concentrated in the hippocampus, and the Purkinje and granular cells of the cerebellum. Exogenous expression of GEFT promotes dendrite outgrowth in hippocampal neurons, resulting in spines with larger size as compared with control spines. In neuroblastoma cells, GEFT promotes the active GTP-bound state of Rac1, Cdc42, and RhoA and increases neurite outgrowth primarily via Rac1. Furthermore, we demonstrated that PAK1 and PAK5, both downstream effectors of Rac1/Cdc42, are necessary for GEFT-induced neurite outgrowth. AP-1 and NF-κB, two transcriptional factors involved in neurite outgrowth and survival, were up-regulated in GEFT-expressing cells. Together, our data suggest that GEFT enhances dendritic spine formation and neurite outgrowth in primary neurons and neuroblastoma cells, respectively, through the activation of Rac/Cdc42-PAK signaling pathways. The Rho family of small GTPases controls a wide range of cellular processes in eukaryotic cells, such as normal cell growth, proliferation, differentiation, gene regulation, actin cytoskeletal organization, cell fate determination, and neurite outgrowth. The activation of Rho-GTPases requires the exchange of GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors. We demonstrate that a newly identified guanine nucleotide exchange factor, GEFT, is widely expressed in the brain and highly concentrated in the hippocampus, and the Purkinje and granular cells of the cerebellum. Exogenous expression of GEFT promotes dendrite outgrowth in hippocampal neurons, resulting in spines with larger size as compared with control spines. In neuroblastoma cells, GEFT promotes the active GTP-bound state of Rac1, Cdc42, and RhoA and increases neurite outgrowth primarily via Rac1. Furthermore, we demonstrated that PAK1 and PAK5, both downstream effectors of Rac1/Cdc42, are necessary for GEFT-induced neurite outgrowth. AP-1 and NF-κB, two transcriptional factors involved in neurite outgrowth and survival, were up-regulated in GEFT-expressing cells. Together, our data suggest that GEFT enhances dendritic spine formation and neurite outgrowth in primary neurons and neuroblastoma cells, respectively, through the activation of Rac/Cdc42-PAK signaling pathways. Neurite outgrowth, a process responsible for neuronal patterning and connections, is crucial for the development of the nervous system (1Park H.T. Wu J. Rao Y. Bioessays. 2002; 24: 821-827Crossref PubMed Scopus (57) Google Scholar). The regulation of neurite outgrowth is determined largely by the organization of the actin cytoskeleton in response to different environmental cues (2Keynes R. Cook G.M. Cell. 1995; 83: 161-169Abstract Full Text PDF PubMed Scopus (174) Google Scholar). The Rho family of small GTPases, which comprises the key regulators of the actin cytoskeleton (3Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3823) Google Scholar), has been shown to mediate the morphological changes that are observed during neuronal development and plasticity such as neurite outgrowth, axonal guidance, and dendrite topology modifications (4Dunaevsky A. Mason C.A. Trends Neurosci. 2003; 26: 155-160Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 5Li Z. Van Aelst L. Cline H.T. Nat. Neurosci. 2000; 3: 217-225Crossref PubMed Scopus (10) Google Scholar, 6Li Z. Aizenman C.D. Cline H.T. Neuron. 2002; 33: 741-750Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 7Luo L. Liao Y.J. Jan L.Y. Jan Y.N. Genes Dev. 1994; 8: 1787-1802Crossref PubMed Scopus (811) Google Scholar, 8Sin W.C. Haas K. Ruthazer E.S. Cline H.T. Nature. 2002; 419: 475-480Crossref PubMed Scopus (367) Google Scholar, 9Threadgill R. Bobb K. Ghosh A. Neuron. 1997; 19: 625-634Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). Members of the Rho family perform distinct roles in the regulation of the actin cytoskeleton. RhoA is responsible for the formation of focal adhesions and the assembly of actin stress fibers, and it has been found to inhibit the formation of neurite outgrowths (10Mackay D.J. Nobes C.D. Hall A. Trends Neurosci. 1995; 18: 496-501Abstract Full Text PDF PubMed Scopus (117) Google Scholar, 11Sebok A. Nusser N. Debreceni B. Guo Z. Santos M.F. Szeberenyi J. Tigyi G. J. Neurochem. 1999; 73: 949-960Crossref PubMed Scopus (109) Google Scholar). Rac1 promotes the formation of membrane lamellae, whereas Cdc42 regulates the outgrowth of filopodia (12Kozma R. Sarner S. Ahmed S. Lim L. Mol. Cell. Biol. 1997; 17: 1201-1211Crossref PubMed Scopus (535) Google Scholar). Both Rac and Cdc42 positively regulate neurite outgrowth (13Nikolic M. Int. J. Biochem. Cell Biol. 2002; 34: 731-745Crossref PubMed Scopus (118) Google Scholar). The Rho-GTPases function as molecular switches, cycling between GTP-bound forms and GDP-bound forms. They are active in the GTP-bound form, and hydrolysis of the GTP by their intrinsic GTPase activity returns them to the GDP-bound inactive state (14Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar). The active/inactive states of these proteins are regulated by a variety of intracellular molecules, predominantly by two classes of proteins: GTPase-activating proteins and guanine nucleotide exchange factors (GEFs) 1The abbreviations used are: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; PAK, p21-activated kinase; MAPK, mitogen-activated protein kinase; RBP, Rho-binding protein.1The abbreviations used are: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; PAK, p21-activated kinase; MAPK, mitogen-activated protein kinase; RBP, Rho-binding protein. (15Narumiya S. J. Biochem. (Tokyo). 1996; 120: 215-228Crossref PubMed Scopus (359) Google Scholar). GTPase-activating proteins catalyze the intrinsic GTPase activity of the Rho proteins, thus inactivating them, whereas GEFs catalyze the exchange of GDP for GTP, thereby activating GTPases. The Rho GEFs are a class of enzymes with high specificity for Rho-GTPases and contain a Dbl homology domain of ∼20 amino acids immediately followed by a pleckstrin homology domain of ∼100 amino acids (16Stam J.C. Collard J.G. Prog. Mol. Subcell. Biol. 1999; 22: 51-83Crossref PubMed Scopus (40) Google Scholar). Dbl homology domains interact directly with Rho-GTPases to catalyze guanine nucleotide exchange (17Arozarena I. Aaronson D.S. Matallanas D. Sanz V. Ajenjo N. Tenbaum S.P. Teramoto H. Ighishi T. Zabala J.C. Gutkind J.S. Crespo P. J. Biol. Chem. 2000; 275: 26441-26448Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 18Worthylake D.K. Rossman K.L. Sondek J. Nature. 2000; 408: 682-688Crossref PubMed Scopus (305) Google Scholar). Pleckstrin homology domains promote the translocation of Dbl-related proteins to plasma membranes (19Whitehead I.P. Campbell S. Rossman K.L. Der C.J. Biochim. Biophys. Acta. 1997; 1332: F1-F23Crossref PubMed Scopus (333) Google Scholar, 20Whitehead I.P. Lambert Q.T. Glaven J.A. Abe K. Rossman K.L. Mahon G.M. Trzaskos J.M. Kay R. Campbell S.L. Der C.J. Mol. Cell. Biol. 1999; 19: 7759-7770Crossref PubMed Google Scholar), as well as participate directly in GTPase binding and regulation of GEF activity (21Snyder J.T. Rossman K.L. Baumeister M.A. Pruitt W.M. Siderovski D.P. Der C.J. Lemmon M.A. Sondek J. J. Biol. Chem. 2001; 276: 45868-45875Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). We recently identified GEFT as a new Rho-family-specific GEF that is highly expressed in the brain, heart, and skeletal muscle (22Guo X. Stafford L.J. Bryan B. Xia C. Ma W. Wu X. Liu D. Songyang Z. Liu M. J. Biol. Chem. 2003; 278: 13207-13215Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 23Liu D. Yang X. Yang D. Songyang Z. Oncogene. 2000; 19: 5964-5972Crossref PubMed Scopus (33) Google Scholar). Unlike many other GEF proteins, GEFT comprises primarily the Dbl homology and pleckstrin homology domains only, with short N- and C-terminal sequences. Expression of GEFT has been shown to promote the formation of lamellipodia, actin microspikes, and filopodia in NIH3T3 cells via activation of the Rho family of small GTPases (22Guo X. Stafford L.J. Bryan B. Xia C. Ma W. Wu X. Liu D. Songyang Z. Liu M. J. Biol. Chem. 2003; 278: 13207-13215Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). A potential splice variant of GEFT, encoding the gene for p63RhoGEF, has also been identified and shown to be highly expressed in both the brain and heart, and it induces a RhoA-dependent stress fiber formation in several cell types (24Souchet M. Portales-Casamar E. Mazurais D. Schmidt S. Leger I. Javre J.L. Robert P. Berrebi-Bertrand I. Bril A. Gout B. Debant A. Calmels T.P. J. Cell Sci. 2002; 115: 629-640Crossref PubMed Google Scholar, 25Lutz S. Freichel-Blomquist A. Rumenapp U. Schmidt M. Jakobs K.H. Weiland T. Naunyn-Schmiedebergs Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar). Using primary hippocampal neurons and Neuro2a (N2A) neuroblastoma cell lines, we examined the role that GEFT plays in the regulation of dendritic spine morphogenesis and neurite outgrowth. In addition, we studied the molecular signaling mechanisms by which GEFT regulates dendritic spine morphogenesis and neurite outgrowth. DNA Constructs—The gene encoding human GEFT was subcloned from a pSPORT-6 vector into the HindIII and SalI sites of a pCMV-Tag2B vector (Stratagene), resulting in the plasmid pCMV-GEFT. RhoA, Cdc42, and Rac1 expression vectors were previously constructed (22Guo X. Stafford L.J. Bryan B. Xia C. Ma W. Wu X. Liu D. Songyang Z. Liu M. J. Biol. Chem. 2003; 278: 13207-13215Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). RhoA T19N, RhoA G14V, Cdc42 T17N, Cdc42 G12V, Rac1 T17V, and Rac1 G12V where obtained from the Guthrie cDNA resource Center. PAK1 constructs were kindly supplied by Dr. Jonathan Chernoff at Fox Chase Cancer Center. PAK5, PAK5 S573N/S602E, and PAK5 K478M were a generous donation from Audrey Minden (Columbia University). Cell Culture, Transfection, and Differentiation—Dentate explants were cultured as previously described (26Wu Y. Wang W. Richerson G.B. J. Neurosci. 2001; 21: 2630-2639Crossref PubMed Google Scholar). Briefly, dentate gyrus and CA3 regions were isolated from the hippocampi of 2- to 4-day-old Sprague-Dawley rats, maintained in culture for 8–25 days on a Matrigel substrate (1:50 dilution, Collaborative Research), and plated on glass coverslips in medium supplemented with B-27 (Invitrogen). Proliferation of non-neuronal cells was prevented by the addition of 2 μm cytosine β-d-arabinofuranoside (Sigma). The explants were transfected with pCMV-GEFT or vector at 7 days in vitro by calcium phosphate precipitation (27Xia Z. Dudek H. Miranti C.K. Greenberg M.E. J. Neurosci. 1996; 16: 5425-5436Crossref PubMed Google Scholar). Co-transfection with enhanced green fluorescent protein (GFP) was used to identified the transfected neurons and visualize the detailed morphology including dendritic spines. Neuro2A (N2A) cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). Cell transfection was performed using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Cells were then allowed to grow for 48 h and assayed, or selected with G418 to produce stable cell lines. For each assay, empty vector was used as a control. Neurite Outgrowth Measurements—To quantify dendrite morphology, the enhanced GFP-expressing neurons were imaged at 21 days in vitro using a high resolution Zeiss LSM 510 Meta system (Zeiss). To quantify the dendritic spines, a 50-μm image of the dendrite from primary or distal dendrites was viewed at high magnification (40×, numerical aperture of 1.4 with additional optic zoom of 2–3), and each individual spine present on the dendrites was manually traced onto an acetate sheet. The spines measured were selected from 30 randomly selected 50-μm dendritic segments, and the spine numbers measured were over 900 for control and 400 for GEFT. The drawings were then scanned into a computer and the maximum length, head width, and shape factor of each spine was calculated automatically with the Metamorph software and logged into Microsoft Excel. The limit set for considering dendritic outgrowth was filopodia greater than 4 μm without a bulbous head (28Fiala J.C. Feinberg M. Popov V. Harris K.M. J. Neurosci. 1998; 18: 8900-8911Crossref PubMed Google Scholar). Spines were defined as a headless protrusion 1–4 μm long or a headed protrusion of any length up to 4 μm. The cumulative percentage graphs include for the spine length, width, and area show the overall spread or distribution of the data for these parameters. A lower magnification (20×, numerical aperture of 0.75) was used to image the overall dendrite morphology. Whole cell morphology was traced onto an acetate sheet and scanned as above. Total dendritic branch length was calculated using Scion software, whereas tips were counted manually. The dendrite data shown are mean (±S.E.), and, using Student's unpaired t test, p < 0.001. Phase contrast images of N2A cells were viewed at ×200 magnification, and images were captured on a charge-coupled device camera mounted on a Nikon Eclipse TS100 microscope using SPOT Advanced Imaging Software. Cells were scored for the percentage of cells expressing neurites, average number of neurites per cell, and average length of neurites. Cells with neurites were defined as cells that possessed at least one neurite of more than one-half the cell body diameter in length. The data presented are the mean of three individual transfected 10-cm2 dishes and are representative of at least three independent experiments. At least 250 cells per transfection were scored for neurite outgrowth. The N2A data shown are mean (±S.E.), and, using Student's unpaired t test, p < 0.05. Immunofluorescence—Fluorescence images for primary rat neurons were captured using confocal microscopy using a high resolution Zeiss LSM 510 Meta system with sequential acquisition. A z series projection of ∼7 to 15 images with 0.5- to 1-μm depth interval, each averaged two times was taken to cover the entire z dimension of the labeled neurons. N2A cells used for immunofluorescence were grown on 0.5% gelatin-coated glass coverslips. The cells were fixed (4% paraformaldehyde, 0.1% Triton X-100), blocked with 0.2% bovine serum albumin, and incubated with a monoclonal antibody against FLAG (M2 monoclonal, Sigma). Double-labeled immunostaining was carried out with the appropriate fluorochrome-conjugated secondary antibodies. Fluorescence images for N2A cells were captured at ×400 on a charge-coupled device camera mounted on an Olympus inverted research microscope using Ultraview imaging software (Olympus, Inc.). Transient Expression of Reporter Gene Assays—N2A cells were transfected using LipofectAMINE (Invitrogen) as described previously. Transfected cells were harvested after 48 h. The resulting cell lysates were analyzed for luciferase activity using enhanced chemiluminescence reagents from Promega, according to the manufacturer's instructions. The reporter constructs for the AP1-Luc and NF-κB-Luc were obtained from K. Guan (University of Michigan). The data presented are the mean of three individual transfected wells, and the experiments were performed at least three times. GST Pull-down Assays—GTPase activation assays were performed by GST-p21 binding domain pull-down assays as described previously (29Bagrodia S. Taylor S.J. Jordon K.A. Van Aelst L. Cerione R.A. J. Biol. Chem. 1998; 273: 23633-23636Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 30Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). Briefly, cells transfected with GEFT or a control plasmid (pCMV-Tag2B) were washed and lysed on the dish in 50 mm Tris (pH 7.5), 500 mm NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10% glycerol, 10 mm MgCl2, 10 μg/ml leupeptin and aprotinin, and 1 mm phenylmethylsulfonyl fluoride. GTP-bound Rac and Cdc42 were pulled down using the GST-p21-binding domain of PAK1 immobilized on glutathione beads, whereas GTP-bound RhoA was pulled down using the GST-bound Rho-binding protein immobilized on glutathione beads. The amounts of active Rac1, Cdc42, and RhoA (GTP-bound form) were detected by Western blot using antibodies against Rac1, Cdc42, or RhoA. PAK Kinase Assay—1 mg of total cell lysate was immunoprecipitated with anti-PAK antibody (Santa Cruz Biotechnology). Samples were resuspended in kinase buffer (30 mm HEPES (pH 7.5), 10 mm MgCl2, 2.5 mm EGTA, 1 mm dithiothreitol, 0.1 mm Na3VO4, 1 mm NaF) and mixed with MgCl2, ATP, myelin basic protein, and 10 μCi of [γ-32P]ATP. The reaction was allowed to proceed and then loaded onto a 12% SDS-PAGE gel. The gel was dried and exposed to x-ray film overnight. In Situ Hybridization—Brains from adult female mice were obtained and sectioned for slides. In situ hybridizations were performed using the Dig Wash and Detection Kit (Roche Diagnostics) according to the manufacturer's instructions. GEFT Is Highly Expressed in the Hippocampus, Granular Layer, and Purkinje Cells of the Adult Mouse Brain—To investigate the potential role of GEFT in neuronal cells and in brain development, we examined the expression of GEFT transcript using in situ hybridization on brain sections of adult mice. As shown in Fig. 1A, GEFT was found to be broadly expressed in all regions of the brain, however, significantly higher expression occurred in both the hippocampus (Fig. 1C) and the Purkinje cells and granular layer of the cerebellum (Fig. 1D). To confirm the specific expression of GEFT, a GEFT sense probe was used as a control in similar in situ hybridization assays. No positive staining was observed using the control sense probe (Fig. 1B). GEFT Promotes Dendritic Spine Formation in Primary Hippocampus Neurons—Due to the high level of GEFT expression in the brain, specifically in the hippocampus, we sought to determine if GEFT might influence axonal outgrowth, dendrite spine formation, and dendritic arbor morphology by examining the effect of GEFT overexpression in rat dentate gyrus explant culture. GEFT significantly alters dendrite morphology in hippocampal neurons (Fig. 2, A and B), however, no noticeable difference was observed in axon length or branching (data not shown). To quantify the effect of GEFT on dendritic arbor complexity, we measured the total dendritic length and terminal tip number. As illustrated in Fig. 2C, there was a significant increase in terminal tip number (as a measure of dendritic branching) for GEFT transfected neurons (8.55 ± 0.77 tips/cell; control: 5.54 ± 0.27 tips/cell), further emphasizing its role in the regulation of dendritic outgrowth. However, the total dendrite length was found to be no different from the control cells (Fig. 2, D–F), suggesting that GEFT may primarily promote dendrite branching. In contrast to the subtle effects on overall dendritic morphology, overexpression of GEFT has more prominent effects on dendritic spines. As shown in Fig. 3, there was a significant increase in mature spine density for GEFT-transfected neurons (19.52 ± 1.59 spines/50-μm dendritic segment) as compared with control neurons (16.2 ± 0.67 spines/50-μm dendritic segment). In addition, the spines from the GEFT-transfected neurons were large in size as demonstrated by a significantly higher length (2.64 ± 0.07 μm), width (1.46 ± 0.02 μm), and area (2.25 ± 0.09 μm2) as compared with control spines (length, 1.85 ± 0.02 μm; width, 1.07 ± 0.01 μm; and area, 1.21 ± 0.01 μm2) (Fig. 3, E–G). Cumulative percentage graphs representing the overall spread or distribution of spine length, width, and area for vector and GEFT-transfected cells demonstrate very evidently an increase in spine length, width, and area for GEFT-transfected neurons (Fig. 3, H–J). Therefore, overexpression of GEFT in hippocampal neurons promotes a significant increment in mature spine density and larger spines with longer length, width, and area.Fig. 3GEFT induces dendritic spine formation in primary rat hippocampal neurons. The two panels illustrate spine formation in GFP-transfected dentate gyrus neurons (A) and GFP-GEFT co-transfected neurons (B). The graphs illustrate mature spine number (C), filopodia number (D), dendritic spine length (E), width (F), and area (G). Cumulative percentage graphs are included for the spine length (H), width (I), and area (J), which show the overall spread or distribution of the data for these parameters. Scale bar = 20 μm (upper panel) and 5 μm (lower panel). The p value for each experiment is <0.001.View Large Image Figure ViewerDownload (PPT) GEFT Induces Neurite Outgrowth in N2A Cells—To study more extensively the role of GEFT in the regulation of neurite outgrowth, we exogenously expressed this gene in a neuroblastoma cell line, N2A, and quantified the morphological changes in neurite outgrowth. FLAG-tagged GEFT was transiently expressed in N2A cells plated on gelatin-coated coverslips, and immunofluorescence was performed using an anti-FLAG antibody to examine the GEFT-mediated morphological changes in neurite outgrowth (Fig. 4A). Transfection efficiency was ∼60%, allowing simultaneous observation of both transfected (bright staining) and non-transfected (nonspecific staining) cells. In non-transfected N2A cells, short neurites were present, but few in number. However, in N2A cells expressing GEFT, neurite outgrowths were significantly increased in number, length, and the degree of neuronal branching. Subsequently, stable cell lines were selected after transfection with either empty vector or GEFT. The expression of FLAG-tagged GEFT in the stable line was confirmed by Western blot analysis using an anti-FLAG antibody (Fig. 4B). We then quantified the morphology, number, and length of neurite outgrowths in N2A cells expressing empty vector or GEFT. Cells with neurites were defined as cells that possessed at least one neurite more than one-half the cell body diameter in length. Exogenous expression of GEFT in N2A cells induced significant changes in neurite morphology compared with control cells expressing vector only (Fig. 4, C and D). Control cells exhibited neurite outgrowths on 27.5 ± 8.2% of the cells (Fig. 5A), whereas GEFT-expressing cells exhibited neurite outgrowths on 49 ± 5.1% of the cells, an almost 2-fold increase for neurite outgrowth in GEFT-expressing cells compared with the control cell line (Fig. 5A). We further measured the average neurite length for control and GEFT-expressing cells (Fig. 5B). The average neurite length in control cells was 52 ± 4.2 μm. In the presence of exogenously expressed GEFT, the average neurite length increased to 103 ± 3.5 μm, resulting in a 2-fold increase in neurite length. In addition, we determined the average number of neurites per cell for vector and GEFT-expressing cells (Fig. 5C). Vector control cells exhibited 3.1 ± 0.6 neurites/cell, whereas GEFT-expressing cells exhibited 5.5 ± 0.9 neurites/cell. Therefore, our data suggest that expression of GEFT induces neurite outgrowth in N2A cells, including a strong increase in the number of cells exhibiting neurite processes, an increase in the number of neurites per cell, and an overall increase in the length of the neurite. GEFT Induces Neurite Outgrowth via Activation of Rac1 in Neuronal Cells—It has been previously shown that exogenous expression of GEFT increases the active forms of both Rac1 and Cdc42 in NIH3T3 cells (22Guo X. Stafford L.J. Bryan B. Xia C. Ma W. Wu X. Liu D. Songyang Z. Liu M. J. Biol. Chem. 2003; 278: 13207-13215Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). To determine which Rho-GTPases mediate GEFT-induced neurite outgrowth of N2A cells in vivo, we compared the amount of GTP-bound forms (active status) of Rac1, Cdc42, and RhoA in N2A cells transfected with GEFT or a control plasmid. To determine the level of GTP-bound Rac1 and Cdc42 in the cells, we utilized a GST-PAK1 fusion protein containing the Rac1/Cdc42 binding domain as an affinity reagent in a GST pull-down assay. Pak1 is a downstream effector of Rac1 and Cdc42, and PAK1 binds only to the active GTP-bound forms of Rac1 and Cdc42 GTPases. To determine the level of GTP-bound Rho in the cells, we utilized a GST-Rho-binding protein fusion protein containing the Rho binding domain as an affinity reagent in a similar fashion as above. Transfection of GEFT in N2A cells increased the GTP-bound active forms of Rac1, Cdc42, and RhoA significantly when compared with the vector control (Fig. 6A). These data suggest that GEFT activates Rac1, Cdc42, and RhoA in the N2A cells by stimulating the guanine nucleotide exchange of the three GTPases. Although GEFT expression induces neuronal outgrowth, a process attributed to the activation of both Rac1 and Cdc42 and inhibited by RhoA, GEFT shows no specificity in catalyzing the GDP- to GTP-bound state for any Rho-GTPase in the N2A cells. It has previously been demonstrated that GEFT is most specific for Rac1 and Cdc42 in NIH3T3 cells, and multiple studies suggest that GEFT could regulate RhoA in a cell-type specific manner (22Guo X. Stafford L.J. Bryan B. Xia C. Ma W. Wu X. Liu D. Songyang Z. Liu M. J. Biol. Chem. 2003; 278: 13207-13215Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 24Souchet M. Portales-Casamar E. Mazurais D. Schmidt S. Leger I. Javre J.L. Robert P. Berrebi-Bertrand I. Bril A. Gout B. Debant A. Calmels T.P. J. Cell Sci. 2002; 115: 629-640Crossref PubMed Google Scholar, 25Lutz S. Freichel-Blomquist A. Rumenapp U. Schmidt M. Jakobs K.H. Weiland T. Naunyn-Schmiedebergs Arch. Pharmacol. 2004; 369: 540-546Crossref PubMed Scopus (42) Google Scholar). Although GEFT may activate RhoA, Rac1, and Cdc42, it is essential to determine which of the Rho-GTPases acts as the effector for downstream induction of neurite outgrowth. Using constitutively active and dominant negative RhoA, Rac1, and Cdc42 mutants, we examined the downstream targets of GEFT in inducing neurite outgrowth by co-transfection of the mutants with GEFT. As shown in Fig. 6 (B and C), induction of neurite outgrowth (measured as number of neurites per cell (Fig. 6B) and neurite length (Fig. 6C)) in both vector and GEFT-expressing N2A cells was observed with the addition of the constitutive active mutants Rac1-G12V and Cdc42-G12V, respectively. As expected, in vector control cells, addition of Rac1-T17N and Cdc42-T17N dominant negative mutants blocked the induction of neurite outgrowths. In GEFT-expressing cells, co-transfection of Rac1 dominant negative mutant (T17N) significantly decreased neurite outgrowth compared with cells expressing GEFT alone or GEFT coexpressing Rac1-T17N, suggesting Rac1 plays a major role in GEFT-mediated neurite outgrowth. However, addition of Cdc42 dominant-negative mutant (Cdc42T17N) to GEFT-expressing cells showed no significant decrease in neurite outgrowth compared with that observed in GEFT cells. Inhibition of RhoA activity by transfection of dominant negative RhoA (T19N) resulted in increased neurite outgrowth in GEFT-expressing cells; however, addition of constitutively active RhoA (G14V) did not abrogate the effects of GEFT overexpression on neurite outgrowth. These data suggest that Rac1 plays the major role in GEFT-induced neurite outgrowth. PAK1 and PAK5 Are Essential for GEFT-induced Neurite Outgrowth—Members of the mammalian p21-activated kinase (PAK) family of serine/threonine kinases constitute effectors for Rac1 and Cdc42, but not RhoA (31Daniels R.H. Hall P.S. Bokoch G.M. EMBO J. 1998; 17: 754-764Crossref PubMed Scopus (256) Google Scholar, 32Dan C. Nath N. Liberto M. Minden A. Mol. Cell. Biol. 2002; 22: 567-577Crossref PubMed Scopus (136) Google Scholar). Two PAK family members, PAK1 and PAK5, have been implicated in neurite outgrowth. PAK1 kinase activity alone has been shown to be insufficient to induce neurite outgrowth, rather, the targeting of PAK1 to the plasma membrane induces the outgrowth of neurite-like structures in PC-12 cells (32Dan C. Nath N. Liberto M. Minden A. Mol. Cell. Biol. 2002; 22: 567-577Crossref PubMed Scopus (136) Google Scholar). PAK5, a brain-specific PAK family member similar to Drosophila MBT (“mushroom body tiny”) protein, has been shown to trigger both filopodium formation and neurite outgrowth in N1E-115 cells (32Dan C. Nath N. Liberto M. Minden A. Mol. Cell. Biol. 2002; 22: 567-577Crossref PubMed Scopus (136) Google Scholar). To investigate whether GEFT-mediated activation of Rac1 and Cdc42 triggers the activation of PAK, the kinase activity of PAK1 was assessed using myelin basic protein as a substrate (Fig. 7A). PAK1 activity was significantly increased in GEFT extracts (lane 2) compared with vector extracts (lane 1), suggesting that exogenous expression of GEFT up-regulates PAK1 activity. To determine whether PAK1 and PAK5, both involved in regulating neurite outgrowth, mediate the promotion of GEFT-induced neurite outgrowth, N2A cells were co-transfected with PAK1 or PAK5 constitutively active and dominant negative" @default.
• W2052796543 created "2016-06-24" @default.
• W2052796543 creator A5008587965 @default.
• W2052796543 creator A5053704104 @default.
• W2052796543 creator A5053927760 @default.
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• W2052796543 date "2004-10-01" @default.
• W2052796543 modified "2023-10-16" @default.
• W2052796543 title "GEFT, A Rho Family Guanine Nucleotide Exchange Factor, Regulates Neurite Outgrowth and Dendritic Spine Formation" @default.
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