A Rac/Cdc42-specific Exchange Factor, GEFT, Induces Cell Proliferation, Transformation, and Migration
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m208896200
ISSN1083-351X
AutoresXiangrong Guo, Lewis J. Stafford, Brad A. Bryan, Chunzhi Xia, Wenbin Ma, Xiushan Wu, Dan Liu, Zhou Songyang, Mingyao Liu,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoThe Rho family of small GTPases, including Rho, Rac, and Cdc42, play essential roles in diverse cellular functions. The ability of Rho family GTPases to participate in signaling events is determined by the ratio of inactive (GDP-bound) and active (GTP-bound) forms in the cell. The activation of Rho family proteins requires the exchange of bound GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors (GEFs). The GEFs have high affinity for the guanine nucleotide-free state of the GTPases and are thought to promote GDP release by stabilizing an intermediate transition state. In this study, we have identified and characterized a new Rac/Cdc42-specific Dbl family guanine nucleotide exchange factor, named GEFT. GEFT is highly expressed in the excitable tissues, including brain, heart, and muscle. Low or very little expression was detected in other nonexcitable tissues. GEFT has specific exchange activity for Rac and Cdc42 in our in vitro GTPase exchange assays and glutathione S-transferase-PAK pull-down assays with GTP-bound Rac1 and Cdc42. Overexpression of GEFT leads to changes in cell morphology and actin cytoskeleton re-organization, including the formation of membrane microspikes, filopodia, and lamilliopodia. Furthermore, expression of GEFT in NIH3T3 cells promotes foci formation, cell proliferation, and cell migration, possibly through the activation of transcriptional factors involved in cell growth and proliferation. Together, our data suggest that GEFT is a Rac/Cdc42-specific GEF protein that regulates cell morphology, cell proliferation, and transformation. The Rho family of small GTPases, including Rho, Rac, and Cdc42, play essential roles in diverse cellular functions. The ability of Rho family GTPases to participate in signaling events is determined by the ratio of inactive (GDP-bound) and active (GTP-bound) forms in the cell. The activation of Rho family proteins requires the exchange of bound GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors (GEFs). The GEFs have high affinity for the guanine nucleotide-free state of the GTPases and are thought to promote GDP release by stabilizing an intermediate transition state. In this study, we have identified and characterized a new Rac/Cdc42-specific Dbl family guanine nucleotide exchange factor, named GEFT. GEFT is highly expressed in the excitable tissues, including brain, heart, and muscle. Low or very little expression was detected in other nonexcitable tissues. GEFT has specific exchange activity for Rac and Cdc42 in our in vitro GTPase exchange assays and glutathione S-transferase-PAK pull-down assays with GTP-bound Rac1 and Cdc42. Overexpression of GEFT leads to changes in cell morphology and actin cytoskeleton re-organization, including the formation of membrane microspikes, filopodia, and lamilliopodia. Furthermore, expression of GEFT in NIH3T3 cells promotes foci formation, cell proliferation, and cell migration, possibly through the activation of transcriptional factors involved in cell growth and proliferation. Together, our data suggest that GEFT is a Rac/Cdc42-specific GEF protein that regulates cell morphology, cell proliferation, and transformation. Jun NH2-terminal kinase guanine nucleotide exchange factors pleckstrin homology glutathioneS-transferase diffuse B-cell lymphoma Dbl homology enhanced retroviral mutagen phosphate-buffered saline serum response element p21-activated protein kinase The Rho-related GTP-binding proteins of the Ras superfamily function as molecular switches in a variety of cellular signaling pathways and regulate diverse cellular functions, including control of cell morphology, cell migration, cell growth and proliferation, actin dynamics, transcriptional activation, apoptosis signaling, and neurite outgrowth (1Ridley A.J. Paterson H.F. Johnston C.L. 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The activation of Rho family proteins requires the exchange of bound GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors (GEFs) and other specific GEFs (27Zheng Y. Trends Biochem. Sci. 2001; 26: 724-732Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar, 28Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar, 29Meller N. Irani-Tehrani M. Kiosses W.B. Del Pozo M.A. Schwartz M.A. Nat. Cell Biol. 2002; 4: 639-647Crossref PubMed Scopus (145) Google Scholar). Like the ligand-activated seven-transmembrane receptors in activation of heterotrimeric G-proteins, the GEFs have high affinity for the guanine nucleotide-free state of the GTPases and are thought to promote GDP release by stabilizing an intermediate transition state (28Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (967) Google Scholar, 30Karnoub A.E. Worthylake D.K. Rossman K.L. Pruitt W.M. Campbell S.L. Sondek J. 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In response to physiological stimuli, the active GTP-bound Rac and Cdc42 interact with the p21 (Rac/Cdc42)-binding domain of PAK, resulting in PAK autophosphorylation and increased kinase activity, and downstream activation of a variety of cellular functions (22Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1442) Google Scholar, 46Frost J.A. Swantek J.L. Stippec S. Yin M.J. Gaynor R. Cobb M.H. J. Biol. Chem. 2000; 275: 19693-19699Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 47Bagrodia S. Cerione R.A. Trends Cell Biol. 1999; 9: 350-355Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 48Sells M.A. Boyd J.T. Chernoff J. J. Cell Biol. 1999; 145: 837-849Crossref PubMed Scopus (326) Google Scholar, 49Xia C. Ma W. Stafford L.J. Marcus S. Xiong W.C. Liu M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6174-6179Crossref PubMed Scopus (58) Google Scholar, 50Jaffer Z.M. Chernoff J. Int. J. 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Overexpression of GEFT in NIH3T3 cells caused transformed phenotypes similar to the activation of Rac1 and Cdc42 by Vav. Furthermore, expression of GEFT induces the formation of lamellipodia, actin microspikes, and filopodia, similar to the activation of Rac and Cdc42 proteins. In addition, GEFT also stimulates the transcriptional activities of SRE, Elk1, and the c-Jun transcription factors. Taken together, our data suggest that GEFT is a specific activator preferentially for Rac1 and Cdc42 GTPases and may play important roles in cell morphology, growth, and proliferation. The mouse GEFT fragment was initially identified by enhanced retroviral mutagen (ERM) strategy (53Liu D. Yang X. Yang D. Songyang Z. Oncogene. 2000; 19: 5964-5972Crossref PubMed Scopus (33) Google Scholar). Briefly, we constructed enhanced retroviral mutagen (ERM) vectors that contained several engineered sequences (e.g. an ERM tag and a splice donor) controlled by a tetracycline-responsive promoter. The ERM vectors were introduced into the NIH3T3 cells. Endogenous genes can thus be randomly activated and tagged in a conditional system. NIH3T3 cells were used to screen for focus-forming genes using the ERM strategy. Full-length cDNAs encoding human GEFTs were obtained by screening human brain library (Clontech). For mammalian expression, cDNAs encoding GEFT were inserted into the HindIII and SalI sites of pCMV-Tag2B (Stratagene), resulting in the plasmid of pCMV-GEFT. For expression and purification of recombinant GEFT in bacteria, GEFT was subcloned in-frame into pQE-31 (Qiagen), generating His-tagged pQE-GEFT. The wild-type full-lengths of the Rho family GTPases, Cdc42, Rac1, and RhoA, were subcloned into theBamHI and SalI sites of pGEX-4T-1, a GST gene fusion vector (Amersham Biosciences), respectively, to produce three GST-fused pGEX vectors. Bacterially expressed His6-tagged GEFT protein and GST fusion GTPases were purified according to the standard procedures of the manufacturers. Escherichia colistrain BL21 was transformed by pQE-GEFT and pGEX vectors, respectively, grown to midlog phase at 37 °C, and then induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3–4 h. For His6-tagged GEFT, the protein was purified by nickel-nitrilotriacetic acid-agarose (Qiagen). For GST fusion GTPases, the proteins were purified by GSH-agarose (Sigma). The GST fusion proteins were in the beads or eluted in the solution containing 50 mm Tris (pH 8.0), 10 mm reduced glutathione (Sigma). All the proteins used in the assays were visualized by Coomassie Blue staining after SDS-polyacrylamide gel electrophoresis. The content of each protein was at least 90% pure. The effects of GEFT on the dissociation of [3H]GDP from the Rho family GTPases were assayed as described previously (37Zhang B. Zhang Y. Wang Z. Zheng Y. J. Biol. Chem. 2000; 275: 25299-25307Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 54van Horck F.P. Ahmadian M.R. Haeusler L.C. Moolenaar W.H. Kranenburg O. J. Biol. Chem. 2001; 276: 4948-4956Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 55Hoshino M. Sone M. Fukata M. Kuroda S. Kaibuchi K. Nabeshima Y. Hama C. J. Biol. Chem. 1999; 274: 17837-17844Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Briefly, each 1 μm eluted GTPases was incubated with 1 μm[3H]GDP at 25 °C in the buffer B containing 50 mm HEPES (pH 7.6), 100 mm NaCl, and 1 mm dithiothreitol in the presence or absence of purified 1.5 μm GEFT. To stabilize the [3H]GDP-bound GTPases, the reaction mixtures were supplemented with 20 mmMgCl2. After a binding equilibrium was reached (∼60 min), the GDP/GTP exchange reactions were initiated by the addition of excess free 400 μm GTP (final concentration). At different time points, the reactions were terminated by filtration of 20 μl of the mixtures through nitrocellulose filters. And the filters were washed twice with the ice-cold buffer B. The amount of the radionucleotides remaining bound to the Rho GTPases (RhoA, Rac1, and Cdc42) were quantified by scintillation counting, and normalized as the percentage of [3H]GDP bound at time 0. For each time point, the samples were assayed in triplicate. HeLa, COS-7, and NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cell transfection was performed using LipofectAMINE (Invitrogene) as previously described according to the manufacturer's instructions (49Xia C. Ma W. Stafford L.J. Marcus S. Xiong W.C. Liu M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6174-6179Crossref PubMed Scopus (58) Google Scholar). Cells were then allowed to grow 48 h. For each assay, control vector encoding LacZ was used as a control. For foci formation analyses, infected NIH3T3 cells were maintained in growth medium for 12–14 days and assayed as previously described (56Abe K. Rossman K.L. Liu B. Ritola K.D. Chiang D. Campbell S.L. Burridge K. Der C.J. J. Biol. Chem. 2000; 275: 10141-10149Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Briefly, NIH3T3 cells were infected with GEFT virus or a vector (pMSCV2.1) control virus for 2 days. Cells were then washed with PBS, counted, and plated as shown in 6-well plates coated with 1 μg/ml collagen. 5 ml of Dulbecco's modified Eagle's medium with 10% fetal bovine serum was added and changed every 3 days. Cells were allowed to grow for 14 days in a 37 °C incubator with a 95:5, air/carbon dioxide mixture. At the end of 14 days, cells were washed once with PBS and stained with crystal violet (0.5%), and the number of foci of transformed cells was then quantitated. Total number of foci in each well was counted with a light microscope and foci numbers were averaged for the three wells. COS-7 cells were transfected by using LipofectAMINE (Invitrogen) as described previously (57Xia C. Bao Z. Yue C. Sanborn B.M. Liu M. J. Biol. Chem. 2001; 276: 19770-19777Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Analyses of the cell lysates of the transiently transfected cells were performed using enhanced chemiluminescence reagents from Promegaas described previously (58Xia C. Bao Z. Tabassam F. Ma W. Qiu M. Hua S. Liu M. J. Biol. Chem. 2000; 275: 20942-20948Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The reporter constructs for AP1-Luc and c-Jun-Luc were obtained from Stratagene. Reporter constructs for Elk1-luc and SAP1-luc were obtained from Dr. K. L. Guan at the University of Michigan. The data presented are the mean of three individual transfected wells and the experiments were performed at least three times. To study the expression patterns of GEFT in different human tissues, a RNA filter comprising poly(A)-selected RNAs of multiple human tissues (Clontech, Inc.) was hybridized with specific 32P-labeled cDNAs as described previously (49Xia C. Ma W. Stafford L.J. Marcus S. Xiong W.C. Liu M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6174-6179Crossref PubMed Scopus (58) Google Scholar, 58Xia C. Bao Z. Tabassam F. Ma W. Qiu M. Hua S. Liu M. J. Biol. Chem. 2000; 275: 20942-20948Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In brief, human GEFT probe were radiolabeled with [α-32P]CTP by nick translation using random primers. Probes (∼4 × 107 cpm/μg) were hybridized with the RNA filter and analyzed according to manufacturers protocol. Immunoprecipitation of individual proteins was carried out as previously described (49Xia C. Ma W. Stafford L.J. Marcus S. Xiong W.C. Liu M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6174-6179Crossref PubMed Scopus (58) Google Scholar). In brief, cell lysates (1 mg of protein) were incubated with antibodies (1–10 μg) at 4 °C for 1 h in a final volume of 1 ml of modified RIPA buffer (10 mm sodium phosphate, pH 7, 1% Triton X-100, 0.1% SDS, 2 mm EDTA, 150 mm NaCl, 50 mm NaF, 0.1 mm sodium vanadate, 4 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) with constant rocking. After the addition of protein A-agarose beads, reactions were incubated at 4 °C for 1 h. Immune complexes were resolved by SDS-PAGE and subjected to immunoblotting for interacting proteins. For fluorescence labeling of the cellular components and observing cell morphology changes, 48 h after transfection, cells were plated on 10 μg/ml fibronectin-coated glass coverslips. Then, cells were fixed with 4% paraformaldehyde for 20 min, blocked with 10% bovine serum albumin, and incubated with monoclonal antibody against FLAG (M2 monoclonal, Sigma). Actin filaments were labeled with rhodamine-conjugated phalloidin (Molecular Probes). Double-label immunostaining was done with appropriate fluorochrome-conjugated secondary antibodies. Fluorescent images of cells were captured on a CCD camera mounted on an Olympus inverted research microscope using Ultraview imaging software (Olympus, Inc.). To determine GEFT binding affinity to the Rho GTPases, 20 μg of His-tagged protein was incubated at 4 °C overnight with 20 μl of GSH-agarose beads loaded with 20 μg of each GTPase, Cdc42, Rac1, or RhoA in the absence of guanine nucleotides. The beads were washed three times with PBS. The bound proteins were separated by SDS-PAGE, and His-tagged GEFT proteins were detected by Western blotting using an anti-His6 monoclonal antibody (Santa Cruz Biotechnology). GTPase activation assays in the cells were performed by GST-p21-binding domain pull-down assays as described previously (59Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (585) Google Scholar, 60Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 61Bagrodia 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 (270) Google Scholar). Briefly, cells transfected with GEFT or a control plasmid (pCMV-LacZ) 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 MgCl2, 10 μg/ml leupeptin and aprotinin, and 1 mm phenylmethylsulfonyl fluoride. GTP-bound Rac1 or Cdc42 was pulled down using the GST-p21-binding domain of PAK1 immobilized on glutathione beads. The amount of active Rac1 and Cdc42 (GTP-bound form) was detected by Western blot using specific antibodies against Rac1 and Cdc42, respectively. Proliferation studies were carried out using the CellTiter96 AQueous One solution cell proliferation assay (Promega). Briefly, cells were transfected with GEFT or a control plasmid. Cells were plated at 500 cells/well and allowed to adhere to the plate. At the indicated time points, the AQueous One solution was added to the samples and measured at 490 nm. Cell migration/motility assays were examined using modified Boyden chambers, as described previously (62Stafford L.J. Xia C. Ma W. Cai Y. Liu M. Cancer Res. 2002; 62: 5399-5404PubMed Google Scholar, 63Banyard J. Anand-Apte B. Symons M. Zetter B.R. Oncogene. 2000; 19: 580-591Crossref PubMed Scopus (140) Google Scholar). Briefly, NIH3T3 cells were stably transfected with GEFT or vector (pCMV-tag2B). The outside of the filters was coated with 1 μg/ml collagen for 1 h and then washed three times with PBS. Filters were then incubated with Dulbecco's modified Eagle's medium with bovine serum albumin for 1 h. Filters were then put into Dulbecco's modified Eagle's medium without fetal bovine serum and with 0.5 ng of mouse basic fibroblast growth factor. NIH3T3 cells expressing the receptor or vector were seeded at 20,000/well on top of the filter. Plates were incubated for 6 h. Excess cells that did not migrate through the filter were removed from the insides of the filters. Cells were then fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and then stained with crystal violet. Stained cells were examined under the microscope. To identify genes responsible to tumorigenesis, an ERM strategy was used to screen for foci-forming genes in NIH3T3 cells (53Liu D. Yang X. Yang D. Songyang Z. Oncogene. 2000; 19: 5964-5972Crossref PubMed Scopus (33) Google Scholar). One of the novel genes, mutagenized by the ERM, has shown strong oncogenic activity and was identified by reverse transcriptase-PCR and direct sequencing. The gene product shows sequence homology to the Dbl family of GEFs, and was named GEFT. Subsequently, we cloned the human and mouse GEFT full-length open reading frame by reverse transcriptase-PCR and by 5′- and 3′-rapid amplification of cDNA ends. The mouse GEFT (mGEFT) sequence is 90% identical to the human GEFT (hGEFT) (Fig. 1A). In contrast to hGEFT, mGEFT possesses an extra NH2-terminal domain. Like other family members of the Dbl proteins, GEFT has an NH2-terminal Rho exchange factor domain (Dbl homology domain, called DH domain) and is followed by a PH domain (Fig.1B). Sequence alignment of Dbl domains from hGEFT and other Dbl-containing proteins shows significant homology in this region, suggesting that GEFT is a potential exchange factor for the Rho family (RhoA, Rac1, and Cdc42) of GTPases (Fig. 1C). A data base search found that GEFT shows 35% sequence identity with human Huntingtin-associated protein-interacting protein (Duo protein) or the spectrin-like Kalirin (64Colomer V. Engelender S. Sharp A.H. Duan K. Cooper J.K. Lanahan A. Lyford G. Worley P. Ross C.A. Hum. Mol. Genet. 1997; 6: 1519-1525Crossref PubMed Scopus (108) Google Scholar, 65Alam M.R. Johnson R.C. Darlington D.N. Hand T.A. Mains R.E. Eipper B.A. J. Bio
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