Src Kinase Regulates the Activation of a Novel FGD-1-related Cdc42 Guanine Nucleotide Exchange Factor in the Signaling Pathway from the Endothelin A Receptor to JNK
2003; Elsevier BV; Volume: 278; Issue: 32 Linguagem: Inglês
10.1074/jbc.m301559200
ISSN1083-351X
AutoresYuki Miyamoto, Junji Yamauchi, Hiroshi Itoh,
Tópico(s)Mechanisms of cancer metastasis
ResumoSmall GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Upon stimulation with extracellular signals, guanine-nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP to shift toward the active forms of small GTPases, recognizing the downstream targets. Here we show that KIAA0793, containing substantial sequence homology with the catalytic Dbl homology domain of the faciogenital dysplasia gene product (FGD1), is a specific GEF for Cdc42. We, therefore, tentatively named it FRG (FGD1-related Cdc42-GEF). Src kinase directly phosphorylates and activates FRG, as Vav family GEFs. Additionally, FRG is involved in the signaling pathway from the endothelin A receptor to c-Jun N-terminal kinase, resulting in the inhibition of cell motility. These results suggest that FRG is a member of Cdc42-GEF and plays an important role in the signaling pathway downstream of G protein-coupled receptors. Small GTPases act as binary switches by cycling between an inactive (GDP-bound) and an active (GTP-bound) state. Upon stimulation with extracellular signals, guanine-nucleotide exchange factors (GEFs) stimulate the exchange of GDP to GTP to shift toward the active forms of small GTPases, recognizing the downstream targets. Here we show that KIAA0793, containing substantial sequence homology with the catalytic Dbl homology domain of the faciogenital dysplasia gene product (FGD1), is a specific GEF for Cdc42. We, therefore, tentatively named it FRG (FGD1-related Cdc42-GEF). Src kinase directly phosphorylates and activates FRG, as Vav family GEFs. Additionally, FRG is involved in the signaling pathway from the endothelin A receptor to c-Jun N-terminal kinase, resulting in the inhibition of cell motility. These results suggest that FRG is a member of Cdc42-GEF and plays an important role in the signaling pathway downstream of G protein-coupled receptors. Rho family small GTPases comprise a large branch within the Ras family of low molecular weight guanine nucleotide-binding proteins (1Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2072) Google Scholar, 2Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 3Schoenwaelder S.M. Burridge L.M. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (651) Google Scholar, 4Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2101) Google Scholar). One of the well known roles of Rho family GTPases is the regulation of the actin cytoskeleton (5Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5228) Google Scholar, 6Kaibuchi K. Kuroda S. Amano M. Annu. Rev. Biochem. 1999; 68: 459-486Crossref PubMed Scopus (891) Google Scholar). Rho family GTPases RhoA, Rac, and Cdc42 induce the formation of stress fibers, lamellipodia, and filopodia, respectively (5Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5228) Google Scholar). Furthermore, Rho family GTPases have been implicated in the control of diverse physiological responses, such as cell proliferation and cell motility as well as in physiopathological processes such as transformation and metastasis (7Ridley A.J. J. Cell Sci. 2001; 114: 2713-2722Crossref PubMed Google Scholar, 8Whitehead I.P. Zohn I.E. Der C.J. Oncogene. 2001; 20: 1547-1555Crossref PubMed Scopus (82) Google Scholar). Rho family GTPases adopt either an active GTP-bound or an inactive GDP-bound conformational state (2Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 3Schoenwaelder S.M. Burridge L.M. Curr. Opin. Cell Biol. 1999; 11: 274-286Crossref PubMed Scopus (651) Google Scholar, 4Van Aelst L. D'Souza-Schorey C. 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The G protein-coupled receptors (GPCRs) are the largest family of human cell-surface receptors (26Strader C.D. Fong T.M. Tota M.R. Underwood D. Dixon R.A. Annu. Rev. Biochem. 1994; 63: 101-132Crossref PubMed Scopus (996) Google Scholar, 27Rohrer D.K. Kobilka B.K. Physiol. Rev. 1998; 78: 32-52Crossref Scopus (95) Google Scholar, 28Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar, 29Luttrell L.M. Daaka Y. Lefkowitz R.J. Curr. Opin. Cell Biol. 1999; 11: 177-183Crossref PubMed Scopus (612) Google Scholar). Some GPCRs activate c-Jun N-terminal kinase (JNK), a subfamily of mitogen-activated protein kinases (28Gutkind J.S. J. Biol. Chem. 1998; 273: 1839-1842Abstract Full Text Full Text PDF PubMed Scopus (692) Google Scholar). This pathway depends on Src kinase and Rho family GTPases, Src kinase often acting upstream of Rho family GTPases (28Gutkind J.S. J. Biol. 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However, the detailed mechanism linking Src kinase to Rho family GTPases remains unclear (34Sah V.P. Seasholz T.M. Sagi S.A. Brown J.H. Annu. Rev. Pharmacol. Toxicol. 2000; 40: 459-489Crossref PubMed Scopus (298) Google Scholar). To clarify the mechanism of GPCR-mediated activation of Rho family GTPases, we performed a BLAST search using Dbl big sister (Dbs), a GEF involved in GPCR-mediated activation of Rho family GTPases (35Yamauchi J. Hirasawa A. Miyamoto Y. Kokubu H. Nishii H. Okamoto M. Sugawara Y. Tsujimoto G. Itoh H. Biochem. Biophys. Res. Commun. 2002; 296: 85-92Crossref PubMed Scopus (12) Google Scholar), as a query. As a result, we found a novel Rho family GEF gene, KIAA0793. The KIAA0793 protein shows sequence similarity to a human faciogenital dysplasia gene product (FGD1), which is a specific activator of Cdc42 (36Zheng Y. Fischer D.J. Santos M.F. Tigyi G. German Pasteris N. Gorski J.L. Xu Y. J. Biol. Chem. 1996; 271: 33169-33172Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) and appears to be a family of related Cdc42-GEFs including mouse Fgd2 and Fgd3 (37German Pasteris N. Nagata K. Hall A. Gorski J.L. Gene (Amst.). 2000; 242: 237-247Crossref PubMed Scopus (36) Google Scholar) and mouse Frabin (38Obaishi H. Nakanishi H. Mandai K. Satoh K. Satoh A. Takahashi K. Miyahara M. Nishioka H. Takaishi K. Takai Y. J. Biol. Chem. 1998; 273: 18697-18700Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). In the present study, we show that KIAA0793 is a specific GEF toward Cdc42, thus termed FRG (FGD1-related Cdc42-GEF). FRG is directly phosphorylated by Src kinase, which increases the Cdc42-GEF activity of FRG. We also demonstrate that FRG is involved in the signaling pathway linking the endothelin A (ETA) receptor to JNK, leading to the inhibition of cell motility. Taken together, our results indicate that FRG is a novel member of Cdc42-GEFs and may act as a direct regulator linking the Src kinase and Rho family GTPases in the chemore-pellant GPCR-JNK-signaling pathway. Antibodies—A mouse monoclonal antibody M2 against a FLAG-peptide was obtained from Sigma-Aldrich. A mouse monoclonal antibody 12CA5 against the hemagglutinin (HA) epitope was purchased from Roche Diagnostics. A mouse monoclonal antibody 1E4 against Aequorea victoria green fluorescence protein was obtained from Medical and Biological Laboratories, Co., Ltd. (Nagoya, Japan). A mouse monoclonal anti-phosphorylated tyrosine antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). A rabbit polyclonal antibody against SRC2 and a mouse monoclonal antibody 26C4 against RhoA were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal antibodies against Rac1 and Cdc42 were purchased from BD Biosciences (San Jose, CA). Anti-mouse and anti-rabbit IgG antibodies conjugated with horseradish peroxidase were obtained from Amersham Biosciences. Inhibitors—PP1 and PP2, inhibitors of Src kinase, were purchased from Biomol (Plymouth Meeting, PA) and Calbiochem-Novabiochem, respectively. Clostridium difficile toxin B, which inhibits Rho family small GTPases, was obtained from Calbiochem-Novabiochem. Endothelin-1 was purchased from Peptide Institute, Inc. (Osaka, Japan). Plasmids—The cDNA of KIAA0793, GenBank™ accession number AB018336, was kindly provided by Drs. F. Miki and T. Nagase (Kazusa DNA Research Institute, Chiba, Japan). The region encoding the KIAA0793 (FRG) protein (1055 amino acids) was subcloned into the mammalian HA tag expression vector pCMV-2HA. The fragments of FRG (ΔDH) and FRG (ΔDHPH) lacking amino acids 501–729 and 501–820 of FRG, respectively, were inserted into pCMV-2HA. The portions encoding the FERM domain (1–300), the proline-rich domain (301–500), the DH and PH domains (491–827), and the second PH domain (821–1055) were amplified from cDNA of FRG as a template and ligated into the mammalian FLAG-tag expression vector pCMV-FLAG. The cDNAs of RhoA, Rac1, and Cdc42 were amplified from total RNA of human kidney 293T cells using the method of reverse transcription polymerase chain reaction and were inserted the Escherichia coli GST-tag expression vector pET42a. pCMV-FLAG-RhoA, pCMV-FLAG-Rac1, pCMVFLAG-Cdc42, pCMV-FLAG-RhoG14V, pCMV-FLAG-RacG12V, pCMVFLAG-Cdc42G12V, pCMV-FLAG-RhoT19N, pCMV-FLAG-RacT17N, pCMV-FLAG-Cdc42T17N, and pCMV-FLAG-MKK4K95R were constructed as described previously (30Yamauchi J. Kaziro Y. Itoh H. J. Biol. Chem. 1999; 274: 1957-1965Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 39Yamauchi J. Hirasawa A. Miyamoto Y. Itoh H. Tsujimoto G. Biochem. Biophys. Res. Commun. 2001; 284: 1199-1203Crossref PubMed Scopus (20) Google Scholar). pUSE-CA-Src (a constitutively activated mutant of c-Src) was purchased from Upstate Biotechnology. The E. coli expression plasmids encoding the RhoA binding domain (RBD) of mDia1 and the Rac1 and Cdc42 binding domain (CRIB) of αPak were constructed as described previously (32Yamauchi J. Itoh H. Shinoura H. Miyamoto Y. Tsumaya K. Hirasawa A. Kaziro Y. Tsujimoto G. Biochem. Biophys. Res. Commun. 2001; 288: 1087-1094Crossref PubMed Scopus (11) Google Scholar, 40Kimura K. Tsuji T. Takada Y. Miki T. Narumiya S. J. Biol. Chem. 2000; 275: 17233-17236Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 41Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). pME-ETA receptor-enhanced green fluorescence protein was generously provided by Dr. T. Sakurai (University of Tsukuba, Ibaraki, Japan) (42Abe Y. Nakayama K. Yamanaka A. Sakurai T. Goto K. J. Biol. Chem. 2000; 275: 8664-8671Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). pGEX2T-c-Jun (amino acids 1–221) and SRα-HA-JNK1 were kindly provided by Dr. M. Karin (University of California, San Diego, CA). All DNA sequences were confirmed using DNA sequencer L-4200L (LI-COR). Recombinant Proteins—Recombinant GST-tagged mDia1RBD, αPakCRIB, Rho family GTPases, and c-Jun proteins were purified using E. coli BL21 (DE3) pLysS, as described previously (32Yamauchi J. Itoh H. Shinoura H. Miyamoto Y. Tsumaya K. Hirasawa A. Kaziro Y. Tsujimoto G. Biochem. Biophys. Res. Commun. 2001; 288: 1087-1094Crossref PubMed Scopus (11) Google Scholar). Cell Culture and Transfection—Human kidney epithelial 293T cells were maintained in Dulbecco's modified Eagle's medium containing 100 μg/ml kanamycin and 10% heat-inactivated fetal bovine serum. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Plasmid DNAs were transfected into cells by the calcium phosphate precipitation method. The final amount of the transfected DNA for a 60-mm dish was adjusted to 25 μg by an empty vector, pCMV. 293T cells were transfected with 1 μg of pCMV-2HA-FRG, 1 μg of pCMV-2HA-FRG-ΔDH, 1 μg of pCMV-2HA-FRG-ΔDHPH, 3 μg of pCMV-FLAG-FRG-FERM, 3 μg of pCMV-FLAG-FRG-proline, 3 μg of pCMV-FLAG-FRG-DHPH, 3 μg of pCMV-FLAG-FRG-2nd PH, 3 μg of pCMV-FLAG-RhoA, 3 μg of pCMV-FLAG-Rac1, 3 μg of pCMV-FLAGCdc42, 3 μg of pCMV-FLAG-RhoG14V, 3 μg of pCMV-FLAG-RacG12V, 3 μg of pCMV-FLAG-Cdc42G12V, 1 μg of pCMV-FLAG-RhoT19N, 1 μg of pCMV-FLAG-RacT17N, 1 μg of pCMV-FLAG-Cdc42T17N, 3 μg of pCMV-FLAG-MKK4K95R, 3 μg of pUSE-CA-Src, 0.3 μg of pME-ETARenhanced green fluorescence protein, or 1 μg of SRα-HA-JNK1. The medium was replaced 24 h after transfection, and cells were starved in a serum-free medium for 24 h before the addition of 100 nm endothelin-1. Immunoprecipitation and Immunoblotting—After the addition of endothelin-1, cells were lysed in 600 μl of lysis buffer A (20 mm HEPESNaOH (pH 7.5), 3 mm MgCl2, 100 mm NaCl, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, 1 μg/ml leupeptin, 1 mm EGTA, 1 mm Na3VO4, 10 mm NaF, 20 mm β-glycerophosphate, and 0.5% Nonidet P-40) for a 60-mm dish. The lysates were centrifuged at 15,000 rpm for 10 min at 4 °C. Aliquots (400 μg) of the supernatants were mixed with protein G-Sepharose CL-4B preabsorbed with 0.5 μg of anti-FLAG or 0.5 μg of anti-HA antibody. The immune complexes were precipitated by centrifugation and washed twice with lysis buffer A and twice with kinase buffer A (20 mm HEPES-NaOH (pH 7.5), 10 mm MgCl2, 1 mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride, 0.1 μg/ml leupeptin, 0.1 mm EGTA, 10 μm Na3VO4, and 2 mm β-glycerophosphate) for JNK, kinase buffer B (20 mm HEPES-NaOH (pH 7.5), 10 mm MgCl2, 3 mm MnCl2, 1 mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride, 0.1 μg/ml leupeptin, 0.1 mm EGTA, 10 μm Na3VO4, and 2 mm β-glycerophosphate) for Src kinase, or an exchange buffer (20 mm HEPES-NaOH (pH 7.5), 5 mm MgCl2, 150 mm NaCl, 1 mm dithiothreitol, 0.1 mm phenylmethanesulfonyl fluoride, 0.1 μg/ml leupeptin, and 3 mm EDTA). To compare the amounts of the immunoprecipitates or expressed proteins in the cell lysates in each transfection, the precipitates or aliquots of the cell lysates were boiled in a Laemmli sample buffer and then separated on 8–15% SDS-polyacrylamide gels. The electrophoretically separated proteins were transferred to polyvinylidene difluoride membranes. The membranes were blocked with phosphate-buffered saline containing 0.1% Tween 20 and 10 mg/ml bovine serum albumin and immunoblotted with antibodies. The bound antibodies were detected using the ECL system (Amersham Biosciences) with anti-mouse or anti-rabbit IgG antibodies conjugated with horseradish peroxidase according to the manufacturer's protocol. Images of protein bands were captured using Adobe Photoshop 5.0 plug-in software and an EPSON GT-7000U scanner. The band intensity of kinases was semiquantified using NIH Image 1.61. Representatives of at least three separate experiments are shown in the figures. Kinase Assays—After the addition of 100 nm endothelin-1 for 20 min, transfected cells were lysed in 600 μl of lysis buffer A for a 60-mm dish, and the lysates were centrifuged as described above. Aliquots (400 μg) of the supernatants were mixed with protein G-Sepharose CL-4B preabsorbed with anti-HA antibody. The activities of the proteins were measured as the 32P radioactivity incorporated into recombinant c-Jun using a BAS 2500 imaging analyzer (Fujifilm, Tokyo, Japan) as described previously (30Yamauchi J. Kaziro Y. Itoh H. J. Biol. Chem. 1999; 274: 1957-1965Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 31Yamauchi J. Kawano T. Nagao M. Kaziro Y. Itoh H. J. Biol. Chem. 2000; 275: 7633-7640Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 32Yamauchi J. Itoh H. Shinoura H. Miyamoto Y. Tsumaya K. Hirasawa A. Kaziro Y. Tsujimoto G. Biochem. Biophys. Res. Commun. 2001; 288: 1087-1094Crossref PubMed Scopus (11) Google Scholar, 33Yamauchi J. Miyamoto Y. Kokubu H. Nishii H. Okamoto M. Sugawara Y. Hirasawa A. Tsujimoto G. Itoh H. FEBS Lett. 2002; 527: 284-288Crossref PubMed Scopus (32) Google Scholar, 38Obaishi H. Nakanishi H. Mandai K. Satoh K. Satoh A. Takahashi K. Miyahara M. Nishioka H. Takaishi K. Takai Y. J. Biol. Chem. 1998; 273: 18697-18700Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Activities were normalized to the amounts of immunoprecipitated JNK, as estimated from these band intensities. Pull-down Assays of Rho Family GTPases—Cells were stimulated with or without 100 nm endothelin-1 for 15 min and then lysed in lysis buffer B (20 mm HEPES-NaOH (pH 7.5), 20 mm MgCl2, 150 mm NaCl, 1 mm dithiothreitol, 1 mm phenylmethanesulfonyl fluoride, 1 μg/ml leupeptin, 1 mm EGTA, 1 mm Na3VO4, 10 mm NaF, and 0.5% Nonidet P-40). To detect active GTP-bound Rho family GTPases in the cell lysates, we performed pull-down assays using recombinant GST-tagged mDia1RBD (for RhoA) and αPakCRIB (for Rac1 and Cdc42) as described previously (32Yamauchi J. Itoh H. Shinoura H. Miyamoto Y. Tsumaya K. Hirasawa A. Kaziro Y. Tsujimoto G. Biochem. Biophys. Res. Commun. 2001; 288: 1087-1094Crossref PubMed Scopus (11) Google Scholar, 40Kimura K. Tsuji T. Takada Y. Miki T. Narumiya S. J. Biol. Chem. 2000; 275: 17233-17236Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 41Benard V. Bohl B.P. Bokoch G.M. J. Biol. Chem. 1999; 274: 13198-13204Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar). Aliquots (400 μg) of the supernatants mixed with glutathione-Sepharose 4B with 10 μg of GST-mDia1RBD or 20 μg of GST-αPakCRIB were precipitated by centrifugation, and complexes were boiled in a Laemmli sample buffer and then separated on 15% SDS-polyacrylamide gels. The separated proteins were immunoblotted using each antibody against Rho family small GTPases. Exchange Assays—After the addition of endothelin-1 for 15 min, cells were lysed in 900 μl of lysis buffer A for a 10-cm dish, and the lysates were centrifuged as described above. Aliquots (800 μg) of the supernatants were used for the exchange assay. For releasing assays, the [3H]GDP-bound Rho family GTPases were obtained by incubation with an exchange buffer containing 125 ng/μl Rho GTPases, 250 ng/μl bovine serum albumin, 5 mm EDTA, and 0.3 μm [3H]GDP (0.3 μCi/μl) at 30 °C for 90 min. The reaction was stopped by adding 5 mm MgCl2, and mixtures were immediately cooled on ice. The immunoprecipitated DH domains of FRG and the FRG protein were incubated in 30 μl of an exchange buffer containing 16 ng/μl Rho GTPases·[3H]GDP, 33 ng/μl bovine serum albumin, and 3 μm cold GDP at 30 °C for 0, 10, 20, and 30 min. The reactions were stopped by adding 1 ml of an ice-cold wash buffer (20 mm HEPES-NaOH (pH 7.5) and 10 mm MgCl2) and filtered through nitrocellulose membranes. The membranes were immediately washed twice with an ice-cold wash buffer and air-dried. The radioactivity remaining on each membrane was measured by an LS6500 liquid scintillation counter (Beckman Coulter Inc., Palo Alto, CA). For the binding assay, the immunoprecipitated DH domain of FRG and the FRG protein were incubated in 30 μl of an exchange buffer containing 16 ng/μl Rho GTPases, 33 ng/μl bovine serum albumin, and 3 μm [32P]GTP (0.1 μCi/μl) at 30 °C for 0, 10, 20, and 30 min. The reactions were stopped by adding an ice-cold wash buffer and filtered through nitrocellulose membranes. The membranes were immediately washed twice with a wash buffer and air-dried. The radioactivity remaining on each membrane was measured as before. Activities were normalized to the amounts of FRG polypeptides in the immunoprecipitates. In Vitro Tyrosine Phosphorylation—Cells were lysed in 900 μl of lysis buffer A for a 10-cm dish, and the lysates were centrifuged, as described above. Aliquots (800 μg) of the supernatants were used for an in vitro phosphorylation reaction using recombinant c-Src (6 units) in 30 μl of kinase buffer B containing 20 μm ATP at 30 °C for 15 min and then chilled on ice. The tyrosine-phosphorylated FRG protein was washed twice with an exchange buffer and used for an exchange reaction for Cdc42. Cell Motility Assay—Cell motility was measured using a 24-well Boyden chamber (BD Biosciences) according to the manufacturer's protocol. Briefly, upper wells with polyethylene terephthalate filters (8-μm pore size) were coated with 10 μg/ml extra cellular matrix E-C-L (Upstate). Serum-starved cells (2 × 105 cells in 500 μl of Dulbecco's modified Eagle's medium per well) were loaded into upper wells, which were immediately plated on the chamber containing 165 nm endothelin-1 (750 μl of Dulbecco's modified Eagle's medium per well). After incubation at 37 °C for 5 h, upper filters were stained with a Diff-Quick staining kit (Biochemical Sciences Inc., Sterling Height, MI) according to the manufacturer's protocol. Cells that had not migrated were wiped away from the inner surface of the upper wells. Using an optical microscope, the number of migrated cells was counted in at least three independent experiments. Statistical Analysis—Statistical analysis was performed using SAS StatView 5.0. Values shown represent the mean ± S.E. from at least three separate experiments. A Student's t test was carried out for intergroup comparisons (*, p < 0.01). KIAA0793 Shows a Significant Homology to Cdc42-specific GEFs—We performed a BLAST search using the DH domain of Dbs because Dbs was implicated in the signaling pathway linking GPCRs to Rho family GTPases (35Yamauchi J. Hirasawa A. Miyamoto Y. Kokubu H. Nishii H. Okamoto M. Sugawara Y. Tsujimoto G. Itoh H. Biochem. Biophys. Res. Commun. 2002; 296: 85-92Crossref PubMed Scopus (12) Google Scholar). The search revealed the appearance of several uncharacterized proteins exhibiting a putative DH domain. One of them, KIAA0793, was of particular interest and further characterized. Fig. 1 shows the structural features of KIAA0793 in comparison with those of other Rho family GEFs. This molecule contains a tandem of DH and two PH domains, the DH domain being closely related to those of CDEP (chondrocyte-derived ezrin-like domain-containing protein) (43Koyano Y. Kawamoto T. Shen M. Yan W. Noshiro M. Fujii K. Kato Y. Biochem. Biophys. Res. Commun. 1997; 241: 369-375Crossref PubMed Scopus (29) Google Scholar, 44Koyano Y. Kawamoto T. Kikuchi A. Shen M. Kurata Y. Tsutsumi S. Fujimoto K. Noshiro M. Fujii K. Kato Y. Osteoarthritis Cartilage. 2001; 9: S64-S68Abstract Full Text PDF PubMed Scopus (14) Google Scholar) (54% identity, 71% similarity), Frabin (38Obaishi H. Nakanishi H. Mandai K. Satoh K. Satoh A. Takahashi K. Miyahara M. Nishioka H. Takaishi K. Takai Y. J. Biol. Chem. 1998; 273: 18697-18700Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) (30% identity, 47% similarity), FGD1 (36Zheng Y. Fischer D.J. Santos M.F. Tigyi G. German Pasteris N. Gorski J.L. Xu Y. J. Biol. Chem. 1996; 271: 33169-33172Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) (28% identity, 51% similarity), Fgd2 (30% identity, 51% similarity), and Fgd3 (37German Pasteris N. Nagata K. Hall A. Gorski J.L. Gene (Amst.). 2000; 242: 237-247Crossref PubMed Scopus (36) Google Scholar) (26% identity, 46% similarity). KIAA0793 contains an N-terminal region exhibiting extensive homology to the FERM domain, also called band 4.1 homology domain of ezrin, radixin, and moesin (ERM) proteins, which function as the cross-linker between plasma membranes and actin filaments (45Bretscher A. Curr. Opin. Cell Biol. 1999; 11: 109-116Crossref PubMed Scopus (331) Google Scholar, 46Tsukita S. Yonemura S. J. Biol. Chem. 1999; 274: 34507-34510Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). KIAA0793 exhibits an additional structural feature, a proline-rich region (amino acids 398–498) (47Cesareni G. Panni S. Nardelli G. Casagnoli L. FEBS Lett. 2002; 513: 38-44Crossref PubMed Scopus (123) Google Scholar). KIAA0793 Activates Cdc42 but Not RhoA and Rac1—To examine which Rho family GTPases are activated by the DH and PH domains of KIAA0793, the ability of KIAA0793 to induce the dissociation of [3H]GDP from recombinant RhoA, Rac1, and Cdc42 proteins was measured. It has been demonstrated that the isolated fragment containing DH and PH domains of Rho family GEFs acts as a limited catalytic domain toward Rho family GTPases (12Olson M.F. Sterpetti P. Nagata K. Toksoz D. Hall A. Oncogene. 1997; 15: 2827-2831Crossref PubMed Scopus (62) Google Scholar, 48Nimnual A.S. Yatsula B.A. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (389) Google Scholar). As shown in Fig. 2A, the isolated DH and PH fragment of KIAA0793 promoted nucleotide exchange o
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