The Formin/Diaphanous-related Protein, FHOS, Interacts with Rac1 and Activates Transcription from the Serum Response Element
2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês
10.1074/jbc.m105162200
ISSN1083-351X
Autores Tópico(s)Muscle Physiology and Disorders
ResumoFHOS is a member of the formin homology (FH) family of proteins and is expressed at high levels in splenic cells. FH proteins link cellular signaling pathways to the actin cytoskeleton and serum response factor-dependent transcription. In these studies, the role of FHOS in Rho family GTPase signaling pathways was analyzed. FHOS interacted with the polybasic domain in the Rac1 C terminus in a guanine nucleotide-independent manner but did not interact with RhoA, Cdc42Hs, Rac2, or Rac3. Intramolecular autoinhibitory interactions between the C terminus of FHOS and an N-terminal region partially overlapping the Rac1 interaction domain were also identified. FHOS truncation mutants lacking the N- or C-terminal autoregulatory domains stimulated transcription of a c-fos serum response element (SRE)-driven reporter. Overexpression of wild-type and mutant (N17 and V12) Rac1 proteins repressed SRE induction by the N-terminal FHOS deletion mutant but not by the C-terminal FHOS deletion mutant. Immunofluorescence studies indicated that the localization of the mutant FHOS proteins might contribute to their differential responses to Rac1. Wild-type FHOS and the N-terminal deletion mutant localized to the perinuclear region and membrane edges. In contrast, the C-terminal FHOS mutants were diffusely localized. These data suggest that FHOS induces transcription from SREs by multiple pathways and that Rac1 may influence the course of some FHOS-induced signaling events. FHOS is a member of the formin homology (FH) family of proteins and is expressed at high levels in splenic cells. FH proteins link cellular signaling pathways to the actin cytoskeleton and serum response factor-dependent transcription. In these studies, the role of FHOS in Rho family GTPase signaling pathways was analyzed. FHOS interacted with the polybasic domain in the Rac1 C terminus in a guanine nucleotide-independent manner but did not interact with RhoA, Cdc42Hs, Rac2, or Rac3. Intramolecular autoinhibitory interactions between the C terminus of FHOS and an N-terminal region partially overlapping the Rac1 interaction domain were also identified. FHOS truncation mutants lacking the N- or C-terminal autoregulatory domains stimulated transcription of a c-fos serum response element (SRE)-driven reporter. Overexpression of wild-type and mutant (N17 and V12) Rac1 proteins repressed SRE induction by the N-terminal FHOS deletion mutant but not by the C-terminal FHOS deletion mutant. Immunofluorescence studies indicated that the localization of the mutant FHOS proteins might contribute to their differential responses to Rac1. Wild-type FHOS and the N-terminal deletion mutant localized to the perinuclear region and membrane edges. In contrast, the C-terminal FHOS mutants were diffusely localized. These data suggest that FHOS induces transcription from SREs by multiple pathways and that Rac1 may influence the course of some FHOS-induced signaling events. formin homology serum response element Diaphanous-related Formin GTPase binding domain polymerase chain reaction glutathioneS-transferase polyacrylamide gel electrophoresis hemagglutinin serum response factor 4-morpholineethanesulfonic acid secreted alkaline phosphatase guanosine 5′-3-O-(thio)triphosphate Dulbecco's modified Eagle's medium fetal bovine serum monoclonal antibody ternary complex factor phosphate-buffered saline Dia-autoregulatory domain Formin homology (FH)1proteins are highly structured proteins and components of Rho family GTPase signaling pathways that affect cytoskeletal organization and induce transcriptional activation of the serum response element (SRE) (1Wasserman S. Trends Cell Biol. 1998; 8: 111-115Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 2Watanabe N. Madaule P. Reid T. Ishizaki T. 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From the N terminus, FH proteins contain a loosely conserved FH3 domain, a GTPase binding domain (GBD), highly conserved FH1 and FH2 domains, a coiled-coil, and an autoregulatory domain (1Wasserman S. Trends Cell Biol. 1998; 8: 111-115Abstract Full Text PDF PubMed Scopus (163) Google Scholar, 2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar, 7Petersen J. Nielsen O. Egel R. Hagan I.M. J. Cell Biol. 1998; 141: 1217-1228Crossref PubMed Scopus (133) Google Scholar, 8Castrillon D.H. Wasserman S.A. Development. 1994; 120: 3367-3377Crossref PubMed Google Scholar, 9Alberts A.S. J. Biol. Chem. 2000; 16: 16Google Scholar). In a subset of FH proteins, the Diaphanous-related Formin (DRF) proteins, the autoregulatory domain forms intramolecular interactions with residues within and/or between the FH3 and GBD (3Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. 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Genet. 1998; 62: 533-541Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). DRF proteins are activated in vivo by Rho family GTPases. Rho-related proteins are a subset of the Ras superfamily of GTPases and include RhoA–E, Rho6, Rho7, Rac1–3, Cdc42, TC10, and TTF. Rho GTPases regulate numerous cellular events, including gene transcription, cell cycle progression, adhesion, actin cytoskeletal organization, cytokinesis, and motility, by cycling between inactive GDP- and active GTP-bound states and relaying signals from membrane receptors to downstream effector molecules (21Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 22Scita G. Tenca P. Frittoli E. Tocchetti A. Innocenti M. Giardina G. Di Fiore P.P. EMBO J. 2000; 19: 2393-2398Crossref PubMed Google Scholar). DRF proteins are downstream effectors of Rho GTPases. Activated Rho GTPases bind to the GBDs of DRFs and relieve intramolecular interactions. At the present time, four FH proteins are known to interact with Rho GTPases. DRF1 and DRF2 bind to RhoA, RhoB, and RhoC (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar, 3Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (727) Google Scholar). DRF2 also interacts with Cdc42Hs, but neither DRF1 nor DRF2 associate with Rac1 (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar, 3Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (727) Google Scholar, 6Tominaga T. Sahai E. Chardin P. McCormick F. Courtneidge S.A. Alberts A.S. Mol. Cell. 2000; 5: 13-25Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). TheSaccharomyces cerevisiae DRF, Bni1p, associates with Rho1p and Cdc42p (13Evangelista M. Blundell K. Longtine M.S. Chow C.J. Adames N. Pringle J.R. Peter M. Boone C. Science. 1997; 276: 118-122Crossref PubMed Scopus (531) Google Scholar, 23Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar). DRFs preferentially interact with GTP-bound Rho proteins and are inhibited by Rho inactivation (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar, 23Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar). The product of formin-related gene in leukocytes (FRL) was recently shown to interact with Rac1 but not with RhoA or Cdc42Hs (15Yayoshi-Yamamoto S. Taniuchi I. Watanabe T. Mol. Cell. Biol. 2000; 20: 6872-6881Crossref PubMed Scopus (85) Google Scholar). Thus, FH proteins appear to interact with specific Rho family GTPases. One effect of Rho family GTPase and DRF activation is the induction of gene transcription from the SRE (5Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 6Tominaga T. Sahai E. Chardin P. McCormick F. Courtneidge S.A. Alberts A.S. Mol. Cell. 2000; 5: 13-25Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). The SRE is a short gene-regulatory sequence that is sufficient to activate transcription of immediate-early genes, such as c-fos and β-actin, in response to growth factor stimulation (24Treisman R. Semin. Cancer Biol. 1990; 1: 47-58PubMed Google Scholar). Maximal activation of the c-fos SRE requires the formation of a complex between the ubiquitous transcription factor, SRF (25Norman C. Runswick M. Pollock R. Treisman R. Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (708) Google Scholar), and certain Ets domain proteins, such as Elk1 and SAP1, that are collectively referred to as ternary complex factors (TCF). TCFs potentiate SRE transcriptional activation when they are phosphorylated by extracellular signal-regulated kinases in response to Ras/Raf or Rac1/Cdc42/PAK signals (21Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar, 26Shaw P.E. Schroter H. Nordheim A. Cell. 1989; 56: 563-672Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 27Marais R. Wynne J. Treisman R. Cell. 1993; 73: 381-393Abstract Full Text PDF PubMed Scopus (1108) Google Scholar, 28Frost J.A. Steen H. Shapiro P. Lewis T. Ahn N. Shaw P.E. Cobb M.H. EMBO J. 1997; 16: 6426-6438Crossref PubMed Scopus (362) Google Scholar). SRF activity is controlled independently of TCF activation and is induced by activated forms of RhoA, Rac1, and Cdc42 (29Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). RhoA-induced activation is essential for SRE-serum responsiveness and requires the FH proteins, DRF1 (mDia1) and DRF2 (mDia2) (5Sotiropoulos A. Gineitis D. Copeland J. Treisman R. Cell. 1999; 98: 159-169Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 6Tominaga T. Sahai E. Chardin P. McCormick F. Courtneidge S.A. Alberts A.S. Mol. Cell. 2000; 5: 13-25Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 9Alberts A.S. J. Biol. Chem. 2000; 16: 16Google Scholar). Rho activation, however, is not required for Rac1 or Cdc42-induced SRF activation (29Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar). Thus, multiple pathways target the SRF and TCF complex at the c-fos SRE, and distinct Rac1 signals affect both subunits of the complex. FHOS is a member of the formin homology (FH) family of proteins that is expressed at high levels in human splenic cells (30Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (47) Google Scholar). FHOS contains all the conserved domains, including FH1, FH2, and FH3 domains, and a coiled-coil, that are characteristic of FH proteins. In this report, the identification of a C-terminal autoregulatory domain and a GTPase binding domain are described. The role of FHOS in Rho GTPase signaling pathways is also examined. In contrast to DRFs (mDia1 and mDia2), which are downstream effectors of GTP-bound RhoA, FHOS binds to Rac1 in a guanine nucleotide-independent manner. Deletion of either the N or C termini created active forms of FHOS that stimulated transcription from the SRE. The N-terminal deletion mutant was localized to membrane ruffles, and its ability to activate the SRE was blocked by overexpression of several Rac1 proteins. These data identify novel functions for FH proteins and Rac1 in the cell. The pCMV5-HA-FHOS full-length and HA-FHOS-(1–1010) expression plasmids were previously described (30Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (47) Google Scholar). For in vitro transcription reactions, these FHOS sequences were excised from pCMV5 with Asp718 andBamHI or Asp718 and SmaI, respectively, and subcloned into pKS-Bluescript (pKS). pCMV5-HA- and pKS-FHOS-(1–421) were constructed by subcloning the Asp718/HindIII fragment from pCMV5-HA-FHOS into the base vectors. Similarly, pKS-FHOS-(1–321) is theAsp718/SalI fragment frompCMV5-HA-FHOS. pKS-FHOS-(1–717) was amplified with Pfu polymerase (Stratagene) frompCMV5-HA-FHOS with gene-specific oligonucleotides (Integrated DNA Technologies, Inc.) containing Asp718 orBamHI restriction sites (GATGGTACCATGGCGGGCGGGGAAGA and TACGGATCCGGTGGCAGTGTGGT AGGCCGATGTTGATG). FHOS-(469–1165) was constructed by amplifying a 248-base pair region ofpKS-FHOS between nucleotides 1408 and 1648 with oligonucleotides containing Asp718 or BglII restriction sites (AAGGTACCATGCCCAATGAGGCGG and CTAGATCTGAAAAGTCCAGGTCC), respectively. The resulting PCR product was subcloned into pKS-FHOS in place of the wild-type 5′-sequence. The fidelity of the Pfu polymerase and subcloning reactions was verified by automated DNA sequencing. FHOS-(1–717) and -(469–1165) were subcloned from pKS into the pCMV5-HA vector (30Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (47) Google Scholar) with Asp718 andBamHI. For yeast two-hybrid assays, pAS-2-1-FHOS-(1–328) and -(1–421) were generated by subcloning theEcoRI/SalI or EcoRI/BamHI fragments from the respective pCMV5-HA-FHOS vectors intopAS2-1 (CLONTECH). FHOS-(491–1165) was created by subcloning the 2-kilobase pair PstI fragment ofpCMV5-HA-FHOS-(1–1165) into pAS2-1. pAS2-1-FHOS-(855–1165) was made by removing the SalI fragment from pAS-2-1-FHOS-(491–1165). To generatepAS2-1-FHOS-(668–1165), the FHOS sequence was amplified by PCR with Pfu Turbo (Stratagene) and oligonucleotides, GTTGAATTCGAACACCTCTTTGAGTC and GTTGGATCCTCCAGGGTCCAGATAGAT (Integrated DNA Technologies). The purified PCR product was then subcloned into the EcoRI/BamHI sites ofpAS2-1. Prokaryotic expression vectors for the GST-GTPase fusion proteins were kindly provided by Shuh Narumiya, pGEX-2T-RhoA, -Rac1, and Cdc42 (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar); Gary Bokoch, pGEX-4T-Rac2 (31Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Abstract Full Text PDF PubMed Google Scholar); Nora Heisterkamp,pGEX-2T-Rac3 (32Haataja L. Groffen J. Heisterkamp N. J. Biol. Chem. 1997; 272: 20384-20388Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar); and Ulla Knaus, pGEX-Rac1→2 and -Rac 2→1 (33Knaus U.G. Wang Y. Reilly A.M. Warnock D. Jackson J.H. J. Biol. Chem. 1998; 273: 21512-21518Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). pZipNeo-RacN17, Cdc42N17, and RhoAN19 expression vectors were obtained from Yi Zheng (34Fischer R.S. Zheng Y. Quinlan M.P. Cell Growth Differ. 1998; 9: 209-221PubMed Google Scholar). Rob Lewis providedCMV-Rac1-myc and pCGT-RacV12 and -RacN17 (35Joneson T. McDonough M. Bar-Sagi D. Van Aelst L. Science. 1996; 274: 1374-1376Crossref PubMed Scopus (232) Google Scholar). GST-GTPase fusion proteins were produced in Escherichia coli, DH5α, during a 3-h induction with 0.1 mmisopropyl-1-thio-β-d-galactopyranoside. Bacteria were lysed by resuspension in Buffer A (10 mm MES, pH 6.5, 150 mm NaCl, 2 mm MgCl2, 0.5 mm EDTA, 0.5% Triton X-100, 5 mmdithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, aprotinin, and pepstatin A) (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar) and sonication. Insoluble material was separated by centrifugation. GST fusion proteins were purified from the lysates with glutathione-Sepharose beads (Amersham Pharmacia Biotech). After washing the beads three times in Buffer A, fusion protein concentrations were estimated by comparison to bovine serum albumin standards in Coomassie-stained SDS-PAGE gels. For reactions in the presence of GDP or GTPγS (Sigma), 10 μm GST-GTPases on Sepharose beads were preloaded overnight with 1 mm guanine nucleotide (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar).35S-Labeled FHOS proteins were in vitrotranscribed and translated with the TNT T3 or T7-Coupled Reticulocyte Lysate Systems (Promega). One-tenth of the lysate was incubated with 400 pmol of each nucleotide-loaded or -unloaded GST-GTPase-beads for 90 min at 4 °C. Beads were washed three times in Buffer A containing the appropriate guanine nucleotide. Proteins were resolved by 7% SDS-PAGE. The lower portion of each gel was stained with Coomassie dye to verify equal loading of the GST-GTPases to the reactions (data not shown). The upper portions of the gels were fixed in 40% methanol and 10% acetic acid, incubated with Amplify (Amersham Pharmacia Biotech), dried, and exposed to film. For immunoprecipitation assays, two 10-cm plates of confluent MDA-231 cells were lysed on ice for 10 min in 1 ml of modified RIPA buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 3 μg/ml aprotinin, 1 μm phenylmethylsulfonyl fluoride). Lysates were precleared with 50 μl of 50% protein G-Sepharose slurry for 30 min at 4 °C. Following centrifugation for 10 min at 4000 rpm, the lysates were collected and incubated overnight with 4 μg of anti-Rac mAb (Upstate Biotechnology, Inc.) or a control IgG mAb (MOPC-21, Sigma). Immune complexes were collected with 50 μl of 50% protein G-Sepharose slurry for 2 h at 4 °C, washed three times with modified RIPA buffer, resolved by 12% SDS-PAGE, and transferred to Immobilon P (Millipore). Rac1 and FHOS were detected by immunoblotting with murine Rac1 (1:1000, UBI) and FHOS-(1:600) (30Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (47) Google Scholar) antibodies diluted in Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat dried milk. Proteins were visualized with SuperSignal West Pico Chemiluminescent Substrate (Pierce) and autoradiography. For yeast two-hybrid assays S. cerevisiae strain Y190 (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, −112 +URA3::GAL→lacZ, LYS2::GAL→HIS3) were co-transformed with the indicated pAS-2-1-FHOS expression plasmid andpACT-FHOS-(904–1165) (30Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (47) Google Scholar). Transformed yeast were selected on SD media lacking leucine (Leu), tryptophan (Trp), and histidine (His). Five individual colonies were restreaked onto SD media lacking His-Trp-Leu (HWL) in the presence or absence of 50 mm 2-aminotriazole. COS7 cells were seeded at a density of 5.2 cells/cm2 in 12-well plates (2 × 104 cells/well). Cells were allowed to adhere and grow for 4–16 h in DMEM (Life Technologies, Inc.) containingl-glutamine, penicillin/streptomycin, and 10% FBS (BioWhittaker). The pSRE, pAP-1, and NFκB luciferase (Luc) reporter plasmids contain three, four, and four tandem copies of the respective promoter element fused to the herpes simplex virus-thymidine kinase TATA-like promoter (CLONTECH). The SRE sequence is identical to the SRE in the c-fos promoter (24Treisman R. Semin. Cancer Biol. 1990; 1: 47-58PubMed Google Scholar). Luciferase reporter plasmids (0.5 μg) were mixed with 100 ng ofpCMV5-secreted alkaline phosphatase (SEAP) and the indicatedpCMV5-HA-FHOS or Rac expression vectors. Cells were transfected by incubation with reporter and expression plasmid mixtures in D-PBS (BioWhittaker) containing 0.5 mg/ml DEAE-dextran for 20 min at 37 °C, followed by a 2.5-h incubation in DMEM containing 5% FBS and 80 μm chloroquine. After removal of the media, cells were shocked for 2.5 min with DMEM containing 10% Me2SO and 5% FBS. The cells were then washed with d-PBS and incubated for 16–40 h in DMEM containing 0.1% FBS. Luciferase activity was measured with the Luciferase Assay Systems (Promega) as instructed by the manufacturer. SEAP activity was measured as described previously (36Berger J. Hauber J. Hauber R. Geiger R. Cullen B.R. Gene (Amst.). 1988; 66: 1-10Crossref PubMed Scopus (581) Google Scholar, 37Westendorf J.J. Yamamoto C.M. Lenny N. Downing J.R. Selsted M.E. Hiebert S.W. Mol. Cell. Biol. 1998; 18: 322-333Crossref PubMed Google Scholar). Luciferase activity was normalized for transfection efficiency with the SEAP values for each sample. Fold activation was determined relative to samples transfected withpCMV5. Values represent the mean of three independent transfections ± standard error of the mean. NIH3T3 were grown on coverslips and transfected with the indicated FHOS expression plasmids with Superfect (Qiagen). C2C12 cells were transduced with the indicatedMSCV-HA-FHOS vector using conventional methods (38Norris P.S. Jepsen K. Haas M. J. Virol. Methods. 1998; 75: 161-167Crossref PubMed Scopus (10) Google Scholar). Forty hours later, cells were fixed with 2% paraformaldehyde, lysed in PBS containing 0.3% Triton X-100, and blocked for 30 min in IF buffer (3% bovine serum albumin, 20 mm MgCl2, 0.3% Tween 20, 1× PBS). Cells were incubated sequentially in IF buffer containing anti-HA mAb (clone 12CA5, Sigma), anti-mouse IgG-Alexa 546 (Molecular Probes), and anti-Rac1-fluorescein isothiocyanate (Upstate Biotechnology, Inc.). Cells were mounted in 90% glycerol containing 0.4% n-propyl gallate. Endogenous FHOS was detected in HeLa cells with FHOS antiserum (30Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (47) Google Scholar). DNA was counterstained for 5 min with Hoescht's dye (5 μg/ml) as indicated. FHOS is a recently described member of the Formin/Diaphanous family of proteins. Formin homology proteins interact with and are downstream effectors of Rho family GTPases (2Watanabe N. Madaule P. Reid T. Ishizaki T. Watanabe G. Kakizuka A. Saito Y. Nakao K. Jockusch B.M. Narumiya S. EMBO J. 1997; 16: 3044-3056Crossref PubMed Scopus (688) Google Scholar, 3Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (727) Google Scholar, 6Tominaga T. Sahai E. Chardin P. McCormick F. Courtneidge S.A. Alberts A.S. Mol. Cell. 2000; 5: 13-25Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 15Yayoshi-Yamamoto S. Taniuchi I. Watanabe T. Mol. Cell. Biol. 2000; 20: 6872-6881Crossref PubMed Scopus (85) Google Scholar). To determine its molecular function, FHOS was tested for its ability to associate with the most frequently studied members of the Rho subfamily of GTPases, Cdc42Hs, Rac1, and RhoA. FHOS interacted with Rac1 but not with Cdc42Hs or RhoA (Fig.1 A). The interactions between FHOS and Rac1 were detected in the presence of either GDP or GTPγS (Fig. 1, A and B) and the absence of guanine nucleotides (Fig. 1 B). FHOS did not associate with Rac2 or Rac3, which are the Rho family GTPases that are most identical to Rac1, in any condition (Fig. 1 B). To verify the interaction between FHOS and Rac1 in vivo, lysates from human breast carcinoma cell line, MDA-231, were immunoprecipitated with Rac1 mAb. FHOS was detected in the Rac1 immune complexes by immunoblotting but not in control immunoprecipitates (Fig. 1 C). Moreover, Rac1 co-localized with FHOS in the cytoplasm of HeLa cells (Fig.1 D). Thus FHOS interacts specifically with the Rho GTPase, Rac1, in an apparently guanine nucleotide-independent manner. The interaction between FHOS and Rac1, but not with Rac2 or Rac3, was somewhat surprising given the extensive sequence similarity between the three proteins (31Knaus U.G. Heyworth P.G. Kinsella B.T. Curnutte J.T. Bokoch G.M. J. Biol. Chem. 1992; 267: 23575-23582Abstract Full Text PDF PubMed Google Scholar, 32Haataja L. Groffen J. Heisterkamp N. J. Biol. Chem. 1997; 272: 20384-20388Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The extreme C termini of Rac proteins, however, are poorly conserved (Fig. 2 A), and thus it was hypothesized that the C terminus of Rac1 may mediate the interaction with FHOS. FHOS was tested for interactions with Rac1 and Rac2 chimeras in which the polybasic regions (amino acids 183–188) of each protein are interchanged (Fig. 2, A andB) (33Knaus U.G. Wang Y. Reilly A.M. Warnock D. Jackson J.H. J. Biol. Chem. 1998; 273: 21512-21518Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). FHOS interacted with GST-Rac1 and the GST-Rac2 chimera containing the Rac1 polybasic domain (Rac2→1) but did not interact with GST, GST-Rac2, or the GST-Rac1 chimera containing six amino acids from Rac2 (Rac1→2) (Fig. 2 C). These data indicate that the polybasic domain in the Rac1 C terminus mediates interactions with FHOS. To determine the region of FHOS that interacted with Rac1, truncated FHOS proteins (Fig.3 A) were incubated with GST or GST-Rac1 in the absence of guanine nucleotides. Rac1 interacted with full-length FHOS-(1–1165) as well as with FHOS proteins (1–717 and 469–1165) that retained the F
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