Biochemical Characterization of the Diaphanous Autoregulatory Interaction in the Formin Homology Protein FHOD1
2005; Elsevier BV; Volume: 281; Issue: 8 Linguagem: Inglês
10.1074/jbc.m509226200
ISSN1083-351X
AutoresAndré Schönichen, Michael Alexander, Judith E. Gasteier, Fanny E. Cuesta, O. Fackler, Matthias Geyer,
Tópico(s)Muscle Physiology and Disorders
ResumoDiaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site. Diaphanous related formins (DRFs) are cytoskeleton remodeling proteins that mediate specific upstream GTPase signals to regulate cellular processes such as cytokinesis, cell polarity, and organelle motility. Previous work on the Rho-interacting DRF mDia has established that the biological activity of DRFs is regulated by an autoinhibitory interaction of a C-terminal diaphanous autoregulatory domain (DAD) with the DRF N terminus. This autoinhibition is released upon competitive binding of an activated GTPase to the N terminus of the DRF. Analyzing autoregulation of the Rac1-interacting DRF FHOD1, we utilized in vitro binding studies to identify a 60-amino acid DAD at the protein C terminus that recognizes an N-terminal formin homology (FH) 3 domain. Importantly, the FH3 domain of FHOD1 does not overlap with the proposed Rac1-binding domain. The FHOD1 DAD was found to contain one functional hydrophobic autoregulatory motif, while a previously uncharacterized basic cluster that is conserved in all DRF family DADs also contributed to the FH3-DAD interaction. Simultaneous mutation of both motifs efficiently released autoinhibition of FHOD1 in NIH3T3 cells resulting in the formation of actin stress fibers and increased serum response element transcription. A second putative hydrophobic autoregulatory motif N-terminal of the DAD belongs to a unique FHOD subdomain of yet undefined function. NMR structural analysis and size exclusion chromatography experiments revealed that the FHOD1 DAD is intrinsically unstructured with a tendency for a helical conformation in the hydrophobic autoregulation motif. Together, these data suggest that in FHOD1, DAD acts as signal sequence for binding to the well folded and monomeric FH3 domain and imply an activation mechanism that differs from competitive binding of Rac1 and DAD to one interaction site. Formin proteins are involved in the regulation of many cytoskeletal processes including cytokinesis, actin cable and stress fiber formation, polarity establishment, neurite outgrowth, and intracellular trafficking (reviewed in Refs. 1Wallar B.J. Alberts A.S. Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar and 2Tanaka K. Biochem. Biophys. Res. Commun. 2000; 267: 479-481Crossref PubMed Scopus (26) Google Scholar). These functions are achieved by their ability to promote F-actin assembly at the filament barbed end and to move processively with the barbed end as it elongates (3Zigmond S.H. Curr. Opin. Cell Biol. 2004; 16: 99-105Crossref PubMed Scopus (204) Google Scholar, 4Watanabe N. Higashida C. 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Formins are large proteins of typically more than 1000 amino acids that are defined by the presence of two conserved regions, namely the formin homology 1 and 2 (FH1 and FH2) 2The abbreviations used are: FH, formin homology; DAD, diaphanous autoregulatory domain; DRF, diaphanous-related formin; FHOD1, formin homology 2 domain-containing protein 1; GBD, GTPase-binding domain; GST, glutathione S-transferase; HA, hemagglutinin; ITC, isothermal titration calorimetry; SRE, serum response element; TRITC, tetramethylrhodamine isothiocyanate; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride. 2The abbreviations used are: FH, formin homology; DAD, diaphanous autoregulatory domain; DRF, diaphanous-related formin; FHOD1, formin homology 2 domain-containing protein 1; GBD, GTPase-binding domain; GST, glutathione S-transferase; HA, hemagglutinin; ITC, isothermal titration calorimetry; SRE, serum response element; TRITC, tetramethylrhodamine isothiocyanate; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride. domains (1Wallar B.J. Alberts A.S. Trends Cell Biol. 2003; 13: 435-446Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Additional conserved domains such as a N-terminal GTPase-binding domain (GBD) and a C-terminal diaphanous autoregulation domain (DAD) were found to constitute a formin subfamily, the diaphanous related formins (DRFs) (8Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Over the last years, DRFs have emerged as a group of proteins with the potential to bridge between G-protein signals and the cytoskeleton via their ability to bind activated small GTPases and to subsequently remodel the cytoskeleton (9Gasman S. Kalaidzidis Y. Zerial M. Nat. Cell Biol. 2003; 5: 195-204Crossref PubMed Scopus (175) Google Scholar, 10Kohno 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, 11Evangelista 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 (527) Google Scholar, 12Habas R. Kato Y. He X. Cell. 2001; 107: 843-854Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar, 13Tominaga 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 (328) Google Scholar).At present, phylogenetic analyses of FH2 domains suggest that metazoan formins fall into seven groups, termed Dia (diaphanous), DAAM (dishevelled-associated activator of morphogenesis), FRL (formin-related gene in leukocytes), FHOD (formin homology domain-containing protein), INF (inverted formin), FMN (formin), and delphilin (14Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2005; 16: 1-13Crossref PubMed Scopus (199) Google Scholar). FHOD1 (previously named FHOS) was initially identified in splenic cells as an interaction partner of the acute myeloid leukemia transcription factor (AML-1B) (15Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (46) Google Scholar). It is ubiquitously expressed and facilitates transcription from the serum response element (SRE) (15Westendorf J.J. Mernaugh R. Hiebert S.W. Gene (Amst.). 1999; 232: 173-182Crossref PubMed Scopus (46) Google Scholar, 16Westendorf J.J. J. Biol. Chem. 2001; 276: 46453-46459Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). An activated form of FHOD1 in which autoinhibition is constitutively released induces the formation of and association with actin stress fibers (16Westendorf J.J. J. Biol. Chem. 2001; 276: 46453-46459Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 17Gasteier J.E. Madrid R. Krautkramer E. Schroder S. Muranyi W. Benichou S. Fackler O.T. J. Biol. Chem. 2003; 278: 38902-38912Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 18Koka S. Neudauer C.L. Li X. Lewis R.E. McCarthy J.B. Westendorf J.J. J. Cell Sci. 2003; 116: 1745-1755Crossref PubMed Scopus (65) Google Scholar). FHOD1 was shown to stimulate cell migration in an integrin-independent manner (18Koka S. Neudauer C.L. Li X. Lewis R.E. McCarthy J.B. Westendorf J.J. J. Cell Sci. 2003; 116: 1745-1755Crossref PubMed Scopus (65) Google Scholar), an effect that may relate to its ability to coordinate actin filaments and microtubules to induce cell elongation (19Gasteier J.E. Schroeder S. Muranyi W. Madrid R. Benichou S. Fackler O.T. Exp. Cell Res. 2005; 306: 192-202Crossref PubMed Scopus (32) Google Scholar). A recently identified homolog, FHOD2, is expressed in heart, kidney, and brain and localizes to nestin intermediate filaments to promote their actin-organizing activity (20Kanaya H. Takeya R. Takeuchi K. Watanabe N. Jing N. Sumimoto H. Genes Cells. 2005; 10: 665-678Crossref PubMed Scopus (39) Google Scholar).The biological activity of DRFs is mediated by its central FH1-FH2 module that nucleates actin filaments and remains bound to the barbed end of the growing filament (21Moseley J.B. Sagot I. Manning A.L. Xu Y. Eck M.J. Pellman D. Goode B.L. Mol. Biol. Cell. 2004; 15: 896-907Crossref PubMed Scopus (222) Google Scholar, 22Higashida C. Miyoshi T. Fujita A. Oceguera-Yanez F. Monypenny J. Andou Y. Narumiya S. Watanabe N. Science. 2004; 303: 2007-2010Crossref PubMed Scopus (247) Google Scholar, 23Romero S. Le Clainche C. Didry D. Egile C. Pantaloni D. Carlier M.F. Cell. 2004; 119: 419-429Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). Structural analyses have provided the molecular basis of a tethered dimer architecture that may allow formins to stair-step on the barbed end of an elongating nascent filament by binding two actins, one permitting monomer binding and the other permitting monomer dissociation (24Shimada A. Nyitrai M. Vetter I.R. Kuhlmann D. Bugyi B. Narumiya S. Geeves M.A. Wittinghofer A. Mol. Cell. 2004; 13: 511-522Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 25Xu Y. Moseley J.B. Sagot I. Poy F. Pellman D. Goode B.L. Eck M.J. Cell. 2004; 116: 711-723Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 26Otomo T. Tomchick D.R. Otomo C. Panchal S.C. Machius M. Rosen M.K. Nature. 2005; 433: 488-494Crossref PubMed Scopus (277) Google Scholar). However, in context of the full-length proteins, DRF molecules are thought to exist in an inactive state due to an intramolecular interaction between the C-terminal autoregulatory DAD and its N-terminal recognition domain (8Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 27Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (720) Google Scholar, 28Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 29Li F. Higgs H.N. J. Biol. Chem. 2005; 280: 6986-6992Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). This interaction requires an regulatory motif (MDxLL) in the DAD (8Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar) and is suggested to mask the conserved FH1 and FH2 domains, thereby autoinhibiting their biological activity. DRF-DADs also contain a highly conserved cluster of positively charged residues with yet unknown relevance to autoregulation (8Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). This autoinhibition is released upon interaction with specific members of the Rho-family GTPases in their activated state (8Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 28Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). Consequently, DRF proteins lacking the DAD or its recognition domain behave as dominant active molecules with substantial actin remodeling activity (8Alberts A.S. J. Biol. Chem. 2001; 276: 2824-2830Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 13Tominaga 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 (328) Google Scholar, 16Westendorf J.J. J. Biol. Chem. 2001; 276: 46453-46459Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 27Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (720) Google Scholar, 30Copeland J.W. Treisman R. Mol. Biol. Cell. 2002; 13: 4088-4099Crossref PubMed Scopus (162) Google Scholar). Recent structural data have shed light on the activation interaction for the mDia1 protein (31Rose R. Weyand M. Lammers M. Ishizaki T. Ahmadian M.R. Wittinghofer A. Nature. 2005; 435: 513-518Crossref PubMed Scopus (214) Google Scholar, 32Otomo T. Otomo C. Tomchick D.R. Machius M. Rosen M.K. Mol. Cell. 2005; 18: 273-281Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The structurally and functionally less well defined FH3 domain (33Petersen J. Nielsen O. Egel R. Hagan I.M. J. Cell Biol. 1998; 141: 1217-1228Crossref PubMed Scopus (131) Google Scholar), also referred to as diaphanous inhibitory domain (29Li F. Higgs H.N. J. Biol. Chem. 2005; 280: 6986-6992Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), was found to interact with the autoregulating DAD (32Otomo T. Otomo C. Tomchick D.R. Machius M. Rosen M.K. Mol. Cell. 2005; 18: 273-281Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), while a small preceding region forms the GTPase-binding subdomain of mDia1 that interacts with the switch I and II regions of the Rho GTPase (31Rose R. Weyand M. Lammers M. Ishizaki T. Ahmadian M.R. Wittinghofer A. Nature. 2005; 435: 513-518Crossref PubMed Scopus (214) Google Scholar). A succeeding three-helix bundle forms a dimerization domain, which is believed to be followed by a coiled-coil region before the proline-rich FH1 domain starts.Several lines of evidence suggest that the molecular mechanisms of autoinhibition for FHOD formins could differ from that of Dia-family formins. First, although the N-terminal regions of mDia1 and FHOD1 are of similar length, there is no apparent homology between the two different subfamily proteins (16.7% identity over 570 residues). In fact, the overall modular domain architecture of FHOD1 and mDia1 seems to be distinct, since the GTP-binding region was suggested to directly precede the FH1 domain in FHOD1 (Ref. 16Westendorf J.J. J. Biol. Chem. 2001; 276: 46453-46459Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar; see also Fig. 1A) while it was mapped to a near N-terminal domain in mDia1 (27Watanabe N. Kato T. Fujita A. Ishizaki T. Narumiya S. Nat. Cell Biol. 1999; 1: 136-143Crossref PubMed Scopus (720) Google Scholar, 28Li F. Higgs H.N. Curr. Biol. 2003; 13: 1335-1340Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 29Li F. Higgs H.N. J. Biol. Chem. 2005; 280: 6986-6992Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 31Rose R. Weyand M. Lammers M. Ishizaki T. Ahmadian M.R. Wittinghofer A. Nature. 2005; 435: 513-518Crossref PubMed Scopus (214) Google Scholar, 32Otomo T. Otomo C. Tomchick D.R. Machius M. Rosen M.K. Mol. Cell. 2005; 18: 273-281Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Moreover, FHOD family proteins interact with Rac1 instead of Rho or CDC42 GTPases (16Westendorf J.J. J. Biol. Chem. 2001; 276: 46453-46459Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 17Gasteier J.E. Madrid R. Krautkramer E. Schroder S. Muranyi W. Benichou S. Fackler O.T. J. Biol. Chem. 2003; 278: 38902-38912Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). In contrast to the mDia-GTPase interaction, binding of FHOD1 to Rac1 is not regulated by the loaded nucleotide state of the GTPase and activated Rac1 fails to induce the full phenotype observed with dominant active FHOD1 (16Westendorf J.J. J. Biol. Chem. 2001; 276: 46453-46459Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 17Gasteier J.E. Madrid R. Krautkramer E. Schroder S. Muranyi W. Benichou S. Fackler O.T. J. Biol. Chem. 2003; 278: 38902-38912Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Finally, also the C-terminal DAD in FHOD1 differs from Dia family DADs by its length and the number of potential autoregulation signals (Fig. 1, B and C). This leads to an ambiguous alignment between these DADs. While current listings (e.g. SMART) provide a lineup with no gaps between the FH2 and DAD, an alternative alignment scheme might assemble the C-terminal motifs for functional similarities (Fig. 1D, left and right panels).This study focused on the biochemical analysis of the autoregulation within human FHOD1. We found that an N-terminal stable domain (FH3, 1-377) directly interacted with the DAD. Mutation of the three proposed DAD consensus motifs showed that only the two C-terminal motifs contributed to FH3 domain binding in vitro and control FHOD1 activity in cells. Structural analysis of the FHOD1 DAD revealed an intrinsically unstructured domain with some tendency for a helical conformation in the hydrophobic consensus motif. The DAD could act as signal sequence for binding to the well folded and monomeric FH3 domain. These data imply that distinct, individually adapted molecular surfaces mediate the autoregulation and activation mechanism of FHOD1 that contains an additional region in between the FH2 domain and DAD.EXPERIMENTAL PROCEDURESProtein Sequence Analysis—Sequence alignments and protein secondary structure predictions were done prior to protein fragmentation using following protein sequence data base entries: FHOD1 (human) Q9Y613, FHOD1 (mouse) AAH60654, FHOD2 (mouse) BAC98303, FHOD3 (human) XP_371114, mDia1 (mouse) O08808, DRF1/hDia1 (human) O60610, mDia3 (mouse) O70566, DRF2/hDia2 (human) O60879, diaphanous (Drosophila) P48608, DRF3/mDia2 (mouse) Q9Z207, and Bni1p (yeast) P41832. Multiple sequence alignments were performed using the MultAlign software (au.expasy.org/). For domain architecture analyses and secondary structure predictions following open access programs were quoted: SMART (smart.embl-heidelberg.de/), Prosite (au.expasy.org/prosite/), and PredictProtein (www.predictprotein.org/).Plasmid Cloning, Protein Expression, and Purification—The coding DNA sequence for human fhod1 (GenBank™ accession code: AF113615) was used to generate fragments thereof by PCR-mediated amplification with primer containing BamHI and EcoRI restriction sites at the 5′- and 3′-ends, respectively. Fragments were cloned in the procaryotic expression vectors pProEx-HTb (Invitrogen) or pGEX-2T-tev (Amersham Biosciences) for protein expression and purification. Codon optimization of the DAD was performed using the mega-primer method for mutagenesis as described previously (34Schulte A. Czudnochowski N. Barboric M. Schonichen A. Blazek D. Peterlin B.M. Geyer M. J. Biol. Chem. 2005; 280: 24968-24977Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Site-directed mutagenesis of DAD consensus motifs was conducted similarly using both sense and antisense oligo nucleotides. Full-length plasmids for cellular transfection assays were cloned in the pEF-HA vector similarly as described (17Gasteier J.E. Madrid R. Krautkramer E. Schroder S. Muranyi W. Benichou S. Fackler O.T. J. Biol. Chem. 2003; 278: 38902-38912Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), which contained an N-terminal HA-epitope for immunostaining.For expression of FHOD1 protein domains the coding plasmids were transformed into Escherichia coli BL21(DE3) cells (Novagen), expressed at 30 °C and induced at an A600 of 0.6 to 1.0 with 0.3 mm IPTG for 5 h growth. For His-tagged proteins cells were fluidized in lysis buffer A (20 mm Tris/HCl, pH 7.6, 500 mm NaCl, 5 mm β-mercaptoethanol) with 20 mm imidazole and cleared by spinning for 45 min at 30,000 × g. The lysate was loaded onto 5 ml of nickel-nitrilotriacetic acid resin (Qiagen) that had been pre-equilibrated with lysis buffer. After washing with 10 volumes of lysis buffer A the protein was eluted with 10 volumes of lysis buffer A using a linear gradient from 20 to 250 mm imidazole. The peak fractions were dialyzed in buffer A, and if required the histidine tag was cleaved off at 4 °C over 12 h with Tev protease. FHOD1 was depleted of the protease and of uncleaved fragments by affinity chromatography. The protein containing flow-through was concentrated and further purified by gel filtration on a S75 column in 20 mm Tris/HCl, pH 7.6, 150 mm NaCl. GST fusion proteins (377-573 and 1104-1164) were expressed as described above and fluidized in 20 mm Tris, 150 mm NaCl, 1 mm EDTA, pH 8.0. After washing with 20 mm Tris/HCl, 1 m NaCl, 1 mm EDTA GST fusion proteins were eluted with 10 mm GSH and cleaved by Tev-protease at 4 °C over 12 h. GSH was removed by gel filtration and GST by GSH affinity column. Uniformly 15N-labeled DAD of FHOD1 was produced in minimal medium containing 15NH4Cl as the sole nitrogen source. Fractions were analyzed by SDS-PAGE and fractions containing FHOD1 proteins (about 98% pure) were concentrated (Amicon filter) and stored at -80 °C. Protein concentrations were determined by Bradford assay (Bio-Rad) and extinction coefficient measurements.GST Pull-down Assays and Western Blotting—For direct interaction assays between various DAD constructs and the N-terminal domains of FHOD1 (1-573), the FH3 domain (1-377), and the proposed GBD domain (377-573), about 2 μg of GST or GST fusion proteins were immobilized on glutathione-Sepharose beads (Amersham Biosciences) and incubated with 10-20 μg of the respective target protein. Binding reactions were performed in 500 μl of buffer solution (20 mm Tris/HCl, pH 7.6, 150 mm NaCl, 1 mm dithioerythritol, 0.1% Nonidet P-40) for 1-3 h at 4 °C or for 0.5 h at room temperature, respectively. Beads were washed three to five times in the same buffer, and bound proteins were analyzed by SDS-PAGE and subsequent Coomassie staining or Western blotting, respectively, using standard protocols. Recombinant proteins were detected with an anti-His antibody (Santa Cruz Biotechnology). Expression of HA-tagged FHOD1 proteins in transfected NIH3T3 cells was analyzed by SDS-PAGE/Western blotting of postnuclear cell lysates with an anti-HA antibody (Santa Cruz Biotechnology).Transfections and Immunofluorescence Microscopy—Functional analyses of FHOD1 proteins were carried out in NIH3T3 cells essentially as described previously (17Gasteier J.E. Madrid R. Krautkramer E. Schroder S. Muranyi W. Benichou S. Fackler O.T. J. Biol. Chem. 2003; 278: 38902-38912Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). For immunofluorescence, cells were plated onto coverglasses overnight and subsequently transfected with a total of 1 μg of DNA using Metafectene (Biontex). 24 h post-transfection, the cells were fixed with 3% paraformaldehyde (15 min at room temperature), permeabilized with phosphate-buffered saline, 0.1% Triton X-100 for 2 min, and blocked with phosphate-buffered saline, 1% bovine serum albumin for 30 min. HA-tagged FHOD1 proteins were revealed by staining with the mouse monoclonal antibody F-7 (Santa Cruz Biotechnology) and appropriate secondary antibodies conjugated with Alexa 488 (Molecular Probes). F-actin was stained with TRITC-conjugated phalloidin (Sigma). Following extensive washing, cells were mounted with Histogel (Linaris), and indirect fluorescence images were monitored with an Olympus IX70 microscope and processed using Adobe Photoshop.SRE Transcription Assay—Activation of the SRE by FHOD1 was quantified in NIH3T3 cells as described previously (17Gasteier J.E. Madrid R. Krautkramer E. Schroder S. Muranyi W. Benichou S. Fackler O.T. J. Biol. Chem. 2003; 278: 38902-38912Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Briefly, luciferase activity of NIH3T3 cells was determined 24 h post-transfection of FHOD1 expression vectors, the 5×SRE-Luc reporter plasmid, and pTK-Renilla with a Luminoskan Ascent luminometer (Thermo Laboratories) using the dual luciferase reporter assay system kit (Promega). SRE firefly luciferase counts were normalized to the activity of the Renilla luciferase internal control and calculated as fold transactivation with the counts for FHOD1-wt arbitrarily set to 1.Isothermal Titration Calorimetry—The thermodynamic parameters of the FHOD1 interaction with wild type and mutant DAD were determined by isothermal titration calorimetry (MCS-ITC, MicroCal). All proteins were dialyzed against ITC buffer (50 mm Hepes, pH 7.2, 100 mm NaCl, 1 mm TCEP), and the DAD fragment (1104-1164) was thermostated in the sample cell at 25 °C at a concentration of 35 μm. The FH3 domain-containing fragment (400 μm) was injected stepwise by volumes of 8 μl from the syringe into the solution. The change in heating power was observed for 4 min until equilibrium was reached before the next injection was started. Further data evaluation was performed using the manufacturers analysis software, yielding ΔG° and ΔH° values with 0.5 kcal/mol errors each and typical errors for dissociation constants of 0.5 μm and somewhat higher for weak binding mutants.Analytical Gel Filtration Chromatography—Analytical gel filtration experiments were performed with a multicomponent Waters 626 LC system (Waters) using a Superdex 75 column (10/30, column volume 25.7 ml, Amersham Biosciences) or a Biosep-SEC-S2000 column (300 × 1.8 mm, Phenomenex) with a separation range for globular proteins from 1 to 300 kDa. The columns were first equilibrated in the respective running puffer following injection of the protein samples. The flow rate was set to 0.5 or 1.0 ml/min for the Superdex or Biosep column, respectively. Elution profiles were monitored by UV absorption at 280 nm. The void volume (V0) was determined with blue dextran (Sigma). The columns were calibrated with the following standards (Bio-Rad): thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.35 kDa). The ratio of elution volume to void volume (Ve/V0) was plotted versus the log(Mr) for each standard to generate a linear calibration curve. FHOD1 samples were dialyzed from frozen stocks into the equilibration buffer, diluted to a concentration of 1 mg/ml each, and injected onto the column at a volume of 90 μl. Protein complexations were incubated for 30 min prior to subjection to the column. The apparent molecular weight of each FHOD1 fragment was determined from the standard curve. Gel filtration experiments were performed repeated times at room temperature.NMR Spectroscopy—NMR experiments with homonuclear or 15N-labeled protein samples were performed on a Varian Inova 600 spectrometer, equipped with a triple resonance probe with shielded Z gradients. Proteins were dissolved in phosphate buffer (20 mm KPi, pH 7.0, 100 mm NaCl, 1 mm TCEP) at a concentration of 0.6 mm and measured at 25 °C in SHIGEMI tubes. Homonuclear two-dimensional experiments and 15N/1H-heteronuclear HSQC experiments were recorded following the Varian NMR suite with typically 2048 × 512 data points. NMR spectra were converted to Bruker format and processed with XWINNMR and evaluated and plotted with Aurelia (35Neidig K.P. Geyer M. Gorler A. Antz C. Saffrich R. Beneicke W. Kalbitzer H.R. J. Biomol. NMR. 1995; 6: 255-270Crossref PubMed Scopus (109) Google Scholar). The assignment strategy was based on the identification of individual resonance spin systems from homonuclear TOCSY experiments following the sequential path with homonuclear two-dimensional and 15N-separated three-dimensional NOESY spectra similarly are described (36Geyer M. Munte C.E. Schorr J. Kellner R. Kalbitzer H.R. J. Mol. Biol. 1999; 289: 123-138Crossref PubMed Scopus (95) Google Scholar).RESULTSIdentification of the Diaphanous Autoregulatory Domain in FHOD1 and Its Interacting Domain—We started to analyze the domain boundaries of the interacting C-terminal DAD and its N-terminal recognition domain by GST pull-down binding assays. To this end we first expressed a construct in Escherichia coli bacterial cells that started C-terminal to the proposed FH2 domain at position 1032 and went up to the C terminus of the protein (Fig. 2A, left lane). Analytical mass spectrometry, however, revealed a proteolytic cleavage or premature truncation of the fragment that corresponded to an apparent mass of 10,046 Da (data not shown). This termination corresponded to the second appearance of a rare AGG codon at amino acid position 1126, which encoded the first arginine residue of the positively charged cluster of the DAD (
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