Identification of a Short Spir Interaction Sequence at the C-terminal End of Formin Subgroup Proteins
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.030320
ISSN1083-351X
AutoresMarkos Pechlivanis, Annette Samol, Eugen Kerkhoff,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoThe actin nucleation factors Spire and Cappuccino interact with each other and regulate essential cellular events during Drosophila oogenesis in a cooperative fashion. The interaction blocks formin actin nucleation activity and enhances the Spire activity. Analogous to Spire and Cappuccino, the mammalian homologs Spir-1 and formin-2 show a regulatory interaction. To get an understanding of the nature of the Spir-formin cooperation, we have analyzed the interaction biochemically and biophysically. Our data shows that the association of Spir-1 and formin-2 is not significantly mediated by binding of the Spir-1-KIND domain to the formin FH2 core domain. Instead, a short sequence motif C-terminal adjacent to the formin-2-FH2 domain could be characterized that mediates the interaction and is conserved among the members of the Fmn subgroup of formins. In line with this, we found that both mammalian Spir proteins, Spir-1 and Spir-2, interact with mammalian Fmn subgroup proteins formin-1 and formin-2. The actin nucleation factors Spire and Cappuccino interact with each other and regulate essential cellular events during Drosophila oogenesis in a cooperative fashion. The interaction blocks formin actin nucleation activity and enhances the Spire activity. Analogous to Spire and Cappuccino, the mammalian homologs Spir-1 and formin-2 show a regulatory interaction. To get an understanding of the nature of the Spir-formin cooperation, we have analyzed the interaction biochemically and biophysically. Our data shows that the association of Spir-1 and formin-2 is not significantly mediated by binding of the Spir-1-KIND domain to the formin FH2 core domain. Instead, a short sequence motif C-terminal adjacent to the formin-2-FH2 domain could be characterized that mediates the interaction and is conserved among the members of the Fmn subgroup of formins. In line with this, we found that both mammalian Spir proteins, Spir-1 and Spir-2, interact with mammalian Fmn subgroup proteins formin-1 and formin-2. Basic cell biological functions such as proliferation, migration, division, and vesicle transport rely on the organization of the actin cytoskeleton. The initiation of actin polymerization from free actin monomers is regulated by actin nucleation factors (NF), 2The abbreviations used are:NFnucleation factorFSIformin Spir interactioneFSIextended FSIFCSfetal calf serumDMEMDulbecco's modified Eagle's mediumTRITCtetramethylrhodamine isothiocyanateDADdiaphanous autoregulatory domainDIDdiaphanous inhibitory domainGSTglutathione S-transferaseGFPgreen fluorescent proteineGFPenhanced GFPTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. which help to overcome the kinetic barrier of spontaneous G-actin nucleation and, thus, catalyze the formation of filamentous actin structures and networks (1Pollard T.D. Annu. Rev. Biophys. Biomol. Struct. 2007; 36: 451-477Crossref PubMed Scopus (713) Google Scholar). To date, three different classes of NFs are described, the ARP2/3 complex, FH2 domain containing NFs of the formin superfamily, and NFs containing one or multiple WH2 domains (Spire/Cordon-bleu/Leiomodin) (2Chesarone M.A. Goode B.L. Curr. Opin. Cell Biol. 2009; 21: 28-37Crossref PubMed Scopus (225) Google Scholar). The formin superfamily is subdivided into seven subfamilies (Dia, FRL, DAAM, Delphilin, INF, FHOD, Fmn) (3Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2005; 16: 1-13Crossref PubMed Scopus (200) Google Scholar). The mechanisms of actin nucleation as well as the regulation of the NFs vary significantly between the three classes (and also show variances in between the distinct superfamilies). Spire and Cappuccino are NFs that belong to the Spire subfamily of WH2 containing nucleators and to the Fmn subfamily of the FH2 domain containing formins, respectively. In contrast to the Arp2/3 complex that nucleates branched filaments, Spire and the formin Cappuccino nucleate unbranched actin filaments (4Quinlan M.E. Heuser J.E. Kerkhoff E. Mullins R.D. Nature. 2005; 433: 382-388Crossref PubMed Scopus (257) Google Scholar).Almost two decades ago it was found that mutants of the two Drosophila NFs (Spire/Cappuccino) have an identical phenotype in early Drosophila oogenesis, i.e. both induce premature ooplasmic streaming (5Manseau L.J. Schüpbach T. Genes Dev. 1989; 3: 1437-1452Crossref PubMed Scopus (176) Google Scholar, 6Theurkauf W.E. Science. 1994; 265: 2093-2096Crossref PubMed Scopus (127) Google Scholar). Later it was shown that both proteins cooperate in the generation of a dynamic actin mesh in the oocyte that prevents premature ooplasmic streaming (7Dahlgaard K. Raposo A.A. Niccoli T. St Johnston D. Dev. Cell. 2007; 13: 539-553Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Spire and Cappuccino do not solely have the same mutant phenotype; the proteins also physically interact and cross-regulate each other. The Cappuccino C-terminal half, encoding the FH2 domain and flanking sequences, enhances the nucleation activity of Spire, whereas the nucleation activity of Cappuccino is decreased in the presence of the Spire-KIND domain (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar).Cappuccino belongs to the Fmn subgroup of formins (3Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2005; 16: 1-13Crossref PubMed Scopus (200) Google Scholar, 9Emmons S. Phan H. Calley J. Chen W. James B. Manseau L. Genes Dev. 1995; 9: 2482-2494Crossref PubMed Scopus (136) Google Scholar). In mammals, two Fmn subgroup members (formin-1, formin-2) and two Spir proteins (Spir-1, Spir-2) exist (3Higgs H.N. Peterson K.J. Mol. Biol. Cell. 2005; 16: 1-13Crossref PubMed Scopus (200) Google Scholar, 10Schumacher N. Borawski J.M. Leberfinger C.B. Gessler M. Kerkhoff E. Gene Expr. Patterns. 2004; 4: 249-255Crossref PubMed Scopus (40) Google Scholar). The formin-2 and spir-1 genes are coexpressed in the developing and adult nervous system, and the proteins interact analogous to their Drosophila counterparts Spire and Cappuccino (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar, 10Schumacher N. Borawski J.M. Leberfinger C.B. Gessler M. Kerkhoff E. Gene Expr. Patterns. 2004; 4: 249-255Crossref PubMed Scopus (40) Google Scholar). Several reports showed the importance of formin-2 in mouse oogenesis and here especially in the positioning of the meiotic spindle (11Azoury J. Lee K.W. Georget V. Rassinier P. Leader B. Verlhac M.H. Curr. Biol. 2008; 18: 1514-1519Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 12Dumont J. Million K. Sunderland K. Rassinier P. Lim H. Leader B. Verlhac M.H. Dev. Biol. 2007; 301: 254-265Crossref PubMed Scopus (145) Google Scholar, 13Leader B. Lim H. Carabatsos M.J. Harrington A. Ecsedy J. Pellman D. Maas R. Leder P. Nat. Cell Biol. 2002; 4: 921-928Crossref PubMed Scopus (265) Google Scholar, 14Schuh M. Ellenberg J. Curr. Biol. 2008; 18: 1986-1992Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Recently it was found that a dynamic actin mesh, as during Drosophila oogenesis, is also required for mouse oogenesis (11Azoury J. Lee K.W. Georget V. Rassinier P. Leader B. Verlhac M.H. Curr. Biol. 2008; 18: 1514-1519Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 14Schuh M. Ellenberg J. Curr. Biol. 2008; 18: 1986-1992Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). The correct localization of the meiotic spindle during mouse oogenesis and the resulting asymmetric division depends on an actin mesh that is built up by formin-2. Myosin-2 generates the pulling forces required for spindle movement (14Schuh M. Ellenberg J. Curr. Biol. 2008; 18: 1986-1992Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Beside the evolutionary conserved roles for the formins Cappuccino and formin-2, Spire family proteins also seem to be evolutionary conserved regulators of oocyte development. Spire genes of the African clawed frog Xenopus (pEg6) and the sea squirt Ciona savignyi (Pem-5) have been identified as maternal genes in the oocyte in analogy to its Drosophila homolog and are proposed to function in polarity during early embryogenesis (15Le Goff C. Laurent V. Le Bon K. Tanguy G. Couturier A. Le Goff X. Le Guellec R. Biol. Cell. 2006; 98: 697-708Crossref PubMed Scopus (14) Google Scholar, 16Satou Y. Satoh N. Dev. Biol. 1997; 192: 467-481Crossref PubMed Scopus (74) Google Scholar).In an initial characterization it was found that the KIND domains of Spir-1/dSpire interact with the C-terminal sequences of formin-2/Cappuccino, which encode the FH2 domains and flanking sequences (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar). To gain a further understanding of the interaction and cross-regulation of the two proteins, we investigated this interaction in detail. The objective of the study was the dissection of the formin-2/Spir-1 interaction and the determination of the structural elements that are responsible for the binding. The dissection revealed a high affinity Spir-1 interaction site of formin-2, which could be mapped to the very C terminus of formin-2 adjacent to its core FH2 domain (formin Spir interaction (FSI) sequence). The FSI sequence is conserved among the members of the Fmn subgroup. Consistently we found that all mammalian members of the two distinct nucleator families, Spir-1/2 and Fmn-1/2, interact with each other.EXPERIMENTAL PROCEDURESCloning, Expression, and PurificationAll Spir and formin constructs were generated by standard cloning techniques using Pfu DNA polymerase from Promega® and restriction enzymes and T4-DNA ligase from New England Biolabs® according to the manufacturers' recommendations. Expression vectors were from GE Healthcare® (pGEX), Invitrogen (pProExHTb, pcDNA3), QIAgen® (pQE80L), and Takara/Clontech® (pEGFP-C1, pAcGFP-C1). The Spir and formin proteins correspond to Swiss-Prot entry numbers Q08AE8–2 (hs Spir-1 (II)), Q8WWL2 (hs Spir-2), Q05860 (mm formin-1 (IV)), and Q9JL04 (mm formin-2). Supplemental Table S1 summarizes constructs, a brief description, fragment boundaries, purification scheme, and their purpose in this study (supplemental Table S1). For prokaryotic expression of proteins, Escherchia coli Rosetta pLysS bacteria were transformed with plasmids encoding for glutathione S-transferase (GST) and His6-tagged Spir and formin proteins, respectively. Bacteria were grown in LB (+100 mg/liter ampicillin and 30 mg/liter chloramphenicol) at 37 °C to an A600 of 0.4–0.6. Protein expression was initiated by induction with 100–300 μm isopropyl 1-thio-β-d-galactopyranoside and allowed to proceed at 20 °C for 12–18 h. Bacteria were lysed by ultrasonication, and soluble proteins were purified with an ÄKTA purifier system (GE Healthcare) using nickel-nitrilotriacetic acid affinity (Ni-NTA HP, GE Healthcare), GSH affinity (GSH-Sepharose FF, GE Healthcare), cation exchange (SP-Sepharose XL, GE Healthcare), and size exclusion (Sephadex G200, GE Healthcare) chromatography according to the manufacturers' recommendations. His and GST tags were cleaved where indicated by tobacco etch virus protease. Proteins were concentrated by ultrafiltration using Amicon Ultra-4 ultracentrifugation devices with molecular mass cut offs of 3,000, 10,000, and 30,000 Da (Millipore®). The final purity of the constructs was estimated by SDS-Tris-PAGE and SDS-Tricine-PAGE using Image J gel analysis software (rsb.info.nih.gov) (supplemental Fig. S4).Fluorophore LabelingFor anisotropy measurements, proteins and peptides were labeled on cysteines with the thiol-reactive maleimidocaproyl-linked BodipyFLTM-fluorophore (Probes®). Proteins were buffer-exchanged into labeling buffer (10 mm Hepes, pH 7.0, 100 mm NaCl, 0.25 mm Tris(2-carboxyethyl)phosphine) using Nap10 columns (GE Healthcare). A 2-fold molar excess of dye was coupled with the protein (1–5 g/liter) for 16–18 h at 4 °C. Excess non-reacted dye was quenched with 20 mm dithioerythritol and subsequently removed using Nap10 columns. The degree of labeling was ∼ 0.2–0.25 for the Fmn peptides and ∼0.75 for the KIND domain.Cell Culture and TransfectionHeLa and HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (FCS, HiClone®), 2 mm l-glutamate (Glu), and 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C and 10% CO2. Cells were transfected with LipofectamineTM/Lipofectamine 2000TM according to the manufacturer's recommendations. Briefly cells were seeded in DMEM supplemented with FCS but without Glu and penicillin/streptomycin 24 h before transfection. At the day of transfection, cells reached a confluency of 80–90%. For HeLa cells (∼8 × 105 cells) 6 μl of Lipofectamine were incubated with 1.4 μg of DNA in DMEM for 20 min; HEK293 and HeLa cells (∼8 × 105 cells) were transfected alternatively with 7.5 μl of Lipofectamine 2000/4 μg of DNA in DMEM). After 5 h the medium was changed to DMEM containing FCS, penicillin/streptomycin, and Glu. Cells were allowed to express proteins for 24–36 h.GST Pulldown AssayFor GST Pulldown experiments with the GST-KIND domains and enhanced green fluorescent protein (eGFP)-tagged formin proteins, ∼50 μg of purified GST/GST fusion proteins bound to 20 μl of GSH-Sepharose 4B resin (GE Healthcare) were used. Cell lysates were prepared by lysing ∼3 × 106 HEK293 cells in 900 μl of pulldown buffer 1 (25 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Nonidet P-40, 10% (v/v) glycerol, 1 mm EDTA, inhibitor mixture (Roche Applied Science)) for 20 min at 4 °C. The lysate was centrifuged at 20,000 × g for 20 min. 50 μg of GSH-Sepharose 4B-bound GST fusion proteins were incubated with the high speed supernatant of the cell lysate for 2 h at 4 °C. Beads were washed 5 times with pulldown buffer 1 and analyzed by PAGE and subsequent Western blot analysis. GFP-tagged proteins were detected with a rabbit A.V-living colors antibody (1 μg/ml; Takara/Clontech), a horseradish peroxidase-linked anti-rabbit secondary antibody (1:5000; GE Healthcare), and the enhanced chemiluminescence kit from GE Healthcare.Analytical Gel FiltrationAnalytical Gel filtration experiments were done in analytical gel filtration buffer (20 mm Tris, pH 8.0, 50 mm KCl, 2 mm dithioerythritol) using a computer-controlled ÄKTATM purifier high performance liquid chromatography equipped with a Sephadex G200 15/30 column (∼23-ml volume) at 6–8 °C. The injection volume of the proteins was 100 μl with concentrations ranging from ∼2 g/liter for the Spir-KIND domains, ∼3.5 g/liter for the formin FH2 domains, and ∼0.6 or 2 g/liter for the formin peptides. The flow rate was kept constant at 0.4 ml/min. Eluted proteins were collected in 1-ml fractions and analyze by PAGE to verify the identity of the proteins in the distinct peak fractions. Data analysis and processing were done with the Unicorn evaluation software (GE Healthcare) and Sigma Plot 9.0 (Systat Software®). Molecular weight calibration of the analytical gel filtration column was done using the Sigma gel filtration molecular mass standard kit 12,000–200,000 Da (cytochrome c, 12.4 kDa; carbonic anhydrase, 29 kDa; bovine serum albumin, 66 kDa; alcohol dehydrogenase, 150 kDa; b-amylase, 200 kDa). The observed molecular masses for the individual proteins and protein complexes can be found in Table 1.TABLE 1Observed masses of Spir/formin constructs and complexes from the analytical gel filtrationProteinTheoretical massObserved massOligomer state/stoichiometryDaDaSpir-1-KIND25.840.4MonomerSpir-2-KIND21.243.3MonomerFmn-2-FH2-FSI52.7123.8/239.5Dimer/tetramerFmn-2-FH251.2113.4/262.3Dimer/tetramerFmn-2-eFSI7.116.6DimerFmn-2-FSI3.88.0DimerFmn-1-FH250.8100.4DimerSpir-1-KIND and Fmn-2-FH2-FSI157.0 (164.2)164.51:2Spir-1-KIND and Fmn-2-eFSI32.9 (57.0)47.41:2Spir-2-KIND and Fmn-2-eFSI28.3 (59.9)51.91:2Spir-1-KIND and Fmn-2-FSI29.6 (48.4)39.01:2 Open table in a new tab Fluorescence AnisotropyFluorescence anisotropy measurements were performed in a Horriba Jobin Yvon® Fluoromax-4 spectrophotometer in anisotropy buffer (10 mm Hepes, pH 7.0, 100 mm NaCl) at 20 °C. The BodipyFLTM fluorophore was excited at 495 nm, and emission was collected at 510 nm, with an integration time of 2 s for the BodipyFLTM-labeled formin peptides (∼100 nm) and 4 s for the BodipyFLTM-labeled Spir-1-KIND domain (∼100 nm). The slit width of the emission and excitation monochromators was set to 2 nm for the BodipyFLTM-labeled formin peptides and 3 nm for the labeled Spir-1-KIND domain, respectively. Each data point of the binding curve is the mean of at least eight collected polarization signals. Data analysis and processing was done with Sigma Plot 9.0 (Systat Software).Equilibrium binding data were fitted according to the equationy=a⋅xb+x(Eq. 1) with a representing the maximum amplitude (Bmax), and b representing the equilibrium constant (Kd). Competition binding experiments were performed as described by Vinson et al. (17Vinson V.K. De La Cruz E.M. Higgs H.N. Pollard T.D. Biochemistry. 1998; 37: 10871-10880Crossref PubMed Scopus (130) Google Scholar).Competition binding curve was fitted according to the equationr=rf+rb−rfKd⋅C+Kd2Kd2⋅R0(Eq. 2) where is the anisotropy, rb if the anisotropy of the Spir-1-KIND·Bodipy-Fmn-2-eFSI complex, rf is the anisotropy of the free Bodipy-labeled eFSI, [C] is the concentration of the unlabeled eFSI, [R0] is the concentration of the free Spir-1-KIND when [C] = 0, Kd is the dissociation constant for the binding of the Bodipy-labeled eFSI to Spir-1-KIND, and Kd2 is the dissociation constant for the binding of the unlabeled eFSI to Spir-1-KIND.Immunostaining and Fluorescence MicroscopyLipofectamineTM- transfected HeLa cells seeded on 13-mm round glass slides were fixed with 3.7% (w/v) paraformaldehyde for 20 min at 4 °C and permeabilized with 0.2% (v/v) Triton X-100 in phosphate-buffered saline for 3.5 min at 20–22 °C. Immunostaining was done by incubating the cells with the mouse anti-Myc 9E10 antibody (Santa Cruz®) at a concentration of 4 μg/ml in phosphate-buffered saline supplemented with 1% (v/v) FCS at room temperature for 60 min. TRITC-conjugated donkey anti-mouse secondary antibody (Dianova®, 1:200 in phosphate-buffered saline + 1% (v/v) FCS) was allowed to react with the primary antibody for 60 min at room temperature. Stained cells were mounted in moviol solution (15% (w/v) moviol, 30% (v/v) glycerol, and 2.25% (w/v) N-propyl gallate in phosphate-buffered saline) and analyzed with a Leica® AF6000LX imaging system equipped with a Leica® HCX PL APO 63×/1.30 glycerol objective and a Leica® DFC350FX camera. Images were deconvoluted (blind) with the deconvolution software from Leica® (Leica Application Suite Advanced Fluorescence) and further processed with Adobe® Photoshop CS.Native Polyacrylamide Gel ElectrophoresisNative polyacrylamide gel electrophoresis analysis was performed with a Laemmli native PAGE system. 20 μm Spir-KIND was incubated for 20 min in the presence or absence of increasing amounts of formin-2 peptides (0–45 μm Fmn-2-eFSI; 0–80 μm Fmn-2-FSI) in anisotropy buffer at 20–22 °C. Subsequent gel electrophoresis was conducted at 150 V for 20 min followed by a 300-V step for 50 min for Spir-1-KIND. Gel electrophoresis for Spir-2-KIND was performed as for Spir-1 except that the second voltage step lasts only 30 min due to the higher negative charge of the Spir-2-KIND domain.RESULTSThe Spir-1-KIND Domain Binds to the Extreme C Terminus of Formin-2The Spir-1 and formin-2 proteins interact directly with each other (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar). The Spir-1-KIND domain and the C-terminal part of the formin-2 protein encoding the FH2 domain and flanking the C-terminal sequences mediate the interaction. Formin-2 actin nucleation activity is blocked by the interaction of the formin with the Spir-KIND domain (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar). To gain more insights into the interaction between the Spir-1-KIND domain and the formin-2 C-terminal half encoding the actin nucleating FH2 domain and flanking sequences, we have investigated this interaction structurally and biochemically.Purified bacterially expressed recombinant proteins have been employed in analytical gel filtration experiments to analyze the complex formation and dissect the sequences necessary for the interaction. In agreement with previous findings (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar), we found that the KIND domain of Spir-1 (Fig. 1B, KIND short) does not adopt a globular shape, as it migrates on the analytical gel filtration faster than expected from its molecular mass (Fig. 2A, black curve, peak 1, Table 1). The purified formin-2 C-terminal part encoding the FH2 domain and flanking C-terminal sequences (Fig. 1A, Fmn-2-FH2-FSI) eluted in fractions corresponding to dimeric and tetrameric proteins as judged by the elution volumes of marker proteins (Fig. 2A, red curve, 1 and 2, Table 1). Mixing the two proteins resulted in a complex formation, which is nicely documented by a shift of the lower molecular mass form in peak 2 in the elution profile toward higher molecular masses (Fig. 2A, green curve, peak 1) and a co-elution of the Spir-1-KIND domain and the Fmn-2-FH2-FSI protein (Fig. 2A, inset, corresponding to green peak 1). Mixing of the two proteins was also accompanied by partial precipitation of the complex, explaining the decreased signal intensity of the peak fraction containing the complex.FIGURE 2.Analytical gel filtration analysis of Spir-1-KIND and formin-2 C-terminal constructs. The Spir-1-KIND domain interacts with formin-2-FH2-FSI (A) but not with a formin-2-FH2 construct lacking the FSI (B). The last 55 amino acids of the C terminus of formin-2 (eFSI) comprising the last predicted α-helix of the FH2 domain and the FSI bind to the KIND domain of Spir-1 (C). Even the last 29 amino acids of the C terminus outside the core FH2 domain of formin-2 (FSI) still bind to the KIND domain of Spir-1 (D). Insets show the presence of Spir- and formin proteins in the peak fractions (depending on the width of the peak 2 or 3 peak fractions, respectively) of the complexes (insets A, C, and D) and no Spir-1-KIND when mixing Spir-1-KIND and Fmn-2-FH2 lacking the conserved FSI (inset B, green curve, peak 2). Black elution curve, Spir; red elution curve, formin; green elution curve, Spir and formin. Colors of the peak numbering and elution profiles correspond. Masses of molecular weight standards: 12.4 kDa. cytochrome c; 29 kDa, carbonic anhydrase; 66 kDa, bovine serum albumin; 150 kDa, alcohol dehydrogenase; 200 kDa, b-amylase. Observed molecular masses for the Spir/formin proteins and complexes can be found in the Table 1; mAU, milliabsorption units; Ve/Vo, elution volume/void volume.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Formins nucleate actin polymerization by the stabilization of an actin oligomer with a dimeric FH2 domain ring structure. Because the interaction of Spir-1-KIND and the formin-2 protein blocks the actin nucleation activity of the formin, we next tested if the Spir-1-KIND domain binds the formin-2-FH2 core domain (Fig. 1A, Fmn-2-FH2). In contrast to the Fmn-2-FH2-FSI protein that formed a complex with the Spir-1-KIND domain, we could not detect a stable complex formation of the formin-2-FH2 core domain with the Spir-1-KIND domain in our gel filtration experiments (Fig. 2B), which is most likely because of a significant decrease in affinity. The elution profile of the mixed proteins was identical with that of the two individual proteins (Fig. 2B, red, black, and green curves). No Spir-1-KIND protein was shifted into the fraction of Fmn-2-FH2 (Fig. 2B, inset, corresponding to green peak 2).This suggests that flanking sequences of the FH2 core domain mediate the interaction. As no stable complex formation of the Spir-1-KIND domain and the core FH2 domain of formin-2 could be detected on the analytical gel filtration, an interaction sequence of the C terminus of formin-2 outside the core FH2 domain was assumed. To test this, we expressed and purified a peptide comprising the last 56 amino acids of formin-2 (Fmn-2-eFSI). This peptide coeluted with the Spir-1-KIND domain on the analytical gel filtration column, indicating the formation of a complex of the Spir-1-KIND domain and the formin-2 C terminus (Fig. 2C, inset corresponding to green peak 1). An even shorter peptide comprising the last 29 amino acids of the C terminus of formin-2 (Fmn-2-FSI) also coeluted with the core KIND domain of Spir-1 in the analytical gel filtration, indicating that the last 29 amino acids provide the main binding site for the Spir-1-KIND domain (Fig. 2D, inset corresponding to green peak 1). One should note that in the case of the small FSI peptide, no shift of the KIND domain to a higher molecular mass could be observed. This indicates that the core binding motif of Fmn-2 either fills a cavity in the KIND domain or tightly wraps around the KIND domain. This assumption is supported by the fact that the complex of the KIND domain and the Fmn-2-eFSI also elutes at lower molecular weights than expected from the cumulative observed masses for the two proteins (see Table 1). In contrast to the results of Quinlan et al. (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar), who deduced from their analytical ultracentrifugation studies a 2:2 stoichiometry of the Spir-formin complex, our analytical gel filtration analysis along with the densitometric analysis of the peak complex fractions on the SDS-PAGE (Fig. 2; insets) suggest a 1:2 stoichiometry of the KIND-formin complex (see Table 1). The discrepancy between the work done by Quinlan et al. (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar) and our work concerning the stoichiometry of the complex may be because of the different methods used and subsequent data interpretation.It is interesting to note that alignments of the very C-terminal sequences of Fmn subgroup proteins from flies, fish, birds, and mammals show a conserved sequence motif within the C-terminal region which we have mapped as an FSI sequence (Fig. 1C). The conserved sequence is unique for Fmn subgroup proteins and could not be detected in other formins. This suggests that next to Drosophila Cappuccino and vertebrate formin-2, the third member of this protein subgroup, the formin-1 protein, also may interact with Spir proteins (see below).To further analyze the interaction of the C terminus of formin-2 with the KIND domain of Spir-1, colocalization studies in HeLa cells were performed in which Spir-1-KIND and formin-2-eFSI were transiently overexpressed (Fig. 3). Analogous to the assays by Quinlan et al. (8Quinlan M.E. Hilgert S. Bedrossian A. Mullins R.D. Kerkhoff E. J. Cell Biol. 2007; 179: 117-128Crossref PubMed Scopus (132) Google Scholar), a membrane-targeted Myc-tagged Spir-1-KIND domain (Myc-Spir-1-KIND-CAAX) was used to relocate a green fluorescence protein-tagged formin-2 C-terminal peptide (pAcGFP-Fmn2-eFSI) to the membrane in HeLa cells. When expressed alone, the GFP-fused Fmn-2-eFSI construct was evenly distributed in the cytoplasm (Fig. 3). Coexpression of AcGFP-Fmn-2-eFSI and the membrane-targeted KIND domain results in a relocalization of the GFP-tagged formin-2 peptide to the plasma membrane, indicating an interaction of the proteins.FIGURE 3.Colocalization of membrane-targeted Myc-Spir-1-KIND and AcGFP-Fmn-2-eFSI in HeLa cells. Proteins were visualized by monitoring the intrinsic fluorescence of the AcGFP-tag (green) or indirect immunostaining of the Myc-tagged KIND domain with anti-Myc antibody and a TRITC-conjugated secondary antibody (red). When expressed alone, AcGFP-Fmn-2-eFSI (green) is distributed evenly in the cytoplasm. Coexpression of AcGFP-Fmn-2-eFSI (green) and a membrane-targeted version of the KIND domain of Spir-1 (Myc-Spir-1-KIND-CAAX (red)) translocates AcGFP-Fmn-2-eFSI to the membrane. Images are deconvoluted (blind) and were further processed by Adobe Photoshop CS.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fluorescence anisotropy measurements provide a very sensitive tool to detect and quantify protein interactions. As we cannot exclude from our gel filtration experiments that the FH2 core domain does not contribute to the overall binding of the KIND domain, we have analyzed the interaction of the Spir-1-KIND domain with the various C-terminal formin constructs. Using a fluorescent BodipyFl-labeled version of the core Spir-1-KIND domain, the interaction of the KIND domain with the core FH2 domain of formin-2 and the FH2 domain plus its C-terminal extension (Fmn-2-FH2, Fmn-2-FH2-FSI) was probed (Fig. 4, A and B). To monitor the interaction of the C terminus of formin-2 (Fmn-2-eFSI, Fmn-2-FSI) and the Spir-1 KIND domain, BodipyFl-labeled C-terminal peptides of formin-2 were used (Fig. 4, C and D). In these fluorescence anisotropy measurement
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