Tuba, a Novel Protein Containing Bin/Amphiphysin/Rvs and Dbl Homology Domains, Links Dynamin to Regulation of the Actin Cytoskeleton
2003; Elsevier BV; Volume: 278; Issue: 49 Linguagem: Inglês
10.1074/jbc.m308104200
ISSN1083-351X
AutoresMarco Antonio Garrido Salazar, Adam V. Kwiatkowski, Lorenzo Pellegrini, Gianluca Cestra, Margaret H. Butler, Kent L. Rossman, Daniel M. Serna, John Sondek, Frank B. Gertler, Pietro De Camilli,
Tópico(s)Retinal Development and Disorders
ResumoTuba is a novel scaffold protein that functions to bring together dynamin with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Tuba binds a variety of actin regulatory proteins, including N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1, and Ena/VASP proteins, via a C-terminal SH3 domain. Direct binding partners include N-WASP and Ena/VASP proteins. Forced targeting of the C-terminal SH3 domain to the mitochondrial surface can promote accumulation of F-actin around mitochondria. A Dbl homology domain present in the middle of Tuba upstream of a Bin/amphiphysin/Rvs (BAR) domain activates Cdc42, but not Rac and Rho, and may thus cooperate with the C terminus of the protein in regulating actin assembly. The BAR domain, a lipid-binding module, may functionally replace the pleckstrin homology domain that typically follows a Dbl homology domain. The properties of Tuba provide new evidence for a close functional link between dynamin, Rho GTPase signaling, and the actin cytoskeleton. Tuba is a novel scaffold protein that functions to bring together dynamin with actin regulatory proteins. It is concentrated at synapses in brain and binds dynamin selectively through four N-terminal Src homology-3 (SH3) domains. Tuba binds a variety of actin regulatory proteins, including N-WASP, CR16, WAVE1, WIRE, PIR121, NAP1, and Ena/VASP proteins, via a C-terminal SH3 domain. Direct binding partners include N-WASP and Ena/VASP proteins. Forced targeting of the C-terminal SH3 domain to the mitochondrial surface can promote accumulation of F-actin around mitochondria. A Dbl homology domain present in the middle of Tuba upstream of a Bin/amphiphysin/Rvs (BAR) domain activates Cdc42, but not Rac and Rho, and may thus cooperate with the C terminus of the protein in regulating actin assembly. The BAR domain, a lipid-binding module, may functionally replace the pleckstrin homology domain that typically follows a Dbl homology domain. The properties of Tuba provide new evidence for a close functional link between dynamin, Rho GTPase signaling, and the actin cytoskeleton. Fission of clathrin-coated and other endocytic vesicles from the plasma membrane involves the cooperation of several membrane-associated proteins, among which the GTPase dynamin plays a key role (1.Conner S.D. Schmid S.L. Nature. 2003; 422: 37-44Crossref PubMed Scopus (3018) Google Scholar, 2.Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (421) Google Scholar). The participation of dynamin in the fission of endocytic vesicles has been established by a variety of experimental approaches in Drosophila, cultured cells, and cell-free systems, although the precise mechanism of fission and the role of dynamin in this reaction remain unclear (3.Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (580) Google Scholar).Several dynamin partners thought to participate in dynamin recruitment or function have been identified (3.Hinshaw J.E. Annu. Rev. Cell Dev. Biol. 2000; 16: 483-519Crossref PubMed Scopus (580) Google Scholar, 4.Gout I. Dhand R. Hiles I.D. Fry M.J. Panayotou G. Das P. Truong O. Totty N.F. Hsuan J. Booker G.W. Campbell I.D. Waterfield M.D. Cell. 1993; 75: 25-36Abstract Full Text PDF PubMed Scopus (482) Google Scholar). At the synapse, where endocytosis plays a key role in the recycling of synaptic vesicle membranes, two prominent dynamin partners are amphiphysin and endophilin (5.David C. McPherson P.S. Mundigl O. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 331-335Crossref PubMed Scopus (349) Google Scholar, 6.Ringstad N. Nemoto Y. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8569-8574Crossref PubMed Scopus (325) Google Scholar, 7.Ramjaun A.R. Micheva K.D. Bouchelet I. McPherson P.S. J. Biol. Chem. 1997; 272: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Amphiphysin has a three-domain structure with an evolutionarily conserved N-terminal module of ∼250 amino acids called the Bin/amphiphysin/Rvs (BAR) 1The abbreviations used are: BARBin/amphiphysin/RvsSH3Src homology-3DHDbl homologyPHpleckstrin homologyHAhemagglutininGSTglutathione S-transferaseRACErapid amplification of cDNA endsmant-N-methylanthraniloyl-MALDI-TOFmatrix-assisted laser desorption ionization time-of-flightEGFPenhanced green fluorescent protein.1The abbreviations used are: BARBin/amphiphysin/RvsSH3Src homology-3DHDbl homologyPHpleckstrin homologyHAhemagglutininGSTglutathione S-transferaseRACErapid amplification of cDNA endsmant-N-methylanthraniloyl-MALDI-TOFmatrix-assisted laser desorption ionization time-of-flightEGFPenhanced green fluorescent protein. domain, a variable central region, and a C-terminal Src homology-3 (SH3) domain that binds dynamin. Endophilin has a similar domain structure (2.Slepnev V.I. De Camilli P. Nat. Rev. Neurosci. 2000; 1: 161-172Crossref PubMed Scopus (421) Google Scholar). Although the N-terminal domain of endophilin is substantially divergent in amino acid composition from the BAR domain of amphiphysin, it shares some similarity at critical sites, leading to its classification as a BAR domain (8.Farsad K. Ringstad N. Takei K. Floyd S.R. Rose K. De Camilli P. J. Cell Biol. 2001; 155: 193-200Crossref PubMed Scopus (482) Google Scholar). 2See smart.embl-heidelberg.de/smart/get_members.pl?WHAT=NRDB_COUNT&NAME=BAR.2See smart.embl-heidelberg.de/smart/get_members.pl?WHAT=NRDB_COUNT&NAME=BAR. Accordingly, the BAR domains of amphiphysin and endophilin share functional similarities because they both can bind and deform lipid bilayers and mediate homo- and heterodimerization (6.Ringstad N. Nemoto Y. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8569-8574Crossref PubMed Scopus (325) Google Scholar, 8.Farsad K. Ringstad N. Takei K. Floyd S.R. Rose K. De Camilli P. J. Cell Biol. 2001; 155: 193-200Crossref PubMed Scopus (482) Google Scholar, 9.Wigge P. Kohler K. Vallis Y. Doyle C.A. Owen D. Hunt S.P. McMahon H.T. Mol. Biol. Cell. 1997; 8: 2003-2015Crossref PubMed Scopus (208) Google Scholar, 10.Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (510) Google Scholar). Dynamin also can bind and deform lipid bilayers, and it has been proposed that endophilin and amphiphysin might help to recruit and possibly assist dynamin in the generation of membrane curvature at endocytic pits (8.Farsad K. Ringstad N. Takei K. Floyd S.R. Rose K. De Camilli P. J. Cell Biol. 2001; 155: 193-200Crossref PubMed Scopus (482) Google Scholar, 10.Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (510) Google Scholar).The closest homologue of amphiphysin and endophilin in Saccharomyces cerevisiae is Rvs167, that forms a stable heterodimer with Rsv161; Rvs167 has a domain structure like amphiphysin, whereas Rvs161 (homologous to the mammalian protein Bin3) possesses only a BAR domain. Mutation of either one or both components of this heterodimer in yeast produces defects in endocytosis and actin function (11.Lombardi R. Riezman H. J. Biol. Chem. 2001; 276: 6016-6022Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Such a dual phenotype is typical of most mutations in actin regulatory and endocytosis genes in yeast (12.Munn A.L. Biochim. Biophys. Acta. 2001; 1535: 236-257Crossref PubMed Scopus (105) Google Scholar). These observations, together with results from a variety of studies in mammalian cells, have suggested a link between endocytosis and actin, although such a link has remained mechanistically elusive (13.Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: F111-F116Crossref PubMed Scopus (351) Google Scholar, 14.Schafer D.A. Curr. Opin. Cell Biol. 2002; 14: 76-81Crossref PubMed Scopus (236) Google Scholar). Foci of actin can often be seen at endocytic sites (15.Merrifield C.J. Feldman M.E. Wan L. Almers W. Nat. Cell Biol. 2002; 4: 691-698Crossref PubMed Scopus (553) Google Scholar), and endocytic vesicles with actin tails have also been observed (16.Merrifield C.J. Moss S.E. Ballestrem C. Imhof B.A. Giese G. Wunderlich I. Almers W. Nat. Cell Biol. 1999; 1: 72-74Crossref PubMed Scopus (264) Google Scholar). Interestingly, these actin tails contain both dynamin and dynamin-interacting proteins (17.Lee E. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 161-166Crossref PubMed Scopus (202) Google Scholar, 18.Orth J.D. Krueger E.W. Cao H. McNiven M.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 167-172Crossref PubMed Scopus (193) Google Scholar).Several binding partners of dynamin are either physically or functionally linked to actin or actin regulatory proteins. These include, among others, syndapin/pacsin and intersectin/DAP160 (13.Qualmann B. Kessels M.M. Kelly R.B. J. Cell Biol. 2000; 150: F111-F116Crossref PubMed Scopus (351) Google Scholar, 14.Schafer D.A. Curr. Opin. Cell Biol. 2002; 14: 76-81Crossref PubMed Scopus (236) Google Scholar). Intersectin, in particular, represents a striking example of a multidomain protein that links dynamin to proteins that directly or indirectly control actin polymerization, such as the phosphatidylinositol-4,5-bisphosphate phosphatase, synaptojanin, N-WASP (neuronal Wiskott-Aldrich syndrome protein), and the Rho family GTPase Cdc42 (19.Hussain N.K. Jenna S. Glogauer M. Quinn C.C. Wasiak S. Guipponi M. Antonarakis S.E. Kay B.K. Stossel T.P. Lamarche-Vane N. McPherson P.S. Nat. Cell Biol. 2001; 3: 927-932Crossref PubMed Scopus (298) Google Scholar, 20.Roos J. Kelly R.B. J. Biol. Chem. 1998; 273: 19108-19119Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). N-WASP is a member of the WASP superfamily of proteins that includes SCAR/WAVE. All members of this family possess a conserved C terminus that can bind to and activate the Arp2/3 complex, a collection of proteins that catalyzes the nucleation of actin filaments (21.Mullins R.D. Curr. Opin. Cell Biol. 2000; 12: 91-96Crossref PubMed Scopus (140) Google Scholar). N-WASP exists predominantly in an autoinhibited state. When activated Cdc42 or the adaptor protein Nck binds to N-WASP, this autoinhibition is relieved. Phosphatidylinositol 4,5-bisphosphate can further activate N-WASP in combination with Cdc42 and Nck (22.Rohatgi R. Ho H.Y. Kirschner M.W. J. Cell Biol. 2000; 150: 1299-1310Crossref PubMed Scopus (489) Google Scholar, 23.Rohatgi R. Nollau P. Ho H.Y. Kirschner M.W. Mayer B.J. J. Biol. Chem. 2001; 276: 26448-26452Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 24.Higgs H.N. Pollard T.D. J. Cell Biol. 2000; 150: 1311-1320Crossref PubMed Scopus (410) Google Scholar). Intersectin functions to bring together N-WASP and, through the guanyl nucleotide exchange activity of its Dbl homology (DH) domain, activated GTP-bound Cdc42, thus activating Arp2/3 and promoting actin nucleation (19.Hussain N.K. Jenna S. Glogauer M. Quinn C.C. Wasiak S. Guipponi M. Antonarakis S.E. Kay B.K. Stossel T.P. Lamarche-Vane N. McPherson P.S. Nat. Cell Biol. 2001; 3: 927-932Crossref PubMed Scopus (298) Google Scholar). The endocytic protein syndapin/pacsin, which, like intersectin, binds both dynamin and N-WASP, can promote N-WASP-dependent actin nucleation (25.Kessels M.M. Qualmann B. EMBO J. 2002; 21: 6083-6094Crossref PubMed Scopus (164) Google Scholar). Thus, intersectin and syndapin function as molecular links between endocytosis and actin assembly.The Arp2/3 complex plays a key role in generating actin-based protrusive forces that can drive the movement of vesicles and intracellular pathogens such as Listeria monocytogenes (26.Welch M.D. Rosenblatt J. Skoble J. Portnoy D.A. Mitchison T.J. Science. 1998; 281: 105-108Crossref PubMed Scopus (409) Google Scholar). Members of the Ena (Enabled)/VASP (vaodilator-stimulated phosphoprotein) protein family (Mena (mammalian Enabled), VASP, and EVL (Ena-VASP-like)) greatly accelerate the actin-based movement of Listeria (27.Laurent V. Loisel T.P. Harbeck B. Wehman A. Grobe L. Jockusch B.M. Wehland J. Gertler F.B. Carlier M.F. J. Cell Biol. 1999; 144: 1245-1258Crossref PubMed Scopus (290) Google Scholar, 28.Geese M. Loureiro J.J. Bear J.E. Wehland J. Gertler F.B. Sechi A.S. Mol. Biol. Cell. 2002; 13: 2383-2396Crossref PubMed Scopus (87) Google Scholar). Ena/VASP proteins are concentrated in the tips of filopodia and lamellipodia and are known to regulate actin filament geometry in protruding lamellipodia (29.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 30.Gertler F.B. Niebuhr K. Reinhard M. Wehland J. Soriano P. Cell. 1996; 87: 227-239Abstract Full Text Full Text PDF PubMed Scopus (559) Google Scholar). At a molecular level, Ena/VASP proteins are thought to antagonize the activity of capping proteins, thereby permitting increased actin filament elongation at barbed ends and decreasing the density of branched structures by an unknown mechanism (29.Bear J.E. Svitkina T.M. Krause M. Schafer D.A. Loureiro J.J. Strasser G.A. Maly I.V. Chaga O.Y. Cooper J.A. Borisy G.G. Gertler F.B. Cell. 2002; 109: 509-521Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 31.Kwiatkowski A.V. Gertler F.B. Loureiro J.J. Trends Cell Biol. 2003; 13: 386-392Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar).A search of protein data bases for BAR domains reveals a large number of BAR domain-containing proteins in a variety of animal and plant species. In most of these proteins, which generally contain a C-terminal SH3 domain, the BAR domain is located in the N terminus. However, in three homologous vertebrate sequences, the BAR domain is not located at the N terminus, but is instead found downstream of a DH domain. Most DH modules characterized thus far are located upstream of a pleckstrin homology (PH) domain, and the lipid-binding properties of the PH domain are thought to play a critical role in the localization and regulation of DH domain activity (32.Rossman K.L. Cheng L. Mahon G.M. Rojas R.J. Snyder J.T. Whitehead I.P. Sondek J. J. Biol. Chem. 2003; 278: 18393-18400Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We have characterized one of these three proteins, which we have named Tuba. In an independent line of study, we identified the mouse homolog of Tuba in a yeast two-hybrid screen using the Ena/VASP protein EVL as bait. Collectively, our study demonstrates that Tuba is a large scaffold protein that binds dynamin and a variety of actin regulatory proteins and that activates Cdc42. Our results suggest that Tuba functions as in important link between dynamin function, Rho GTPase signaling, and actin dynamics regulated by WASP/WAVE superfamily and Ena/VASP proteins.EXPERIMENTAL PROCEDURESAntibodies and Reagents—The following antibodies were used in this study: rat anti-hemagglutinin (HA) monoclonal epitope (clone 3F10, Roche Applied Science), anti-dynamin polyclonal antibody DG1 (our laboratory) and monoclonal antibody Hudy-1 (Upstate Biotechnology, Inc.), anti-amphiphysin-1 monoclonal antibody-3 (33.Floyd S.R. Porro E.B. Slepnev V.I. Ochoa G.C. Tsai L.H. De Camilli P. J. Biol. Chem. 2001; 276: 8104-8110Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and anti-N-WASP polyclonal antibody (gifts of P. Aspenstrom (Ludvig Institute for Cancer Research, Uppsala, Sweden) and M. W. Kirschner (Harvard Medical School)), anti-CR16 polyclonal antibody (gift of H. Y. Ho and M. W. Kirschner), anti-WAVE1 polyclonal antibody (gift of John Scott, Vollum Institute), anti-Mena monoclonal antibody (our laboratory), anti-green fluorescent protein polyclonal antibody (Clontech), anti-actin monoclonal antibody (Sigma), anti-synaptotagmin monoclonal antibody (gift of Reinhard Jahn, Max-Planck Institute for Biological Chemistry, Göttingen, Germany), and anti-GM-130 antibody (Graham Warren, Yale University). Anti-Tuba polyclonal antibodies were generated by injecting rabbits with a glutathione S-transferase (GST) fusion protein encompassing the final 292 amino acids of human Tuba. The antibodies were affinity-purified on the antigen coupled to SulfoLink beads (Pierce) according to the manufacturer's instructions. A GST fusion protein of the PH domain of phospholipase Cδ was kind gift of Antonella De Matteis (Consorzio Mario Negri Sud, Italy). A GST fusion protein of the BAR domain of amphiphysin-1 was described previously (10.Takei K. Slepnev V.I. Haucke V. De Camilli P. Nat. Cell Biol. 1999; 1: 33-39Crossref PubMed Scopus (510) Google Scholar). The KIAA1010 clone was obtained from the Kazusa Institute. 5′-Rapid Amplification of cDNA Ends (RACE)—To obtain the full-length sequence of Tuba from the KIAA1010 clone, human skeletal muscle Marathon-Ready cDNAs (Clontech) were utilized for 5′-RACE using KIAA1010-specific primers and the Advantage 2 PCR enzyme system (Clontech). Based on this sequence, a full-length clone was generated by PCR using probes corresponding to the 5′- and 3′-ends of the Tuba sequence and human brain Marathon-Ready cDNAs (Clontech) as a template. Nucleotide sequencing confirmed the sequences of KIAA1010 and of the N-terminal region of the protein obtained by 5′-RACE with the exception of the absence in KIAA1010 of 40 amino acids in the second half of the BAR domain (see Fig. 1A). Multiple clones generated by PCR in different amplification cycles yielded only sequences including the 40 amino acids. The nucleotide sequence of human Tuba has been deposited in the GenBank™/EBI Data Bank under accession number AY196211.Yeast Two-hybrid Screen—Full-length EVL was used as bait in the LexA two-hybrid system (Clontech) to probe an embryonic day 19 mouse library. Two independent clones of Tuba comprising amino acids 1502–1577 and 1092–1577 (see Fig. 1A) were identified as strong interactors. Full-length murine Tuba was constructed by ligating the larger of the two clones with fragments generated by reverse transcription-PCR using Tuba-specific primers and a mouse cDNA library. The nucleotide sequence of mouse Tuba has been deposited in the GenBank™/EBI Data Bank under accession number AY383729.Affinity Chromatography—GST or GST fusion proteins of SH3 domain-containing regions of Tuba were bound to glutathione beads (Amersham Biosciences) and incubated with a Triton X-100-solubilized rat brain extract. Bound material was recovered by centrifugation, followed by elution with SDS and separation by SDS-PAGE.For biochemical analysis of the interaction of the SH3-6 domain of Tuba with actin regulatory proteins in non-neuronal cell extracts, D7 fibroblastic cells, which lack endogenous expression of all Ena/VASP proteins, were used (34.Bear J.E. Loureiro J.J. Libova I. Fassler R. Wehland J. Gertler F.B. Cell. 2000; 101: 717-728Abstract Full Text Full Text PDF PubMed Scopus (373) Google Scholar). Uninfected or infected D7 cells were grown to confluency and extracted in Nonidet P-40 lysis buffer. Cell extracts were clarified by centrifugation and used for affinity chromatography experiments as described above. 10–15 μg of GST or GST fusion protein was incubated with 1 mg of lysate.Immunocytochemistry of Brain Tissue—Immunofluorescence of frozen rat brain sections was performed by standard procedures on formaldehyde-perfused brains. Anti-Tuba immunogold labeling was performed on lysed synaptosomes embedded in agarose, followed by Epon embedding and thin sectioning as described (35.De Camilli P. Harris Jr., S.M. Huttner W.B. Greengard P. J. Cell Biol. 1983; 96: 1355-1373Crossref PubMed Scopus (373) Google Scholar).Guanine Nucleotide Exchange Assays—Exchange assays using bacterially expressed and purified Rho GTPases were performed essentially as described (36.Snyder J.T. Worthylake D.K. Rossman K.L. Betts L. Pruitt W.M. Siderovski D.P. Der C.J. Sondek J. Nat. Struct. Biol. 2002; 9: 468-475Crossref PubMed Scopus (189) Google Scholar). In particular, 2 μm RhoA(C190S), Rac1(C188S), or Cdc42(C188S) was added to buffer containing 20 mm Tris (pH 7.5), 100 mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 10% glycerol, and 400 nm mant-GTP (Molecular Probes, Inc.) and allowed to equilibrate for 5 min before adding the indicated concentrations of His-tagged Tuba DH domain or 200 nm His-tagged Vav2 DH-PH fragment. Increased fluorescence indicative of mant-GTP binding to GTPases was monitored using a PerkinElmer LS-55 spectrophotometer (λex = 360 nm and λem = 440 nm, slits = 5/5 nm) thermostatted to 25 °C. Fluorescence was normalized to the initial value at the start of the experiment. A fragment containing the DH and BAR domains of Tuba is insoluble upon expression in Escherichia coli, preventing a comparison of exchange rates between this larger portion of Tuba and the isolated DH domain of Tuba.Transferrin Uptake—Chinese hamster ovary cells were transfected with pcHA (pcDNA3-based vector)-full-length Tuba or pcHA-Tuba SH3-1,2,3,4 using FuGENE 6 transfection reagent (Roche Applied Science) and incubated overnight. They were then washed with serum-free medium and incubated in serum-free medium for 24 h in the presence of Alexa-transferrin (Molecular Probes, Inc.) during the last 7 min before a brief wash with phosphate-buffered saline, followed by fixation with 4% formaldehyde. Cells were stained by immunofluorescence according to standard procedures.Peptide Binding—Overlapping SPOTs peptides corresponding to the proline-rich regions of mouse N-WASP (amino acids 217–399), SCAR/WAVE1 (amino acids 274–402), EVL (amino acids 157–210), and Mena (amino acids 277–351) were custom-synthesized by Sigma. All peptides are 12-mers offset by three amino acids, and each spot carries an equivalent amount of peptide (5 nmol) covalently attached to the membrane. A negative control peptide was added (no. 118 on the blot) that contains a known proline-rich binding site for Ena/VASP proteins that does not bind SH3 domains. The Tuba SH3-6 domain in pGEX-2TK was labeled by phosphorylating with protein kinase A and [γ-32P]ATP. The relative intensity of each spot was calculated by converting the PhosphorImager scan into an 8-bit image and measuring the average pixel intensity in a 74-pixel circle centered over each spot in Scion Image. This value was then divided by the background (the average intensity value of the 10 lowest spots) for comparison.Mitochondrial Targeting of the Tuba SH3-6 Domain—The mouse Tuba SH3-6 domain was fused in-frame to a modified retroviral expression vector downstream of an ATG start codon and upstream of a fusion between DsRed2 and the mitochondrial targeting sequence from the L. monocytogenes protein ActA (excluding Ena/VASP-binding regions) (37.Pistor S. Chakraborty T. Niebuhr K. Domann E. Wehland J. EMBO J. 1994; 13: 758-763Crossref PubMed Scopus (153) Google Scholar). This construct, referred to as SH3-6-mito, was used to transiently transfect CAD cells. Cells were plated onto poly-d-lysine-coated coverslips 24 h post-transfection. The medium was replaced with serum-free medium to induce differentiation 3–4 h after plating. Cells were incubated for an additional 24–36 h before fixation with 4% formaldehyde. Previously established procedures were used to visualize cells by immunofluorescence.Miscellaneous—We carried out Western and Northern blotting following standard procedures. For Northern blotting, human multipletissue RNA blots were obtained from Clontech. For multiple-tissue Western blotting, tissues were isolated and prepared as described previously (38.McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar). Far-Western assays and gel overlays were carried out using standard procedures. Immunoprecipitations were carried out as described previously (38.McPherson P.S. Garcia E.P. Slepnev V.I. David C. Zhang X. Grabs D. Sossin W.S. Bauerfeind R. Nemoto Y. De Camilli P. Nature. 1996; 379: 353-357Crossref PubMed Scopus (486) Google Scholar).RESULTSTuba Is a Novel BAR Domain-containing Protein—A BLAST search for proteins containing a domain related to the BAR domain of amphiphysin-1 revealed a large number of sequences. In one (KIAA1010), the putative BAR domain is not located at the N terminus of the protein as in most other sequences, but downstream of a DH domain (Fig. 1A). Although the overall homology to the amphiphysin-1 BAR domain is limited (24% identical and 39% similar) (Fig. 1B), similarities are concentrated in regions generally conserved among the Bin/amphiphysin family. This region in KIAA1010 is currently identified as a BAR domain by protein module-recognizing algorithms such as those of the Pfam and SMART programs.We undertook 5′-RACE using human cDNAs from muscle and brain to isolate a full-length protein corresponding to KIAA1010. This protein, which we have named Tuba in line with the tradition of naming large synaptic proteins after musical instruments (39.Cases-Langhoff C. Voss B. Garner A.M. Appeltauer U. Takei K. Kindler S. Veh R.W. De Camilli P. Gundelfinger E.D. Garner C.C. Eur. J. Cell Biol. 1996; 69: 214-223PubMed Google Scholar), comprises 1577 amino acids with a predicted molecular mass of 178 kDa. The corresponding gene is located on human chromosome 10. The domain structure of Tuba (Fig. 1A) includes four N-terminal SH3 domains (referred to as SH3-1, SH3-2, SH3-3, and SH3-4), a predicted coiled-coil domain, a DH domain, the BAR domain, and two additional C-terminal SH3 domains (SH3-5 and SH3-6). In addition, a proline-rich low complexity region containing putative SH3 domain-binding sites is present upstream of the coiled-coil region (Fig. 1A).In an independent line of study, a yeast two-hybrid screen aimed at identifying ligands for the Ena/VASP family protein EVL from an embryonic mouse library led to the isolation of two independent clones highly homologous to the C terminus of KIAA1010 (Fig. 1A) (data not shown). Reverse transcription-PCR using an embryonic mouse cDNA library was carried out to obtain the full-length gene, which turned out to be the mouse ortholog of Tuba. Mouse Tuba is 70% identical and 77% similar to human Tuba, has a similar domain structure, and is encoded by a gene located on mouse chromosome 19.Searches through genomic and expressed sequence tag data bases revealed numerous expressed sequence tags to two genes that encode proteins homologous to the C-terminal half of Tuba in both human and mouse. We have named these proteins Tuba2 and Tuba3 (Fig. 1A). Tuba2 is located on human chromosome 4 and mouse chromosome 3, and Tuba3 is located on human chromosome 5 and mouse chromosome 8. Tuba2 is 41% identical and 60% similar to Tuba, and Tuba3 is 25% identical and 41% similar to Tuba. Tuba2 is 31% identical and 48% similar to Tuba3. Recent searches have also revealed what appears to be an alternately spliced form of Tuba that is similar in structure to Tuba2 and Tuba3 (Fig. 1A).A putative ortholog of Tuba, GEI-18 (GEX interactor-18), was identified in Caenorhabditis elegans (Fig. 1A) (40.Tsuboi D. Qadota H. Kasuya K. Amano M. Kaibuchi K. Biochem. Biophys. Res. Commun. 2002; 292: 697-701Crossref PubMed Scopus (27) Google Scholar). Two alternate transcripts of GEI-18 are described that comprise the N- and C-terminal halves of the protein. Although not recognized by Pfam or SMART, the region C-terminal of the DH domain in GEI-18 appears to be very similar to a BAR domain. A comparison of the BAR domains of the Tuba family of proteins with each other and human amphiphysin-1 is shown in Fig. 1B.Tuba Is Ubiquitous—Northern blot analysis of human tissues with a probe corresponding to the C terminus of Tuba (nucleotides 4035–4540) revealed two transcripts of 7.3 and 6 kb (Fig. 2A) whose levels varied in different tissues. A probe directed against the N-terminal half of the protein (nucleotides 1246–1696) recognized only the larger transcript (data not shown). A third transcript of 4.5 kb was observed in a number of mouse tissues with a larger probe corresponding to the C terminus of mouse Tuba (nucleotides 2971–4742) (data not shown). This Tuba mRNA likely encodes only the second half of the protein, i.e. a Tuba splice variant similar in domain structure to the homologous proteins Tuba2 and Tuba3 (Fig. 1A). When tested by Western blotting against various rat tissues, affinity-purified antibodies generated against the C terminus of Tuba recognized a band at the expected molecular mass of full-length Tuba (∼180 kDa). The band was the strongest in testis, followed by brain, heart, liver, spleen, and lung. In addition, the same antibodies recognized lower molecular mass bands at ∼105 and 75 kDa with differential tissue distribution that may represent alternatively spliced forms of Tuba or proteolytic C-terminal fragments (Fig. 2B). The 75-kDa band may also represent cross-reactivity of the antibodies against either Tuba2 or Tuba3, whose molecular masses are predicted to be in this range. Collectively, these data indicate that Tuba has a broad tissue distribution and may exist in multiple isoforms.Fig. 2Ubiquitous expression of Tuba transcripts and their protein products.A, multiple-tissue Northern blot using a probe directed against the 3′-end of Tuba. B, multiple-tissue Western blot of rat tissue post-nuclear supernatants with affinity-purified Tuba-specific antibodies. Sk., skeletal.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tuba Is Found at the Synapse—To determine the localization of Tuba in brain, where dynamin participates in the clathrin-mediated endocytosis of s
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