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The Activity of the Vinculin Binding Sites in Talin Is Influenced by the Stability of the Helical Bundles That Make Up The Talin Rod

2006; Elsevier BV; Volume: 281; Issue: 11 Linguagem: Inglês

10.1074/jbc.m508058200

ISSN

1083-351X

Autores

B. Patel, Alexandre R. Gingras, Audrey A. Bobkov, L. Miya Fujimoto, Man Zhang, Robert Liddington, Daniela Mazzeo, Jonas Emsley, Gordon C. K. Roberts, Igor Barsukov, David R. Critchley,

Tópico(s)

Cellular transport and secretion

Resumo

The talin rod contains ∼11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a Kd of ∼9 μm. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (Kd ∼ 0.14 μm), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs. The talin rod contains ∼11 vinculin binding sites (VBSs), each defined by hydrophobic residues in a series of amphipathic helices that are normally buried within the helical bundles that make up the rod. Consistent with this, talin failed to compete for binding of the vinculin Vd1 domain to an immobilized talin polypeptide containing a constitutively active VBS. However, talin did bind to GST-Vd1 in pull-down assays, and isothermal titration calorimetry measurements indicate a Kd of ∼9 μm. Interestingly, Vd1 binding exposed a trypsin cleavage site in the talin rod between residues 898 and 899, indicating that there are one or more active VBSs in the N-terminal part of the talin rod. This region comprises a five helix bundle (residues 482-655) followed by a seven-helix bundle (656-889) and contains five VBSs (helices 4, 6, 9, 11, and 12). The single VBS within 482-655 is cryptic at room temperature. In contrast, talin 482-889 binds Vd1 with high affinity (Kd ∼ 0.14 μm), indicating that one or more of the four VBSs within 656-889 are active, and this likely represents the vinculin binding region in intact talin. In support of this, hemagglutinin-tagged talin 482-889 localized efficiently to focal adhesions, whereas 482-655 did not. Differential scanning calorimetry showed a strong negative correlation between Vd1 binding and helical bundle stability, and a 755-889 mutant with a more stable fold bound Vd1 much less well than wild type. We conclude that the stability of the helical bundles that make up the talin rod is an important factor determining the activity of the individual VBSs. The cytoskeletal protein talin is of a number of proteins including filamin (1Calderwood D.A. Huttenlocher A. Kiosses W.B. Rose D.M. Woodside D.G. Schwartz M.A. Ginsberg M.H. Nat. Cell Biol. 2001; 3: 1060-1068Crossref PubMed Scopus (181) Google Scholar), α-actinin (2Otey C.A. Vasquez G.B. Burridge K. Erickson B.W. J. Biol. Chem. 1993; 268: 21193-21197Abstract Full Text PDF PubMed Google Scholar), tensin (3Calderwood D.A. Fujioka Y. de Pereda J.M. Garcia-Alvarez B. Nakamoto T. Margolis B. McGlade C.J. Liddington R.C. Ginsberg M.H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2272-2277Crossref PubMed Scopus (328) Google Scholar, 4Torgler C.N. Narasimha M. Knox A.L. Zervas C.G. Vernon M.C. Brown N.H. Dev. Cell. 2004; 6: 357-369Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and ILK (5Zervas C.G. Gregory S.L. Brown N.H. J. Cell Biol. 2001; 152: 1007-1018Crossref PubMed Scopus (232) Google Scholar) implicated in coupling integrins to F-actin in cellular junctions with the extracellular matrix. Talin was originally described as a protein localized in cell extracellular matrix junctions (focal adhesions (FAs)) 2The abbreviations used are: FA, focal adhesion; PIP, phosphatidylinositol phosphate; VBS, vinculin-binding site; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; DSC, differential scanning calorimetry; PBS, phosphate-buffered saline; ITC, isothermal titration calorimetry; HA, hemagglutinin.2The abbreviations used are: FA, focal adhesion; PIP, phosphatidylinositol phosphate; VBS, vinculin-binding site; ELISA, enzyme-linked immunosorbent assay; GST, glutathione S-transferase; DSC, differential scanning calorimetry; PBS, phosphate-buffered saline; ITC, isothermal titration calorimetry; HA, hemagglutinin. and membrane ruffles in cultured cells (6Burridge K. Connell L. J. Cell Biol. 1983; 97: 359-367Crossref PubMed Scopus (262) Google Scholar) and was subsequently shown to bind integrins in gel filtration studies (7Horwitz A. Duggan K. Buck C. Beckerle M.C. Burridge K. Nature. 1986; 320: 531-533Crossref PubMed Scopus (818) Google Scholar). Its role as a key component of FAs is supported by antibody microinjection experiments (8Bolton S.J. Barry S.T. Mosley H. Patel B. Jockusch B.M. Wilkinson J.M. Critchley D.R. Cell Motil. Cytoskeleton. 1997; 36: 363-376Crossref PubMed Scopus (39) Google Scholar, 9Nuckolls G.H. Romer L.H. Burridge K. J. Cell Sci. 1992; 102: 753-762Crossref PubMed Google Scholar), laser ablation studies (10Sydor A.M. Su A.L. Wang F.-S. Xu A. Jay D.G. J. Cell Biol. 1996; 134: 1197-1207Crossref PubMed Scopus (87) Google Scholar), antisense RNA down-regulation (11Albiges-Rizo C. Frachet P. Block M.R. J. Cell Sci. 1995; 108: 3317-3329Crossref PubMed Google Scholar), and Tln gene disruption studies in cells in culture (12Jiang G. Giannone G. Critchley D.R. Fukumoto E. Sheetz M.P. Nature. 2003; 424: 334-337Crossref PubMed Scopus (355) Google Scholar, 13Giannone G. Jiang G. Sutton D.H. Critchley D.R. Sheetz M.P. J. Cell Biol. 2003; 163: 409-419Crossref PubMed Scopus (218) Google Scholar, 14Priddle H. Hemmings L. Monkley S. Woods A. Patel B. Sutton D. Dunn G.A. Zicha D. Critchley D.R. J. Cell Biol. 1998; 142: 1121-1133Crossref PubMed Scopus (148) Google Scholar). Knock-out studies in mouse (15Monkley S.J. Zho X.-H. Kinston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fassler R. Dev. Dyn. 2000; 219: 560-574Crossref PubMed Scopus (170) Google Scholar), Drosophila (16Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev. Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), and Caenorhabditis elegans (17Cram E.J. Clark S.G. Schwarzbauer J.E. J. Cell Sci. 2003; 116: 3871-3878Crossref PubMed Scopus (88) Google Scholar) confirm that talin is essential for a wide spectrum of integrin-mediated developmental events including gastrulation in the mouse (15Monkley S.J. Zho X.-H. Kinston S.J. Giblett S.M. Hemmings L. Priddle H. Brown J.E. Pritchard C.A. Critchley D.R. Fassler R. Dev. Dyn. 2000; 219: 560-574Crossref PubMed Scopus (170) Google Scholar), although the situation is complicated by the discovery of a second talin gene (Tln2) in mammals (18Monkley S.J. Pritchard C.A. Critchley D.R. Biochem. Biophys. Res. Commun. 2001; 286: 880-885Crossref PubMed Scopus (87) Google Scholar). Talin is an elongated (∼60 nm) flexible protein (19Winkler J. Lunsdorf H. Jockusch B.M. Eur. J. Biochem. 1997; 243: 430-436Crossref PubMed Scopus (45) Google Scholar), reportedly an anti-parallel dimer (20Isenberg G. Goldmann W.H. FEBS Letts. 1998; 426: 165-170Crossref PubMed Scopus (31) Google Scholar). The globular N-terminal talin head contains a FERM domain (residues 86-400) with binding sites for the cytoplasmic domains of the integrin β-subunit (21Calderwood D.A. Zent R. Grant R. Rees D.J.G. Hynes R.O. Ginsburg M.H. J. Biol. Chem. 1999; 274: 28071-28704Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar, 22Garcia-Alvarez B. de Pereda J.M. Calderwood D.A. Ulmer T.S. Critchley D. Campbell I.D. Ginsberg M.H. Liddington R.C. Mol. Cell. 2003; 11: 49-58Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar) and also layilin (a hyaluronan receptor) (23Borowsky M.L. Hynes R.O. J. Cell Biol. 1998; 143: 429-442Crossref PubMed Scopus (109) Google Scholar), F-actin (24Lee H.S. Bellin R.M. Walker D.L. Patel B. Powers P. Liu H. Garcia-Alvarez B. de Pereda J.M. Liddington R.C. Volkmann N. Hanein D. Critchley D.R. Robson R.M. J. Mol. Biol. 2004; 343: 771-784Crossref PubMed Scopus (78) Google Scholar), and two signaling proteins, focal adhesion kinase (23Borowsky M.L. Hynes R.O. J. Cell Biol. 1998; 143: 429-442Crossref PubMed Scopus (109) Google Scholar) and the type 1γ661 isoform of PI4′5′-kinase (25Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (375) Google Scholar, 26Ling K. Doughman R.L. Iyer V.V. Firestone A.J. Bairstow S.F. Mosher D.F. Schaller M.D. Anderson R.A. J. Cell Biol. 2003; 163: 1339-1349Crossref PubMed Scopus (126) Google Scholar, 27Di Paolo G. Pellegrini L. Letinic K. Cestra G. Zoncu R. Voronov S. Chang S. Guo J. Wenk M.R. De Camilli P. Nature. 2002; 420: 85-89Crossref PubMed Scopus (372) Google Scholar, 28Barsukov I.L. Prescot A. Bate N. Patel B. Floyd D.N. Bhanji N. Bagshaw C.R. Letinic K. Di Paolo G. De Camilli P. Roberts G.C. Critchley D.R. J. Biol. Chem. 2003; 278: 31202-31209Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 29de Pereda J.M. Wegener K.L. Santelli E. Bate N. Ginsberg M.H. Critchley D.R. Campbell I.D. Liddington R.C. J. Biol. Chem. 2005; 280: 8381-8386Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), both of which are implicated in regulating FA dynamics. The talin rod, which is responsible for dimer formation (30Muguruma M. Nishimuta S. Tomisaka Y. Ito T. Matsumara S. J. Biochem. (Tokyo). 1995; 117: 1036-1042Crossref PubMed Scopus (42) Google Scholar), contains a conserved C-terminal actin-binding site (31Hemmings L. Rees D.J.G. Ohanian V. Bolton S.J. Gilmore A.P. Patel N. Priddle H. Trevithick J.E. Hynes R.O. Critchley D.R. J. Cell Sci. 1996; 109: 2715-2726Crossref PubMed Google Scholar), a second integrin-binding site (32Tremuth L. Kreis S. Melchior C. Hoebeke J. Ronde P. Plancon S. Takeda K. Kieffer N. J. Biol. Chem. 2004; 279: 22258-22266Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and several binding sites for the cytoskeletal protein vinculin (33Bass M.D. Smith B.J. Prigent S.A. Critchley D.R. Biochem. J. 1999; 341: 257-263Crossref PubMed Scopus (66) Google Scholar), which itself has multiple binding partners including F-actin (34Jockusch B.M. Rudiger M. Trends Cell Biol. 1996; 6: 311-315Abstract Full Text PDF PubMed Scopus (92) Google Scholar). Integrin signaling via focal adhesion kinase/Src promotes binding of talin to PIP kinase (26Ling K. Doughman R.L. Iyer V.V. Firestone A.J. Bairstow S.F. Mosher D.F. Schaller M.D. Anderson R.A. J. Cell Biol. 2003; 163: 1339-1349Crossref PubMed Scopus (126) Google Scholar). This activates PIP kinase, and results in translocation of the talin-PIP kinase complex to the plasma membrane (25Ling K. Doughman R.L. Firestone A.J. Bunce M.W. Anderson R.A. Nature. 2002; 420: 89-93Crossref PubMed Scopus (375) Google Scholar). PIP2 has been shown to activate the integrin binding site(s) in talin (35Martel V. Racaud-Sultan C. Dupe S. Marie C. Paulhe F. Galmiche A. Block M.R. Albiges-Rizo C. J. Biol. Chem. 2001; 276: 21217-21227Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and the talin head has in turn been shown to activate integrins (36Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (953) Google Scholar, 37Kim M. Carman C.V. Springer T.A. Science. 2003; 301: 1720-1725Crossref PubMed Scopus (633) Google Scholar), suggesting a model in which the localized production of PIP2 drives the assembly of integrintalin-F-actin complexes (38Critchley D.R. Biochem. Soc. Trans. 2004; 32: 831-836Crossref PubMed Scopus (148) Google Scholar) competent to engage the extracellular matrix. Recruitment of additional components might then facilitate the stabilization and maturation of these complexes into FAs. One protein that has been implicated in stabilizing FAs is vinculin. Down-regulation of vinculin leads to smaller FAs and enhances cell motility (39Rodriguez Fernandez J.L. Geiger B. Salomon D. Benzeev A. J. Cell Biol. 1993; 122: 1285-1294Crossref PubMed Scopus (166) Google Scholar), whereas overexpression of vinculin leads to larger FAs and suppresses motility (40Rodriguez Fernanedez J.L. Geiger B. Salomon D. Ben Ze'ev A. Cell Motil. Cytoskeleton. 1992; 22: 127-134Crossref PubMed Scopus (128) Google Scholar). Studies on vinculin knock-out cells are consistent with these data (41Coll J.-L. Ben Ze'ev A. Ezzell R.M. Rodriguez-Fernandez J.L. Baribault H. Oshima R.G. Adamson E.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9161-9165Crossref PubMed Scopus (174) Google Scholar, 42Volberg T. Geiger B. Kam Z. Pankov R. Simcha I. Sabanay H. Coll J.-L. Adamson E. Ben-Ze'ev A. J. Cell Sci. 1995; 108: 2253-2260Crossref PubMed Google Scholar, 43Xu W. Coll J.-L. Adamson E.D. J. Cell Sci. 1998; 111: 1535-1544Crossref PubMed Google Scholar, 44Xu W. Baribault H. Adamson E.D. Development. 1998; 125: 327-337Crossref PubMed Google Scholar). Moreover, vinculin is recruited to FAs in response to applied mechanical force, supporting the view that it plays a role in stabilizing these structures (45Galbraith C.G. Yamada K.M. Sheetz M.P. J. Cell Biol. 2002; 159: 695-705Crossref PubMed Scopus (713) Google Scholar). Using SDS-PAGE blot assays (46Gilmore A.P. Wood C. Ohanian V. Jackson P. Patel B. Rees D.J.G. Hynes R.O. Critchley D.R. J. Cell Biol. 1993; 122: 337-347Crossref PubMed Scopus (65) Google Scholar) and yeast two hybrid deletion mutants analysis (33Bass M.D. Smith B.J. Prigent S.A. Critchley D.R. Biochem. J. 1999; 341: 257-263Crossref PubMed Scopus (66) Google Scholar, 47Bass M.D. Patel B. Barsukov I.G. Fillingham I.J. Mason R. Smith B.J. Bagshaw C.R. Critchley D.R. Biochem. J. 2002; 362: 761-768Crossref PubMed Scopus (50) Google Scholar), we previously identified three vinculin binding sites (VBSs) in the talin rod, each defined by a single amphipathic helix. More recent data using synthetic peptides equivalent to each of the predicted 62 helices that make up the talin rod (48Mclachlan A.D. Stewart M. Hynes R.O. Rees D.J.G. J. Mol. Biol. 1994; 235: 1278-1290Crossref PubMed Scopus (43) Google Scholar) indicate that there are at least eight additional VBSs (49Gingras A.R. Ziegler W.H. Frank R. Barsukov I.L. Roberts G.C. Critchley D.R. Emsley J. J. Biol. Chem. 2005; 280: 37217-37224Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Crystal structures show that the hydrophobic groups on one face of a talin VBS helix become embedded in a hydrophobic groove in the vinculin head (50Izard T. Evans G. Borgon R.A. Rush C.L. Bricogne G. Bois P.R. Nature. 2004; 427: 171-175Crossref PubMed Scopus (193) Google Scholar, 51Izard T. Vonrhein C. J. Biol. Chem. 2004; 279: 27667-27678Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar), but our recent structural studies (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar, 53Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) show that these vinculin binding determinants in talin are normally buried in the core of a series of amphipathic helical bundles that make up the talin rod (48Mclachlan A.D. Stewart M. Hynes R.O. Rees D.J.G. J. Mol. Biol. 1994; 235: 1278-1290Crossref PubMed Scopus (43) Google Scholar). In the present study we have sought to establish whether some or all of the VBSs in the talin rod are cryptic or constitutively active. Protein Expression and Purification—cDNAs encoding mouse talin 482-636, 482-655, 482-789, and 755-889 and the chicken vinculin Vd1 domain (residues 1-258) were cloned into the expression vector pET-15b (Novagen, Cambridge Bioscience, Cambridge), whereas a cDNA encoding mouse talin 482-889 was cloned into pET-151/D-TOPO (Invitrogen). Recombinant proteins were purified as described previously (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar, 53Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and proteins concentrations were determined using the CB Protein Assay (Calbiochem). cDNAs encoding human talin 1 (resides 1-2541) and the talin 1 rod domain (residues 453-2541) were generated by PCR using pET30a constructs encoding the N-terminal talin head and the talin rod as templates (kindly provided by Dr. Stephen C.-T. Lam, University of Illinois, Chicago, IL). PCR products were cloned into pET30a (Novagen) between the NdeI and EagI sites such that the encoded proteins were expressed with a C-terminal His tag. Constructs were authenticated by DNA sequencing and shown to match the human talin 1 cDNA sequence in GenBank™ (accession number BC042923). Human talin 1 and the talin 1 rod were expressed in Escherichia coli BL21(DE3). In brief, cells (1 liter) were grown at 37 °C to an A600 nm 0.6 and induced with 0.2 mm isopropyl 1-thio-d-galactopyranoside at 15 °C overnight. The cell pellet was resuspended into 35 ml of lysis buffer (20 mm Tris (pH 7.4), 0.4 m NaCl) with 1 mm β-mercaptoethanol and lysed by passing through a French press (Thermo Electron). The soluble cell lysate was then recovered and passed through a 5-ml HiTrap affinity column (Amersham Biosciences) charged with Ni2+. Fractions containing the required protein were combined, concentrated, and further purified by size exclusion chromatography (Superdex 200 10/30, Amersham Biosciences) in buffer containing 20 mm sodium phosphate (pH 7.5), 150 mm NaCl, and 0.1 mm EDTA. The relationship of the various talin polypeptides used in this study to full-length talin is illustrated in Fig. 1. Binding of the Vinculin Vd1 Domain to Talin—The relative affinities of talin and talin polypeptides for the vinculin Vd1 domain was measured by competitive ELISA as described previously (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar). In brief, NUNC immunoplates (F96-maxisorp) coated with a talin 482-636 polypeptide were incubated with the GST-vinculin Vd1 domain at a concentration of 5 nm (54Bakolitsa C. Cohen D.M. Bankston L.A. Bobkov A.A. Cadwell G.W. Jennings L. Critchley D.R. Craig S.W. Liddington R.C. Nature. 2004; 430: 583-586Crossref PubMed Scopus (282) Google Scholar) in the presence of increasing amounts of talin or recombinant talin polypeptides. Binding of GST-vinculin or GST alone was determined using a rabbit polyclonal anti-GST coupled to horseradish peroxidase (Santa Cruz). Binding of talin to GST-Vd1 was also measured using a pull-down assay as described previously (47Bass M.D. Patel B. Barsukov I.G. Fillingham I.J. Mason R. Smith B.J. Bagshaw C.R. Critchley D.R. Biochem. J. 2002; 362: 761-768Crossref PubMed Scopus (50) Google Scholar). Analytical gel filtration chromatography of recombinant talin polypeptides and vinculin Vd1 (1-258) was performed using Superdex-75 (10/30) (Amersham Biosciences) at room temperature. The column was pre-equilibrated and run in 20 mm Tris (pH 8.0), 200 mm NaCl, and 2 mm dithiothreitol at a flow rate of 0.8 ml/min. In each case, 0.5-ml fractions were collected and analyzed using a 15% SDS-PAGE gel and stained using the GelCode blue reagent (Pierce). Calorimetry—Differential scanning calorimetry (DSC) experiments were performed on a N-DSC II differential scanning calorimeter (Calorimetry Sciences Corp, Provo, UT) at the scanning rate of 1000/min under 3.0 atm of pressure. Before measurement, protein samples were dialyzed against PBS. The dialysis buffer was used as the reference solution. Vd1 and talin polypeptides were used at concentrations between 18.0 and 25 μm. Isothermal titration calorimetry (ITC) was performed on a VP-ITC calorimeter from Microcal (Northampton, MA). 8-μl aliquots of solution containing 0.8-1.0 mm vinculin Vd1 (residues 1-258) were injected into the cell containing 40-100 μm talin or the talin polypeptide. In each experiment 37 injections were made. The experiments were performed at 23 °C. Before ITC titrations, all protein samples were dialyzed against PBS buffer. Experimental data were analyzed using Microcal Origin software provided by the ITC manufacturer (Microcal, Northampton, MA). Circular Dichroism Spectroscopy—CD spectra were recorded using a Jasco J-715 spectropolarimeter equipped with a Jasco PTC-348WI temperature control unit. Far-UV CD spectra were recorded at 20 °C over the wavelength range 200-250 nm in a quartz cell of 0.1-cm path length (scan rate 50 nm·min-1). Proteins were dissolved in 20 mm sodium phosphate (pH 6.5), 50 mm NaCl at concentrations of 25 μm. The mean residue molar ellipticity [θ] (deg × cm2 × dmol-1) was calculated according to the formula [θ] = θ/(n × 10 × CM × l), where θ is the measured ellipticity in degrees, n is the number of peptide bonds (residue), l is the path length in centimeters, and CM is the molar protein concentration. The factor 10 originates from the conversion of the molar concentration to the dmol·cm-3 concentration unit. For urea denaturation studies, proteins were dissolved in 20 mm sodium phosphate, pH 6.5, 50 mm NaCl containing 0, 0.8, 1.6, 4.0, or 5.6 m urea. Expression of HA-tagged Talin Polypeptides in NIH3T3 Cells—cDNAs encoding C-terminal HA-tagged talin polypeptides 482-636, 482-655, 482-789, and 482-889 were synthesized by PCR using the mouse talin-1 cDNA (55Rees D.J.G. Ades S.E. Singer S.J. Hynes R.O. Nature. 1990; 347: 685-689Crossref PubMed Scopus (240) Google Scholar) as template and the following primers from Invitrogen: 482, 5′-CGGGATCCATGCGAGGACACATGCCACCT-3′; 636, 5′-GGAATTCTCAAAGAGCGTAATCTGGAACATCGTATGGGTAGTTCTGACGAG-3′; 655, 5′-GGAATTCTCAAAGAGCGTAATCTGGAACATCGTATGGGTAAATTTGCTGCAACAGCTC-3′; 789, 5′-GGAATTCTCAAAGAGCGTAATCTGGAACATCGTATGGGTAGGCGTGGGCCTTCACGTG-3′; 889, 5′-GGAATTCTCAAAGAGCGTAATCTGGAACATCGTATGGGTATCGCTGCTGCTGTTCCTC-3′. PCR products were first cloned into pPCR-Script Amp SK(+) (Stratagene) and then subcloned into the expression vector pcDNA3 (Invitrogen) using BamHI and EcoRI sites. The authenticity of the cloned cDNAs was confirmed by sequencing. The constructs were transfected -2 μg of cDNA/6 μl of FuGENE 6 (Roche Applied Science) into 3 × 104 NIH3T3 cells grown on glass coverslips and cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum. After 16 h at 37 °C, cells were fixed for 10 min in 4% paraformaldehyde/PBS, permeabilized for 5 min in 0.2% (v/v) Triton X-100/PBS, and quenched for 10 min in 50 mm NH4Cl/PBS. After 1 h of incubation in 1% bovine serum albumin/PBS, cells were incubated for 20 min with the anti-HA antibody (Santa Cruz Biotechnologies), washed 3 times with 0.02% bovine serum albumin/PBS, and then incubated for 20 min with fluorescein isothiocyanate-conjugated secondary antibody (Southern Biotechnology Associates, Inc.). F-actin was visualized by staining with phalloidin-Texas Red (Molecular Probes) for 20 min. After 3 washes in 0.02% bovine serum albumin/PBS, coverslips were mounted on glass slides using Prolong Antifade Reagent (Molecular Probes) and inspected using a Nikon TE300 inverted microscope and Openlab 4.0.2 software. The VBSs in Talin Are in a Low Affinity State—Our recent biochemical (33Bass M.D. Smith B.J. Prigent S.A. Critchley D.R. Biochem. J. 1999; 341: 257-263Crossref PubMed Scopus (66) Google Scholar, 47Bass M.D. Patel B. Barsukov I.G. Fillingham I.J. Mason R. Smith B.J. Bagshaw C.R. Critchley D.R. Biochem. J. 2002; 362: 761-768Crossref PubMed Scopus (50) Google Scholar) and structural studies (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar, 53Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) show that the ∼11 VBSs in the talin rod (49Gingras A.R. Ziegler W.H. Frank R. Barsukov I.L. Roberts G.C. Critchley D.R. Emsley J. J. Biol. Chem. 2005; 280: 37217-37224Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) (Fig. 1) are each contained within a single amphipathic helix. Binding is determined by a series of hydrophobic residues on one face of the helix, but these are normally buried in the core of the helical bundles that make up the talin rod (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar, 53Fillingham I. Gingras A.R. Papagrigoriou E. Patel B. Emsley J. Critchley D.R. Roberts G.C. Barsukov I.L. Structure (Camb). 2005; 13: 65-74Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). To investigate whether any of the VBSs in intact talin are active, we set up a competitive ELISA-type assay. Because the talin-binding site in the N-terminal region of vinculin is normally masked by an intramolecular interaction with the C-terminal vinculin tail (54Bakolitsa C. Cohen D.M. Bankston L.A. Bobkov A.A. Cadwell G.W. Jennings L. Critchley D.R. Craig S.W. Liddington R.C. Nature. 2004; 430: 583-586Crossref PubMed Scopus (282) Google Scholar, 56Johnson R.P. Craig S.W. J. Biol. Chem. 1994; 269: 12611-12619Abstract Full Text PDF PubMed Google Scholar), we used a GST-vinculin fusion protein containing just residues 1-258 (GST-Vd1) to assay talin binding. Similarly, because the single VBS contained within the N-terminal region the talin rod (residues 482-655, a 5-helix bundle) is cryptic (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar), we used a construct (residues 482-636) in which the VBS has been activated by deletion of the C-terminal helix. We then measured the ability of GST-Vd1 to bind to talin 482-636 deposited on plastic in the presence of increasing concentrations of talin or talin fragments. As shown previously (52Papagrigoriou E. Gingras A.R. Barsukov I.L. Bate N. Fillingham I.J. Patel B. Frank R. Ziegler W.H. Roberts G.C. Critchley D.R. Emsley J. EMBO J. 2004; 23: 2942-2951Crossref PubMed Scopus (131) Google Scholar), preincubation of GST-Vd1 with increasing concentrations of talin 482-636 in solution progressively inhibited binding to talin 482-636 on plastic, with an IC50 of ∼1 nm, whereas talin 482-655 was completely without effect (Fig. 2A). Interestingly, talin purified from turkey gizzard was also unable to inhibit GST-Vd1 binding, indicating that the VBSs in intact talin are cryptic or in a low affinity state (Fig. 2A). The integrin-binding sites in talin are also reportedly cryptic and can be activated by PIP2 (35Martel V. Racaud-Sultan C. Dupe S. Marie C. Paulhe F. Galmiche A. Block M.R. Albiges-Rizo C. J. Biol. Chem. 2001; 276: 21217-21227Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar) or by cleavage of the talin head from the rod (57Yan B. Calderwood D.A. Yaspan B. Ginsberg M.H. J. Biol. Chem. 2001; 276: 28164-28170Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Preincubation of talin with PIP2 did not activate the VBSs in talin (Fig. 2A), although the purified talin rod (but not the head) did show significantly more Vd1 binding activity than intact talin (Fig. 2A). The above assay provides a measure of the relative affinities of Vd1 for talin and talin fragments and does not necessarily indicate that intact talin cannot bind vinculin. To investigate this, we incubated purified talin with GST or GST-Vd1 and carried out a pull down assay. Talin binding was analyzed by SDS-PAGE followed by Western blotting using the TD77 monoclonal antibody that recognizes the extreme C terminus of talin (8Bolton S.J. Barry S.T. Mosley H. Patel B. Jockusch B.M. Wilkinson J.M. Critchley D.R. Cell Motil. Cytoskeleton. 1997; 36: 363-376Crossref PubMed Scopus (39) Google Scholar). The results clearly establish that talin and talin rod fragments are able to bind to GST-Vd1 (Fig. 2B). To obtain a direct measure of the binding affinities, we used ITC (Table 1). At 23 °C, Vd1 bound talin with a Kd of ∼9 μm, and PIP2 had relatively little effect on binding (Kd ∼5 μm). The talin rod bound Vd1 with a slightly higher affinity (Kd = 2.4 μm),

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