Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online before print May 1, 2007, 10.1110/ps.072798707
Protein Science (2007), 16:1223-1229. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow All Versions of this Article:
ps.072798707v1
16/6/1223    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McCleverty, C. J.
Right arrow Articles by Liddington, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McCleverty, C. J.
Right arrow Articles by Liddington, R. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

PROTEIN STRUCTURE REPORT

Structure of the PTB domain of tensin1 and a model for its recruitment to fibrillar adhesions

Clare J. McCleverty1,3, Diane C. Lin2, and Robert C. Liddington1

1 Cancer Center, Burnham Institute for Medical Research, La Jolla, California 92037, USA
2 Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA

(RECEIVED January 29, 2007; FINAL REVISION March 1, 2007; ACCEPTED March 1, 2007)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgments
 References
 
Tensin is a cytoskeletal protein that links integrins to the actin cytoskeleton at sites of cell-matrix adhesion. Here we describe the crystal structure of the phosphotyrosine-binding (PTB) domain of tensin1, and show that it binds integrins in an NPxY-dependent fashion. Alanine mutagenesis of both the PTB domain and integrin tails supports a model of integrin binding similar to that of the PTB-like domain of talin. However, we also show that phosphorylation of the NPxY tyrosine, which disrupts talin binding, has a negligible effect on tensin binding. This suggests that tyrosine phosphorylation of integrin, which occurs during the maturation of focal adhesions, could act as a switch to promote the migration of tensin–integrin complexes into fibronectin-mediated fibrillar adhesions.

Keywords: structure/function studies; crystallography; calorimetry; mutagenesis (site-directed and general); docking proteins; surface plasmon resonance


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and Methods
 Acknowledgments
 References
 
Cells bind to the extracellular matrix (ECM) by means of the integrin family of plasma membrane receptors (Hynes 2002). Integrins make direct bonds to ECM proteins and indirect bonds to the actin cytoskeleton via a number of cytoskeletal proteins, including tensin, talin, vinculin, and paxillin (Zamir et al. 1999). These complexes provide a scaffold for signaling enzymes that regulate many aspects of cellular behavior. Geiger (Zaidel-Bar et al. 2003) has described three stages in the development of cell-ECM contacts: "focal contacts" are short-lived structures containing integrin {alpha}Vbeta3, which transform into classical "focal adhesions" upon activation of RhoA, and contain both integrins {alpha}Vbeta3 and {alpha}5beta1; a third type of contact, "fibrillar adhesions," is formed when integrin {alpha}5beta1 (the fibronectin receptor) translocates from focal adhesions (Pankov et al. 2000).

The cytoskeletal protein tensin is absent from focal contacts but is recruited to focal adhesions and plays a key role in the establishment of fibrillar adhesions (Zaidel-Bar et al. 2003). Tensin contains binding sites for actin in the N terminus that crosslink actin filaments, and a central domain that caps the barbed ends of F-actin (Lo et al. 1994; Chuang et al. 1995). The C-terminal region contains a Src homology 2 (SH2) domain that mediates binding to tyrosine-phosphorylated proteins, such as phosphoinositide 3-kinase, Cas, and focal adhesion kinase (Auger et al. 1996), and a PTB domain that is required for focal adhesion targeting (Chen and Lo 2003). Three isoforms of tensin have been identified—tensin1, tensin2, and tensin3—which are homologous in their N- and C-terminal domains but divergent in their central regions (Chen et al. 2002; Cui et al. 2004). In addition, a fourth family member, cten, lacks the N-terminal actin-binding domain (Lo and Lo 2002).

The factors governing the dynamics and maturation of cell-ECM contacts are poorly understood. Once activated, talin plays a key role in the early stages, by engaging integrin beta-subunit cytoplasmic tails (Garcia-Alvarez et al. 2003; Wegener et al. 2007). This interaction disrupts {alpha}beta tail association and promotes a conformational change in the extracellular domains of integrins that leads to enhanced affinity for ECM proteins (Vinogradova et al. 2002; Kim et al. 2003; Tadokoro et al. 2003; Xiao et al. 2004), a process termed "inside-out" signaling. A second major player is the tyrosine kinase, Src (or a close relative), which binds constitutively to the C terminus of integrin beta-tails, and is activated by intermolecular phosphorylation when integrins cluster at cell-ECM adhesion sites (Arias-Salgado et al. 2003), leading to a variety of downstream events termed "outside-in" signaling. The activity of Src also disrupts the linkage between talin and integrin via tyrosine phosphorylation of the integrin NPxY motif (Tapley et al. 1989), and promotes the binding of talin to the enzyme, phosphatidylinositol phosphate kinase type 1-{gamma} (PIPKI{gamma}), a key activator of proteins involved in focal adhesion assembly, including talin (Ling et al. 2003).

We present here the crystal structure of the tensin1 PTB domain, together with in vitro binding studies and mutagenesis showing that tensin1 binds to integrins in a talin-like but phosphorylation-independent fashion, and we propose a model in which the migration of tensin from focal adhesions to form fibrillar adhesions is promoted by a switch in integrin-binding preferences upon Src phosphorylation.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and Methods
 Acknowledgments
 References
 
Structure of the tensin1 PTB domain
Crystallization trials with residues 1605–1744 of chicken tensin1 yielded crystals that diffracted to low resolution. Limited proteolysis identified a fragment, residues 1605–1738, which produced high-quality crystals, and its structure was determined at 1.5 Å resolution using multiple anomalous diffraction (MAD) from a selenomethionine derivative (Table 1). The tensin1 PTB domain adopts a mostly canonical PTB fold (Fig. 1). It is most similar to the PTB domain of X11: thus, they both have an inserted beta1' strand and {alpha}1 helix between strands beta1 and beta2, and the central beta sandwiches overlap closely (RMSD = 0.93 Å for C{alpha} atoms). In common with other PTB domains, tensin1 lacks the loop found in talin between strands beta1 and beta2 that is critical for membrane association and integrin activation (Wegener et al. 2007). A unique feature of the tensin1 domain is an insertion of 14 residues between the beta5 and beta6 strands, which forms one side of a pocket that we propose binds to a tryptophan residue from the integrin tail (see below). The loops connecting strands beta3-beta4, beta5-beta6, and beta6-beta7 are partially disordered (the last is implicated in phosphotyrosine binding; see below) with relatively high B values, indicating that these are flexible regions within the domain.


View this table:
[in this window]
[in a new window]

  		Table 1. Data collection and refinement statistics

 


Figure 1
View larger version (57K):
[in this window]
[in a new window]

  		Figure 1. Structure of the tensin PTB domain. (A) Stereo C{alpha} plot of the tensin1 PTB domain with every 10th residue, and N and C termini, labeled. The unstructured beta6-beta7 loop is shown schematically in green. (B) Ribbon diagram of the tensin1 PTB domain, with secondary structural elements labeled. A model of the integrin peptide is also shown (in red), including the side-chains of the NPxY tyrosine and the tryptophan residue at the –8 position. (C) Structure-based sequence alignment of the PTB domains of tensin family members, the talin PTB-like domain (Garcia-Alvarez et al. 2003), and other PTB domains (X11 [Zhang et al. 1997], Numb [Li et al. 1998], Dab1 [Stolt et al. 2003], Shc [Zhou et al. 1995b], IRS-1 [Eck et al. 1996; Zhou et al. 1996], and Dok1 [Shi et al. 2004]). beta-strands are colored red; {alpha}-helices are blue. Residues involved in peptide binding are underlined, and those that directly bind phosphotyrosine are boxed. In the tensin homologs, two basic residues from the beta6-beta7 loop proposed to engage the phosphotyrosine are boxed in green. Numbers in parentheses for X11, Dab1, and Shc indicate the size of sequence insertions that have been removed for clarity. The asterisk in the X11 sequence marks a 19-residue insertion that is also not shown.

 
The tensin1 PTB domain binds integrin cytoplasmic tails
PTB domains characteristically recognize ligands bearing an NPxY motif, and show specificity for residues that lie upstream of this motif. Tensin1 colocalizes predominantly with ECM-bound {alpha}5beta1 integrins in fibrillar adhesions, as well as with focal adhesions containing {alpha}Vbeta3 integrin (Katz et al. 2000). Integrin beta subunit cytoplasmic tails contain two NPxY motifs, and tensin2 has previously been shown to bind integrin beta-tails in vitro (Calderwood et al. 2003). Using surface plasmon resonance (SPR), we investigated the binding of the tensin1 PTB domain to peptides derived from the beta1A and beta3 integrin tails (Table 2; Supplemental Fig. 1). We used the longer domain containing the authentic C terminus (residues 1605–1744), which we found to bind integrin ~10-fold more strongly than the shorter domain used for crystallization. We found that tensin1 has a clear preference for the first, membrane-proximal, NPxY motif (over the membrane-distal motif) for both the beta1A and beta3 tails (residues 779–790 and 738–749, respectively). The affinities, in the low micromolar range, are similar for the beta1A and beta3 tails, and are comparable with other PTB-peptide interactions, such as Numb-Nak (Zwahlen et al. 2000), IRS-1-IL4 (Zhou et al. 1996), SNT-FGFR1 (Dhalluin et al. 2000), Dok1-RET (Shi et al. 2004), and Dab1-ApoER2 (Stolt et al. 2003). Binding is, however, significantly weaker than the value of Kd = 100 nM estimated for talin-integrin binding (Calderwood et al. 1999).


View this table:
[in this window]
[in a new window]

  		Table 2. Binding affinities of beta1 and beta3 integrin peptides for the tensin1 PTB domain

 
The NPxY motif typically adopts a reverse beta-turn that is critical for binding PTB domains. It was previously shown that mutating the NPxY tyrosine to alanine eliminated binding of integrin to talin (Calderwood et al. 2002), and we found the same result with tensin1 (Table 2). Some PTB domains, such as Shc and IRS-1 (Zhou et al. 1995a; Eck et al. 1996), require phosphorylation of the NPxY tyrosine for strong binding, while others, notably talin, are inhibited by phosphorylation (Tapley et al. 1989). A third group, including X11, Numb, and Dab1 (Zhang et al. 1997; Zwahlen et al. 2000; Stolt et al. 2003), is indifferent to the state of phosphorylation. We found that the tensin1 PTB domain falls into the third group: thus, it binds to unphosphorylated and phosphorylated beta1 and beta3 integrin tails with nearly identical affinity (Table 2). PTB domains that bind phosphotyrosine typically possess a pocket lined with basic residues that coordinate the phosphate moiety. A structure-based alignment of PTB domains (Fig. 1C) shows that two basic residues within the beta6-beta7 loop of tensin1, Arg1704 and Lys1705 (conserved in tensin2 and tensin3), are in similar positions to the phosphotyrosine-coordinating residues in the Shc, IRS-1 and Dok1, and X11 PTB domains. The beta6-beta7 loop is disordered in the tensin1 PTB crystal structure, indicating that it is mobile, which may contribute to the ability of the domain to accommodate either phosphorylated or unphosphorylated tyrosine. The analogous loop in talin is acidic (Garcia-Alvarez et al. 2003), consistent with disruption of integrin binding by tyrosine phosphorylation.

PTB domains display ligand-binding specificities for hydrophobic residues upstream of the NPxY motif. For example, Shc, Numb, and Dab1 PTB domains require a hydrophobic residue at position –5 with respect to the tyrosine (Zhou et al. 1995a; Zwahlen et al. 2000; Stolt et al. 2003), and the IRS-1 PTB domain favors hydrophobic residues at position –6 and –8 (Eck et al. 1996). Trp at position –8 in the beta3 integrin tail has been shown to form a critical interaction with the talin PTB domain (Garcia-Alvarez et al. 2003), and a model of the tensin1–integrin interaction (see below) pointed to the existence of an appropriate pocket for the tryptophan. We therefore mutated this Trp to Ala in both the beta1 and beta3 tails, and found in both cases a substantial (30- to 50-fold) loss of binding to the tensin1 PTB domain (Table 2), supporting an important role for Trp (–8) in the interaction.

Model of tensin1 PTB domain binding integrin tail
Based on our crystal structure and peptide binding studies, we built a model of the tensin1–integrin complex (Figs. 1B, 2A). As templates we used two crystal structures: the talin–beta3 integrin complex (Protein Data Bank [PDB] code 1MK9) (Fig. 2B) and, owing to the structural similarity of their PTB domains, the X11–betaAPP complex (PDB code 1X11). We built the beta3-integrin NPIY motif with a typical beta-turn conformation, placing the Tyr (+0) in a pocket adjacent to the basic beta6-beta7 loop. This enables the NPxY Asn, whose side-chain stabilizes the integrin beta-turn, to form H-bonds with the main-chain nitrogens of two residues in the beta4-beta5 loop (Leu1672 and Val1675), while the aromatic ring of Tyr (+0) interacts with Thr1676. To test this model, we mutated Thr1676 to alanine; SPR measurements (at a single protein concentration) indicated a substantial loss of binding to the beta1A tail (Supplemental Fig. 2). As noted above, the C-terminal residues of tensin (1739–1744) enhance integrin binding; it is likely that these residues become ordered in the presence of the integrin tail and create a pocket for the NPIY Ile, as has been observed with the PTB domains of IRS-1 and Shc (Eck et al. 1996; Farooq et al. 2003).


Figure 2
View larger version (46K):
[in this window]
[in a new window]

  		Figure 2. PTB-integrin models. Surface representations of the tensin1-integrin model (A) and the talin-integrin crystal structure (B) (Garcia-Alvarez et al. 2003), colored by electrostatic potential (blue for positive, red for negative). The integrin peptides are shown as sticks and colored by atom type. Integrin residue numbers are in black (and numbered with respect to the NPxY tyrosine in parentheses); in A, tensin residue numbers are given in green. Figures were generated with MOLSCRIPT (Kraulis 1991), RASTER3D (Kraulis 1991; Merritt and Murphy 1994), and PyMol (http://www.pymol.org).

 
We next built the three residues upstream of the NPIY motif such that they make canonical main-chain H-bonds to augment the central beta-sheet. The side-chain of tensin1 Asp1679 disrupts further upstream beta-sheet interactions, although in the other tensin homologs, Asp1679 is replaced by smaller residues (Ser and Gly) that may be less disruptive. Moving further upstream, a hydrophobic pocket is evident at an appropriate location to accept the bulky side-chain of Trp (–8). The pocket is formed by residues from the inserted helix, {alpha}1 (Pro 1624 and Ile1627), from the end of the beta5-strand (Phe1677 and Asp1679), and by Pro1682 from the beginning of the loop, beta5-beta6, that is unique to tensin. To test the role of this pocket, we made alanine mutations to three residues lining the pocket (Pro1624, Pro1682, and Phe1677). Consistent with our model, all mutations significantly reduced integrin binding as judged by SPR (Supplemental Fig. 2). However, in the case of F1682A, although the mutant behaved normally during purification, its melting profile measured by differential scanning calorimetry (DSC) indicated a substantial reduction in stability, which may contribute to the observed reduction in peptide binding (Supplemental Fig. 3). The pocket is invariant (except for Asp1679) in tensin homologs, as is most of the integrin-binding surface, suggesting that all three tensin isoforms will bind integrin. Unlike tensin1, the tensin2 PTB domain has a preference for beta3 over beta1A (Calderwood et al. 2003). Although we cannot immediately explain the structural basis for the different integrin preferences, we note they are consistent with the distinct cellular localizations of the two proteins: thus, tensin2 localizes to fibrillar adhesions (which predominantly contain integrin {alpha}5beta1 rather than {alpha}vbeta3) less efficiently than does tensin1 (Chen et al. 2002). Finally, we note that the distance between the NPxY binding pocket and the tryptophan binding pocket in our tensin–integrin model requires that the intervening residues are in a fully extended conformation (Fig. 2A). In the case of talin, the two pockets are closer together, and the intervening residues bulge out from the side of the PTB domain; i.e., there is slack in the system (Fig. 2B). This would explain why the beta2 integrin tail, whose intervening sequence is one residue shorter than other integrin tails, can bind to talin but not to tensin (Calderwood et al. 2003).

Implications for cell-matrix dynamics
Our analysis reveals similar but distinct structural determinants for the binding of integrin to tensin vis-à-vis talin. The relatively low binding affinity of tensin1 for integrin (Kd~µM) is consistent with the finding that the PTB domain is necessary but not sufficient for focal adhesion targeting (Chen and Lo 2003); that is, high-affinity attachment to cell adhesion sites presumably requires the combinatorial binding of distinct elements of the adhesion site to other domains within the full-length protein. Although we have shown that tensin and talin bind to the same site on the integrin, direct competition is initially unlikely since talin binds with much higher affinity in the absence of phosphorylation (Calderwood et al. 1999). However, upon phosphorylation of the NPxY tyrosine, while the affinity for talin is greatly reduced, we showed that the affinity for tensin is unaffected; that is, we predict that the relative affinities will be reversed. As noted above, during the maturation of focal contacts, integrin tails are phosphorylated by Src, which should trigger the release of talin from integrins (Tapley et al. 1989; Ling et al. 2003). This would make integrin available for tensin binding and thus potentially facilitate the translocation of tensin-bound {alpha}5beta1 integrin to fibrillar adhesions. In support of this model, we note that in Src-null cells, which have low levels of phosphorylation at focal adhesions, tensin fails to segregate into fibrillar adhesions (Volberg et al. 2001).


   Materials and Methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
Acknowledgments
 References
 
Protein expression and purification
DNA sequences corresponding to residues 1605–1744 and 1605–1738 of chicken tensin1 were amplified by PCR and cloned into pET-15b (Novagen) vectors and confirmed by di-deoxy sequencing. Protein was expressed in the Escherichia coli strain BL21 (DE3). Following induction of protein expression with 0.5 mM IPTG for 3 h at 37°C, cell harvest, and lysis by sonication, the protein was purified using a Ni-affinity column (Pharmacia). The His tag was cleaved with thrombin and removed by dialysis against 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT. The purified proteins contain four additional residues (Gly-Ser-His-Met) from the vector at the N terminus. Selenomethionine (SeMet)-labeled protein (residues 1605–1738) was expressed and purified as for the native protein. Single amino acid mutations were generated in the PTB domain (residues 1605–1744) using the QuikChange site-directed mutagenesis kit (Stratagene) and verified by DNA sequencing. All mutants were expressed and purified as for the wild type. To test the effects of the mutations on protein folding, melting profiles proteins were determined using DSC (Supplemental Fig. 3). All peptides were synthesized by United Biochemical Research.

Crystallography
Tensin1 PTB domain (residues 1605–1738) was concentrated to 15 mg/mL in an ultrafiltration cell (Amicon) prior to crystallization. Crystals grew at 4°C by sitting-drop vapor diffusion, using 2 µL protein and 2 µL precipitant (40% PEG 600 and 0.1 M CHES at pH 9.5), and belong to space group I4 with unit cell dimensions a = 75.7 Å, c = 48.7 Å. The asymmetric unit contains one PTB domain and 48% solvent. Diffraction data were collected at 100 K using flash-cooled crystals stabilized in 42% PEG 600 and 0.1 M CHES (pH 9.5). Data from a single native crystal and multiple wavelength anomalous diffraction (MAD) data at three wavelengths near the Se absorption edge from a SeMet-substituted crystal were collected at the SSRL beamline 9-2 (Table 1). All data were processed with DENZO and SCALEPACK (Otwinowski and Minor 1997). The scaled data were input into SOLVE (Terwilliger and Berendzen 1999), and two selenium atoms were located in the asymmetric unit. Phases (calculated to 1.8 Å resolution using SOLVE) were refined by solvent flattening with RESOLVE (Terwilliger and Berendzen 1999). An atomic model (~75% complete) was built with RESOLVE and completed manually using TURBO-FRODO (Roussel and Cambillau 1991). The model was refined with CNS (Brünger et al. 1998) to 1.5 Å resolution (Table 1). After initial simulated annealing, the model was further refined with iterative cycles of manual fitting, conjugate gradient minimization, and isotropic B factor refinement. Further refinement included addition of 107 solvent molecules. The final model converged to an RWORK of 0.21 and RFREE value of 0.23 (calculated with 5% of reflections omitted from refinement). Poor electron density was observed for Gly1605 at the N terminus, residues Arg1662–Phe1666 in the beta3-beta4 loop, Lys1689–G1694 in the beta5-beta6 loop, and Arg1704–Thr1710 in the beta6-beta7 loop. The final model was evaluated with PROCHECK (Laskowski et al. 1993): 88.3% of the main-chain torsion angles (non-glycine) lie in the most favored regions of the Ramachandran plot, with none in disallowed regions. The coordinates and structure factors have been deposited in the PDB with accession code 1WVH. Coordinates of the tensin–integrin model are available from the authors.

Peptide binding studies
SPR studies were carried out on a Biacore 3000 Biosensor (Biacore AB). Peptides were immobilized on a CM5 sensor chip (Biacore) via the amine coupling method using N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linking, as directed by the manufacturer. A blank flow cell that had been EDC/NHS-activated and ethanolamine-blocked was used as a reference. Tensin1 PTB domain was dialyzed extensively into running buffer containing 20 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM DTT, and 0.005% polysorbate 20. Steady-state experiments were performed by injecting the protein over the chip at 10 µL/min for 4 min. The chip surface was regenerated with 1.0 M NaCl. Typically, sensorgrams were obtained at six different concentrations for each experiment, and each set of titrations included a running buffer injection as an additional blank (Supplemental Fig. 1). All data were analyzed using BIAevaluation 3.0, subtracting binding to the blank flow cell and the blank buffer injection to account for any nonspecific binding. For each sensorgram, the peak response levels achieved at steady-state were plotted against analyte concentration. This plot was fitted to a single site binding equation (Langmuir isotherm) to determine dissociation constant (KD) values. All data are the average of at least three independent experiments. Tensin1 mutant binding experiments (Supplemental Fig. 2) were carried out using immobilized integrin beta1A peptide (residues 779–790) at a single fixed protein concentration (10 µM) and thus give only a qualitative measure of binding affinity.


   Footnotes
 
3 Present address: Protein Sciences, Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Dr., San Diego, CA 92121, USA. Back

Supplemental material: see www.proteinscience.org

Reprint requests to: Robert C. Liddington, Cancer Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA; e-mail: rlidding{at}burnham.org; fax: (858) 713-9925.

Abbreviations: PTB, phosphotyrosine-binding (domain); ECM, extracellular matrix; SH2, Src homology 2; PIPKI{gamma}, phosphatidylinositol phosphate kinase type I{gamma} ; EDC, N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS, N-hydroxysuccinimide; RMSD, root mean square deviation; SPR, surface plasmon resonance; DSC, differential scanning calorimetry.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.072798707.


   Acknowledgments
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgments
References
 
We thank the NIH Cell Migration Consortium (to R.C.L.) and the NIH GM22289 (S.L.) for financial support, and the beamline support team at the SSRL, a national synchrotron facility supported by the NIH and DOE.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and Methods
 Acknowledgments
 References
 
Arias-Salgado, E.G., Lizano, S., Sarkar, S., Brugge, J.S., Ginsberg, M.H., and Shattil, S.J. 2003. Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc. Natl. Acad. Sci. 100: 13298–13302.[Abstract/Free Full Text]

Auger, K.R., Songyang, Z., Lo, S.H., Roberts, T.M., and Chen, L.B. 1996. Platelet-derived growth factor-induced formation of tensin and phosphoinositide 3-kinase complexes. J. Biol. Chem. 271: 23452–23457.[Abstract/Free Full Text]

Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54: 905–921.[CrossRef][Medline]

Calderwood, D.A., Zent, R., Grant, R., Rees, D.J.G., Hynes, R.O., and Ginsberg, M.H. 1999. The talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation. J. Biol. Chem. 274: 28071–28074.[Abstract/Free Full Text]

Calderwood, D.A., Yan, B., de Pereda, J.M., Garcia-Alvarez, B., Fujioka, Y., Liddington, R.C., and Ginsberg, M.H. 2002. The phosphotyrosine binding (PTB)-like domain of talin activates integrins. J. Biol. Chem. 277: 21749–21758.[Abstract/Free Full Text]

Calderwood, D.A., Fujioka, Y., de Pereda, J.M., Garcia-Alvarez, B., Nakamoto, T., Margolis, B., McGlade, C.J., Liddington, R.C., and Ginsberg, M.H. 2003. Integrin beta cytoplasmic domain interactions with phosphotyrosine-binding domains: A structural prototype for diversity in integrin signaling. Proc. Natl. Acad. Sci. 100: 2272–2277.[Abstract/Free Full Text]

Chen, H. and Lo, S.H. 2003. Regulation of tensin-promoted cell migration by its focal adhesion binding and Src homology domain 2. Biochem. J. 370: 1039–1045.[CrossRef][Medline]

Chen, H., Duncan, I.C., Bozorgchami, H., and Lo, S.H. 2002. Tensin1 and a previously undocumented family member, tensin2, positively regulate cell migration. Proc. Natl. Acad. Sci. 99: 733–738.[Abstract/Free Full Text]

Chuang, J.Z., Lin, D.C., and Lin, S. 1995. Molecular cloning, expression, and mapping of the high affinity actin-capping domain of chicken cardiac tensin. J. Cell Biol. 128: 1095–1109.[Abstract/Free Full Text]

Cui, Y., Liao, Y.C., and Lo, S.H. 2004. Epidermal growth factor modulates tyrosine phosphorylation of a novel tensin family member, tensin3. Mol. Cancer Res. 2: 225–232.[Abstract/Free Full Text]

Dhalluin, C., Yan, K., Plotnikova, O., Lee, K.W., Zeng, L., Kuti, M., Mujtaba, S., Goldfarb, M.P., and Zhou, M.M. 2000. Structural basis of SNT PTB domain interactions with distinct neurotrophic receptors. Mol. Cell 6: 921–929.[CrossRef][Medline]

Eck, M.J., Dhe-Paganon, S., Trub, T., Nolte, R.T., and Shoelson, S.E. 1996. Structure of the IRS-1 PTB domain bound to the juxtamembrane region of the insulin receptor. Cell 85: 695–705.[CrossRef][Medline]

Farooq, A., Zeng, L., Yan, K.S., Ravichandran, K.S., and Zhou, M.M. 2003. Coupling of folding and binding in the PTB domain of the signaling protein Shc. Structure 11: 905–913.[Medline]

Garcia-Alvarez, B., de Pereda, J.M., Calderwood, D.A., Ulmer, T.S., Critchley, D., Campbell, I.D., Ginsberg, M.H., and Liddington, R.C. 2003. Structural determinants of integrin recognition by talin. Mol. Cell 11: 49–58.[CrossRef][Medline]

Hynes, R.O. 2002. Integrins: Bidirectional, allosteric signaling machines. Cell 110: 673–687.[CrossRef][Medline]

Katz, B.Z., Zamir, E., Bershadsky, A., Kam, Z., Yamada, K.M., and Geiger, B. 2000. Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions. Mol. Biol. Cell 11: 1047–1060.[Abstract/Free Full Text]

Kim, M., Carman, C.V., and Springer, T.A. 2003. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins. Science 301: 1720–1725.[Abstract/Free Full Text]

Kraulis, P.J. 1991. MOLSCRIPT—a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24: 946–950.[CrossRef]

Laskowski, R.J., MacArthur, N.W., Moss, D.S., and Thornton, J.M. 1993. PROCHECK: A program to check stereochemical quality of protein structures. J. Appl. Crystallogr. 26: 283–290.[CrossRef]

Li, S.C., Zwahlen, C., Vincent, S.J., McGlade, C.J., Kay, L.E., Pawson, T., and Forman-Kay, J.D. 1998. Structure of a Numb PTB domain-peptide complex suggests a basis for diverse binding specificity. Nat. Struct. Biol. 5: 1075–1083.[CrossRef][Medline]

Ling, K., Doughman, R.L., Iyer, V.V., Firestone, A.J., Bairstow, S.F., Mosher, D.F., Schaller, M.D., and Anderson, R.A. 2003. Tyrosine phosphorylation of type I{gamma} phosphatidylinositol phosphate kinase by Src regulates an integrin-talin switch. J. Cell Biol. 163: 1339–1349.[Abstract/Free Full Text]

Lo, S.H. and Lo, T.B. 2002. Cten, a COOH-terminal tensin-like protein with prostate restricted expression, is down-regulated in prostate cancer. Cancer Res. 62: 4217–4221.[Abstract/Free Full Text]

Lo, S.H., Janmey, P.A., Hartwig, J.H., and Chen, L.B. 1994. Interactions of tensin with actin and identification of its three distinct actin-binding domains. J. Cell Biol. 125: 1067–1075.[Abstract/Free Full Text]

Merritt, E.A. and Murphy, M.E.P. 1994. Raster3D version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr. D50: 869–873.

Otwinowski, Z. and Minor, W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307–326.

Pankov, R., Cukierman, E., Katz, B.Z., Matsumoto, K., Lin, D.C., Lin, S., Hahn, C., and Yamada, K.M. 2000. Integrin dynamics and matrix assembly: Tensin-dependent translocation of {alpha}5beta1 integrins promotes early fibronectin fibrillogenesis. J. Cell Biol. 148: 1075–1090.[Abstract/Free Full Text]

Roussel, A. and Cambillau, C. 1991. Silicon graphics geometry partners directory. p. 86. Silicon Graphics, Mountain View, CA.

Shi, N., Ye, S., Bartlam, M., Yang, M., Wu, J., Liu, Y., Sun, F., Han, X., Peng, X., Qiang, B., et al. 2004. Structural basis for the specific recognition of RET by the Dok1 phosphotyrosine binding domain. J. Biol. Chem. 279: 4962–4969.[Abstract/Free Full Text]

Stolt, P.C., Jeon, H., Song, H.K., Herz, J., Eck, M.J., and Blacklow, S.C. 2003. Origins of peptide selectivity and phosphoinositide binding revealed by structures of disabled-1 PTB domain complexes. Structure 11: 569–579.[Medline]

Tadokoro, S., Shattil, S.J., Eto, K., Tai, V., Liddington, R.C., de Pereda, J.M., Ginsberg, M.H., and Calderwood, D.A. 2003. Talin binding to integrin beta tails: A final common step in integrin activation. Science 302: 103–106.[Abstract/Free Full Text]

Tapley, P., Horwitz, A., Buck, C., Duggan, K., and Rohrschneider, L. 1989. Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts. Oncogene 4: 325–333.[Medline]

Terwilliger, T.C. and Berendzen, J. 1999. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55: 849–861.[CrossRef][Medline]

Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E.F., and Qin, J. 2002. A structural mechanism of integrin {alpha}IIbbeta3 "inside-out" activation as regulated at its cytoplasmic face. Cell 110: 587–597.[CrossRef][Medline]

Volberg, T., Romer, L., Zamir, E., and Geiger, B. 2001. pp60(c-src) and related tyrosine kinases: A role in the assembly and reorganization of matrix adhesions. J. Cell Sci. 114: 2279–2289.[Medline]

Wegener, K.L., Partridge, A.W., Han, J., Pickford, A.R., Liddington, R.C., Ginsberg, M.H., and Campbell, I.D. 2007. Structural basis of integrin activation by talin. Cell 128: 171–182.[CrossRef][Medline]

Xiao, T., Takagi, J., Coller, B.S., Wang, J.H., and Springer, T.A. 2004. Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics. Nature 432: 59–67.[CrossRef][Medline]

Zaidel-Bar, R., Ballestrem, C., Kam, Z., and Geiger, B. 2003. Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J. Cell Sci. 116: 4605–4613.[Abstract/Free Full Text]

Zamir, E., Katz, B.Z., Aota, S., Yamada, K.M., Geiger, B., and Kam, Z. 1999. Molecular diversity of cell-matrix adhesions. J. Cell Sci. 112: 1655–1669.[Abstract]

Zhang, Z., Lee, C.H., Mandiyan, V., Borg, J.P., Margolis, B., Schlessinger, J., and Kuriyan, J. 1997. Sequence-specific recognition of the internalization motif of the Alzheimer's amyloid precursor protein by the X11 PTB domain. EMBO J. 16: 6141–6150.[CrossRef][Medline]

Zhou, M.M., Ravichandran, K.S., Olejniczak, E.F., Petros, A.M., Meadows, R.P., Sattler, M., Harlan, J.E., Wade, W.S., Burakoff, S.J., and Fesik, S.W. 1995a. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378: 584–592.[CrossRef][Medline]

Zhou, M.M., Ravichandran, K.S., Olejniczak, E.F., Petros, A.M., Meadows, R.P., Sattler, M., Harlan, J.E., Wade, W.S., Burakoff, S.J., and Fesik, S.W. 1995b. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature 378: 584–589.[CrossRef][Medline]

Zhou, M.M., Huang, B., Olejniczak, E.T., Meadows, R.P., Shuker, S.B., Miyazaki, M., Trub, T., Shoelson, S.E., and Fesik, S.W. 1996. Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain. Nat. Struct. Biol. 3: 388–393.[CrossRef][Medline]

Zwahlen, C., Li, S.C., Kay, L.E., Pawson, T., and Forman-Kay, J.D. 2000. Multiple modes of peptide recognition by the PTB domain of the cell fate determinant Numb. EMBO J. 19: 1505–1515.[CrossRef][Medline]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?



This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow All Versions of this Article:
ps.072798707v1
16/6/1223    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McCleverty, C. J.
Right arrow Articles by Liddington, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McCleverty, C. J.
Right arrow Articles by Liddington, R. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS