Title: Kindlin-1 and -2 Directly Bind the C-terminal Region of β Integrin Cytoplasmic Tails and Exert Integrin-specific Activation Effects
Abstract: Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Talin binding to integrin β tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin β tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin β tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr795), a preceding threonine-containing region (Thr788 and Thr789) of the integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp612 and kindlin-2 Trp615) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin β3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous α5β1 or stably expressed αIIbβ3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate αIIbβ3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate β1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects. Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Talin binding to integrin β tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin β tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with β1 and β3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin β tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr795), a preceding threonine-containing region (Thr788 and Thr789) of the integrin β1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp612 and kindlin-2 Trp615) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin β3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous α5β1 or stably expressed αIIbβ3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate αIIbβ3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate β1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects. Integrins are a family of αβ heterodimeric transmembrane receptors that mediate cell adhesion to extracellular matrix, cell surface, or soluble protein ligands and modulate a variety of intracellular signaling cascades. A key feature of integrins is their ability to dynamically regulate their affinity for extracellular ligands. In a tightly regulated process generally termed integrin activation, intracellular signals that impinge upon the β subunit cytoplasmic tail induce conformational rearrangements in the integrin extracellular domains, increasing the binding affinity for extracellular ligands (1.Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (381) Google Scholar, 2.Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6687) Google Scholar, 3.Luo B. Carman C. Springer T. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1198) Google Scholar). Ligand-bound integrins then recruit additional signaling, adaptor, and cytoskeletal proteins to the integrin cytoplasmic domains, providing mechanical connections to the actin cytoskeleton and a link to a variety of signal transduction pathways (2.Hynes R.O. Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6687) Google Scholar, 3.Luo B. Carman C. Springer T. Annu. Rev. Immunol. 2007; 25: 619-647Crossref PubMed Scopus (1198) Google Scholar, 4.Harburger D. Calderwood D. J. Cell Sci. 2009; 122: 159-163Crossref PubMed Scopus (612) Google Scholar, 5.Zaidel-Bar R. Itzkovitz S. Ma'ayan A. Iyengar R. Geiger B. Nat. Cell Biol. 2007; 9: 858-867Crossref PubMed Scopus (857) Google Scholar, 6.Liu S. Calderwood D.A. Ginsberg M.H. J. Cell Sci. 2000; 113: 3563-3571Crossref PubMed Google Scholar, 7.Evans E. Calderwood D. Science. 2007; 316: 1148-1153Crossref PubMed Scopus (388) Google Scholar, 8.Ginsberg M. Partridge A. Shattil S. Curr. Opin. Cell Biol. 2005; 17: 509-516Crossref PubMed Scopus (382) Google Scholar). Recent years have seen significant advances in our understanding of integrin activation. Notable among these is the identification of the actin- and integrin-binding protein talin as a key integrin activator (1.Calderwood D.A. J. Cell Sci. 2004; 117: 657-666Crossref PubMed Scopus (381) Google Scholar, 9.Critchley D. Gingras A. J. Cell Sci. 2008; 121: 1345-1347Crossref PubMed Scopus (166) Google Scholar). The 50-kDa talin head contains the principal integrin-binding site, and expression of the talin head is sufficient to activate β1 and β3 integrins (10.Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 11.Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). The talin head contains a FERM (four point one ezrin radixin moesin) domain. FERM domains consist of trefoil arrangement of three subdomains (F1, F2, and F3). The phosphotyrosine-binding domain-like F3 subdomain of the talin FERM directly binds a conserved NP(I/L)Y motif in integrin β tails, and this interaction is necessary for integrin activation in vitro and in vivo (10.Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 12.Tadokoro 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, 13.Nieswandt B. Moser M. Pleines I. Varga-Szabo D. Monkley S. Critchley D. Fässler R. J. Exp. Med. 2007; 204: 3113-3118Crossref PubMed Scopus (196) Google Scholar, 14.Petrich B. Fogelstrand P. Partridge A. Yousefi N. Ablooglu A. Shattil S. Ginsberg M. J. Clin. Investig. 2007; 117: 2250-2259Crossref PubMed Scopus (104) Google Scholar, 15.Wegener K.L. Partridge A.W. Han J. Pickford A.R. Liddington R.C. Ginsberg M.H. Campbell I.D. Cell. 2007; 128: 171-182Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, 16.Garcia-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, 17.Calderwood D.A. Yan B. de Pereda J.M. Alvarez B.G. Fujioka Y. Liddington R.C. Ginsberg M.H. J. Biol. Chem. 2002; 277: 21749-21758Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 18.Petrich B.G. Marchese P. Ruggeri Z.M. Spiess S. Weichert R.A. Ye F. Tiedt R. Skoda R.C. Monkley S.J. Critchley D.R. Ginsberg M.H. J. Exp. Med. 2007; 204: 3103-3111Crossref PubMed Scopus (215) Google Scholar, 19.Czuchra A. Meyer H. Legate K.R. Brakebusch C. Fassler R. J. Cell Biol. 2006; 174: 889-899Crossref PubMed Scopus (87) Google Scholar). However, although abundant evidence supports the importance of talin binding to integrin β tails during integrin activation, differences in sensitivity of integrins to talin activation and submaximal activation by overexpressed talin suggested that other activating factors may cooperate with talin (10.Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 20.Ma Y.Q. Qin J. Plow E.F. J. Thromb. Haemostasis. 2007; 5: 1345-1352Crossref PubMed Scopus (145) Google Scholar). In an attempt to identify and characterize potential co-activators, we investigated the kindlin family of FERM domain-containing proteins. Kindlin family proteins (21.Larjava H. Plow E.F. Wu C. EMBO Rep. 2008; 9: 1203-1208Crossref PubMed Scopus (200) Google Scholar) were first characterized in nematodes where the sole Caenorhabditis elegans kindlin, UNC-112, was identified in an embryonic screen for defective motility and shown to be essential for the assembly of proper cell-matrix adhesion structures, where it normally co-localized with β integrin (22.Williams B.D. Waterston R.H. J. Cell Biol. 1994; 124: 475-490Crossref PubMed Scopus (265) Google Scholar, 23.Mackinnon A.C. Qadota H. Norman K.R. Moerman D.G. Williams B.D. Curr. Biol. 2002; 12: 787-797Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 24.Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (164) Google Scholar). UNC-112 is conserved across many species, because the nematode, fly, and human homologs are ∼60% similar (∼41% identical) over their entire length (24.Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. J. Cell Biol. 2000; 150: 253-264Crossref PubMed Scopus (164) Google Scholar). Humans express three known homologs of UNC-112: kindlin-1 (Kindlerin, URP1, and FERMT1), kindlin-2 (Mig2 and mig-2), and kindlin-3 (Mig2B and URP2) (25.Weinstein E.J. Bourner M. Head R. Zakeri H. Bauer C. Mazzarella R. Biochim. Biophys. Acta. 2003; 1637: 207-216Crossref PubMed Scopus (67) Google Scholar, 26.Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 27.Boyd R.S. Adam P.J. Patel S. Loader J.A. Berry J. Redpath N.T. Poyser H.R. Fletcher G.C. Burgess N.A. Stamps A.C. Hudson L. Smith P. Griffiths M. Willis T.G. Karran E.L. Oscier D.G. Catovsky D. Terrett J.A. Dyer M.J. Leukemia. 2003; 17: 1605-1612Crossref PubMed Scopus (65) Google Scholar). Kindlin-1 and -2 are most closely related, sharing 60% identity and 74% similarity, whereas kindlin-3 shares 53% identity and 69% similarity to kindlin-1 and 49% identity and 67% similarity to kindlin-2 (28.Ussar S. Wang H.V. Linder S. Fassler R. Moser M. Exp. Cell Res. 2006; 312: 3142-3151Crossref PubMed Scopus (197) Google Scholar). The kindlin proteins all contain a predicted Pleckstrin homology domain and a FERM domain that is most closely related to the talin FERM domain, particularly within the integrin-binding F3 subdomain (29.Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Based on this sequence similarity we proposed that kindlin FERM domains may directly bind integrin β tails, and we previously showed that kindlin-1 could be pulled down from cell lysates using recombinant integrin β1 and β3 tails and that kindlin-1 co-localized with integrins in focal adhesions (29.Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). A similar localization was reported for kindlin-2 (26.Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 30.Shi X. Ma Y.Q. Tu Y. Chen K. Wu S. Fukuda K. Qin J. Plow E.F. Wu C. J. Biol. Chem. 2007; 282: 20455-20466Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), and recent reports provided clear evidence implicating kindlin-2 and kindlin-3 in regulation of integrin activation (31.Moser M. Nieswandt B. Ussar S. Pozgajova M. Fassler R. Nat. Med. 2008; 14: 325-330Crossref PubMed Scopus (521) Google Scholar, 32.Ma Y.Q. Qin J. Wu C. Plow E.F. J. Cell Biol. 2008; 181: 439-446Crossref PubMed Scopus (266) Google Scholar, 33.Montanez E. Ussar S. Schifferer M. Bosl M. Zent R. Moser M. Fassler R. Genes Dev. 2008; 22: 1325-1330Crossref PubMed Scopus (325) Google Scholar). Here, we have used integrin pulldown assays to demonstrate direct binding of full-length kindlin-1 to the cytoplasmic tails of β1A and β3 integrins and to identify key binding residues within the integrin tails and the kindlin F3 subdomain. We confirm that these interactions are important for recruiting kindlin-1 to focal adhesions and show that, contrary to expectations, overexpressed kindlin-1 or -2 inhibit β1 and β3 integrin activation. Overexpressed kindlin-1 or -2 can, however, cooperate with expressed talin head to activate β3 but not β1 integrins. We therefore provide the first data suggesting that kindlin-1 and -2 effects on integrin activation may show β subunit specificity. Antibodies—Ligand-mimetic anti-αIIbβ3 PAC1 (BD Biosciences), anti-hamster α5β1 PB1 (Developmental Studies Hybridoma Bank), anti-talin 8d4 (Sigma), goat polyclonal anti-human kindlin-2 (Santa Cruz Biotechnology), rabbit polyclonal anti-GST 3The abbreviations used are: GST, glutathione S-transferase; CHO, Chinese hamster ovary; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorter; MFI, mean fluorescence intensity. (Chemicon), mouse anti-FLAG (Sigma-Aldrich), and goat anti-GFP (Rockland) were purchased. Generation of anti-human kindlin-1 was previously described (29.Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The anti-αIIbβ3 monoclonal antibody D57 has been described previously (34.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (573) Google Scholar). The α5β1-specific inhibitor 3F compound was kindly provided by Dr. Kessler (35.Heckmann D. Meyer A. Marinelli L. Zahn G. Stragies R. Kessler H. Angew Chem. Int. Ed Engl. 2007; 46: 3571-3574Crossref PubMed Scopus (81) Google Scholar). GST-fibronectin type III repeats 9-11 (FN9-11) have been described previously (36.Calderwood D.A. Tai V. Di Paolo G. De Camilli P. Ginsberg M.H. J. Biol. Chem. 2004; 279: 28889-28895Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Protein Production—Recombinant His-tagged human integrin cytoplasmic tail model proteins were produced and purified as previously described (37.Lad Y. Harburger D.S. Calderwood D.A. Methods Enzymol. 2007; 426: 69-84Crossref PubMed Scopus (31) Google Scholar). Human kindlin-1 and -2 full length (amino acids 1-677 and 1-680, respectively), ΔFERM (amino acids 1-181 and 1-183, respectively), and FERM (amino acids 193-677 and 195-680, respectively) constructs were generated by polymerase chain reaction from human cDNAs generously provided by Mary Beckerle (University of Utah) and subcloned into pGEX4T-3 (Amersham Biosciences) and pEGFP-C1 (BD Biosciences), pFLAG-CMV2 (Sigma-Aldrich), or pDsRed monomer (Clontech) mammalian expression vectors. Point mutations were introduced by QuikChange™ site-directed mutagenesis (Stratagene). All of the inserts were verified by DNA sequencing. GST fusion proteins were produced in Escherichia coli BL21 cells or Rosetta cells (Novagen) and purified on glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. GFP-fused mouse talin1 head (amino acids 1-433) was generated as previously described (10.Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Binding Assays and Analysis—Binding assays using recombinant integrin cytoplasmic tails bound to His-bind resin (Novagen) were performed as previously described (37.Lad Y. Harburger D.S. Calderwood D.A. Methods Enzymol. 2007; 426: 69-84Crossref PubMed Scopus (31) Google Scholar). Briefly, we expressed integrin β tails in a pET15b vector containing an N-terminal His tag sequence followed by a thrombin cleavage site, a cysteine-residue linker, a coiled-coil-forming helical sequence, a four-residue glycine spacer, and the integrin cytoplasmic domain (human β1A residues 751-798 or β3 residues 715-762). Integrin constructs were expressed in BL21 cells (Novagen), purified, and coated to Ni2+-charged nitrilotriacetic acid resin (Novagen) as previously described (37.Lad Y. Harburger D.S. Calderwood D.A. Methods Enzymol. 2007; 426: 69-84Crossref PubMed Scopus (31) Google Scholar). For FLAG fusion proteins, Chinese hamster ovary (CHO) cells were transiently transfected with 3 μg of DNA using Lipofectamine™ (Invitrogen), and the cells were harvested 24 h later and lysed. Cell lysates or bacterially purified proteins were incubated with integrin tails bound to beads (24 h for lysates and 2 h for purified proteins), washed, resuspended in SDS sample buffer, heated for 5 min at 95 °C, and analyzed for binding on 4-20% Tris-glycine SDS-polyacrylamide gradient gel (Invitrogen). The loading percentage lanes in the Western blot images represent input lysates or purified protein as a percentage of total mixed with integrin tails and serve as a reference for relative protein amounts loaded into the assay. Immunofluorescence—NIH 3T3 cells were transiently transfected with 3 μg of indicated cDNAs using Lipofectamine™ (Invitrogen). 24 h after transfection, the cells were detached and allowed to readhere and spread on fibronectin-coated (5 μg/ml) coverslips. After 4 h of plating, the cells were fixed, permeabilized, and stained with anti-vinculin as described previously (38.Kiema T. Lad Y. Jiang P. Oxley C.L. Baldassarre M. Wegener K.L. Campbell I.D. Ylanne J. Calderwood D.A. Mol. Cell. 2006; 21: 337-347Abstract Full Text Full Text PDF PubMed Scopus (307) Google Scholar, 39.Lad, Y., Jiang, P., Ruskamo, S., Harburger, D. S., Ylanne, J., Campbell, I. D., and Calderwood, D. A. (2008) J. Biol. Chem.Google Scholar). Analysis of Integrin Activation—The activation state of endogenous α5β1 or stably overexpressed αIIbβ3 integrins in CHO cells transiently expressing DsRed-tagged kindlins and/or GFP-talin head was assessed in three-color FACS assays using a modification of previously described methods (10.Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 12.Tadokoro 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, 36.Calderwood D.A. Tai V. Di Paolo G. De Camilli P. Ginsberg M.H. J. Biol. Chem. 2004; 279: 28889-28895Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). FACS data analysis was carried out using FlowJo FACS analysis software and statistical analysis using Graphpad Prism. Briefly, to assess α5β1 activation CHO cells were co-transfected with the indicated GFP and DsRed expression constructs using Lipofectamine™ 2000 (Invitrogen), and 24 h later the cells were suspended and incubated with biotinylated recombinant GST-FN9-11 in the presence or absence of integrin inhibitor (1 mm RGD peptide (Sigma) or 0.1 μm 3F α5β1 inhibitor (35.Heckmann D. Meyer A. Marinelli L. Zahn G. Stragies R. Kessler H. Angew Chem. Int. Ed Engl. 2007; 46: 3571-3574Crossref PubMed Scopus (81) Google Scholar)). For each preparation of biotinylated GST-FN9-11, the effective concentration was determined by titration. The cells were washed, and bound FN9-11 was detected with allophycocyanin-conjugated streptavidin. FN9-11 binding to doubly transfected (GFP-positive and DsRed-positive) cells was assessed on a FACSCalibur instrument (BD Biosciences). The α5β1 integrin expression was assessed in parallel by staining with antibody PB1 (40.Brown P.J. Juliano R.L. Science. 1985; 228: 1448-1451Crossref PubMed Scopus (131) Google Scholar). The α5β1 activation index was defined as AI = (F - Fo)/(Fintegrin), where F is the geometric mean fluorescence intensity (MFI) of FN9-11 binding, Fo is the MFI of FN9-11 binding in presence of inhibitor, and Fintegrin is the normalized MFI of PB1 binding to transfected cells. The activation state of αIIbβ3 integrins was assessed as described above but using the ligand-mimetic anti-αIIbβ3 monoclonal antibody PAC1 (11.Calderwood D.A. Zent R. Grant R. Rees D.J. Hynes R.O. Ginsberg M.H. J. Biol. Chem. 1999; 274: 28071-28074Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar, 12.Tadokoro 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, 36.Calderwood D.A. Tai V. Di Paolo G. De Camilli P. Ginsberg M.H. J. Biol. Chem. 2004; 279: 28889-28895Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 41.O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) in place of biotinylated FN9-11. αIIbβ3 integrin expression was assessed in parallel by staining with D57 antibody (34.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (573) Google Scholar). Bound PAC1 was detected using Alexa 647 fluorophore-conjugated goat anti-mouse IgM (Invitrogen). Activation of αIIbβ3 in doubly transfected (GFP-positive and Red-positive) cells was quantified in three-color flow cytometric assays, and an activation index was calculated as defined above where F is the MFI of PAC1 binding, Fo is the MFI of PAC1 binding in presence of RGD peptide, and Fintegrin is the MFI of D57 binding to transfected cells. Integrin expression ratio was defined for double expressing cells as follows: Integrin expression ratio = (Ftrans)/(Funtrans), where Ftrans is the geometric MFI of PB1 or D57 binding to double expressing cells, and Funtrans is the MFI of PB1 or D57 binding to untransfected cells. Kindlin-1 and -2 Bind Directly to β1A Integrin Tails—Our initial investigation of kindlin-integrin interactions demonstrated binding of FLAG-tagged full-length kindlin-1 from CHO cell lysates to recombinant models of β1A integrin cytoplasmic tails (29.Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Here we extended this analysis to kindlin-2 and showed that both full-length kindlin-1 and -2 bound β1A tails specifically because no binding of an irrelevant control FLAG-tagged protein (major vault protein (42.Kolli S. Zito C.I. Mossink M.H. Wiemer E.A. Bennett A.M. J. Biol. Chem. 2004; 279: 29374-29385Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar)) was observed, and the kindlins did not bind to αIIb tails (Fig. 1B). Binding of endogenous talin from the cell lysate to β1A but not αIIb tails was examined as an additional specificity control (Fig. 1B, upper panel). To determine whether the interaction between kindlins and integrin β tails is direct, we expressed and purified full-length kindlin-1 and -2 as GST fusion proteins from E. coli. Despite using a range of optimization approaches, including testing various bacterial strains, different induction conditions, and different growth temperatures, the yields of soluble GST-fused kindlin proteins were low (∼0.05 mg/liter of bacterial culture). These proteins were nonetheless sufficient for use in protein-protein interaction assays and revealed direct binding of kindlin-1 and -2 to β1A but not αIIb tails (Fig. 1, C and D). We were unable to quantify the affinity of the kindlin-integrin interaction because dose-response curves did not reach saturation at the available protein concentrations (supplemental Fig. S1). Nonetheless these assays suggest that kindlin-integrin interactions are direct but of relatively low affinity. The FERM Domain in Kindlin-1 and -2 Is Required for Binding to Integrin β1A Cytoplasmic Tails—To determine whether the kindlin FERM domain is sufficient for β1A binding, we expressed the isolated FLAG-tagged kindlin-1 and -2 FERM domains in CHO cells. Cell lysate pulldown assays using recombinant β1A tails revealed binding of kindlin-1-FERM and kindlin-2-FERM to β1A tails (Fig. 2, A and B). This binding was specific because no interaction with the αIIb tails was observed (data not shown) but was much less than that seen for the binding of full-length kindlins. The kindlin FERM domain is necessary for integrin binding because deletion of the FERM domain completely abrogated kindlin-1 and -2 binding to β1A tails (Fig. 2C). Thus the FERM domain of kindlin-1 and -2 is required for interaction with β1A integrin tails. This is consistent with the results of Shi et al. (30.Shi X. Ma Y.Q. Tu Y. Chen K. Wu S. Fukuda K. Qin J. Plow E.F. Wu C. J. Biol. Chem. 2007; 282: 20455-20466Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), who previously reported that the kindlin-2-FERM was required for β tail binding. However, although the kindlin FERM domain may be sufficient for integrin binding, the presence of more N-terminal portions of the protein appears to contribute to the interaction. A Partially Conserved Site in the F3 Subdomain of the Kindlin-1 and -2 FERM Domain Is Required for Binding to β1A Tails—Similarity between the talin FERM domain and the kindlin-1 FERM domain, particularly within the F3 subdomain (29.Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), prompted us to test whether kindlin-1 binds integrin β tails via its F3 subdomain, as talin does. We have shown that Arg358, Trp359, and Ile396 in the talin1-F3 subdomain are important for binding to integrin β tails, whereas Lys357 plays a lesser role (10.Bouaouina M. Lad Y. Calderwood D.A. J. Biol. Chem. 2008; 283: 6118-6125Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 12.Tadokoro 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, 16.Garcia-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). We therefore mutated the corresponding residues in the F3 subdomain of FLAG-kindlin-1 and tested binding to β1A tails. As shown in Fig. 3 (A and B), K610A, W612A, and I651A mutations strongly inhibited binding to β1A tails, whereas a Q611A substitution (predicted to correspond to talin Arg358) had no effect. Furthermore, when the W612A was introduced into GST-kindlin-1, the mutation was sufficient to strongly inhibit direct kindlin-1 binding to β1A tails (Fig. 3E). We also introduced corresponding mutations into kindlin-2 and tested their effect on β1A binding (Fig. 3, C, D, and F). As was seen for kindlin-1, a Q614A mutation in kindlin-2 did not substantially inhibit β1A binding, whereas W615A mutations strongly inhibited binding. However, in the case of kindlin-2, I654A mutation did not inhibit β1A binding. Together this reveals that, as was seen for talin-β tail interactions, the F3 subdomain is important for kindlin binding to β1A tails and that residues on a similar portion of the molecule are involved. However, the differences in specific residues whose mutation inhibits the interaction suggest subtle differences between the kindlin-1, kindlin-2, and talin interactions with β tails. Integrin Binding Activity Correlates with Kindlin-1 Targeting to Focal Adhesions—Focal adhesions are integrin-rich sites where adherent cells make strong contacts with the surrounding matrix (43.Brakebusch C. Fassler R. Cancer Metastasis Rev. 2005; 24: 403-411Crossref PubMed Scopus (176) Google Scholar). Previous studies have shown that kindlin-1 and -2 localize to focal adhesions (26.Tu Y. Wu S. Shi X. Chen K. Wu C. Cell. 2003; 113: 37-47Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 29.Kloeker S. Major M.B. Calderwood D.A. Ginsberg M.H. Jones D.A. Beckerle M.C. J. Biol. Chem. 2004; 279: 6824-6833Abstract Full Text Full Text PDF P