Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.
Shattil SJ, Kim C, Ginsberg MH (2010) The final steps of integrin activation: the end game. Nat Rev Mol Cell Biol 11: 288–300.
[3]
Bertagnolli ME, Locke SJ, Hensler ME, Bray PF, Beckerle MC (1993) Talin distribution and phosphorylation in thrombin-activated platelets. J Cell Sci 106(Pt 4): 1189–1199.
[4]
Goult BT, Bate N, Anthis NJ, Wegener KL, Gingras AR, et al. (2009) The structure of an interdomain complex that regulates talin activity. J Biol Chem 284: 15097–15106.
[5]
Goksoy E, Ma YQ, Wang X, Kong X, Perera D, et al. (2008) Structural basis for the autoinhibition of talin in regulating integrin activation. Mol Cell 31: 124–133.
[6]
Calderwood DA (2004) Integrin activation. J Cell Sci 117: 657–666.
[7]
Roberts GC, Critchley DR (2009) Structural and biophysical properties of the integrin-associated cytoskeletal protein talin. Biophys Rev 1: 61–69.
[8]
Harburger DS, Calderwood DA (2009) Integrin signalling at a glance. J Cell Sci 122: 159–163.
[9]
Chrzanowska-Wodnicka M, Smyth SS, Schoenwaelder SM, Fischer TH, White GC 2nd (2005) Rap1b is required for normal platelet function and hemostasis in mice. J Clin Invest 115: 680–687.
[10]
Cifuni SM, Wagner DD, Bergmeier W (2008) CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin alphaIIbbeta3 in platelets. Blood 112: 1696–1703.
[11]
Watanabe N, Bodin L, Pandey M, Krause M, Coughlin S, et al. (2008) Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin alphaIIbbeta3. J Cell Biol 181: 1211–1222.
[12]
Han J, Lim CJ, Watanabe N, Soriani A, Ratnikov B, et al. (2006) Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3. Curr Biol 16: 1796–1806.
[13]
Lee HS, Lim CJ, Puzon-McLaughlin W, Shattil SJ, Ginsberg MH (2009) RIAM activates integrins by linking talin to ras GTPase membrane-targeting sequences. J Biol Chem 284: 5119–5127.
[14]
Kalli AC, Wegener KL, Goult BT, Anthis NJ, Campbell ID, et al. (2010) The structure of the talin/integrin complex at a lipid bilayer: an NMR and MD simulation study. Structure 18: 1280–1288.
[15]
Anthis NJ, Wegener KL, Ye F, Kim C, Goult BT, et al. (2009) The structure of an integrin/talin complex reveals the basis of inside-out signal transduction. Embo J 28: 3623–3632.
[16]
Lau TL, Kim C, Ginsberg MH, Ulmer TS (2009) The structure of the integrin alphaIIbbeta3 transmembrane complex explains integrin transmembrane signalling. Embo J 28: 1351–1361.
[17]
Ye F, Hu G, Taylor D, Ratnikov B, Bobkov AA, et al. (2010) Recreation of the terminal events in physiological integrin activation. J Cell Biol 188: 157–173.
[18]
Moser M, Nieswandt B, Ussar S, Pozgajova M, Fassler R (2008) Kindlin-3 is essential for integrin activation and platelet aggregation. Nat Med 14: 325–330.
[19]
Moser M, Bauer M, Schmid S, Ruppert R, Schmidt S, et al. (2009) Kindlin-3 is required for beta2 integrin-mediated leukocyte adhesion to endothelial cells. Nat Med 15: 300–305.
[20]
Moser M, Legate KR, Zent R, Fassler R (2009) The tail of integrins, talin, and kindlins. Science 324: 895–899.
[21]
Ma YQ, Qin J, Wu C, Plow EF (2008) Kindlin-2 (Mig-2): a co-activator of beta3 integrins. J Cell Biol 181: 439–446.
[22]
Harburger DS, Bouaouina M, Calderwood DA (2009) Kindlin-1 and -2 directly bind the C-terminal region of beta integrin cytoplasmic tails and exert integrin-specific activation effects. J Biol Chem 284: 11485–11497.
[23]
Shi X, Ma YQ, Tu Y, Chen K, Wu S, et al. (2007) The MIG-2/integrin interaction strengthens cell-matrix adhesion and modulates cell motility. J Biol Chem 282: 20455–20466.
[24]
Tu Y, Wu S, Shi X, Chen K, Wu C (2003) Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 113: 37–47.
[25]
Ye F, Petrich BG (2011) Kindlin: helper, co-activator, or booster of talin in integrin activation? Curr Opin Hematol 18: 356–360.
[26]
Vu TK, Hung DT, Wheaton VI, Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64: 1057–1068.
[27]
Hughes PE, Diaz-Gonzalez F, Leong L, Wu C, McDonald JA, et al. (1996) Breaking the integrin hinge. A defined structural constraint regulates integrin signaling. J Biol Chem 271: 6571–6574.
[28]
O'Toole TE, Katagiri Y, Faull RJ, Peter K, Tamura R, et al. (1994) Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol 124: 1047–1059.
[29]
Shattil SJ, Hoxie JA, Cunningham M, Brass LF (1985) Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation. J Biol Chem 260: 11107–11114.
[30]
Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR (1999) Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 103: 879–887.
[31]
Kao FT, Puck TT (1968) Genetics of somatic mammalian cells, VII. Induction and isolation of nutritional mutants in Chinese hamster cells. Proc Natl Acad Sci U S A 60: 1275–1281.
[32]
DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, et al. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol Cell Biol 7: 379–387.
[33]
Todaro GJ, Green H (1963) Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17: 299–313.
[34]
Yao F, Svensjo T, Winkler T, Lu M, Eriksson C, et al. (1998) Tetracycline repressor, tetR, rather than the tetR-mammalian cell transcription factor fusion derivatives, regulates inducible gene expression in mammalian cells. Hum Gene Ther 9: 1939–1950.
[35]
Tadokoro S, Shattil SJ, Eto K, Tai V, Liddington RC, et al. (2003) Talin binding to integrin beta tails: a final common step in integrin activation. Science 302: 103–106.
[36]
Qin XF, An DS, Chen IS, Baltimore D (2003) Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A 100: 183–188.
[37]
Arias-Salgado EG, Lizano S, Sarkar S, Brugge JS, Ginsberg MH, et al. (2003) Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc Natl Acad Sci U S A 100: 13298–13302.
[38]
Arias-Salgado EG, Lizano S, Shattil SJ, Ginsberg MH (2005) Specification of the direction of adhesive signaling by the integrin beta cytoplasmic domain. J Biol Chem 280: 29699–29707.
[39]
Brass LF (2009) Molecular basis for platelet activation. In: Hoffman R, Benz E, Shattil S, Furie B, Silberstein L, McGlave P, Heslop H, editors. Hematology. Basic Principles and Practice. pp. 1793–1803. 5 Ed.
[40]
Bottcher RT, Lange A, Fassler R (2009) How ILK and kindlins cooperate to orchestrate integrin signaling. Curr Opin Cell Biol 21: 670–675.
[41]
Bledzka K, Bialkowska K, Nie H, Qin J, Byzova T, et al. (2010) Tyrosine phosphorylation of integrin beta3 regulates kindlin-2 binding and integrin activation. J Biol Chem 285: 30370–30374.
[42]
Malinin NL, Plow EF, Byzova TV (2010) Kindlins in FERM adhesion. Blood 115: 4011–4017.
[43]
Lemmon MA (2007) Pleckstrin homology (PH) domains and phosphoinositides. Biochem Soc Symp 81–93.
[44]
Qu H, Tu Y, Shi X, Larjava H, Saleem MA, et al. (2011) Kindlin-2 regulates podocyte adhesion and fibronectin matrix deposition through interactions with phosphoinositides and integrins. J Cell Sci 124: 879–891.
[45]
O'Toole TE, Loftus JC, Plow EF, Glass AA, Harper JR, et al. (1989) Efficient surface expression of platelet GPIIb-IIIa requires both subunits. Blood 74: 14–18.
[46]
Choy MK, Movassagh M, Siggens L, Vujic A, Goddard M, et al. (2010) High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-kappaB-complex-dependent gene expression in human heart failure. Genome Med 2: 37.
[47]
Ammoun S, Cunliffe CH, Allen JC, Chiriboga L, Giancotti FG, et al. (2010) ErbB/HER receptor activation and preclinical efficacy of lapatinib in vestibular schwannoma. Neuro Oncol 12: 834–843.
[48]
Hiatt SM, Shyu YJ, Duren HM, Hu CD (2008) Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans. Methods 45: 185–191.
[49]
Kerppola TK (2008) Bimolecular fluorescence complementation: visualization of molecular interactions in living cells. Methods Cell Biol 85: 431–470.
[50]
Kodama Y, Hu CD (2010) An improved bimolecular fluorescence complementation assay with a high signal-to-noise ratio. Biotechniques 49: 793–805.
[51]
Calderwood DA, Zent R, Grant R, Rees DJ, Hynes RO, et al. (1999) The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation. J Biol Chem 274: 28071–28074.
[52]
Bouaouina M, Goult BT, Huet-Calderwood C, Bate N, Brahme NN, et al. (2012) A conserved lipid-binding loop in the Kindlin FERM F1 domain is required for Kindlin-mediated alphaIIbbeta3 integrin Co-activation. J Biol Chem. in press.
[53]
Perera HD, Ma YQ, Yang J, Hirbawi J, Plow EF, et al. (2011) Membrane binding of the N-terminal ubiquitin-like domain of kindlin-2 is crucial for its regulation of integrin activation. Structure 19: 1664–1671.
[54]
Bialkowska K, Ma YQ, Bledzka K, Sossey-Alaoui K, Izem L, et al. (2010) The integrin co-activator Kindlin-3 is expressed and functional in a non-hematopoietic cell, the endothelial cell. J Biol Chem 285: 18640–18649.
