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Differences in self-association between kindlin-2 and kindlin-3 are associated with differential integrin binding

2020; Elsevier BV; Volume: 295; Issue: 32 Linguagem: Inglês

10.1074/jbc.ra120.013618

ISSN

1083-351X

Autores

Yasmin A. Kadry, Eesha M. Maisuria, Clotilde Huet-Calderwood, David Calderwood,

Tópico(s)

Glycosylation and Glycoproteins Research

Resumo

The integrin family of transmembrane adhesion receptors coordinates complex signaling networks that control the ability of cells to sense and communicate with the extracellular environment. Kindlin proteins are a central cytoplasmic component of these networks, directly binding integrin cytoplasmic domains and mediating interactions with cytoskeletal and signaling proteins. The physiological importance of kindlins is well established, but how the scaffolding functions of kindlins are regulated at the molecular level is still unclear. Here, using a combination of GFP nanotrap association assays, pulldown and integrin-binding assays, and live-cell imaging, we demonstrate that full-length kindlins can oligomerize (self-associate) in mammalian cells, and we propose that this self-association inhibits integrin binding and kindlin localization to focal adhesions. We show that both kindlin-2 and kindlin-3 can self-associate and that kindlin-3 self-association is more robust. Using chimeric mapping, we demonstrate that the F2PH and F3 subdomains are important for kindlin self-association. Through comparative sequence analysis of kindlin-2 and kindlin-3, we identify kindlin-3 point mutations that decrease self-association and enhance integrin binding, affording mutant kindlin-3 the ability to localize to focal adhesions. Our results support the notion that kindlin self-association negatively regulates integrin binding. The integrin family of transmembrane adhesion receptors coordinates complex signaling networks that control the ability of cells to sense and communicate with the extracellular environment. Kindlin proteins are a central cytoplasmic component of these networks, directly binding integrin cytoplasmic domains and mediating interactions with cytoskeletal and signaling proteins. The physiological importance of kindlins is well established, but how the scaffolding functions of kindlins are regulated at the molecular level is still unclear. Here, using a combination of GFP nanotrap association assays, pulldown and integrin-binding assays, and live-cell imaging, we demonstrate that full-length kindlins can oligomerize (self-associate) in mammalian cells, and we propose that this self-association inhibits integrin binding and kindlin localization to focal adhesions. We show that both kindlin-2 and kindlin-3 can self-associate and that kindlin-3 self-association is more robust. Using chimeric mapping, we demonstrate that the F2PH and F3 subdomains are important for kindlin self-association. Through comparative sequence analysis of kindlin-2 and kindlin-3, we identify kindlin-3 point mutations that decrease self-association and enhance integrin binding, affording mutant kindlin-3 the ability to localize to focal adhesions. Our results support the notion that kindlin self-association negatively regulates integrin binding. The phenotypes of kindlin knockout or depletion in mouse, Drosophila, and Caenorhabditis elegans clearly establish the general importance of kindlins for integrin function (1Montanez E. Ussar S. Schifferer M. Bosl M. Zent R. Moser M. Fassler R. Kindlin-2 controls bidirectional signaling of integrins.Genes Dev. 2008; 22 (18483218): 1325-133010.1101/gad.469408Crossref PubMed Scopus (330) Google Scholar, 2Moser M. Nieswandt B. Ussar S. Pozgajova M. Fässler R. Kindlin-3 is essential for integrin activation and platelet aggregation.Nat. Med. 2008; 14 (18278053): 325-33010.1038/nm1722Crossref PubMed Scopus (534) Google Scholar, 3Ussar S. Moser M. Widmaier M. Rognoni E. Harrer C. Genzel-Boroviczeny O. Fässler R. Loss of kindlin-1 causes skin atrophy and lethal neonatal intestinal epithelial dysfunction.PLoS Genet. 2008; 4: e100028910.1371/journal.pgen.1000289Crossref PubMed Scopus (173) Google Scholar, 4Catterson J.H. Heck M.M.S. Hartley P.S. Fermitins, the orthologs of mammalian kindlins, regulate the development of a functional cardiac syncytium in Drosophila melanogaster.PLoS ONE. 2013; 8: e6295810.1371/journal.pone.0062958Crossref PubMed Scopus (9) Google Scholar, 5Bai J. Binari R. Ni J.Q. Vijayakanthan M. Li H.S. Perrimon N. RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance.Development. 2008; 135: 1439-144910.1242/dev.012849Crossref PubMed Scopus (57) Google Scholar, 6Rogalski T.M. Mullen G.P. Gilbert M.M. Williams B.D. Moerman D.G. The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane.J. Cell Biol. 2000; 150 (10893272): 253-26410.1083/jcb.150.1.253Crossref PubMed Scopus (165) Google Scholar). Furthermore, loss-of-function mutations in two of the three mammalian kindlins (kindlin-1 and kindlin-3) result in human diseases associated with altered cell adhesion (7Siegel D.H. Ashton G.H.S. Penagos H.G. Lee J.V. Feiler H.S. Wilhelmsen K.C. South A.P. Smith F.J.D. Prescott A.R. Wessagowit V. Oyama N. Akiyama M. Al Aboud D. Al Aboud K. Al Githami A. et al.Loss of kindlin-1, a human homolog of the Caenorhabditis elegans actin-extracellular-matrix linker protein UNC-112, causes Kindler syndrome.Am. J. Hum. Genet. 2003; 73 (12789646): 174-18710.1086/376609Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 8Mory A. Feigelson S.W. Yarali N. Kilic S.S. Bayhan G.I. Gershoni-Baruch R. Etzioni A. Alon R. Kindlin-3: a new gene involved in the pathogenesis of LAD-III.Blood. 2008; 112 (18779414): 259110.1182/blood-2008-06-163162Crossref PubMed Scopus (114) Google Scholar, 9Malinin N.L. Zhang L. Choi J. Ciocea A. Razorenova O. Ma Y.-Q. Podrez E.A. Tosi M. Lennon D.P. Caplan A.I. Shurin S.B. Plow E.F. Byzova T.V. A point mutation in KINDLIN3 ablates activation of three integrin subfamilies in humans.Nat. Med. 2009; 15 (19234460): 313-31810.1038/nm.1917Crossref PubMed Scopus (278) Google Scholar), whereas mutations in kindlin-2, or changes in its expression level, are associated with cancer (10Zhao T. Guan L. Yu Y. Pei X. Zhan J. Han L. Tang Y. Li F. Fang W. Zhang H. Kindlin-2 promotes genome instability in breast cancer cells.Cancer Lett. 2013; 330 (23211537): 208-21610.1016/j.canlet.2012.11.043Crossref PubMed Scopus (23) Google Scholar, 11Shen Z. Ye Y. Kauttu T. Seppänen H. Vainionpää S. Wang S. Mustonen H. Puolakkainen P. Novel focal adhesion protein kindlin-2 promotes the invasion of gastric cancer cells through phosphorylation of integrin β1 and β3.J. Surg. Oncol. 2013; 108 (23857544): 106-11210.1002/jso.23353Crossref PubMed Scopus (37) Google Scholar). However, while kindlins clearly bind integrin cytoplasmic tails and act as adaptor proteins that regulate integrin adhesive function and connect integrins to cytoskeletal and signaling networks (12Kadry Y.A. Calderwood D.A. Chapter 22: Structural and signaling functions of integrins.Biochim. Biophys. Acta. 2020; 1862: 18320610.1016/j.bbamem.2020.183206Crossref PubMed Scopus (61) Google Scholar, 13Calderwood D.A. Campbell I.D. Critchley D.R. Talins and kindlins: Partners in integrin-mediated adhesion.Nat. Rev. Mol. Cell Biol. 2013; 14 (23860236): 503-51710.1038/nrm3624Crossref PubMed Scopus (385) Google Scholar), how kindlin activity is regulated is only partially understood. Kindlins share a conserved 4.1-ezrin-radixin-moesin (FERM) domain architecture that is distinguished from other FERM domains by the presence of an F0 subdomain, the inclusion of a long flexible loop in the F1 subdomain, and the insertion of a pleckstrin homology (PH) domain into the F2 subdomain (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar, 15Meves A. Stremmel C. Gottschalk K. Fässler R. The Kindlin protein family: new members to the club of focal adhesion proteins.Trends Cell Biol. 2009; 19 (19766491): 504-51310.1016/j.tcb.2009.07.006Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 16Bouaouina M. Goult B.T. Huet-Calderwood C. Bate N. Brahme N.N. Barsukov I.L. Critchley D.R. Calderwood D.A. A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation.J. Biol. Chem. 2012; 287 (22235127): 6979-699010.1074/jbc.M111.330845Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 17Yates L.A. Füzéry A.K. Bonet R. Campbell I.D. Gilbert R.J.C. Biophysical analysis of kindlin-3 reveals an elongated conformation and maps integrin binding to the membrane-distal β-subunit NPXY motif.J. Biol. Chem. 2012; 287 (22989875): 37715-3773110.1074/jbc.M112.415208Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). It is believed that the importance of kindlins in integrin-mediated signaling stems from their role as scaffolds for mediating protein–protein interactions (13Calderwood D.A. Campbell I.D. Critchley D.R. Talins and kindlins: Partners in integrin-mediated adhesion.Nat. Rev. Mol. Cell Biol. 2013; 14 (23860236): 503-51710.1038/nrm3624Crossref PubMed Scopus (385) Google Scholar, 18Rognoni E. Ruppert R. Fassler R. The kindlin family: functions, signaling properties and implications for human disease.J. Cell Sci. 2016; 129 (26729028): 17-2710.1242/jcs.161190Crossref PubMed Scopus (143) Google Scholar), and considerable efforts have been directed toward identifying kindlin binding partners (1Montanez E. Ussar S. Schifferer M. Bosl M. Zent R. Moser M. Fassler R. Kindlin-2 controls bidirectional signaling of integrins.Genes Dev. 2008; 22 (18483218): 1325-133010.1101/gad.469408Crossref PubMed Scopus (330) Google Scholar, 12Kadry Y.A. Calderwood D.A. Chapter 22: Structural and signaling functions of integrins.Biochim. Biophys. Acta. 2020; 1862: 18320610.1016/j.bbamem.2020.183206Crossref PubMed Scopus (61) Google Scholar, 19Dong J.M. Tay F.P.L. Swa H.L.F. Gunaratne J. Leung T. Burke B. Manser E. Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.Sci. Signal. 2016; 9: 1-1410.1126/scisignal.aaf3572Crossref Scopus (60) Google Scholar). Among the best-characterized kindlin interactions is the F3 subdomain-mediated binding to integrin β tails (1Montanez E. Ussar S. Schifferer M. Bosl M. Zent R. Moser M. Fassler R. Kindlin-2 controls bidirectional signaling of integrins.Genes Dev. 2008; 22 (18483218): 1325-133010.1101/gad.469408Crossref PubMed Scopus (330) Google Scholar, 14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar, 20Harburger D.S. Bouaouina M. Calderwood D.A. Kindlin-1 and -2 directly bind the C-terminal region of β integrin cytoplasmic tails and exert integrin-specific activation effects.J. Biol. Chem. 2009; 284 (19240021): 11485-1149710.1074/jbc.M809233200Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). This interaction, which has been structurally characterized (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar), is essential for normal integrin activation and for correct targeting of kindlins to focal adhesions (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar, 20Harburger D.S. Bouaouina M. Calderwood D.A. Kindlin-1 and -2 directly bind the C-terminal region of β integrin cytoplasmic tails and exert integrin-specific activation effects.J. Biol. Chem. 2009; 284 (19240021): 11485-1149710.1074/jbc.M809233200Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 21Ma Y.Q. Qin J. Wu C. Plow E.F. Kindlin-2 (Mig-2): a co-activator of β3 integrins.J. Cell Biol. 2008; 181 (18458155): 439-44610.1083/jcb.200710196Crossref PubMed Scopus (270) Google Scholar). Kindlins also bind several other focal adhesion proteins (12Kadry Y.A. Calderwood D.A. Chapter 22: Structural and signaling functions of integrins.Biochim. Biophys. Acta. 2020; 1862: 18320610.1016/j.bbamem.2020.183206Crossref PubMed Scopus (61) Google Scholar, 13Calderwood D.A. Campbell I.D. Critchley D.R. Talins and kindlins: Partners in integrin-mediated adhesion.Nat. Rev. Mol. Cell Biol. 2013; 14 (23860236): 503-51710.1038/nrm3624Crossref PubMed Scopus (385) Google Scholar, 18Rognoni E. Ruppert R. Fassler R. The kindlin family: functions, signaling properties and implications for human disease.J. Cell Sci. 2016; 129 (26729028): 17-2710.1242/jcs.161190Crossref PubMed Scopus (143) Google Scholar) including integrin-linked kinase (ILK), a pseudokinase important for linking integrins to the actin cytoskeleton. The kindlin–ILK interaction, which is mediated by the kindlin F2PH subdomain and the ILK pseudokinase domain, also contributes to recruitment and retention of kindlin and ILK at focal adhesions (22Kadry Y.A. Huet-Calderwood C. Simon B. Calderwood D.A. Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading.J. Cell Sci. 2018; 131: jcs22118410.1242/jcs.221184Crossref PubMed Scopus (25) Google Scholar, 23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar, 24Fukuda K. Bledzka K. Yang J. Perera H.D. Plow E.F. Qin J. Molecular basis of kindlin-2 binding to integrin-linked kinase pseudokinase for regulating cell adhesion.J. Biol. Chem. 2014; 289 (25160619): 28363-2837510.1074/jbc.M114.596692Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It has recently been proposed that in addition to other protein–protein interactions, kindlins may also self-associate (form kindlin-kindlin interactions) (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar), and that this may be important in kindlin-mediated integrin signaling. Whereas crystal structures of full-length kindlins are not yet available, the structure of a truncated kindlin-2 lacking the large F1 loop and the PH domain revealed a domain-swapped dimer (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar). However, whether this dimeric form is physiologically relevant remains unclear, especially because the reported spontaneous monomer-dimer transition in solution is unfavorable, occurring on the order of days in vitro (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar). Despite this, kindlin self-association has emerged as an attractive mechanistic model for how kindlins may promote integrin signaling (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar); indeed, kindlins have been implicated in driving integrin clustering (25Ye F. Petrich B.G. Anekal P. Lefort C.T. Kasirer-Friede A. Shattil S.J. Ruppert R. Moser M. Fässler R. Ginsberg M.H. The mechanism of kindlin-mediated activation of integrin αIIbβ3.Curr. Biol. 2013; 23 (24210614): 2288-229510.1016/j.cub.2013.09.050Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). However, it is currently unclear if kindlin self-association occurs in full-length kindlins in mammalian cells, whether or not other kindlin isoforms besides kindlin-2 can self-associate, and the impact of self-association on integrin binding and focal adhesion localization. Despite sharing a conserved domain organization, the three mammalian kindlins exhibit specific functions and interactions (18Rognoni E. Ruppert R. Fassler R. The kindlin family: functions, signaling properties and implications for human disease.J. Cell Sci. 2016; 129 (26729028): 17-2710.1242/jcs.161190Crossref PubMed Scopus (143) Google Scholar, 23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar, 26Plow E.F. Qin J. The kindlin family of adapter proteins: a past, present, and future prospectus.Circ. Res. 2019; 124 (30653443): 202-20410.1161/CIRCRESAHA.118.314362Crossref PubMed Scopus (16) Google Scholar, 27Bandyopadhyay A. Rothschild G. Kim S. Calderwood D.A. Raghavan S. Functional differences between kindlin-1 and kindlin-2 in keratinocytes.J. Cell Sci. 2012; 125 (22328497): 2172-218410.1242/jcs.096214Crossref PubMed Scopus (42) Google Scholar). Previous studies from our laboratory and others have demonstrated that the widely expressed kindlin-2 and hematopoietic-specific kindlin-3 exhibit functional differences that go beyond expression patterns. For example, when expressed in Chinese hamster ovary (CHO) cells or bovine aortic endothelial cells (BAEC) plated on fibronectin, kindlin-2 localizes to focal adhesions, whereas kindlin-3 does not (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar, 28Meller J. Rogozin I.B. Poliakov E. Meller N. Bedanov-Pack M. Plow E.F. Qin J. Podrez E.A. Byzova T.V. Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness.Mol. Biol. Cell. 2015; 26 (25540429): 786-79610.1091/mbc.E14-08-1294Crossref PubMed Scopus (14) Google Scholar). These functional disparities can be traced, in part, to differential binding to β1 integrins and to ILK and the ILK-PINCH-parvin (IPP) complex (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar, 28Meller J. Rogozin I.B. Poliakov E. Meller N. Bedanov-Pack M. Plow E.F. Qin J. Podrez E.A. Byzova T.V. Emergence and subsequent functional specialization of kindlins during evolution of cell adhesiveness.Mol. Biol. Cell. 2015; 26 (25540429): 786-79610.1091/mbc.E14-08-1294Crossref PubMed Scopus (14) Google Scholar). Here, we extend these studies to include comparison of kindlin self-association. We report that full-length kindlin-2 and kindlin-3 can each self-associate in mammalian cells but that kindlin-3 self-associates to a greater extent than kindlin-2. We identify domains important for mediating self-association and, through analysis of kindlin-3 mutants, present evidence suggesting that kindlin self-association impairs integrin binding and inhibits focal adhesion localization. Although the crystal structure of kindlin-2 lacking the PH domain and a portion of the F1 subdomain (kindlin-2ΔPHΔF1loop) suggested that kindlin-2ΔPHΔF1loop dimerizes (14Li H. Deng Y. Sun K. Yang H. Liu J. Wang M. Zhang Z. Lin J. Wu C. Wei Z. Yu C. Structural basis of kindlin-mediated integrin recognition and activation.Proc. Natl. Acad. Sci. U S A. 2017; 114 (28739949): 9349-935410.1073/pnas.1703064114Crossref PubMed Scopus (99) Google Scholar), it was unclear whether full-length kindlin-2 or the related kindlin-3 could self-associate in mammalian cells. To test this, we employed a coimmunoprecipitation (co-IP) assay using GFP-nanotrap beads and coexpressed GFP-tagged and FLAG-tagged kindlins (22Kadry Y.A. Huet-Calderwood C. Simon B. Calderwood D.A. Kindlin-2 interacts with a highly conserved surface of ILK to regulate focal adhesion localization and cell spreading.J. Cell Sci. 2018; 131: jcs22118410.1242/jcs.221184Crossref PubMed Scopus (25) Google Scholar, 29Rothbauer U. Zolghadr K. Muyldermans S. Schepers A. Cardoso M.C. Leonhardt H. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins.Mol. Cell. Proteomics. 2008; 7 (17951627): 282-28910.1074/mcp.M700342-MCP200Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). First, to assess the ability of kindlin-2 to self-associate, FLAG-kindlin-2 was coexpressed with either GFP-kindlin-2 or GFP (as a negative control) in HEK293T cells (Fig. 1, A and B). In parallel, kindlin-3 self-association was assessed by coexpressing FLAG-kindlin-3 with either GFP-kindlin-3 or GFP (as a negative control). Cells were lysed and incubated with GFP-nanotrap beads to purify the GFP-tagged protein, and the amount of associating FLAG-tagged protein was assessed by immunoblotting (Fig. 1A). Whereas negligible amounts of FLAG-kindlin-3 copurified with GFP, readily detectable levels of FLAG-kindlin-3 copurified with GFP-kindlin-3 (Fig. 1, B and C). Similarly, FLAG-kindlin-2 copurified with GFP-kindlin-2 but consistently less FLAG-kindlin-2 associated with GFP-kindlin-2 than in the case of kindlin-3 (Fig. 1, B and C). This suggests that both kindlin-2 and kindlin-3 can self-associate in mammalian cells but that kindlin-3 self-associates to a greater extent than kindlin-2 (Fig. 1, B and C). We note that while these experiments clearly show that kindlins can assemble into larger complexes, they cannot provide information on the stoichiometry of the complex or whether kindlin–kindlin interactions are direct. Kindlin-3 shares the same conserved domain architecture and 67% amino acid sequence similarity with kindlin-2 (20Harburger D.S. Bouaouina M. Calderwood D.A. Kindlin-1 and -2 directly bind the C-terminal region of β integrin cytoplasmic tails and exert integrin-specific activation effects.J. Biol. Chem. 2009; 284 (19240021): 11485-1149710.1074/jbc.M809233200Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). Therefore, we tested whether kindlin-3 could associate with kindlin-2. Notably, despite their similarity, GFP-kindlin-3 could associate with FLAG-kindlin-3 but not with FLAG-kindlin-2 (Fig. 2, A and B). This allowed us to map the subdomains of kindlin-3 that are important for self-association by evaluating the ability of FLAG-kindlin chimeras to restore or disturb association with GFP-kindlin-3. Two sets of chimeric FLAG-kindlins were tested: loss-of-function chimeras consisting of a FLAG-kindlin-3 backbone with inserted subdomain(s) of kindlin-2 intended to disturb association with GFP-kindlin-3 and gain-of-function chimeras composed of a FLAG-kindlin-2 backbone with inserted subdomain(s) of kindlin-3 intended to restore association with GFP-kindlin-3. Chimeric kindlins were named using four characters to denote the F0, F1, F2PH, and F3 regions, respectively, with each number denoting the kindlin isoform from which each subdomain originates (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar), and chimeric kindlins were functionally characterized as previously described (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). We first tested the ability of the FLAG-K2233 and FLAG-K3322 chimeras to associate with GFP-kindlin-3 to pinpoint whether the N- or C-terminal subdomains of kindlin-3 are most important for association with GFP-kindlin-3 (Fig. 2C). In the GFP nanobody co-IP assay, FLAG-K3322 was severely impaired in association with GFP-kindlin-3 (Fig. 2, D and E), suggesting that replacement of the F2PH and F3 subdomains of kindlin-3 is detrimental to the ability of kindlin-3 to associate. In contrast, despite consistently expressing less well than FLAG-kindlin-3, when corrected for input material, FLAG-K2233 was enhanced in association with GFP-kindlin-3 (Fig. 2, D and E). We note that these chimeric kindlins are likely to be correctly folded, as we have previously shown that they bind β1 integrin tails and ILK (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). Furthermore, K3322, which exhibits reduced association with kindlin-3, shows robust targeting to focal adhesions (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). Together, these results suggest that the C-terminal region of kindlin-3 (F2PH and F3 subdomains) is important for association of FLAG-kindlin-3 with GFP-kindlin-3: replacing this region with that from kindlin-2 (K3322) impairs the ability to associate with GFP-kindlin-3, whereas inserting this region into kindlin-2 (K2233) restores and enhances association. To determine which subdomain(s) in the C-terminal region of kindlin-3 are important for self-association, association between GFP-kindlin-3 and a series of FLAG-tagged loss-of-function chimeras was examined (Fig. 3, A and B). Four FLAG-tagged loss-of-function chimeras were tested: K3332, K3323, kindlin-3 with the F2 subdomain of kindlin-3 (K3 F2 swap), and kindlin-3 with the PH subdomain of kindlin-2 (K3 PH swap). The FLAG-K3332 chimera associated with GFP-kindlin-3; in contrast, FLAG-K3323 was severely impaired in association with GFP-kindlin-3 (Fig. 3, A and B). We consider this unlikely to be because of misfolding, as K3323 can bind β1 integrins and ILK and targets to focal adhesions (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). Swapping only the PH domain (K3 PH swap) also strongly impaired association with GFP-kindlin-3 (Fig. 3, A and B). Like kindlin-3, the PH swap construct fails to target to focal adhesions, but when expressed as a GFP fusion protein it does not form aggregates, suggesting that it is not misfolded (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). Thus, the F2PH subdomain, and particularly the PH domain, is important for the ability of kindlin-3 to self-associate in mammalian cells, as replacement of the kindlin-3 F2PH with the F2PH of kindlin-2 cannot fully support self-association. Further support for the importance of the F2PH subdomain for kindlin-3 self-association was provided when we evaluated association between GFP-kindlin-3 and a series of FLAG-tagged gain-of-function chimeras (Fig. 3, C and D). We observed that FLAG-K2232 associated with GFP-kindlin-3; however, we note that this chimera only partially restored association (Fig. 3, C and D). Meanwhile, FLAG-K2223 did not associate with GFP-kindlin-3 (Fig. 3, C and D). These chimeras bind β1 integrins and ILK, suggesting that they are correctly folded (23Huet-Calderwood C. Brahme N.N. Kumar N. Stiegler A.L. Raghavan S. Boggon T.J. Calderwood D.A. Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation.J. Cell Sci. 2014; 127 (25086068): 4308-432110.1242/jcs.155879Crossref PubMed Scopus (53) Google Scholar). In addition, neither the FLAG K2 F2 swap nor PH swap chimeras associated with GFP-kindlin-3 (Fig. 3, C and D). Although the loss-of-f

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