Differences in Binding to the ILK Complex Determines Kindlin Isoform

ß 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4308–4321 doi:10.1242/jcs.155879 RESEARCH ARTICLE Differences in binding to the ILK complex determines kindlin isoform adhesion localization and integrin activation Clotilde Huet-Calderwood1,*, Nina N. Brahme2,*, Nikit Kumar2, Amy L. Stiegler1, Srikala Raghavan1,3, Titus J. Boggon1 and David A. Calderwood1,2,` ABSTRACT et al., 2013; Shattil et al., 2010; Ye et al., 2012). Once activated or engaged by ECM molecules, integrin receptors can cluster to Kindlins are essential FERM-domain-containing focal adhesion (FA) form focal adhesions (FAs) (Harburger and Calderwood, 2009; proteins required for proper integrin activation and signaling. Wehrle-Haller, 2012). These complex multi-protein assemblies Despite the widely accepted importance of each of the three contain a large array of cytoskeletal, scaffolding and signaling mammalian kindlins in cell adhesion, the molecular basis for their proteins (Geiger and Zaidel-Bar, 2012; Harburger and function has yet to be fully elucidated, and the functional differences Calderwood, 2009) and serve as anchorage points for the between isoforms have generally not been examined. Here, we cytoskeleton and sites at which the cell senses and applies report functional differences between kindlin-2 and -3 (also known mechanical force. as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 Kindlins are a family of proteins that bind to the integrin localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not cytoplasmic tail and that are crucial for normal integrin regulation kindlin-3, can rescue a5b1 integrin activation defects in kindlin-2- and signaling (Calderwood et al., 2013; Karako¨se et al., 2010; knockdown fibroblasts. Using chimeric kindlins, we show that the Larjava et al., 2008). Loss of the mainly epithelial kindlin-1 (also relatively uncharacterized kindlin-2 F2 subdomain drives FA known as FERMT1) leads to Kindler syndrome, manifesting in targeting and integrin activation. We find that the integrin-linked skin blistering and ulcerative colitis (Siegel et al., 2003; Ussar kinase (ILK)–PINCH–parvin complex binds strongly to the kindlin-2 et al., 2008). Knockout of the ubiquitously expressed kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated (also known as FERMT2) halts murine development at the peri- kindlin-2, we establish that efficient kindlin-2-mediated integrin implantation stage due to cell detachment from the basement activation and FA targeting require binding to the ILK complex. membrane (Montanez et al., 2008). Finally, loss of the Thus, ILK-complex binding is crucial for normal kindlin-2 function hematopoietic-specific kindlin-3 (also known as FERMT3) and differential ILK binding contributes to kindlin isoform specificity. causes leukocyte adhesion deficiency type III (LADIII) (Kuijpers et al., 2009; Malinin et al., 2009; Moser et al., 2009; KEY WORDS: Focal adhesion, Integrin, Integrin activation, Svensson et al., 2009), which is characterized by immune defects Integrin-linked kinase, Kindlin and severe bleeding. Impaired integrin activation is a common feature of kindlin disease or knockout phenotypes, strongly implicating all three INTRODUCTION kindlins in integrin activation and integrin-mediated processes. Integrins are essential heterodimeric transmembrane adhesion This is supported by studies of knockout and knockdown cells receptors that mediate bi-directional signaling between cells and and by overexpression experiments (Dowling et al., 2008; the extracellular matrix (ECM) (Harburger and Calderwood, Harburger et al., 2009; Ma et al., 2008; Malinin et al., 2009; 2009). As such, integrins govern a range of physiological Moser et al., 2009; Moser et al., 2008; Shi et al., 2007; Ussar processes including cell migration, tissue organization, et al., 2008). For example, when kindlin-2 is depleted, cells angiogenesis, hemostasis and the immune response. Integrins exhibit defects in b1 and b3 integrin activation, FA formation and can be found in an ‘inactive’ state with relatively low affinity for cell spreading (Montanez et al., 2008; Pluskota et al., 2011; Shi ECM ligands or in an ‘active’ conformation with high ligand- et al., 2007). Similar defects are reported following knockout or binding affinity (Calderwood, 2004a). Integrin activation (the knockdown of kindlin-1 (Ussar et al., 2008), and loss of kindlin-3 conversion from inactive to active states) can be triggered by results in impaired activation of b1, b2 and b3 integrins (Moser binding to ECM or by intracellular signaling events that et al., 2009; Moser et al., 2008). However, although all three culminate in the binding of the cytoskeletal adaptor protein kindlin isoforms are key regulators of cell adhesion, they also talin to the cytoplasmic tail of the integrin b subunit (Calderwood appear to have isoform-specific functions. For instance, endogenous kindlin-2 is unable to fully compensate for loss of kindlin-1 in the skin or for non-functional kindlin-3 in 1Department of Pharmacology, Yale University, New Haven, CT 06520, USA. hematopoietic cells (Bialkowska et al., 2010; Lai-Cheong et al., 2Department of Cell Biology, Yale University, New Haven, CT 06520, USA. 3Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, Karnataka 2008; Malinin et al., 2009; Moser et al., 2009; Ussar et al., 2008). 560065, India. Furthermore, although all three isoforms bind to the integrin b *These authors contributed equally to this work subunit cytoplasmic tails, differences in subcellular and tissue `Author for correspondence ([email protected]) localization have been observed. For example, whereas kindlin-1 and kindlin-2 both target to FAs in adherent cells, kindlin-1 Received 28 April 2014; Accepted 2 July 2014 targets preferentially to avb6-rich adhesions in b1-knockout cells Journal of Cell Science 4308 RESEARCH ARTICLE Journal of Cell Science (2014) 127, 4308–4321 doi:10.1242/jcs.155879 (Bandyopadhyay et al., 2012), and kindlin-3 does not localize to domain complex, mapped the ILK-binding site to the kindlin F2 FAs (Bialkowska et al., 2010; Ussar et al., 2006). The molecular domain, and identified a point mutation that strongly inhibited basis for the differences in isoform localization and function is ILK binding. By using kindlin-2–kindlin-3 chimeras in which the generally not understood. overall kindlin domain structure was preserved, and kindlin Despite recent progress (Ye et al., 2013), the mechanisms by mutants defective in binding the ILK complex, we showed that which kindlins affect integrin activation and signaling remain to strong ILK-complex binding supports FA targeting and b1 be fully determined. Kindlins are made up of an atypical FERM integrin activation. Overall, our work shows that ILK drives (band 4.1, ezrin, radixin, moesin) domain composed of four kindlin subcellular localization and kindlin-mediated integrin subdomains (F0, F1, F2 and F3) with a pleckstrin homology (PH) activation, and provides insight into how kindlin and ILK domain nested within the F2 subdomain (Calderwood et al., 2013; contribute to integrin signaling. Goult et al., 2009). The lack of recognizable enzymatic domains suggests that kindlins act as scaffold or adaptor proteins. RESULTS Consistent with this, the F3 subdomain contains a binding site GFP-tagged kindlin-1 and -2, but not kindlin-3, target to b1- for integrin b-tails, and this binding is required for kindlins to integrin-rich FAs in CHO cells enhance talin-mediated integrin activation (Harburger et al., To compare the ability of the three mammalian kindlins to 2009; Ma et al., 2008; Moser et al., 2008; Ussar et al., 2008). accumulate in FAs, N-terminally GFP-tagged kindlin-1, -2 and -3 However, subdomain deletion and mutagenesis studies show that were each overexpressed in CHO cells plated on fibronectin and kindlin function, and localization to adhesions, also requires the their localization was assessed by fluorescence microscopy. As F0, F1 and PH domains, at least in part because of membrane- previously reported (Bouaouina et al., 2012a; Goult et al., 2009; binding sites in these domains (Bouaouina et al., 2012a; Harburger et al., 2009), GFP–kindlin-1 and -2 were readily Harburger et al., 2009; Liu et al., 2011; Perera et al., 2011; Xu detected in FAs at 24 h after replating (Fig. 1A). In clear contrast, et al., 2013; Yates et al., 2012). The inability of isolated kindlin GFP–kindlin-3 was always cytoplasmic and did not target to FAs, subdomains to support integrin activation or signaling has made regardless of expression level (Fig. 1A). We assessed the extent characterization of the kindlin mechanism of action difficult. of FA targeting by quantifying blindly the percentage of cells in Besides integrin, integrin-linked kinase (ILK) is one of the few which the GFP-tagged protein clearly colocalizes with vinculin- known kindlin-binding proteins (Calderwood et al., 2013). ILK is rich FAs (Fig. 1B). GFP–kindlin-1 and -2 targeted to FAs in most a FA protein essential for development and normal integrin expressing cells, and kindlin-2 consistently targeted better than signaling (Friedrich et al., 2004; Sakai et al., 2003). ILK was kindlin-1 (99% and 86%, respectively). However, we could not originally identified as a binding partner for integrin b-tails in a observe GFP–kindlin-3 targeting to FAs. As expected (Harburger yeast two-hybrid screen (Hannigan et al., 1996), and is composed et al., 2009; Montanez et al., 2008; Ussar et al., 2008), the of an N-terminal ankyrin repeat domain (ILK-ARD) (Chiswell integrin-binding defective mutants GFP–kindlin-1-W612A and et al., 2008) followed by a C-terminal kinase domain (ILK-KD) GFP–kindlin-2-W615A failed to accumulate in FAs (Fig. 1B), (Fukuda et al., 2009). The kinase domain lacks key catalytic confirming the specificity of our assays and demonstrating that residues, and ILK is generally thought to be a pseudokinase integrin-binding is required for GFP–kindlin-1 and -2 (Fukuda et al., 2009) functioning primarily as an adaptor protein.

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