RESEARCH ARTICLE

Kindlin-2 interacts with a highly conserved surface of ILK toregulate focal adhesion localization and cell spreadingYasmin A. Kadry1, Clotilde Huet-Calderwood1, Bertrand Simon1 and David A. Calderwood1,2,‡

ABSTRACTThe integrin-associated adaptor proteins integrin-linked kinase (ILK)and kindlin-2 play central roles in integrin signaling and control ofcell morphology. A direct ILK–kindlin-2 interaction is conservedacross species and involves the F2PH subdomain of kindlin-2 and thepseudokinase domain (pKD) of ILK. However, complete understandingof the ILK–kindlin-2 interaction and its role in integrin-mediated signalinghas been impeded by difficulties identifying the binding site for kindlin-2on ILK.We used conservation-guidedmapping to dissect the interactionbetween ILK and kindlin-2 and identified a previously unknown bindingsite for kindlin-2 on the C-lobe of the pKD of ILK. Mutations at this siteinhibit binding to kindlin-2 while maintaining structural integrity of thepKD. Importantly, kindlin-binding-defective ILKmutants exhibit impairedfocal adhesion localization and fail to fully rescue the spreading defectsseen in ILK knockdown cells. Furthermore, kindlin-2 mutants withimpaired ILK binding are also unable to fully support cell spreading.Thus, the interaction between ILK and kindlin-2 is critical for cellspreading and focal adhesion localization, representing a key signalingaxis downstream of integrins.

This article has an associated First Person interview with the first authorof the paper.

KEY WORDS: Integrin-linked kinase, Kindlin-2, Integrin

INTRODUCTIONCell adhesion to the extracellular matrix is a spatially and temporallyregulated process governed by the dynamic assembly ofcomplexes between transmembrane heterodimeric integrin adhesionreceptors and cytoplasmic regulators (Morse et al., 2014). Hetero-dimerization of the 18 α- and 8 β-integrin subunits to form 24 αβ-integrin combinations drives ligand-binding specificity (Humphries,2006) with signaling activity determined by the recruitment ofspecific binding partners to the cytoplasmic tails (Iwamoto andCalderwood, 2015). These complexes facilitate an essential linkbetween the intracellular and extracellular environment, signaling bi-directionally through clusters of activated ligand-bound integrins thatare associated with cytoplasmic regulators and cytoskeletal adaptorsin foci known as focal adhesions (Anthis and Campbell, 2011).Integrin-linked kinase (ILK) is a key component of focal

adhesions, acting as a non-catalytic scaffold that serves as a hubfor integrin-mediated signaling (Lange et al., 2009; Wickström

et al., 2010). Loss of ILK is lethal at the embryonic stage in mice,and loss or depletion of ILK in cultured mammalian cells leads todefects in cell spreading and focal adhesion formation (Friedrichet al., 2004; Fukuda et al., 2003; Sakai et al., 2003). ILK comprisesan N-terminal ankyrin repeat domain (ARD) and a C-terminalpseudokinase domain (pKD) (Chiswell et al., 2008; Fukuda et al.,2009). ILK forms a tripartite complex with particularly interestingnew cysteine-histidine rich protein (PINCH, officially known asLIMS) through its ARD (Chiswell et al., 2008) and parvin through itspKD (Fukuda et al., 2009) that is known as the ILK–PINCH–parvin(IPP) complex. The IPP complex is important for stabilization of thethree proteins as well as their cellular functions (Stiegler et al., 2013;Zhang et al., 2002). Mammals have two PINCH isoforms, PINCH1and PINCH2 (LIMS1 and LIMS2, respectively), and three parvinisoforms, α-parvin, β-parvin and γ-parvin (PARVA, PARVB andPARVG, respectively), with PINCH1 and α-parvin being the mostwidely expressed across development and cell type (Nikolopoulos andTurner, 2000; Stanchi et al., 2005; Tu et al., 2001; Yamaji et al., 2001).The distinct signaling pathways downstream of ILK and the IPPcomplex remain largely un-elucidated, although the IPP complex hasbeen shown to interact with many other signaling proteins such aspaxillin (Nikolopoulos and Turner, 2002), Nck2, a regulatory adaptorof receptor tyrosine kinases (Velyvis et al., 2003), and kindlins (Fukudaet al., 2014; Huet-Calderwood et al., 2014; Mackinnon et al., 2002;Qadota et al., 2014).

Kindlins are a family of key cytoplasmic regulators of integrinactivation, acting by direct interaction with the cytoplasmic tail ofthe β integrin subunit (Calderwood et al., 2013; Harburger et al.,2009; Li et al., 2017; Qadota et al., 2012). Kindlins are composed ofan atypical 4.1, ezrin, radixin, moesin (FERM) domain that – inaddition to the classic F1, F2 and F3 subdomains found in all FERMdomains – is hallmarked by an N-terminal F0 lobe, a large flexibleF1 insertion and a pleckstrin homology (PH) domain that splits theF2 subdomain to generate a module that we have termed F2PH(Calderwood et al., 2013; Li et al., 2017). The kindlin familyconsists of three isoforms (kindlin-1, kindlin-2 and kindlin-3) thatexhibit restricted expression and non-overlapping functions.Kindlin-2 (officially known as FERMT2) is the ubiquitouslyexpressed isoform and loss of kindlin-2 expression in mice isembryonic lethal (Montanez et al., 2008). In cultured murinefibroblasts, kindlin-2 deficiency impairs cell spreading and celladhesion (Böttcher et al., 2017; Huet-Calderwood et al., 2014;Montanez et al., 2008; Theodosiou et al., 2016).

The interaction between ILK and kindlin-2 is conserved acrossspecies, and was first found in Caenorhabditis elegans (C. elegans)using a yeast two-hybrid screen in which the kindlin orthologueUNC-112, used as bait, interacted with PAT-4, the orthologue ofILK (Mackinnon et al., 2002). In mammals, ILK and kindlin-2 alsoform a complex (Fukuda et al., 2014; Huet-Calderwood et al., 2014;Montanez et al., 2008). Biochemical and biophysical studies haveshown binding of highly conserved leucine residues – located atReceived 6 June 2018; Accepted 17 September 2018

1From the Department of Pharmacology, Yale University, New Haven CT 06510,USA. 2Department of Cell Biology, Yale University, New Haven CT 06510, USA.

‡Author for correspondence (david.calderwood@yale.edu)

Y.A.K., 0000-0002-4209-7416; B.S., 0000-0002-1825-053X; D.A.C., 0000-0002-0791-4142

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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs221184. doi:10.1242/jcs.221184

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the hydrophobic face of an amphipathic helix within the F2PHsubdomain of kindlin-2 – to an unidentified region on the pKD ofILK (ILK-pKD) (Fukuda et al., 2014; Huet-Calderwood et al.,2014). Overexpression studies have shown that mutation of one ormore of the conserved leucine residues to alanines on this helix,which inhibits binding to the ILK-pKD, impairs cell spreading(Fukuda et al., 2014). However, difficulties in identifying thebinding site for kindlin-2 on the ILK-pKD have hindered furtherunderstanding of the interaction and its role in signalingdownstream of integrins.Here we report the identification of a novel binding interface

for kindlin-2 on the ILK-pKD, allowing us to investigate thefunctional significance of the ILK–kindlin-2 interaction. Weshow that mutations on helix-αH in the C-lobe of the ILK-pKDimpair the interaction with kindlin-2 without perturbing α-parvinbinding or structural integrity of the ILK-pKD. This binding siteis distinct from a recently reported kindlin-2 binding interface inthe N-lobe of the ILK-pKD (Guan et al., 2018), and we found thatthe reported double glycine for arginine substitutions (R243Gand R334G) at that site destabilize the interaction of theILK-pKD with α-parvin, potentially disrupting interaction withkindlin-2 indirectly. We demonstrate that mutations on helix-αHof the ILK-pKD impair the ability of GFP-tagged ILK(GFP–ILK) to localize to focal adhesions and disrupt the abilityof GFP–ILK to fully rescue spreading defects observed in ILK-knockdown HeLa cells. Similarly, we show that a kindlin-2mutant impaired in binding to ILK is unable to fully rescuespreading defects caused by depletion of kindlin-2, solidifyingthe role of the kindlin-2-ILK interaction in cell spreading andfocal adhesion signaling.

RESULTSMutation of a highly conserved hydrophobic patch on theILK-pKD impairs binding to the F2PH domain of kindlin-2Previouswork has demonstrated that the ILK-pKDbinds to a series ofhighly conserved leucine residues, including L357 of human kindlin-2; these residues lie along the non-polar face of an amphipathic helixin the F2PH subdomain of kindlin-2 (Fukuda et al., 2014; Huet-Calderwood et al., 2014). Therefore, we hypothesized that kindlin-2interacts with a conserved, hydrophobic patch on the ILK-pKD. Toidentify potential kindlin-2-binding surfaces on the ILK-pKD, weused ILK sequences from 37 species including C. elegans and Homosapiens (Table S1), and the previously reported crystal structure of thehuman ILK-pKD in complex with the second calponin homologydomain (CH2) of α-parvin (α-parvin-CH2) bound to MgATP (PDBID: 3KMW) (Fukuda et al., 2009) to generate a conservation surfacemap with the ConSurf server (http://consurf.tau.ac.il; Landau et al.,2005). We initially identified two surfaces (surface 1 and 2) withclusters of highly conserved residues (Fig. 1A). We also selected athird, less well conserved surface on the lateral face of the ILK-pKDthat may accommodate the helical fragment of the F2PH, which bindsthe ILK-pKD (surface 3) (Fig. 1A) (Fukuda et al., 2014). Next, wegenerated a map of the coulombic surface potential of the ILK-pKDto identify patches with neutral surface potential, a proxy forhydrophobicity, using Chimera software (https://www.cgl.ucsf.edu/chimera/; Pettersen et al., 2004) (Fig. 1B). We noticed that all threeselected surfaces lie on hydrophobic patches. Importantly, none of theselected candidate kindlin-binding surfaces overlap with the bindinginterface for α-parvin or the ATP-binding site on the ILK-pKD(Fukuda et al., 2009). In order to disrupt the potential non-polarinteraction with the kindlin-2 F2PH, we mutated selected non-polar,solvent-exposed residues on each surface to either an aspartic acid or

glutamic acid (Fig. 1C). On surface 1, we generated substitutionmutations of isoleucine, phenylanaline and serine (I244D, F245Dand S246D) on a loop at the C-terminus of theαChelix. For surface 2,we replaced I427 with glutamic acid (I427E) on helix-αH and onsurface 3 we replaced F287, which resides on a loop between helix-αD and helix-αE, with D (F287D).

As ILK protein stability is crucially dependent on associationwithparvin (Fukuda et al., 2003)we co-expressed FLAG-taggedα-parvin(FLAG–α-parvin) and GFP–ILK or ILKmutants in chinese hamsterovary (CHO) cells and assessed the ability of purified recombinantglutathione S-transferase (GST) fused to kindlin-2 F2PH (GST–kindlin-2 F2PH) to pull down GFP–ILK from cell lysates. Aspreviously reported (Huet-Calderwood et al., 2014), kindlin-2 F2PHbound GFP–ILK and FLAG–α-parvin in a specific ILK-dependentmanner, as when GFP was co-expressed with FLAG-α-parvin,negligible GFP or FLAG–α-parvin binding was detected (Fig. 2A,B). Specificity was further confirmed using GST–kindlin-2 F2PHL357A (F2PH L/A), which contains a mutation in the previouslyidentified ILK-binding site (Fukuda et al., 2014; Huet-Calderwoodet al., 2014) as a negative control. All five ILK mutants expressedwell at the expected molecular mass, and non-specific binding toGST–kindlin-2 F2PH L/A was minimal (Fig. 2C). While surface 1and 3 mutants exhibited approximately wild-type levels of bindingto kindlin-2 F2PH, the I427E mutant on surface 2 abolishedbinding (Fig. 2C,D). Importantly, the binding of FLAG–α-parvin tothe GST–kindlin-2 F2PH only occurred concomitantly with thebinding to GFP–ILK, consistent with the fact that the interaction isspecificallymediated by ILK (Fukuda et al., 2014;Huet-Calderwoodet al., 2014). We also tested a more conservative I427A mutation, inwhich the isoleucine side chain was removed but no negative chargewas introduced and found that this also impaired binding to GST–kindlin-2 F2PH in pulldown assays (Fig. S1A,B). In summary, thesedata suggest that I427 on helix-αH of ILK-pKD is involved in theinteraction between the ILK-pKD and kindlin-2.

Additional mutations in helix-αH on surface 2 impair bindingto kindlin-2To further map the kindlin-binding surface on the ILK-pKD, were-examined the ILK-pKD–α-parvin-CH2 co-crystal structure(Fukuda et al., 2009) to look for additional surface-exposedresidues close to I427. We selected the well-conserved K423 andthe poorly conserved K426 on helix-αH, as well as the more-distantbut conserved hydrophobic residue I413 on the linker between helix-αG and helix-αH (Fig. 3A). We tested the effects of charge reversaland charge removal mutations at selected lysine residues (i.e. K423D,K423A, K426D or K426A), and the effect of replacing I413 with acharged residue (I413D). When co-expressed with FLAG-α-parvin,GFP–ILK K423D and GFP–ILK K423A were severely impaired inbinding to GST–kindlin-2 F2PH, while the K426D and K426Amutations exhibited a moremodest inhibition (Fig. 3B,C). The I413Dmutation had no marked effect on binding to GST–kindlin-2 F2PH(Fig. 3B,C). This suggests that the highly conserved K423, which liesalong the same face of helix-αH as I427, is also involved in theinteraction between the ILK-pKD and the kindlin-2 F2PH, and thatαH is a key mediator of the interaction with the kindlin-2 F2PH.

We, and others, previously localized the ILK-binding site in thekindlin-2 F2PH to leucine residues in a helical region in the linkerbetween the F2 and PH domains, spanning residues 329-368(Fukuda et al., 2014; Huet-Calderwood et al., 2014). Therefore, tofurther confirm the specificity of our results, we performed pull-down assays with purified GST–kindlin-2 329-368. Consistentwith our kindlin-2 F2PH data, mutants GFP–ILK I427E and

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GFP–ILK K423D were severely impaired in binding to GST–kindlin-2 329-368, while K426 mutants had a partial effect, andGFP–ILK I413D bound at levels comparable to those of GFP–ILK(Fig. 3D,E). Again, the L357A (L/A) mutations in the kindlin-2protein strongly perturbed ILK binding. Finally, we also confirmedthat GFP–ILK I427E and K423D were impaired in their ability tobind to purified full-length GST–kindlin-2 in pulldowns from celllysate (Fig. 3F,G). Thus, the highly conserved surface-exposed ILKresidues I427 and K423 are important for kindlin-2 binding,apparently by interacting with kindlin-2 residues 329-368 in theF2PH domain.Prior studies have highlighted kindlin-2 residues L353 and L357

within the helical linker region as important for binding ILK

(Fig. S2A) (Fukuda et al., 2014; Huet-Calderwood et al., 2014) butour data, showing the importance of ILK residue K423, suggest thatcharged kindlin residues are also important in the interaction withhelix-αH of the ILK-pKD.We, therefore, re-examined the sequenceof the helical region looking for negatively charged sidechains thatmight interact with ILK residue K423. We tested the importance ofE354 and E358 in kindlin-2 by generating individual charge-reversal mutations (E354K and E358K) and an E354K/E358Kdouble mutant (GST–kindlin-2 F2PH EE/KK). In pulldown assayswith GST–kindlin-2 F2PH, we found that the E354K and E358Kmutations partially impaired binding to GFP–ILK, and the EE/KKdouble mutation severely inhibited binding of GFP–ILK (Fig. S2B,C). Thus, kindlin-2 residues E354 and E358, in addition to L353

Fig. 1. Selection of highly conserved, hydrophobic patches on the ILK-pKD by surface mapping. (A) ConSURF (Landau et al., 2005) surface mapgenerated from 37 species of ILK-pKDmapped onto the previously determined crystal structure of the ILK-pKD in complex with α-parvin-CH2 (gray ribbon) boundto MgATP (not visible in orientations shown), generated with Chimera software (Pettersen et al., 2004), and shown in two different orientations related bya 60° rotation as indicated (PDB ID: 3KMW). Schematic representing a top-down view of the complex to show the relative orientation of α-parvin-CH2 to the ILK-pKD (left). Color scale (bottom of panel), with positions for which the conservation score was assigned with low confidence indicated in light yellow. Color-codedsurface is shown at 50% transparency, with ribbon structure in black. N- and C-termini are indicated. (B) Coulombic surface map indicating the electrostaticpotential was generated by using Chimera software (Pettersen et al., 2004) for each orientation of the ILK-pKD–α-parvin-CH2 complex shown in Fig. 1A.Color scale (bottom of panel) is given in units of kcal mol−1 e−1 at 298 K. (C) Ribbon diagram of selected regions from the ConSURF map shown in A. Residuesselected for mutagenesis are shown as ball-and-stick display.

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and L357, are important for the interaction between kindlin-2 andILK. To test whether the effect of these charge-swap mutationscould be reversed by a compensatory charge swap in ILK,we assessed binding of these GST–kindlin-2 F2PH mutants toGFP–ILK K423D (Fig. S2B,C). While GFP–ILK K423D wasstrongly impaired in binding to GST–kindlin-2 E358K and GST–kindlin-2 F2PH EE/KK, its binding to GST–kindlin-2 E354K wasslightly enhanced (Fig. S2B,C). This raises the possibility of aninteraction between K423 in ILK and E354 in kindlin-2, although adirect interaction between these residues cannot be confirmedwithout high-resolution structural information.

Kindlin-2-binding-deficient ILK-pKD mutants co-purify withα-parvin-CH2 and are structurally stableILK stability and function require binding to the CH2 domain ofα-parvin (Fukuda et al., 2009). To test whether the effect of ILKI427E and K423D mutations on kindlin binding might be mediatedby destabilization of the ILK-pKD and/or disruption of theILK-pKD–α-parvin-CH2 interaction, we co-purified recombinantGST-ILK-pKD (wild type or mutant) in complex with α-parvin-CH2 by co-expressing the GST-ILK-pKD and α-parvin-CH2 inEscherichia coli. The GST-fusion complex was purified byglutathione affinity chromatography and, after removal of the

GST tag with tobacco etch virus protease (TEV), further purified byResource S cation exchange chromatography. The wild-type ILK-pKD and the I427E and K423D point mutants each co-purified withα-parvin-CH2 through both chromatography steps (Fig. 4A). Thus,like wild-type ILK-pKD, the two helix-αH mutants are stablyassociated with α-parvin-CH2. To assess protein stability, wemeasured the apparent melting temperature of the purified proteincomplexes by differential scanning fluorimetry (DSF) usingSYPRO orange fluorescence. Visual inspection of the firstderivatives of fluorescence revealed similar melting profiles forthe wild-type and mutant complexes (Fig. 4B), and non-linearfitting of the thermal denaturation data to identify the midpoint ofunfolding (Tm) (Huynh and Partch, 2015) revealed similar Tm

values for the wild-type, I427E and K423D ILK-pKD–α-parvin-CH2 complexes in multiple experiments (Fig. 4C,D). These valuesare comparable to the previously reported Tm for the ILK-pKD–α-parvin-CH2 complex (Murphy et al., 2014). Furthermore, by usingthe purified ILK-pKD–α-parvin-CH2 complexes described above,we assessed direct binding to GST–kindlin-2 constructs in pulldownassays. This showed that, as seen in the lysate pulldown assays(Fig. 2C,D and Fig. 3B-E), the I427E andK423Dmutant complexesare severely impaired in binding to GST–kindlin-2 F2PH and GST–kindlin-2 329-368 (Fig. 4E-H). Thus, the inability of the ILK-pKD

Fig. 2. I427E mutation of αH in surface 2of ILK-pKD impairs binding of GFP–ILKtoGST–kindlin-2 F2PH. (A) GFPor GFP–ILK co-expressed with FLAG–α-parvin inCHO cells bound to glutathione bead-immobilized GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L357A (L/A) as a negativecontrol detected by immunoblotting. Onerepresentative blot for each constructtested is shown. The lane labeled ‘3%’

indicates 3% of input lysate. Bead loadingwas visualized by Ponceau S staining.(B) Quantification of GFP or GFP–ILKbinding to GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A (mean±s.e.m.; n=4).(C) Representative immunoblots forpulldown of GFP–ILK mutants co-expressed with FLAG–α-parvin in CHOcell lysates by GST–kindlin-2 F2PH orGST–kindlin-2 F2PH L/A. The lane labeled‘3%’ indicates 3% of input lysate. Beadloading was visualized by Ponceau Sstaining. (D) Quantification of binding ofGFP–ILK and GFP–ILK mutants to GST–kindlin-2 F2PH or GST–kindlin-2 F2PHL/A (mean±s.e.m.; n≥3); *P<0.005(Student’s t-test). Pulldown quantificationgraphs are shown as bar charts withindividual data points plotted (dots).

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Fig. 3. Additional residues on αH in the ILK-pKD are implicated in the interactionwith kindlin-2. (A) Ribbon diagram of helix-αHand surrounding residues inthe ILK-pKD–α-parvin-CH2 co-crystal structure (PDB ID: 3KMW) generated with Chimera software (Pettersen et al., 2004). Residues selected for mutation arelabeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same color scale as shown in Fig. 1A. (B,C) Pulldownof GFP–ILK or GFP–ILK mutants by GST–kindlin-2 F2PH and GST–kindlin-2 F2PH L357A (L/A) from CHO cell lysate co-overexpressing FLAG–α-parvinassessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n≥3; *P<0.001 (Student’s t-test). (D,E) Pulldown of GFP–ILK or GFP–ILKmutantsfrom CHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 329-368 or GST–kindlin-2 329-368 L/A were assessed by representativeimmunoblots (D) and quantified (E); mean±s.e.m.; n≥3; *P≤0.0006. (F,G) Pulldown of GFP–ILK or GFP–ILK mutants from CHO cell lysate co-overexpressingFLAG–α-parvin using GST or GST–kindlin-2 were assessed in representative immunoblots (F) and quantified (G); mean±s.e.m.; n=4; *P≤0.0001 (Student’st-test). Pulldown quantification graphs are shown as bar charts with individual data points plotted (dots). GST- protein loading is indicated by Ponceau S staining.

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I427E and K423D mutants to bind to kindlin-2 F2PH cannot beexplained by instability of the ILK-pKD or a defect in α-parvinbinding and is, instead, likely to be due to direct disruption of aconserved binding surface on the C-lobe of ILK-pKD for a highlyconserved helical fragment in the kindlin-2 F2PH.

Mutations at a recently reported kindlin-2 binding interfaceon the ILK-pKD perturb α-parvin bindingWhile our study was underway, it was reported that substitutionsR243G and R334G in the N-lobe of the ILK-pKD inhibit binding tokindlin-2 F2PH (Guan et al., 2018). These residues are in the

vicinity of our surface 1 mutants (Fig. 5A). While our surface 1mutants had little impact on GFP–ILK binding to kindlin-2 F2PH(Fig. 2C,D), we confirmed that the R243G and R334G doublemutant (GFP-ILK RR/GG) is strongly impaired in binding(Fig. 5B,C). However, when we attempted to purify recombinantGST-ILK-pKD RR/GG from bacteria co-expressing α-parvin-CH2,we were unable to obtain stable soluble material (data notshown). This prevented us from directly assessing the proteinstability of the purified ILK-pKD RR/GG–α-parvin-CH2 mutantcomplex as we had done for the helix-αH mutants (Fig. 4B,C).We, therefore, utilized immunoprecipitation to assess association of

Fig. 4. ILK-pKD mutants that are defective inbinding kindlin-2 co-purify with α-parvin-CH2 and are structurally stable. (A)Coomassie protein stain of purified non-mutantILK-pKD, ILK-pKD I427E and K423D ILK-pKD–α-parvin-CH2 complexes. (B) Negativederivatives of SYPRO Orange fluorescencewith respect to temperature for the non-mutantILK-pKD, ILK-pKD I427E and K423D ILK-pKD–α-parvin-CH2 complexes from onerepresentative experiment. (C) NormalizedSYRPO Orange fluorescence data fitted to aBoltzmann sigmoid by using Graphpad Prismsoftware for the purified ILK-pKD–α-parvin-CH2complexes from one representative experiment.Statistical fit parameters: non-mutant ILK-pKD:r2=0.99, V50=49.7±0.2 (V50±s.e.m.); ILK-pKDI427E: r2=0.99, V50=49.7±0.2; ILK-pKDK423D: r2=0.95, V50=50.4±0.4. (D) Meltingtemperature extracted from the half-maximalpoint of the fitted fluorescence data (mean±s.e.m.; n=3); ns, not significant, P>0.05(Student’s t-test). (E,F) Pulldown of purified ILK-pKD–α-parvin-CH2 complexes by immobilizedGST–kindlin-2 F2PH or GST–kindlin-2 F2PHL357A (L/A) assessed by representativeimmunoblots (E) and quantified (F) (mean±s.e.m.; n≥3); *P≤0.0001 (Student’s t-test).(G,H) Pulldown of purified ILK-pKD–α-parvin-CH2 complexes by immobilized GST–kindlin-2329-368 or GST–kindlin-2 329-368 L/Aassessed by representative immunoblots(G) and quantified (H); mean±s.e.m.; n≥3;P≤0.0001 (Student’s t-test). Pulldownquantification is shown as bar charts withindividual data points plotted (dots). GST-protein loading for pulldowns is indicated byPonceau S staining.

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co-overexpressed GFP–ILK RR/GG with FLAG–α-parvin andincluded GFP–ILK I427E as a control. We also included theadditional control of the K220M substitution mutant of GFP–ILK(GFP–ILK K220M), originally generated as a kinase-dead mutant

but now known to disrupt α-parvin binding (Lange et al., 2009).Using a GFP-nanotrap (Rothbauer et al., 2008), we precipitatedGFP–ILK, assessed the amount of co-precipitating FLAG–α-parvinby immunoblotting and calculated the ratio of co-precipitated

Fig. 5. R243G/R334G double mutation of GFP–ILK (GFP–ILK RR/GG) impairs binding of the ILK to α-parvin. (A) Ribbon diagram of selected regions in theILK KD–α-parvin-CH2 complex co-crystal structure (PDB ID: 3KMW) surrounding I244, F245, and S246, generated with Chimera software (Pettersen et al.,2004). Residues selected for mutagenesis are labeled and shown as a ball-and-stick representation. Conservation coloring is indicated using the same colorscale as shown in Fig. 1A. (B,C) Pulldown of GFP–ILK or GFP–ILK RR/GG from CHO cell lysates co-overexpressing FLAG–α-parvin using GST–kindlin-2F2PH or GST–kindlin-2 F2PH L357A (L/A) assessed by representative immunoblots (B) and quantified (C); mean±s.e.m.; n=3; *P≤0.0001 (Student’s t-test).(D) Diagram of the GFP nanotrap experiment. GFP–ILK was purified from lysate of cells co-expressing GFP–ILK and FLAG-α-parvin. The amount of co-purifyingFLAG–α-parvin was assessed by immunoblotting and the FLAG:GFP ratio for each construct was calculated. (E,F) GFP-nanotrap co-purification of GFP–ILKconstructs co-expressed FLAG–α-parvin in CHO cells was assessed by immunoblot from one experiment (E) and quantified (F) as a raw FLAG:GFP ratio.Dots represent single data points for each construct tested. The lane labeled ‘2%’ indicates the 2% input of lysate. (G) The FLAG:GFP ratio for each GFP or GFP–ILK construct tested is expressed relative to the FLAG:GFP ratio of the GFP–ILK control within each experiment, which is set to 1. The dataset includes theexperiment shown in E and F (mean±s.e.m.; n≥4); *P≤0.0015; ns, statistically not significant, P>0.05 (Student’s t-test). (H,I) Pulldown of GFP–ILK K220M fromCHO cell lysate co-overexpressing FLAG–α-parvin using GST–kindlin-2 F2PH or GST–kindlin-2 F2PH L/A assessed by representative immunoblot (H) andquantified (I); mean±s.e.m.; n=3; *P≤0.0001. Pulldown and co-purification quantification graphs are shown as bar charts with individual data points plotted (dots).GST-protein loading control for pulldown experiments is indicated by Ponceau S staining.

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FLAG–α-parvin to precipitated GFP–ILK (FLAG:GFP ratio)(Fig. 5D). Fig. 5E shows representative immunoblots from oneexperiment, with quantification of the raw FLAG:GFP ratios shownin Fig. 5F. Pooled data frommultiple experiments, where the FLAG:GFP ratio was normalized to the ratio of the GFP–ILK control ineach experiment, are shown in Fig. 5G. Although this approachcannot provide the stoichiometry of ILK–kindlin-2 complexes, asexpected from our experiments with purified proteins (Fig. 4A),GFP–ILK I427E co-precipitated an amount of FLAG–α-parvin thatwas comparable to that of wild-type GFP–ILK, while GFP–ILKK220M precipitated significantly less FLAG–α-parvin (Fig. 5E-G).Notably, GFP–ILK RR/GG also co-precipitated substantiallyless α-parvin than GFP–ILK wild type and GFP–ILK I427E(Fig. 5E-G). The crystallographically defined α-parvin-binding site,and ILK residues R243 and R334 lie on opposite sides of theILK-pKD (Fukuda et al., 2009), yet our data clearly suggest thatα-parvin binding is perturbed when these residues are mutated toglycines. Since α-parvin binding is required for ILK structuralstability, it is possible that the impaired binding ability of the RR/GG mutant to kindlin-2 is the indirect result of its reduced ability tobind α-parvin. This is consistent with our inability to producesoluble ILK-pKD RR/GG in E. coli (data not shown). Notably,GFP–ILKK220M, another parvin-binding defective mutant (Langeet al., 2009), is also impaired in binding to GST–kindlin-2 F2PH inpulldown experiments (Fig. 5H,I), supporting the idea thatdisruption of the ILK–α-parvin interaction indirectly impairskindlin binding, possibly by destabilization of the ILK-pKD.

Kindlin-2 binding facilitates ILK localization to focaladhesionsTo test whether the interaction with kindlin-2 is required for thelocalization of ILK to focal adhesions, we utilized total internalreflection fluorescence (TIRF) microscopy to visualize thelocalization of GFP–ILK in live cells. We generated stable CHOcell lines expressing mCherry-paxillin as a marker for focaladhesions. We transiently overexpressed GFP–ILK or GFP–ILK

mutants in these cells concomitantly with FLAG-α-parvin, platedthem on fibronectin-coated glass-bottom dishes and examined thelocalization of GFP–ILK mutants to paxillin-containing focaladhesions by TIRF and epifluorescence microscopy. AlthoughGFP–ILK localized to focal adhesions when conductingepifluorescence microscopy, GFP, GFP–ILK I427E and GFP–ILK K423D exhibited barely detectable focal adhesion targeting(Fig. 6). When the same cells were imaged by TIRF microscopy toenhance visualization at the cell-matrix interface, GFP–ILK was veryclearly detected in focal adhesions, while GFP alone was not; andalthough GFP–ILK I427E and GFP–ILK K423D were observed insome focal adhesions, they appeared to target muchmoreweakly thanwild-type GFP–ILK (Fig. 6). GFP–ILK K426D, which only partiallyinhibited binding to kindlin-2 in pulldown experiments,and GFP–ILK I413D, which had no strong effect on kindlin-2binding, localized to focal adhesions when visualized by eitherepifluorescence or TIRF microscopy (Fig. 6). The kindlin-bindingdefective I427A mutant is also strongly impaired in its localization tofocal adhesions in these cells (Fig. S3A), suggesting that changes incharge distribution along helix-αH do not explain the defect in focaladhesion targeting imparted by surface 2 mutations. Consistent withprior reports, GFP–ILK RR/GG, which is impaired in binding toparvin and kindlin, exhibits strongly impaired focal adhesionlocalization (Fig. S3B and Guan et al., 2018). In addition, GFP–ILK K220M is also impaired in focal adhesion targeting (Fig. S3B).Together, these results suggest that binding of kindlin-2 to ILKfacilitates the localization of ILK to focal adhesions.

Interaction between kindlin-2 and ILK is required forspreading of HeLa cellsBoth kindlin-2 and ILK have been implicated in cell spreading(Friedrich et al., 2004; Montanez et al., 2008; Sakai et al., 2003;Stanchi et al., 2009; Theodosiou et al., 2016). We, therefore,evaluated the role of the ILK–kindlin-2 interaction in cell spreading.To generate ILK-deficient cells, we infected HeLa cells with alentivirally delivered shRNA construct targeting human ILK

Fig. 6. GFP–ILK mutants that are impaired in kindlin-2 binding localize poorly to focal adhesions. CHO cells stably expressing mCherry-paxillin weretransiently co-transfected with FLAG–α-parvin and either GFP alone, GFP–ILK or one of the GFP–ILK mutants. Six hours after replating on fibronectin-coatedglass-bottom dishes, live cells were imaged by epifluorescence (EPI) and/or TIRF microscopy as indicated. Images in each channel were linearly and uniformlyadjusted, and cropped for clarity. Scale bar: 20 µm.

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(shILK) or a scrambled shRNA (shScr) control. Immunoblottingshowed only ∼10% residual ILK protein in shILK-infected ILKknockdown HeLa (shILK cells) and, as expected, protein levels ofα-parvin and PINCH1– components of the IPP complex –were alsosubstantially diminished (Fig. 7A,B). Kindlin-2 levels were onlymodestly impacted (Fig. 7A,B). Morphologically, shILK cellsspread poorly on fibronectin and formed few, small vinculin-containing focal adhesions (Fig. 7C). This was not simply due todelayed spreading of shILK cells as the phenotype was still evident18 h after cell plating (Fig. 7C). To investigate the importance ofinteractions between ILK and kindlin for the spreading of HeLacells, we transiently re-expressed shILK-resistant GFP–ILK or oursurface 2 GFP–ILK mutants (I427E, K423D, K426D and I413D) inshILK cells, and plated the cells on fibronectin-coated coverslips.

Cells were fixed and stained with phalloidin 18 h after plating andCellProfiler version 2.0 (Kamentsky et al., 2011) was used to calculateareas of GFP-positive cells across multiple fields of view and multiplereplicate experiments using the phalloidin stain to identify the celloutline. Immunoblotting confirmed that all constructs expressed attheir expected molecular mass (Fig. S4A). We clearly observed aprofound spreading defect in shILK cells expressing GFP, which wasfully reversed in shILK cells expressingGFP–ILK (Fig. 7D). Surface 2mutant GFP–ILK I413D, for which kindlin-2 binding was notsignificantly altered, and GFP–ILK K426D, for which only partialinhibition of kindlin-2 binding was produced, were also able to fullyrescue shILK cell spreading (Fig. 7D). Notably, neither GFP–ILKI427E nor GFP–ILK K423D, which both strongly impair binding tokindlin-2, were able to fully rescue the spreading defect of shILK cells

Fig. 7. The ILK–kindlin-2 interaction is important for normalcell spreading in HeLa cells. (A) Immunoblotting of shScr andshILK HeLa cells to show protein levels of ILK, kindlin-2, α-parvin,PINCH1 and vinculin. (B) Bar graph showing residual proteinlevels in shILK cells calculated relative to those in shScr cells(mean±s.e.m.); individual data points are indicated (dots; n≥4).(C) Immunofluorescence staining of endogenous vinculin in fixedshScr or shILK HeLa cells spread on fibronectin-coated glasscoverslips and acquired by epifluorescence microscopy. Scalebars: 20 µm. (D) CellProfiler quantification of GFP-positive cellareas pooled across three independent experiments shown asbox and whiskers plots indicating 10th and 90th percentile range.n=155 shScr+GFP cells, 169 shILK+GFP cells, 154 shILK+GFP–ILK cells, 162 shILK+I427E cells, 143 shILK+K423D cells, 137shILK+K426D cells, 125 shILK+I413D cells; *, significantlydifferent from shScr+GFP calculated using one-way ANOVA andTukey’s correction for multiple comparisons (P≤0.01); ns,statistically not significant; P>0.05. (E) Immunoblotting of shScrand shK2HeLa cells to show protein levels of kindlin-2, ILK, and β-tubulin. (F) Bar graph showing residual kindlin-2 or ILK proteinlevels in shK2 cells calculated relative to those in shScr cells(mean±s.e.m.); individual data points are indicated (dots; n≥3).(G) Immunofluorescence staining of endogenous vinculin in fixedshScr or shK2 HeLa cells spread on fibronectin-coated glasscoverslips and acquired by epifluorescence microscopy. Scalebars: 20 µm. (H) CellProfiler quantification of GFP-positive cellsareas pooled across three independent experiments; n=143shScr+GFP cells, 121 shK2+GFP cells, 145 shK2+GFP-K2 cells,130 shK2+GFP-K2 LA cells. Data are shown as box and whiskersplot, with whiskers indicating the 10th and 90th percentile range.*, significantly different from shScr+GFP calculated using one-way ANOVA and Tukey’s correction for multiple comparisons(P≤0.015); ns, statistically not significant. Microscopy imageswere linearly and uniformly adjusted for clarity.

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(Fig. 7D), suggesting that kindlin binding contributes to ILK functionin cell spreading.Notably,whenwe examined the localization ofGFP–ILK in live shILK HeLa cells using mCherry-Paxillin as a marker forfocal adhesions, GFP–ILK K423D and GFP–ILK I427E clearlylocalized to focal adhesions in these cells, although targeting appearedweaker compared to that of GFP–ILK (Fig. S5). This is consistent withthe notion that, under conditions of reduced competition fromendogenous ILK, GFP–ILK can be recruited to focal adhesions in akindlin-2-independent manner (Huet-Calderwood et al., 2014).Nonetheless, despite targeting to focal adhesions, the kindlin-binding defective ILK was unable to fully support cell spreading.To further test the importance of kindlin-ILK interactions in cell

spreading, we knocked down kindlin-2 in HeLa cells (shK2 cells).Despite screening five shRNAs that target kindlin-2, the bestspecific knockdown we achieved was ∼70% depletion and thishairpin was used in further experiments (Fig. 7E,F). Kindlinknockdown had minimal effect on ILK expression levels (Fig. 7E,F).Nonetheless, similar to shILK cells, shK2 cells spread poorly onfibronectin, and formed only small vinculin-containing adhesions(Fig. 7G). We expressed GFP or knockdown-resistant GFP-kindlin-2or GFP–kindlin-2 L357A (L/A) in these cells and used Cell Profiler2.0 (Kamentsky et al., 2011) to quantify the area ofGFP-positive cellsacross multiple biological replicates. We found that shK2 cellstransiently expressing GFP were smaller than shScr cells; this defectwas fully rescuable with GFP-kindlin-2 but not with the ILK-bindingdefective GFP–kindlin-2 L/A (Fig. 7H), despite both constructsmigrating at their expected size in immunoblots (Fig. S4B). Together,with our results in reconstituted ILK knockdown cells, this stronglysupports the conclusion that kindlin-2-ILK interactions are essentialfor normal cell spreading.

DISCUSSIONKindlins and ILK are key components of cell-matrix adhesions withessential roles in cell adhesion and spreading (Calderwood et al.,2013; Li et al., 1999). Kindlins make direct interactions with theβ-integrin cytoplasmic tail (Calderwood et al., 2013) and therecognition that kindlins also directly bind ILK (Fukuda et al., 2014;Huet-Calderwood et al., 2014) highlighted a potential kindlin-mediated linkage between integrins and the IPP complex. However,a thorough understanding of the kindlin-ILK interaction and itsimportance in integrin-mediated signaling has been hindered by aninability to identify the binding site for kindlin-2 on ILK and, hence,to generate mutations that selectively impair kindlin binding. In thisstudy, we used a combination of conservation-guided surfacemapping of the ILK-pKD, biochemical analysis of the kindlin-2-ILK interaction, and functional studies in knockdown cells to showthat (1) kindlin-2 interacts with highly conserved residues on helix-αH in the C-lobe of the ILK-pKD, (2) efficient localization of ILKto focal adhesions requires binding to kindlin-2 and, (3) the ILK–kindlin-2 interaction is important for normal cell spreading.Focusing on highly conserved hydrophobic patches on the

surface of the ILK-pKD, we identified three candidate kindlin-binding sites that we termed surface 1, 2 and 3. Mutagenesislocalized the interaction to surface 2 on the C-lobe of the pKD andrevealed key residues on helix-αH, i.e. I427 and K423, with theadjacent K426 playing a less important role. We demonstrate thathelix-αH mutations that disrupt binding to kindlin-2 did not impairthe association of ILK with α-parvin, a requirement for stability(Fukuda et al., 2009), and maintained structural integrity of the ILK-pKD–α-parvin-CH2 complex when assessed by DSF. Furthermore,even somewhat conservative ILK I427A mutations perturbedkindlin-2 binding, indicating that the effect is not simply due to

changing the surface charge in this area. Notably, helix-αH has beenimplicated in protein–protein interactions in other kinase domains.For example, an important component of activation of the epidermalgrowth factor receptor (EGFR) involves formation of an asymmetricdimer through hydrophobic interactions between the N-lobe of onekinase domain and residues in the C-lobe of its partner kinasedomain that includes residues on helix-αH (Zhang et al., 2006). ILKis a catalytically incompetent scaffold (Fukuda et al., 2009); thus,we propose that helix-αH is important not for activation but forbinding kindlin-2.

Previous studies have proposed other potential kindlin-2interaction surfaces on the ILK-pKD. Most recently, a bindinginterface for the ILK-binding helical fragment in the kindlin-2F2PH (Fukuda et al., 2014; Huet-Calderwood et al., 2014) wasidentified on the N-lobe of the ILK-pKD by rigid-body docking(Guan et al., 2018). The reported binding surface is formed by thehighly conserved R243 and the less-well conserved R334 (Guanet al., 2018). These residues lie near the surface 1 residues that wefound to have a minimal effect on kindlin-2 binding; but weconfirmed that the reported GFP-ILK RR/GG double mutant (Guanet al., 2018) impaired kindlin-2 binding in our pulldown assays andILK targeting to focal adhesions in cells. Unfortunately, we wereunable to obtain soluble purified GST-ILK RR/GG in complex withα-parvin-CH2 from E. coli, preventing us from assessing thestability of the mutant ILK-pKD (data not shown). Co-immunoprecipitation assays from transfected mammalian cells didhowever indicate that the GFP-ILK RR/GG mutation significantlyimpaired association with FLAG-α-parvin. R243 and R334 arelocated far from the crystallographically defined parvin-binding site(Fukuda et al., 2009) but we suggest that the double RR/GGmutation alters ILK confirmation or stability indirectly, resulting inimpaired parvin and kindlin binding. In support of this idea, wefound that a K220M mutation known to indirectly disrupt ILK–α-parvin association (Lange et al., 2009) is also severely impaired inbinding to kindlin-2. Thus, although we cannot definitively rule outthe possibility that the interface formed by R243 and R334 isinvolved in the interaction with another part of the kindlin-2 F2PH,we clearly show that mutations on helix–αH selectively impairbinding to the conserved helical fragment in the kindlin-2 F2PH,while maintaining association with α-parvin and integrity of theILK-pKD. Furthermore, spatial separation of the helix-αH residuesthat we identified as being important for binding kindlin-2, andR243 and R334, make it unlikely that the ILK-binding helicalfragment of kindlin-2 could engage both sites simultaneously. Wealso note that, although not discussed or tested further, in the priordocking study two additional models showed the kindlin-2 helicalfragment binding to helix-αH in the ILK-pKD (Guan et al., 2018),further supporting a role for helix-αH in kindlin binding.

Notably, studies with the C. elegans orthologues of kindlin(UNC-112) and ILK (PAT-4) have also investigated the ILK-kindlin interface (Mackinnon et al., 2002; Qadota et al., 2012,2014). A yeast two-hybrid screen of UNC-112 (kindlin) mutantsdefective in PAT-4 (ILK) binding identified a D382V mutant in thelinker between the F2 and PH domains of UNC-112 (Qadota et al.,2012). This region contains the ILK-binding site in mammaliankindlins (Fukuda et al., 2014; Huet-Calderwood et al., 2014) butD382 corresponds to highly conserved S351 in mammaliankindlins. However, a S351V mutation in kindlin-2 does notinhibit ILK binding (Huet-Calderwood et al., 2014), indicatingdifferences in the mammalian and C. elegans interactions. A yeasttwo-hybrid suppressor mutation screen for PAT-4 mutants thatrestore binding to UNC-112 D382V suggested two large potential

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interaction surfaces for UNC-112 (Qadota et al., 2014). The firstmaps to a region in the N-lobe of the ILK-pKD overlapping with oursurface 1 and includes the C. elegans PAT-4 residues I261 and F262that correspond to human ILK residues I244 and F245. However, asin our pulldown assays – where I244D or F245D mutants of ILKbound kindlin-2 at near wild-type levels – PAT4 I261N or F262Lmutants still bound UNC-112 (Qadota et al., 2014). The secondinterface maps to the C-lobe of the ILK-pKD and includes tworesidues on helix-αH: M440 (human residue M425) and A433(human residue S418). In human ILK, M425 is located on theburied face of αH, and S418 lies on the N-terminal cap of helix-αHoriented away from the key kindlin-binding residues K423 and I427identified in our study, which are conserved in PAT-4 (R438 andI442 respectively). While the mechanisms by which suppressormutations facilitate PAT-4 binding to mutant UNC-112 are unclear,their localization supports our conclusion that the ILK helix-αH isimportant for binding kindlin. The role of residues in surface 1appears more complex, as no surface 1 mutations that selectivelyimpair kindlin binding have been identified. However, the fact thatthis region has been identified as a potential kindlin-binding site inindependent studies (Guan et al., 2018; Qadota et al., 2014)suggests that residues in surface 1 may be important for overallpseudokinase domain conformation.Our observation that ILKmutants impaired in binding to kindlin-2

are hindered in their ability to localize to focal adhesions is consistentwith a linear model where integrin β subunit cytoplasmic tails bindand recruit kindlin, which in turn recruits ILK and the IPP complex.However, additional data indicate that this model is overly simplisticas our data, and that of others, shows that kindlin and ILK are co-dependent because kindlin mutations that inhibit ILK binding alsoimpair kindlin accumulation at adhesions in mammalian cells and inC. elegans muscle cells in vivo (Fukuda et al., 2014; Huet-Calderwood et al., 2014; Qadota et al., 2012). It has been proposedthat ILK binding induces conformational changes in kindlin thatexpose the integrin-binding site allowing kindlin to recruit ILK toadhesions (Qadota et al., 2012) which may explain the reliance ofboth kindlin and ILK on binding for recruitment to adhesions, butdirect evidence of such a conformational change is currently notavailable. Understanding kindlin and ILK targeting to adhesions isfurther complicated by the observation that kindlins (through the F0and F2-PH domains) and the IPP complex (through the CH2 domainof parvin) both bind paxillin, and that these interactions are alsoimportant for their targeting to adhesions (Gao et al., 2017; Stiegleret al., 2012; Theodosiou et al., 2016;Wang et al., 2008). Nonetheless,our observation that mutants in ILK or kindlin-2 that disrupt the ILK-kindlin complex also fail to fully rescue spreading defects in ILK orkindlin-2 knockdown cells further underscores the importance of thiscomplex. Dissection of the specific downstream signaling along theILK–kindlin-2 axis that promotes normal cell spreading will be thesubject of future studies.In summary, we have mapped the interaction between kindlin-2

and ILK to highly conserved residues on helix-αH of the ILK-pKD,and established that the interaction between kindlin-2 and ILK isimportant for their correct localization to focal adhesions and fornormal cell spreading.

MATERIALS AND METHODSAntibodiesPrimary antibodies against GFP (Rockland, catalog #601-101-215;Limerick, PA) (used at 1:1000), ILK (Cell Signaling, catalog #3862;Danvers, MA) (used at 1:1000), FLAG (Sigma, catalog #F1804; St. Louis,MO) (used at 1:1000), vinculin (Sigma, catalog #V9131) (used at 1:10,000),

kindlin-2 (Proteintech, catalog #11453-1-AP; Rosemont, IL) (used at1:1000), α-parvin (Cell Signaling, catalog #8190) (used at 1:1000),PINCH1 (Proteintech, catalog #55336-1-AP) (used at 1:1000), carbonylreductase (Santa Cruz Biotechnology, catalog #sc-70212; Dallas, Texas)(used at 1:1000) and β-tubulin (Developmental Studies Hybridoma Bank,catalog #E7; Iowa City, Iowa) (used at 1:1000) as well as IRDye-conjugatedsecondary antibodies (Li-Cor; Lincoln, NE) (used at 1:10,000) werepurchased from commercial sources.

ConstructsVectors encoding N-terminally GFP-tagged human ILK and kindlin-2, GFPalone, FLAG-tagged human α-parvin, GST-tagged human ILK-pKD, andHis-FLAG-tagged human α-parvin-CH2 were as previously described (Huet-Calderwood et al., 2014; Stiegler et al., 2012, 2013). Point mutations weregenerated by QuikChange site directed mutagenesis following themanufacturer’s instructions (Stratagene; La Jolla, CA). A pLENTI CMVHygro lentiviral paxillin-mCherry expression vector was previouslydescribed (Huet-Calderwood et al., 2017). All constructs were verified byDNA sequencing. pLKO expression vectors containing shRNAs (Scramble:catalog no. SHC002; human ILK: TRCN0000199983, targetsequence 5′-GCAATGACATTGTCGTGAAGG-3′; human kindlin-2:TRCN0000127493, target sequence 5′-CCAGGGCTTAACCTATATGAA-3′ were purchased from Sigma.

Cell culture and transfectionHeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)with 9% fetal bovine serum (FBS), sodium pyruvate, and non-essentialamino acids (NEAA) (Gibco Laboratories; Gaithersburg, MD) at 37°C in5% CO2, and were obtained from colleagues at Yale University. CHO cells,which have been engineered to stably express integrin αIIbβ3 and describedpreviously (O’Toole et al., 1994), were cultured in DMEM with 9% FBS,sodium pyruvate, and NEAA at 37°C in 5% CO2. CHO cells weretransiently transfected with linear polyethylenimine (PEI, MW 25,000)(Polysciences, Inc.; Warrington, PA), and HeLa cells were transientlytransfected with Lipofectamine 2000 (Invitrogen; Waltham, MA). Cellswere regularly tested for mycoplasma using the MycoAlert mycoplasmadetection kit (Lonza; Basel, Switzerland).

Recombinant protein production and purificationGST-ILK-pKD in complex with His-FLAG–α-parvin-CH2 was producedin BL21(De3) E. coli cells (Millipore Sigma) as previously described (Huet-Calderwood et al., 2014). The complex was co-purified by glutathioneaffinity chromatography using glutathione-sepharose 4b (GE Healthcare;Chicago, IL), and eluted with reduced glutathione. The GST- and His-tagswere removed by cleavage with purified tobacco etch virus protease (TEV).Following affinity tag removal, the ILK-pKD–α-parvin-CH2 complex wasanalyzed and purified by Resource S cation exchange chromatography (GEHealthcare). Fractions containing the complex were pooled, concentratedusing Amicon Ultra centrifugal filter units (Sigma), snap frozen in liquidnitrogen, and stored at –80°C for subsequent usage. Recombinant GST–kindlin-2 F2PH and GST–kindlin-2 F2PH 329-368 were purified aspreviously described (Huet-Calderwood et al., 2014).

GFP nanotrap purificationGFP binding protein, derived from a llama single-chain antibody(Rothbauer et al., 2008), was produced in BL21 RIPL competent E. coli(Millipore Sigma). Cultures (1 l) were induced with 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) at 16°C for 20 h. Bacterial pellets wereharvested by centrifugation, and re-suspended in lysis buffer consisting of20 mM HEPES (pH 7.2), 250 mM NaCl, 10% glycerol, cOmplete EDTA-free protease inhibitor (Roche; Indianapolis, IN), and 1 mg ml−1 lysozyme.The lysate was incubated on a rotary mixer for 1 h at 4°C and then sonicated.After clarification by centrifugation, the lysate was loaded onto a pre-equilibrated Ni-NTA column (Millipore Sigma), and bound protein waseluted with 20 mM HEPES (pH 7.2), 250 mM NaCl, 5% glycerol, and200 mM imidazole. Fractions containing protein were pooled and dialyzedinto PBS at 4°C. After dialysis, the material was fractionated using a HiLoadSuperdex S200 preparative grade size-exclusion chromatography column

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(GE Healthcare). Appropriate fractions were pooled, and snap frozen inliquid nitrogen. Covalent coupling was done according to manufacturerinstructions. In brief, aliquots were thawed on ice, and 1 M NaHCO3 (pH8.3) added to a final concentration of 200 mM. After clarification bycentrifugation, soluble protein was incubated with NHS-activated FastFlowsepharose (GE Healthcare) for 24 h at 4C. The reaction was quenched with0.1 M Tris-HCl (pH 8.5) for 4 h at room temperature. Beads were washedwith 0.1 M sodium acetate (pH 4.5), 500 mM NaCl and PBS andre-suspended in a 50% slurry in PBS with 0.04% NaN3 and cOmpleteEDTA-free protease inhibitor (Roche), and subsequently stored at 4°C.

Lentiviral knockdown and overexpressionLentiviruses were produced by transfecting HEK293T cells with packagingvectors psPAX2 (viral proteins Gag and Rev under the SV40 promoter;Addgene plasmid #12260, a gift from Didier Trono (École PolytechniqueFédérale de Lausanne, Lausanne, Switzerland) and pMD2.G (viral proteinVSV-G expressed under the CMV promoter; Addgene plasmid #12259, a giftfrom D. Trono) together with the pLKO shRNA or pLenti-Hygro mCherry-paxillin constructs. Viral supernatant was harvested 48 and 72 h aftertransfection, and filtered with a 0.45-µm low-protein-binding filter. Cell lineswere transduced by incubating cells with viral supernatant diluted 1:5 in cellculture media supplemented with 8 µg ml−1polybrene (Sigma) for 16 h.pLKO-infectedHeLa cells were selected with 1 µgml−1 puromycin for∼72 h,until non-infected control cells were dead. pLenti-Hygro-infected HeLa cellsand CHO cells were, respectively, selected with 250 µg ml−1 and 350 µg ml−1

hygromycin B for 5 days, until non-infected control cells were dead.To calculate the residual protein level in ILK and kindlin-2 knockdown

cells, the densitometric signal obtained from immunoblots was normalizedto an internal loading control (β-tubulin or vinculin) for each protein; thisratio was then normalized to that of shScramble cells to calculate the residualprotein level.

GST-pulldown and GST-binding experimentsFor pulldown experiments from cell lysate performed with GST–kindlin-2F2PH, GST–kindlin-2 329-368, and GST–kindlin-2, GFP–ILK wasoverexpressed together with FLAG–α-parvin in CHO cells by transienttransfection with PEI. Cells were lysed in buffer X (1 mM NaVO4, 50 mMNaF, 40 mM sodium pyrophosphate, 50 mMNaCl, 150 mM sucrose, 10 mMPIPES (pH 6.8) containing 0.5% Triton X-100, 0.2% deoxycholic acid andcOmplete EDTA-free protease inhibitor cocktail (Roche) for 15 min at 4°C.After clarification by centrifugation, lysatewas diluted in bufferX-T (buffer Xwith 0.05% Triton X-100), and incubated with GST–kindlin-2 F2PH, GST–kindlin-2 329-368 or GST–kindlin-2 coupled to Glutathione Sepharose 4b.Lysate was also incubated with GST–kindlin-2 F2PH L/A or GST–kindlin-2329-368 L/A, or GST only as a negative control. Incubations were carried outfor 2 h while rocking at 4°C. Beads were then washed three times with bufferX-T, and bound proteins fractionated by reducing SDS-PAGE and analyzedby immunoblotting. Immunoblots were imaged on an Odyssey InfraredImaging System (Li-Cor), and analyzed using Image Studio Lite (Li-Cor).Ponceau S stains were imaged using a UVP EpiChemI II Darkroom equippedwith a CCD camera (UVP; Upland, CA). For quantification of binding fromimmunoblots, the fluorescence intensity of the band corresponding to boundmaterial was quantified as fraction of the fluorescence of the 3% inputmaterialband for each condition. The binding of the internal positive control (e.g.GFP–ILKbinding toGST–kindlin-2 F2PH,GST–kindlin-2 329-368 orGST–kindlin-2) was set to 1, and the binding in all other conditions was expressedrelatively within each experiment. Bead loading was verified by staining thenitrocellulose membrane with Ponceau S.

For pulldowns performed with recombinant ILK-pKD–α-parvin-CH2, thecomplex was diluted in buffer X-T to a final concentration of ∼27 nM.Diluted protein was incubated with Glutathione-bead immobilized GST–kindlin-2 F2PH or GST–kindlin-2 F2PH 329-368 (and relevant negativecontrol) for 2 h rocking at 4°C. Beads werewashed three times with buffer X-T, and bound proteins fractionated by reducing SDS-PAGE and analyzed byimmunoblotting in the same fashion. Specific binding was calculated fromimmunoblots by subtracting the fluorescence intensity for binding to thenegative control beads from that of the positive control (e.g. ILK-pKDbinding to GST–kindlin-F2PH or GST–kindlin-2 329-368). This value was

then quantified as fraction of the fluorescence of the 3% input material bandfor each condition. The binding of the internal positive control (e.g. ILK-pKDbinding toGST–kindlin-2 F2PH orGST–kindlin-2 329-368)was set to 1, andthe binding in all other conditions was expressed relatively within eachexperiment. Bead loading was verified by staining the nitrocellulosemembrane with Ponceau S.

Differential scanning fluorimetry (DSF)Recombinant purified ILK-pKD-α-parvin-CH2 complex was diluted in50 mM Tris, 150 mM NaCl pH 8.0, to a final concentration of 4 µM in aPCRmulti-plate (BioRad; Hercules, CA). 5000× SYPRO orange protein gelstain (Invitrogen) was then diluted to a final concentration of 2.5× into theprotein-buffer mixture immediately before the assay. The multi-plate wasinserted into a BioRad CFX384 Touch Real-time PCR Machine (BioRad),and emission monitored using the FAM filter. Following a short step at 4°C,temperature steps of 1°C/min were performed from 5°C to 95°C. Raw data,as well as the negative first derivatives of fluorescence emission, wereexported into Microsoft Excel (Microsoft, Redmond, WA).

Non-linear fitting of DSF melting curvesRaw fluorescence data were exported from the BioRad CFX384 Touch real-time PCR Machine into a Microsoft Excel spreadsheet. The maximumnumerical fluorescence for each condition was identified, and the datatruncated beginning two points after this maximum point (Huynh andPartch, 2015). The data were exported into GraphPad Prism (GraphPadSoftware; La Jolla, CA), and data within each condition were normalized toa value between 0–100, where 0 represents the smallest value in the datasetand 100 the largest. These normalized data were then fitted to a Boltzmannsigmoid function by non-linear regression, using a least-squares-basedmethod (ordinary fit) in GraphPad Prism (GraphPad Software).

Total internal reflection fluorescence microscopyFor total internal reflection fluorescence (TIRF) microscopy, 35 mm glassbottom microwell dishes with a 14 mm microwell diameter (MatTekCorporation; Ashland, MA) were coated with 5 µg ml−1 bovine plasmafibronectin (Sigma) for 18 h at 37°C. Cells (CHO cells stably expressingmCherry-paxillin and transiently transfected with GFP–ILK or GFP–ILKmutant and FLAG–α-parvin or shILKHeLa cells stably expressing mCherry-paxillin and transiently transfected with GFP, GFP–ILK or GFP–ILKmutant)were plated on the coated glass bottom dishes in DMEM without glutamineand Phenol Red (Gibco Laboratories) but supplemented with 9% FBS,sodium pyruvate, NEAA, and GlutaMAX supplement (Gibco Laboratories).Cells were live imaged 6 h (CHO) or 18 h (shILK HeLa) later in atemperature- and CO2-controled environmentOkoLab (OkoLab; Burlingame,CA) chamber mounted onto a Nikon Ti-2 Eclipse microscope (Nikon; Tokyo,Japan) equippedwith amotorized Ti-LA-HTIRFmodulewith LUN4 488 and561 nm lasers (15 mW), using a CFI Plan Apo Lambda 100× Oil TIRFobjective and a Prime95B RoHS cMOS Camera (pixel size=110 nm)(Photometrics; Tuscon, AZ). Images were acquired and processed with theNIS-Elements AR software and ImageJ.

Immunofluorescence and quantification of the cell areaFor cell spreading experiments, transiently transfected HeLa cells were platedon glass coverslips that had been coated with 5 µg ml−1 bovine plasmafibronectin (Sigma) for 2 h at 37°C, and allowed to spread for 18 h at 37°C in5%CO2. Cells were then washed with PBS, fixed with 4% paraformaldehydein PBS for 5 min, permeabilized with PBS containing 0.1% Triton X-100 for3 min, quenched and blocked with 50 mM NH4Cl, 0.2% BSA and 0.1%Triton X-100 in PBS (PMZ-T) for 30 min at room temperature. Coverslipswere stainedwithAlexa Fluor 647 phalloidin at a 1:500 dilution in PMZ-T for30 min (Invitrogen). Coverslips were washed three times with PBS, rinsed inmilliQ H2O and mounted in FluorSave Reagent (Millipore Sigma).

To quantify cell areas,multiple fields of view containingGFP-positive cellswere imaged on each coverslip across multiple biological replicates using aNikon Ti inverted microscope equipped with a ×40 immersion oil objective.Cell Profiler version 2.0 (Kamentsky et al., 2011) was used to measure cellareas. Phalloidin staining was used to identify cells and measure cell area, andthe daa were pooled across multiple biological replicates.

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StatisticsStatistical tests to calculate P values were performed using Prism software.Two-tailed Student’s t-test or one-way ANOVA using Tukey’s correctionfor multiple comparisons were performed as indicated in figure legends.

AcknowledgementsThe authors thank Amy Stiegler for her help with purification of the ILK-pKD–α-parvin-CH2 complexes. We also thank Josie Bircher for her contributions at thebeginning of this work, and members of the Calderwood lab for helpful discussionsand comments.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsY.A.K. designed and performed experiments, analyzed data, and wrote thepaper. C.H.-C. performed experiments and provided expertise. B.S. providedessential reagents and expertise. D.A.C. designed experiments, analyzed data,and wrote the paper.

Author contributions metadataConceptualization: Y.A.K., D.A.C.; Investigation: Y.A.K., C.H.-C., B.S.; Writing -original draft: Y.A.K.; Writing - review & editing: Y.A.K., C.H.-C., B.S., D.A.C.;Visualization: Y.A.K.; Supervision: D.A.C.; Project administration: D.A.C.; Fundingacquisition: D.A.C.

FundingThis work was supported by the National Institutes of Health grants R01-GM068600and R01-NS085078 (to D.A.C). Y.A.K is supported by a National ScienceFoundation Graduate Research Fellowship (DGE1122492). Deposited in PMC forrelease after 12 months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.221184.supplemental

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