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. 2018 Oct 26;9(1):4465.
doi: 10.1038/s41467-018-06906-7.

Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion

Julia Vaynberg  1 , Koichi Fukuda  1 , Fan Lu  1   2 , Katarzyna Bialkowska  1 , Yinghua Chen  3 , Edward F Plow  1 , Jun Qin  4   5
Affiliations

Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion

Julia Vaynberg et al. Nat Commun. .

Abstract

Dynamic communication between integrin-containing complexes (focal adhesions, FAs) and actin filaments is critical for regulating cell adhesion. Pseudokinase ILK plays a key role in this process but the underlying mechanism remains highly elusive. Here we show that by recruiting FA adaptors PINCH and Parvin into a heterotrimeric complex (IPP), ILK triggers F-actin filament bundling - a process known to generate force/mechanical signal to promote cytoskeleton reassembly and dynamic cell adhesion. Structural, biochemical, and functional analyses revealed that the F-actin bundling is orchestrated by two previously unrecognized WASP-Homology-2 actin binding motifs within IPP, one from PINCH and the other from Parvin. Strikingly, this process is also sensitized to Mg-ATP bound to the pseudoactive site of ILK and its dysregulation severely impairs stress fibers formation, cell spreading, and migration. These data identify a crucial mechanism for ILK, highlighting its uniqueness as a pseudokinase to transduce non-catalytic signal and regulate cell adhesion.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
IPP interaction with F-actin. a Schematic organization of IPP based on structural data. ILK binds to PINCH LIM1 via its ankyrin domain and α-Parvin CH2 via its pseudokinase domain, respectively. The Wiscott–Aldrich syndrome protein (WASP) homology domain (WH2) motifs are highlighted in PINCH and α-Parvin. b A representative gel filtration profile of the purified IPP complex by Superose 6 10/300 GL size exclusion chromatography column (GE healthcare). The eluted peak is overlaid with an elution curve of standard molecular weight proteins (dot lines). c Co-sedimentation of IPP at dose-dependent amounts in the presence/absence of F-actin. The F-actin was incubated at 2.3 μM constant concentration with increasing concentrations of each test sample in 5% glycerol containing protein buffer. Representative gels with Coomassie stain are shown. M marker proteins, S supernatant, P pellets
Fig. 2
Fig. 2
Mapping and structural analysis of the major actin binding site in PINCH-1. a Overall, 0.1 mM 1H-15N HSQC of LIM5-T in the absence (black) and presence (red) of 0.2 mM AP-actin showing that LIM5-T binds to G-actin. Strongly perturbed residues in LIM5-T by the actin binding are labeled, which primarily involve PINCH-1 C-terminal tail. b Co-sedimentation data (pellets in duplicates) showing LIM5-T but not LIM5 alone binds to F-actin. c (Top) NMR structure of LIM5-T and the actin binding surface derived from the chemical shift mapping. Note that the H2 helix that packs against the LIM domain as reflected in all calculated structures. Residues undergoing the largest chemical shift changes upon interaction with AP-actin are shown with side chains. Two zinc atoms are shown as gray spheres. (Bottom) Superposition of 20 lowest energy structures of LIM5-T. d 0.1 mM 1H-15N HSQC of C-terminal LIM5-T 4A mutant (F307A/L311A/K312A/K313A) in the absence (black) and presence (red) of 0.2 mM AP-actin showing that the mutations drastically reduced the actin binding to LIM5-T
Fig. 3
Fig. 3
Identification of actin-binding WH2 motifs in PINCH-1 and α-Parvin. a Sequence alignment of PINCH-1 C-terminal tail and α--terminus with representative WH2 motifs found in other proteins showing the presence of distinct WH2 motifs in PINCH and Parvin. b 0.1 mM 1H-15N HSQC of α-Parvin-N in the absence and presence of 0.2 mM AP-actin showing that α-Parvin-N has potent binding to G-actin (left panel). Mutation of putative G-actin-binding residues L37A/R39A/R40A/K41A/K42A (α-Parvin 5 A) drastically reduces actin binding (right panel). c Co-sedimentation assay showing α-Parvin-N binds to F-actin potently. +, ++, +++ correspond to the concentration of α-Parvin-N at 7.7 μM, 23.0 μM, and 76.7 μM, respectively
Fig. 4
Fig. 4
IPP triggers F-actin bundle formation. a Selected microscopic image showing the uniform F-actin filament network in the presence of the buffer alone. b Selected microscopic image showing F-actin bundles in the presence of IPP. c A high resolution image of IPP-induced F-actin bundle detected by confocal microscopy. d Selected image showing that IPP-4A mutations led to drastically reduced F-actin bundle formation as compared with (b). e and f. Quantitative comparison on the number (e) and size (f) of the F-actin bundles induced by IPP and IPP-4A, respectively. Bar = 100 μm except in (c) where bar = 50 μm
Fig. 5
Fig. 5
Cellular defects by PINCH-1 4A mutation. a Overexpression experiment. HEL cells expressing EGFP-PINCH-1 WT or -1 4A cells were stimulated with 800 nM PMA for 10 min, spread on 20 μg ml−1 fibrinogen for 1 h, fixed and stained with Alexa 647-phalloidin to visualize actin and anti-vinculin antibodies to mark FAs. Transfected cells were identified by EGFP fluorescence. Top panels. WT EGFP-PINCH-1 promotes the formation of F-actin stress fibers. By contrast, the stress fibers were substantially disrupted in the cells expressing the mutant PINCH-1-4A (bottom panels). Scale bar, 10 µm. b Quantitative analysis of inhibition of cell spreading by PINCH-4A as compared with the WT PINCH-1. Cells were spread on fibrinogen as described in (a). The areas of EGFP-positive cells were measured using ImageJ software. *P ≤ 0.001. In total, 150 cells were quantified in each sample. c CRISPR/Cas9-based knock-in mutation experiment. HeLa clone (2F5) with frame-shifted PINCH deletion mutant, PINCH-∆C (2F5) was transfected by pEGFP vector (V), pEGFP-PINCH wildtype (WT) or pEGFP-PINCH mutant (4A) and sorted for 2 h spreading on fibronectin coated coverslips (10 µg cm−2). Focal adhesions and stress fibers are clearly disrupted by PINCH 4A mutation. Cells were fixed and stained for GFP, ILK, and F-actin. Images were then captured with confocal microscope, under 63x magnification. Scale bar, 20 µm. d Quantification of cell spreading with physiological level of PINCH WT/4A expression (P1) in HeLa clone (2F5). Spreading area of 44 cells from each group were quantified with Image Pro plus software based on F-actin staining. Significance was calculated by t test. **P < 0.01. The box of the boxplot illustrated the upper and lower quartile for each population (EGFP vector, EGFP-PINCH1-WT and EGFP-PINCH1-4A). Median of spreading area is marked by a horizontal line within the box. The attached whisker indicates the range, and the discrete points (•) are the outliers
Fig. 6
Fig. 6
ILK L207W mutation disrupts the F-actin bundling. a (Top left) Structural comparison of the ILK KLD bound to α-Parvin CH2 and (colored in gray; PDB ID 3KMW) and its comparison with the ILK mutant form (colored in blue). The superposition of the mutant ILK KLD (267 aligned Cα atoms) to the ATP-bound (PDB ID 3KMW) and -free (PDB ID 3KMU) forms shows overall similarities with root-mean-square deviations of 0.58 Å and 0.47 Å, respectively. A small conformational change is observed in the ATP-binding site of the mutant ILK KLD likely due to mutation or distinct crystal packing. (Top right) Close-up view of the ATP-binding sites of the ILK KLD between ATP-bound wild type (gray) and deficient mutant (blue). Mg, ATP, L207 in the wild type, and W207 in the mutant ILK KLDs are highlighted in ball and stick models. (Bottom) Close-up stereo view of the loss-of-ATP-binding mutation site in the ILK KLD. The 2Fo-Fc electron density map contoured at 1 σ is shown in gray mesh. The Fo–Fc omit map calculated from the mutant structure without the residue (W207), contoured at 3.5 σ, is overlaid (red mesh). Selected residues in the ATP-binding site are labeled. b Representative microscopic image showing that IPP L207W impaired F-actin bundle formation (no larger bundles) as compared with the WT IPP in (c). Selected microscopic image showing F-actin bundles in the presence of WT IPP. Bar = 100  μm. d Quantitative comparison of the F-actin bundle sizes of the randomly selected 20 slides showing the mutation dramatically reduced the F-actin bundle sizes (red squares) as compared with those induced by WT IPP (blue triangles)
Fig. 7
Fig. 7
Overexpression of ILK L207W causes defects in cell spreading and migration. a HEL cells expressing EGFP-ILK WT or EGFP-ILK L207W were stimulated with 800 nM PMA for 10 min, spread on 20 μg ml−1 fibrinogen for 1 h, fixed and stained with Alexa 647-phalloidin to visualize actin and anti-vinculin antibodies to mark focal adhesions. Transfected cells were identified with EGFP fluorescence. (Top panels) WT EGFP-ILK promotes formation of F-actin stress fibers. By contrast, the stress fibers were substantially disrupted in cells expressing the mutant ILK (bottom panels). Scale bar, 10 µm. b Quantitative analysis of inhibition of cell spreading by ILK L207W as compared to the WT ILK. The areas of EGFP-positive cells were measured using ImageJ software. *P ≤ 0.001. In total, 150 cells were quantified in each sample. The box of the boxplot illustrated the upper and lower quartile for each population (EGFP vector, EGFP-ILK-WT and EGFP-ILK L207W). Median of spreading area is marked by a horizontal line within the box. The attached whisker indicates the range, and the discrete points (•) are the outliers.  c Overexpression of WT ILK but not the L207W mutant in HeLa cells promotes cell migration significantly. Images were captured after 10 h migration, under 10x magnification. d Quantitative change of cell migration with WT ILK versus ILK L207W mutant. The relative fold change of migrated cells was calculated from average of five randomly picked fields for each insert. The results were obtained from three independent experiments. Values are given as mean ± SD
Fig. 8
Fig. 8
Cellular defects by knock-in mutation (ILK L207W). a Comparison of focal adhesions and stress fibers between WT ILK containing HeLa cells and ILK L207W knock-in cells. b Western blot of whole-cell lysates from CRISPR generated clones showing that ILK L207W had little effect on the expression of ILK, PINCH, and Parvin. c, d Quantification of focal adhesion number (c) and size (d) by WT ILK vs ILK L207W. e Cell spreading defects by ILK 207W. The box of the boxplot illustrated the upper and lower quartile for each population (WT ILK and ILK L207W). Median is marked by a horizontal line within the box. The attached whisker indicates the range, and the discrete points (•) are the outliers. f WT ILK but not the L207W mutant in HeLa cells promotes cell migration significantly. Images were captured after 10 hours migration, under 10x magnification (left and middle panels). Quantitative change of cell migration by WT ILK versus ILK L207W mutant (right panel). The migrated cell number was calculated from average of five randomly picked fields for each insert. The results were obtained from three independent experiments. Values are given as mean ± S.E.M .Significance was calculated by t test or Mann–Whitney rank sum test based on the normality of data. Scale bar, 20 µm. ***P < 0.001. **P < 0.01
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