The pseudoactive site of ILK is essential for its binding to alpha-Parvin and localization to focal adhesions

Abstract

Integrin-linked kinase (ILK) plays a pivotal role in connecting transmembrane receptor integrin to the actin cytoskeleton and thereby regulating diverse cell-adhesion-dependent processes. The kinase domain (KD) of ILK is indispensable for its function, but the underlying molecular basis remains enigmatic. Here we present the crystal structure of the ILK KD bound to its cytoskeletal regulator, the C-terminal calponin homology domain of alpha-parvin. While maintaining a canonical kinase fold, the ILK KD displays a striking pseudoactive site conformation. We show that rather than performing the kinase function, this conformation specifically recognizes alpha-parvin for promoting effective assembly of ILK into focal adhesions. The alpha-parvin-bound ILK KD can simultaneously engage integrin beta cytoplasmic tails. These results thus define ILK as a distinct pseudokinase that mechanically couples integrin and alpha-parvin for mediating cell adhesion. They also highlight functional diversity of the kinase fold and its "active" site in mediating many biological processes.

Figure 1. Structure of ILK KD and its Comparison with PKA

(A) Ribbon drawing of the ILK KD structure (blue) in the presence of MgATP. ATP and the activation segment are colored in magenta and green, respectively. The CH2 structure is removed from the panel for simplicity. (B) Structural comparison of the ATP-binding pocket between ILK and PKA. Left: Close-up view of the active site of PKA (PDB entry 1ATP) in the presence of ATP (magenta) and two Mn atoms (purple sphere). D166, K168, and N171 in the catalytic loop and the DFG aspartate (D184) are depicted in stick models. Right: Close-up view of the ATP-binding site in the ILK structure. Residues that are structurally equivalent to those in PKA are depicted in stick models. The DVK motif in the activation segment is highlighted in green, and the K341 residue is depicted in stick and surface models. (C) Comparison of the ATP conformations bound to ILK and PKA. The adenine-ring and ribose are superposed, and the β-γ phosphate groups bound in ILK and PKA are colored in blue and yellow, respectively. (D) Comparison of the ATP-binding sites between ILK and PKA. Left: The overall structure of the ATP-bound PKA. The residues that contact ATP within 3.8 Å are colored. The ATP molecule is shown in stick model (magenta) with transparent surface. Center top: Close-up view of the ATP-binding residues in PKA. Center bottom: Close-up view of the ATP-binding residues in ILK. Right: The overall structure of the ATP-bound ILK. (E) Comparison of the ATP-binding P-loop dynamics. Left: The glycine-rich P-loop movement in PKA upon ATP binding. The structures of ATP-bound PKA and its apo form (PDB entry 1J3H) are colored in yellow and white, respectively. The selected C_α atoms in the P-loop are rendered in spheres. Center: The P-loop conformation of the ILK KD. The coordinates of the ILK KD in the MgATP-bound (blue) and free (white) are superimposed, and the selected triplet residues that are structurally equivalent to those in the PKA catalytic loop are depicted. The ATP molecule is superimposed with the 2.0-Å F_o-F_c omit electron density map contoured at 4σ level. Right: Comparison of the P-loop and the catalytic loop of PKA with their equivalent regions of ILK. The PKA-bound ATP (yellow) is overlaid at the adenine-ring and ribose groups onto the ILK-bound ATP (blue) by a rigid-body superposition. See also Figure S1.

Figure 2. Structural Comparison of the Activation Segment

(A) An orthogonal view of the activation segment. Left: The activation segment (green) in ILK KD. The side chains of S343 and E238 (αC-helix) are depicted in stick models. Right: The activation segment (yellow) in PKA. (B) Divergent activation segment in the ILK KD structure. Left: Overall tube model of the ILK KD and the location of the activation segment that can interact with a cluster of hydrophobic residues in the N-lobe. Right: A detailed view of hydrophobic and polar interactions formed between the activation segment and the N-lobe (<4 Å). See also Figure S2.

Figure 3. Biochemical Analysis of the ILK Kinase Activity

(A) Representative in vitro radioactive kinase assay data using bacterially purified recombinant full-length human ILK. The protein substrates are myelin basic protein (MW: 20K), myelin basic protein + CH2, CH2 alone, maltose binding protein (MBP)-fused integrin β1 CT, and MBP-fused integrin β3 CT. None of these substrates were phosphorylated as compared to the positive controls using bacterially purified PKA (SignalChem) and MEK (Millipore). The kinase reaction was also performed by adding 50 μM PIP3 (Echelon) or full-length α-parvin, but no effect was observed (data not shown). Mg concentration was also varied but no difference was observed (data not shown). (B) Effect of the S343D mutation on ILK activity. The S343D mutant of either full-length ILK or ILK KD did not show any phosphorylation on myelin basic protein. Addition of 50 μM PIP3 into these kinase reactions also had no effect (not shown). Several other ILK mutants, A319D that mimics D166 in PKA, A319D/S343D, triple mutant A319D/N321K/S343D (N321K mimics K168 in PKA), were also examined to see if they may restore the kinase activity. However, none of them phosphorylated MBP (data not shown). Since ILK has multiple degraded catalytic features including distorted ATP/Mg binding, degenerate catalytic loop, and an unusual activation loop, simply converting some catalytic residues are apparently insufficient to recover the kinase activity. A similar case was found in STRADα pseudokinase, where converting key residues did not restore the kinase activity of the protein (Zeqiraj et al., 2009). (C) Kinase activity of partially purified ILK from chicken-tissue in the absence and presence of CH2. The exogenous substrate myelin basic protein was phosphorylated by the partially purified chicken kinase extract containing ILK. However, the phosphorylation was neither enhanced nor inhibited by addition of recombinant CH2. CH2 was not phosphorylated by ILK (lane 6 from left). The MBP-fused integrin β1 or β3 CTs were not phosphorylated either (data not shown). See also Figure S3.

Figure 4. Effect of ILK on Akt Serine 473 Phosphorylation

(A) Effect of bacterially expressed full-length human ILK on Akt phosphorylation (S473). Note that the inactive GST-Akt (SignalChem) used as the substrate exhibits a basal phosphorylation on S473 (lane 5 from left) compared to the same amount of active Akt (lane 6 from left). Active Akt was prepared using the GST-fused active Akt (SignalChem) as a positive control (1 μg) in the same reaction buffer (50 μL in volume) without adding ILK and substrate. (B) Effect of chicken-tissue purified ILK on the Akt S473 phosphorylation. The amount of chicken ILK is shown in parentheses. (C) Overexpression of ILK in HEK 293 cells and the co-immunoprecipitation analysis. (D) Effect of the ILK immunoprecipitants on S473 phosphorylation of Akt. The fractions of FLAG-ILK (lane 1) and control FLAG (lane 2) immunoprecipitates, or anti-FLAG M2-conjugated beads (lane 3) were incubated with 1 μg of GST-Akt and analyzed by Western blotting with anti-phospho Akt S473 specific antibody. Note that the GST-fused Akt exhibits a basal phosphorylation on S473 (lane 3). No further phosphorylation of Akt on S473 was observed during the kinase reaction by the ILK immunoprecipitant (lane 1) as compared to the control by the FLAG only immunoprecipitant (lane 2). See also Figure S4.

Figure 5. Determinants of the Pseudosubstrate Recognition by ILK

(A) Overall architecture of the ILK KD (blue) bound to CH2 (yellow). The activation segment is colored in green. The ATP molecule and magnesium ion are depicted in stick (magenta) and sphere (white) models, respectively. (B) Open-book view of the binding interface of the ILK KD-CH2 complex. Left panel: CH2-binding surface (gray) on ILK KD. The bound CH2 is shown in loop model (yellow) and ILK KD in surface model (blue). Right: ILK-binding surface (gray) on CH2. The bound ILK KD is shown in loop model (blue) and CH2 in surface model (yellow). All residues in the interface are labeled. (C) Involvement of the ILK αG-helix and activation loop in binding to CH2. Left: Ribbon model of the ILK KD/CH2 complex. The CH2 is depicted by the transparent surface model. Right: Close-up view of the hydrophobic and polar interactions between the G-helix in the ILK KD and α-parvin CH2. The ILK KD-binding hydrophobic patch on the CH2 is depicted in stick models and colored in yellow on the transparent surface model of CH2. M350 and P353 in the C-terminal activation segment that interact with CH2 are depicted in green stick and transparent models. (D) Identification of the critical residues involved in the ILK-CH2 interface. Left: Normal expression of GFP-tagged wild type and the mutant forms of ILK. GFP tagged wild type and M402A/K403A mutant forms of ILK were immunoprecipitated from HeLa cells expressing the corresponding ILK proteins with an anti-GFP antibody. Right: Evaluation of the α-parvin binding. The immunoprecipitates were analyzed by Western blotting with anti-α-parvin antibody. (E-H) Focal adhesion localization of wild type and M402A/K403A mutant forms of ILK. HeLa cells expressing GFP-tagged wild type (E and F) and M402A/K403A mutant (G and H) forms of ILK were plated on fibronectin coated cover slips. The cells were stained with a mouse monoclonal antibody recognizing migfilin (as a marker of FAs) and Rhodamine Red^TX-conjugated anti-mouse IgG antibodies. The GFP-tagged wild type (E) and mutant (G) forms of ILK and migfilin (F and H) were observed under a fluorescence microscope. Bar indicates 10 μm. See also Figure S5.

Figure 6. Integrin-Binding to ILK

(A) Binding of the ILK KD-CH2 complex to GST-integrin β1 CT or β3 CT using pull-down assays. Western blots are shown for the bound ILK KD with anti-ILK monoclonal antibody or the GST-integrin CTs and control GST with anti-GST monoclonal antibody. (B) Control experiment of no direct binding of CH2 to integrin CTs. (C) Binding of the ILK KD-α-parvin (full-length) complex to the integrin CTs. Parvin was detected using anti-parvin antibody raised against the parvin N-terminal region, showing that it is associated with the ILK/integrin interaction by binding to ILK. (D) Control experiment of no direct parvin-binding to integrins. (E) Binding of full-length ILK to GST-integrin β1 CT or β3 CT. The Western blotting analysis is shown for the bound full-length ILK with anti-ILK monoclonal antibody. (F) Control experiment showing no direct binding of MBP-fused PINCH LIM1-2 to the integrin CTs. All the binding experiments were repeated three times. See also Figure S6.

Figure 7. Hydrophobic spine motifs in active kinases and ILK

(A) Overall structure of the ILK KD and location of the hydrophobic spine motifs. The hydrophobic residues in the regulatory (R) and catalytic (C) spines are depicted in stick models rendered in the transparent surfaces colored in green and blue, respectively. The conserved αF-helix and the aspartate residue D374 are highlighted. (B) Close-up view of the R- and C-spine motifs in the ILK KD apo form. (C) Close-up view of the R- and C-spine motifs in the ILK KD bound to Mg and ATP. ATP has no effect on the spines. (D) Close-up view of the R- and C-spine motifs in inactive protein kinase CDK2 (PDB entry 1HCL) (apo form). (E) Close-up view of the R- and C-spine motifs in the active CDK2 (PDB entry 1FIN) bound to ATP. Note that the R-spine motif is disrupted in (D), as compared to those in ILK KD in (C) and active CDK2 in (E). (F) Overlay of the R-, C-spine motifs, and other key segments between the ATP-bound ILK KD and the active CDK2, showing a similar spine formation between ILK and active CDK2 kinase. See also Figure S7.