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. 2011 Jun 17;286(24):21886-95.
doi: 10.1074/jbc.M111.240093. Epub 2011 Apr 26.

Biochemical, proteomic, structural, and thermodynamic characterizations of integrin-linked kinase (ILK): cross-validation of the pseudokinase

Koichi Fukuda  1 , James D R Knight, Grzegorz Piszczek, Rashmi Kothary, Jun Qin
Affiliations

Biochemical, proteomic, structural, and thermodynamic characterizations of integrin-linked kinase (ILK): cross-validation of the pseudokinase

Koichi Fukuda et al. J Biol Chem. .

Abstract

Integrin-linked kinase (ILK) is one of the few evolutionarily conserved focal adhesion proteins involved in diverse cell adhesion-dependent physiological and pathological responses. Despite more than a decade of studies and extensive literature, the kinase function of ILK is controversial. ILK contains a highly degraded kinase active site but it has been argued that ILK may be an unusual manganese (Mn)-dependent serine-threonine kinase that targets specific substrates such as glycogen synthase kinase-3β (GSK-3β). In this study, we have tackled this issue by a systematic bottom-up biochemical, proteomic, structural, and thermodynamic analysis of ILK. We show that recombinant ILK from either bacteria or mammalian cells exhibits no kinase activity on GSK-3β in the presence of either Mn(2+) or the conventional kinase co-factor Mg(2+). A comprehensive and unbiased whole cell-based kinase assay using entire mammalian CG-4 and C2C12 cell lysate did not identify any specific ILK substrates. High resolution crystallographic structure analysis further confirmed that the Mn-bound ILK adopts the same pseudo active site conformation as that of the Mg-bound ILK. More importantly, thermodynamic analysis revealed that the K220M mutation, previously thought to inactivate ILK by disrupting ATP binding, significantly impairs the structural integrity and stability of ILK, which provides a new basis for understanding how this mutation caused renal agenesis, a failure of fetal kidney development. Collectively, our data provide strong evidence that ILK lacks intrinsic kinase function. It is a bona fide pseudokinase that likely evolved from an ancestral catalytic counterpart to act as a distinct scaffold to mediate protein-protein interactions during focal adhesion assembly and many other cellular events.

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Figures

FIGURE 1.
FIGURE 1.
Tests of GSK-3β phosphorylation by recombinant ILK expressed in bacteria or mammalian cells. A, non-radioactive in vitro GSK-3 kinase assay by a dose-dependent addition of bacterially expressed and purified full-length human ILK. The kinase reaction was carried out in the presence of either magnesium or manganese as cofactor. The phosphorylation of GSK-3 was examined by Western blot analysis with anti-phospho-specific antibody. GST-fused full-length active AKT was used as a positive control for the phosphorylation of GSK-3. B, in vitro GSK-3β kinase assay by a dose-dependent addition of Myc-tagged human ILK expressed in HEK293. Notice the very weak phosphorylation of GSK-3β in the presence of Mg2+ but not Mn2+, which is in contrast to data from Maydan et al. (26). C, in vitro protein-protein binding interaction between recombinant mammalian ILK and α-parvin CH2 by pull-down assay. The Myc-tagged ILK was potently bound to the GST-fused CH2 but not to the GST alone. D, kinase inhibition assay for the phosphorylation of GSK-3 by a dose-dependent addition of CH2. Notice that the phosphorylation of GSK-3β was not affected by CH2 at all, suggesting that the phosphorylation of GSK-3 was not mediated by Myc-tagged ILK but rather by some contaminating kinase (see Fig. 2).
FIGURE 2.
FIGURE 2.
Test of GSK-3β phosphorylation by Myc-tagged ILK that was further purified by GST-PINCH LIM1–2 affinity chromatography. Each bacterially purified GST-fused PINCH LIM1–2 (residues 1–127) or GST (∼25 μg each) was immobilized on 40 μl of glutathione-Sepharose 4B (GE Healthcare) and equilibrated in pull-down binding buffer as described under “Experimental Procedures.” The mammalian Myc-tagged ILK (1.4 μg) was added in each affinity bead, and the reaction mixtures were incubated at 4 °C by a rotor for 2 h. The beads were extensively washed with binding buffer. The bound proteins were eluted in 60 μl of 20 mm reduced glutathione in binding buffer and designated as the GST affinity co-purified Myc-tagged ILK. Each 10 μl of eluent was analyzed by non-radioactive in vitro phosphorylation assay of GSK-3β using Western blot analysis with phosphospecific antibody, as described under “Experimental Procedures”.
FIGURE 3.
FIGURE 3.
Proteomic analysis of ILK kinase function. A, in vitro radioactive kinase assay with bacterial ILK. Endogenous kinase activity from CG4 cell lysate was blocked as described under “Experimental Procedures.” A kinase assay buffer containing [32P]ATP was added to 100 μg of kinase-inactive lysate, followed by the addition of a kinase (or buffer only without kinase for control). After SDS-PAGE, autoradiography was performed. Lane 1 from left, control (without bacterial ILK); lane 2, 500 ng of p38α; lane 3, 2 μg of bacterial ILK with Mg2+ as a cofactor; lane 4, 2 μg of bacterial ILK with Mn2+ as a cofactor. Similar results were obtained with C2C12 cell lysate (data not shown). B, in vitro radioactive kinase assay with mammalian ILK. C2C12 cell lysate treatment, kinase assay, and autoradiography were performed as above and described under “Experimental Procedures.” Lane 1 from left, control (without mammalian ILK); lane 2, 1 μg of mammalian ILK; lane 3, 1 μg of mammalian ILK in the presence of bacterially purified His-tagged parvin CH2 protein for kinase inhibition analysis (1 μg). The CH2 protein was preincubated with mammalian ILK for 10 min prior to performing the kinase assay. Notice that several 32P-incorporated lysate substrates (marked with asterisks) were detected by autoradiography; however, the band intensities are almost at the background level indicating some low level contamination by some unknown kinase as also found in Fig. 1 and supplemental Figs. S1 and S3. This is confirmed in lane 3 where this activity was not inhibited by CH2 that strongly binds to the p + 1 substrate site of ILK (14).
FIGURE 4.
FIGURE 4.
Crystal structure analysis of the ILK KD bound to CH2 in the presence of manganese and ATP. A, an overlay of the crystal structures of the ILK KD/CH2 complexes between MnATP (colored in purple blue) and MgATP bound (colored in red) forms. B, close-up view of the MnATP-binding sites in the ILK KD. The ATP molecule and manganese ion are shown in stick (colored in magenta) and sphere models, respectively. The ATP molecule is superimposed with the 1Fo-Fc omit density map contoured at 4σ level. The contacting residues and atoms of the ILK KD to the ATP molecule were calculated using the maximum distance cutoff of 4 Å by CNS. C, superposition of the ATP-binding ILK residues between MnATP (colored in purple blue) and MgATP (colored in red) bound forms.
FIGURE 5.
FIGURE 5.
Thermodynamic profile for the binding of the ILK KD/CH2 complex to ATP using ITC. Top panel shows raw data of injection profile after baseline correction. Bottom panel shows integration plots (heat released) for each injection, along with a solid line of non-linear least-squares fit for the data.
FIGURE 6.
FIGURE 6.
Comparison of structures between ILK KD and PKA in the presence/absence of MnATP. Left, superposition of the crystal structures of the ILK KD (colored in purple blue) and PKA (colored in yellow). Right, close-up views of the MnATP-binding sites and the conserved ATP-binding lysine residues before and after ATP-binding.
FIGURE 7.
FIGURE 7.
Thermodynamic properties of wild type and the K220M mutant of the ILK KD bound to CH2. A, thermal denaturation curves of the ILK KD/CH2 complex between apo (colored in black) and ATP-bound (colored in red) forms by fluorescence analysis. B, an overlay of far-UV CD spectra of the recombinant ILK KD/CH2 complex between wild-type ILK (colored in black) and the K220M mutant (colored in red). C, an overlay of normalized thermal unfolding profiles of the recombinant ILK KD/CH2 complex between the wild type (colored in black) and the K220M mutant (colored in red). D, thermal denaturation curves of the recombinant ILK KD/CH2 complex between the wild type (colored in black) and the K220M mutant (colored in red) by tryptophan fluorescence analysis. C and D demonstrate that K220M is substantially less stable than the WT ILK.
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