title: Biochemical, proteomic, structural, and thermodynamic characterizations of integrin-linked kinase (ILK): cross-validation of the pseudokinase.
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.
authors.affiliation: Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Koichi
authors.initials: K
authors.lastname: Fukuda
authors.forename: James D R
authors.initials: JD
authors.lastname: Knight
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kothary
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
mesh_terms: D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D005982:Glutathione Transferase / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D008345:Manganese; D051379:Mice; D010766:Phosphorylation; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D013816:Thermodynamics; D000099200:Scaffold Protein ILK
chemical_list: D005982:Glutathione Transferase; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D008345:Manganese; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D000099200:Scaffold Protein ILK; C000605507:Gsk3b protein, mouse