Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 286, Issue 24, Pages 21886-21895Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M111.240093
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Funding
- National Institute of Health
- Canadian Institutes of Health Research
- Multiple Sclerosis Society of Canada
- Kirschstein-NRSA (the National Institutes of Health)
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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-3 beta (GSK-3 beta). 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-3 beta in the presence of either Mn2+ or the conventional kinase co-factor Mg2+. 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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