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 (GSK-3). 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 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.The adhesion of cells to the extracellular matrix (ECM) 3 is primarily mediated by heterodimeric integrin transmembrane receptors. Upon activation, the integrin extracellular domain adheres to the ECM by binding to numerous ligands such as fibronectin and fibrinogen. However, for cells to firmly adhere to the ECM, integrins must connect to the backbone of the cell, the actin cytoskeleton, via its cytoplasmic tails (CTs) (1-2). Such connection is mediated by the supramolecular focal adhesion (FAs) complex. The reassembly/disassembly of FAs can lead to dynamic cell adhesion processes such as cell migration and spreading, which are fundamental for a variety of physiological responses. Over the past decades, major effort has been made to search for proteins involved in FA assembly and regulation. Integrin-linked kinase (ILK) was discovered in 1996 as a binding partner of integrin  CTs (3) and was found to critically regulate FAs (4). Genetic and genomic analyses have established that ILK is one of the few essential and evolutionarily conserved proteins coupling integrins to actin (5-7), and its dysregulation has been linked to major human diseases such as heart failure (8 -10) and cancer (11). ILK has a molecular mass of ϳ51 kDa containi...