The long term goal of this renewal proposal is to gain deep mechanistic understanding of integrin-mediated cell adhesion that occurs in virtually every human life process. Integrins are a family of 24 heterodimeric (α/β) transmembrane receptors differentially expressed in cells and tissues. Being expressed in inactive form, integrins are activated via a distinct process called “inside-out” signaling where cellular stimulation elicits intracellular signals to induce global conformational change of the receptors to bind extracellular matrix (ECM) ligands. However, for cells to firmly adhere to ECM, or change shape, or migrate as required by many physiological processes, the activated integrins need to further communicate with intracellular cytoskeleton (“outside-in” signaling) via the formation of large protein complexes called focal adhesions (FAs). Understanding the mechanism of such bi-directional signaling phenomenon has been the central topic of cell adhesion biology ever since the discovery of integrins in middle 1980s. While much has been learned about the integrin “inside-out” signaling, how FAs are assembled to mediate the integrin “outside-in” signaling still remains highly elusive due to its complex and dynamic nature. To this end, we have been focusing on integrin-linked kinase (ILK) - a major FA regulator and initiator of the integrin outside-in signaling. ILK was long thought to function as a Ser/Thr kinase to phosphorylate integrin β cytoplasmic tail (CT) to trigger signaling. However, our recent studies challenged the longstanding dogma by showing that ILK is a non-catalytic pseudokinase yet can transmit mechanical signaling linking integrin to actin to regulate cytoskeleton reorganization and cell adhesion. Interestingly, ILK was also shown to mediate multiple important biochemical signaling events yet much of the published data were misinterpreted by considering ILK as a kinase with exact mechanisms being unknown. This proposal will rigorously address this issue with a central hypothesis that an ILK-centered heterotrimer called IPP spatiotemporally recruits distinct signaling enzymes to integrin adhesion sites and trigger signaling. In Aim1 we will investigate a longstanding puzzle of ILK-mediated Ser/Thr kinase AKT1 signaling. Aim2 will elucidate the mechanism of ILK-mediated small GTPase Rac1 signaling. Aim3 will develop potent ILK inhibitor that targets at the pseudoactive site of ILK to regulate the IPP stability, which will help to deepen the understanding of the ILK signaling events. Overall, our proposed studies reflect a strong momentum of our program towards establishing a new paradigm of how ILK acts as a multi-functional pseudokinase to spatiotemporally regulate integrin outside- in signaling events to promote dynamic cell adhesion and diverse adhesion-dependent physiological processes. These studies, along with the development of potent ILK inhibitor, may also transform the diagnosis and treatment of ILK-associated dis...