Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase with key roles in the regulation of cell adhesion migration, proliferation and survival. In cancer FAK is a major driver of invasion and metastasis and its upregulation is associated with poor patient prognosis. FAK is autoinhibited in the cytosol, but activated upon localisation into a protein complex, known as focal adhesion complex. This complex forms upon cell adhesion to the extracellular matrix (ECM) at the cytoplasmic side of the plasma membrane at sites of ECM attachment. FAK is anchored to the complex via multiple sites, including direct interactions with specific membrane lipids and connector proteins that attach focal adhesions to the actin cytoskeleton. In migrating cells, the contraction of actomyosin stress fibres attached to the focal adhesion complex apply a force to the complex, which is likely transmitted to the FAK protein, causing stretching of the FAK molecule. In this review we discuss the current knowledge of the FAK structure and how specific structural features are involved in the regulation of FAK signalling. We focus on two major regulatory mechanisms known to contribute to FAK activation, namely interactions with membrane lipids and stretching forces applied to FAK, and discuss how they might induce structural changes that facilitate FAK activation.

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.

Significance: Nonreceptor tyrosine kinases are major players in cell signaling. Among them, focal adhesion kinase (FAK) is the key integrator of signals from growth factors and cell adhesion. In cancer, FAK is frequently overexpressed, and by promoting adhesion to the tumor stroma and ECM, FAK provides important signals for tumor invasion and metastasis. Although autoinhibitory mechanisms have previously been described and the players involved in FAK regulation are largely known, on a mechanistic level, FAK activation is currently not understood. Here, we present a multidisciplinary approach demonstrating a multistep mechanism resulting in FAK activation. This mechanistic insight enables the design of alternative strategies for the discovery of potential anticancer drugs that inhibit both catalytic and scaffolding functions of FAK with high specificity.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.