Structural and mechanistic insights into mechanoactivation of focal adhesion kinase

Abstract

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 lowforce 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.atomic force microscopy | mechanobiology | focal adhesion signaling | protein kinase regulation | single-molecule force spectroscopy

Characterization of CaMKIIα holoenzyme stability

“…The crystals were obtained without any nucleotide added; however, an additive ( p ‐coumaric acid) was resolved in the ATP‐binding site. As we suspected, and is true for other kinases, 27 ATP/MgCl 2 binding stabilizes the kinase domain by 4°C (Figure 3b). Adding MgCl 2 without ATP may also have an effect on stability, which will be tested in future studies.…”

FAK Structure and Regulation by Membrane Interactions and Force in Focal Adhesions

“…On the other hand, unfolding of the ATP loaded kinase domain was observed only above 50 pN, hence even peak forces in focal adhesions do not result in FAK deactivation. In order to obtain an atomic view of FAK stretching, experimental pulling of FAK by AFM was correlated to corresponding force-probe molecular dynamics simulations performed via the same attachment points [88]. Overall simulations agreed well with experiments, with FERM-kinase detachment occurring prior to any FAK unfolding over a range of loading rates.…”

“…In agreement with such a scenario, vinculin is shown to move away from the membrane towards the actin regulatory layer during myosin II mediated focal adhesion maturation [87]. Further, using atomic force microscopy (AFM) based single molecule force spectroscopy, it was shown that forces applied to FAK molecules via the KAKTLRK lipid binding site in the FERM domain and the C-terminus of the kinase domain (the FAT domain was not included in these studies) results in rupture of autoinhibitory interactions between the FERM and kinase domain [88]. A force peak observed only with wild-type FAK but not the constitutively open mutant of FAK was observed at 25 pN using a retraction speed of the AFM cantilever of 12,800 nm/s.…”

Single‐Molecule Manipulation in Zero‐Mode Waveguides

“…For other proteins, as for example in focal adhesions, which are assumed to bear force‐regulatory functions our technique can help to characterize them by providing both biochemical and biomechanical information. [ 32,33 ] Our drift correcting, automated workflow, allows for measuring several days without interruption. This enables probing of an even larger number of ZMWs and will thus further improve statistical power.…”