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Structural basis of Focal Adhesion Kinase activation on lipid membranes.

ABSTRACT: Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.

SUBMITTER: Acebron I

PROVIDER: S-EPMC7527928 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Structural basis of Focal Adhesion Kinase activation on lipid membranes.

Project description:Focal Adhesion Kinase (FAK) is a key component of the membrane-proximal signaling layer in focal adhesions, regulating important processes, including cell migration, proliferation and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to rearrange and interact itself with the membrane in an orientation that places the active site towards the membrane. In this conformation the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and cellular activity including cancer cell invasion, whilst stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner.

| S-SCDT-EMBOJ-2020-104743 | biostudies-other

Structural basis for the autoinhibition of focal adhesion kinase.

Project description:Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation.

| S-EPMC2077847 | biostudies-literature

The structural basis for Ras activation of PI3K? lipid kinase.

Project description:PI3K? is a principal Ras effector that phosphorylates PIP2 to PIP3 in the PI3K/Akt/mTOR pathway. How Ras activates PI3K has been unclear: is Ras' role confined to PI3K recruitment to the membrane or does Ras activation also involve allostery? Recently, we determined the mechanism of PI3K? activation at the atomic level. We showed the vital role and significance of conformational change in PI3K? activation. Here, by a 'best-match for hydrogen-bonding pair' (BMHP) computational protocol and molecular dynamics (MD) simulations, we model the atomic structure of KRas4B in complex with the Ras binding domain (RBD) of PI3K?, striving to understand the mechanism of PI3K? activation by Ras. Point mutations T208D, K210E, and K227E disrupt the KRas4B-RBD interface in the models, in line with the experiments. We identify allosteric signaling pathways connecting Ras to RBD in the p110? subunit. However, the observed weak allosteric signals coupled with the detailed mechanism of PI3K? activation make us conclude that the dominant mechanistic role of Ras is likely to be recruitment and restriction of the PI3K? population at the membrane. Thus, RTK recruits the PI3K? to the membrane and activates it by relieving its autoinhibition exerted by the nSH2 domain, leading to exposure of the kinase domain, which permits PIP2 binding. Ras recruitment can shift the PI3K? ensemble toward a population where the kinase domain surface and the active site position and orientation favor PIP2 insertion. This work helps elucidate Ras-mediated PI3K activation and explores the structural basis for Ras-PI3K? drug discovery.

| S-EPMC6556208 | biostudies-literature

Structural basis of focal adhesion localization of LIM-only adaptor PINCH by integrin-linked kinase.

Project description:The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK.PINCH complex (28 kDa, K(D) approximately 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.

| S-EPMC2645833 | biostudies-literature

Focal adhesion kinase modulates cell adhesion strengthening via integrin activation.

Project description:Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell-ECM forces.

| S-EPMC2675629 | biostudies-literature

Focal Adhesion Kinase Dependent Expression

2013-09-17 | GSE43873 | GEO

Focal Adhesion Kinase Dependent Expression

Project description:The goal of this study was to identify transcripts, which are differentially regulatulated in the presence and absence of Focal Adhesion Kinase. As Focal Adhesion Kinase activity can depend upon cell density (Snijder et al. Nature 2009), biological replicates where cells, were seeded very sparsely or confluently, were used. Focal Adhesion Kinase Knockout (ATCC CRL-2644) and Rescue Cells (Sieg et al. 1998, clone DA2) were seeded at two different concentrations. Replicas refer to biological replicates, performed on different days. Only one single technical replicate has been done per biological replicate.

2013-09-17 | E-GEOD-43873 | biostudies-arrayexpress

Structural basis for paxillin binding and focal adhesion targeting of ?-parvin.

Project description:?-Parvin is a cytoplasmic adaptor protein that localizes to focal adhesions where it interacts with integrin-linked kinase and is involved in linking integrin receptors to the cytoskeleton. It has been reported that despite high sequence similarity to ?-parvin, ?-parvin does not bind paxillin, suggesting distinct interactions and cellular functions for these two closely related parvins. Here, we reveal that ?-parvin binds directly and specifically to leucine-aspartic acid repeat (LD) motifs in paxillin via its C-terminal calponin homology (CH2) domain. We present the co-crystal structure of ?-parvin CH2 domain in complex with paxillin LD1 motif to 2.9 Å resolution and find that the interaction is similar to that previously observed between ?-parvin and paxillin LD1. We also present crystal structures of unbound ?-parvin CH2 domain at 2.1 Å and 2.0 Å resolution that show significant conformational flexibility in the N-terminal ?-helix, suggesting an induced fit upon paxillin binding. We find that ?-parvin has specificity for the LD1, LD2, and LD4 motifs of paxillin, with K(D) values determined to 27, 42, and 73 ?M, respectively, by surface plasmon resonance. Furthermore, we show that proper localization of ?-parvin to focal adhesions requires both the paxillin and integrin-linked kinase binding sites and that paxillin is important for early targeting of ?-parvin. These studies provide the first molecular details of ?-parvin binding to paxillin and help define the requirements for ?-parvin localization to focal adhesions.

| S-EPMC3463362 | biostudies-literature

Structural and mechanistic insights into mechanoactivation of focal adhesion kinase.

Project description: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.

| S-EPMC6452671 | biostudies-literature

Structural features of the focal adhesion kinase-paxillin complex give insight into the dynamics of focal adhesion assembly.

Project description:The C-terminal region of focal adhesion kinase (FAK) consists of a right-turn, elongated, four-helix bundle termed the focal adhesion targeting (FAT) domain. The structure of this domain is maintained by hydrophobic interactions, and this domain is also the proposed binding site for the focal adhesion protein paxillin. Paxillin contains five well-conserved LD motifs, which have been implicated in the binding of many focal adhesion proteins. In this study we determined that LD4 binds specifically to only a single site between the H2 and H3 helices of the FAT domain and that the C-terminal end of LD4 is oriented toward the H2-H3 loop. Comparisons of chemical-shift perturbations in NMR spectra of the FAT domain in complex with the binding region of paxillin and the FAT domain bound to both the LD2 and LD4 motifs allowed us to construct a model of FAK-paxillin binding and suggest a possible mechanism of focal adhesion disassembly.

| S-EPMC2279287 | biostudies-literature

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