Structural and mechanistic insights into mechanoactivation of focal adhesion kinase

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.

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pH sensing by FAK-His58 regulates focal adhesion remodeling

The Journal of Cell Biology ◽

10.1083/jcb.201302131 ◽

2013 ◽

Vol 202 (6) ◽

pp. 849-859 ◽

Cited By ~ 53

Author(s):

Chang-Hoon Choi ◽

Bradley A. Webb ◽

Michael S. Chimenti ◽

Matthew P. Jacobson ◽

Diane L. Barber

Keyword(s):

Focal Adhesion ◽

Conformational Changes ◽

Cancer Metastasis ◽

Ph Sensor ◽

Focal Adhesions ◽

Cell Spreading ◽

Initial Step ◽

Ph Sensing ◽

Cellular Processes ◽

Dynamics Simulations

Intracellular pH (pHi) dynamics regulates diverse cellular processes, including remodeling of focal adhesions. We now report that focal adhesion kinase (FAK), a key regulator of focal adhesion remodeling, is a pH sensor responding to physiological changes in pH. The initial step in FAK activation is autophosphorylation of Tyr397, which increased with higher pHi. We used a genetically encoded biosensor to show increased pH at focal adhesions as they mature during cell spreading. We also show that cells with reduced pHi had attenuated FAK-pY397 as well as defective cell spreading and focal adhesions. Mutagenesis studies indicated FAK-His58 is critical for pH sensing and molecular dynamics simulations suggested a model in which His58 deprotonation drives conformational changes that may modulate accessibility of Tyr397 for autophosphorylation. Expression of FAK-H58A in fibroblasts was sufficient to restore defective autophosphorylation and cell spreading at low pHi. These data are relevant to understanding cancer metastasis, which is dependent on increased pHi and FAK activity.

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Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

Molecular Biology of the Cell ◽

10.1091/mbc.7.8.1209 ◽

1996 ◽

Vol 7 (8) ◽

pp. 1209-1224 ◽

Cited By ~ 358

Author(s):

A P Gilmore ◽

L H Romer

Keyword(s):

Focal Adhesion Kinase ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Umbilical Vein ◽

Adhesion Formation ◽

Focal Adhesions ◽

Kinase Domain ◽

Glutathione S Transferase ◽

Umbilical Vein Endothelial Cells ◽

And Function

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.

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Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

10.1101/2021.10.12.464151 ◽

2021 ◽

Author(s):

Hugo Arellano-Santoyo ◽

Rogelio A Hernandez-Lopez ◽

Emma Stokasimov ◽

Ray YR Wang ◽

David Pellman ◽

...

Keyword(s):

Molecular Dynamics ◽

Single Molecule ◽

Conformational Changes ◽

Intracellular Transport ◽

Structural Features ◽

Cellular Processes ◽

Tubulin Binding ◽

Dynamics Simulations ◽

Spatiotemporal Control ◽

Unique Ability

The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

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Differential activation of focal adhesion kinase, Rho and Rac by the ninth and tenth FIII domains of fibronectin

Journal of Cell Science ◽

10.1242/jcs.112.17.2937 ◽

1999 ◽

Vol 112 (17) ◽

pp. 2937-2946

Author(s):

N.A. Hotchin ◽

A.G. Kidd ◽

H. Altroff ◽

H.J. Mardon

Keyword(s):

Growth Factor ◽

Focal Adhesion Kinase ◽

Signaling Pathways ◽

Focal Adhesion ◽

Cell Attachment ◽

Cell Types ◽

Integrin Receptors ◽

3T3 Cells ◽

Swiss 3T3 ◽

Kinase Phosphorylation

Fibronectins are widely expressed extracellular matrix ligands that are essential for many biological processes. Fibronectin-induced signaling pathways are elicited in diverse cell types when specific integrin receptors bind to the ninth and tenth FIII domains, FIII9-10. Integrin-mediated signal transduction involves activation of signaling pathways of the growth factor-dependent Ras-related small GTP-binding proteins Rho and Rac, and phosphorylation of focal adhesion kinase. We have dissected the requirement of FIII9 and FIII10 for Rho and Rac activity and phosphorylation of focal adhesion kinase in BHK fibroblasts and Swiss 3T3 cells. We demonstrate that FIII10 supports cell attachment but does not induce phosphorylation of focal adhesion kinase. In Swiss 3T3 cells, growth factor-independent phosphorylation of focal adhesion kinase and downstream adhesion events are dependent upon the presence of FIII9 in the intact FIII9-10 pair, whereas FIII10-mediated focal adhesion kinase phosphorylation requires a synergistic signal from growth factors. Furthermore, FIII10 is able to elicit cellular responses mediated by Rho, but not Rac, whereas FIII9-10 can elicit both Rho- and Rac-mediated responses. We propose that activation of specific integrin subunits by the FIII10 and FIII9-10 ligands elicits distinct signaling events. This may represent a general molecular mechanism for activation of receptor-specific signaling pathways by a multi-domain ligand.

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Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00444.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. H627-H638 ◽

Cited By ~ 17

Author(s):

Ana Maria Manso ◽

Seok-Min Kang ◽

Sergey V. Plotnikov ◽

Ingo Thievessen ◽

Jaewon Oh ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Adult Mouse ◽

Cardiac Fibroblasts ◽

Focal Adhesions ◽

Protein Levels ◽

Tyrosine Kinase 2 ◽

A Cell ◽

And Migration ◽

Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

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Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions

Journal of Cellular Physiology ◽

10.1002/jcp.22084 ◽

2010 ◽

pp. n/a-n/a ◽

Cited By ~ 14

Author(s):

David W. Dumbauld ◽

Heungsoo Shin ◽

Nathan D. Gallant ◽

Kristin E. Michael ◽

Harish Radhakrishna ◽

...

Keyword(s):

Cell Adhesion ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Focal Adhesions

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Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline‐rich tyrosine kinase 2‐beta: Insights from molecular dynamics simulations

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.25992 ◽

2020 ◽

Vol 89 (1) ◽

pp. 29-52

Author(s):

Eleni Michael ◽

Savvas Polydorides ◽

Vasilis J. Promponas ◽

Paris Skourides ◽

Georgios Archontis

Keyword(s):

Molecular Dynamics ◽

Tyrosine Kinase ◽

Molecular Dynamics Simulations ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Tyrosine Kinase 2 ◽

Dynamics Simulations

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Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms

Biochemical Journal ◽

10.1042/bj3480119 ◽

2000 ◽

Vol 348 (1) ◽

pp. 119-128 ◽

Cited By ~ 16

Author(s):

Madeleine TOUTANT ◽

Jeanne-Marie STUDLER ◽

Ferran BURGAYA ◽

Alicia COSTA ◽

Pascal EZAN ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Fluorescent Protein ◽

Focal Adhesions ◽

Src Family Kinases ◽

Critical Residue ◽

Green Fluorescent ◽

And Function

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

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Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions

Biochemical Journal ◽

10.1042/bj3240653 ◽

1997 ◽

Vol 324 (2) ◽

pp. 653-658 ◽

Cited By ~ 44

Author(s):

Laura LUO ◽

Tony CRUZ ◽

Christopher McCULLOCH

Keyword(s):

Signal Transduction ◽

Focal Adhesion ◽

Cell Attachment ◽

Interleukin 1 ◽

Primary Cultures ◽

Adhesion Formation ◽

Focal Adhesions ◽

Ion Concentration ◽

Significant Difference ◽

In Cells

The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261–8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100±3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183±30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P > 0.2; 233±90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74±11 nM above baseline), and 1 μM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136±39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1β. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 °C, in cells maintained for 3 h at 4 °C, in cells incubated with BSA beads for 3 h at 37 °C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1β showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.

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