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

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.

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Degraded Collagen Fragments Promote Rapid Disassembly of Smooth Muscle Focal Adhesions That Correlates with Cleavage of Pp125FAK, Paxillin, and Talin

The Journal of Cell Biology ◽

10.1083/jcb.147.3.619 ◽

1999 ◽

Vol 147 (3) ◽

pp. 619-630 ◽

Cited By ~ 172

Author(s):

Neil O. Carragher ◽

Bodo Levkau ◽

Russell Ross ◽

Elaine W. Raines

Keyword(s):

Smooth Muscle ◽

Focal Adhesion ◽

Type I Collagen ◽

Focal Adhesions ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Type I ◽

Focal Adhesion Complex ◽

Adhesion Complex

Active matrix metalloproteinases and degraded collagen are observed in disease states, such as atherosclerosis. To examine whether degraded collagen fragments have distinct effects on vascular smooth muscle cells (SMC), collagenase-digested type I collagen was added to cultured human arterial SMC. After addition of collagen fragments, adherent SMC lose their focal adhesion structures and round up. Analysis of components of the focal adhesion complex demonstrates rapid cleavage of the focal adhesion kinase (pp125FAK), paxillin, and talin. Cleavage is suppressed by inhibitors of the proteolytic enzyme, calpain I. In vitro translated pp125FAK is a substrate for both calpain I– and II–mediated processing. Mapping of the proteolytic cleavage fragments of pp125FAK predicts a dissociation of the focal adhesion targeting (FAT) sequence and second proline-rich domain from the tyrosine kinase domain and integrin-binding sequence. Coimmunoprecipitation studies confirm that the ability of pp125FAK to associate with paxillin, vinculin, and p130cas is significantly reduced in SMC treated with degraded collagen fragments. Further, there is a significant reduction in the association of intact pp125FAK with the cytoskeletal fraction, while pp125FAK cleavage fragments appear in the cytoplasm in SMC treated with degraded collagen fragments. Integrin-blocking studies indicate that integrin-mediated signals are involved in degraded collagen induction of pp125FAK cleavage. Thus, collagen fragments induce distinct integrin signals that lead to initiation of calpain-mediated cleavage of pp125FAK, paxillin, and talin and dissolution of the focal adhesion complex.

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The Mechanosensing and Global DNA Methylation of Human Osteoblasts on MEW Fibers

Nanomaterials ◽

10.3390/nano11112943 ◽

2021 ◽

Vol 11 (11) ◽

pp. 2943

Author(s):

Pingping Han ◽

Cedryck Vaquette ◽

Abdalla Abdal-hay ◽

Sašo Ivanovski

Keyword(s):

Dna Methylation ◽

Osteogenic Differentiation ◽

Focal Adhesion ◽

Adhesion Formation ◽

Focal Adhesions ◽

Global Dna Methylation ◽

Focal Adhesion Complex ◽

Osteogenic Induction ◽

Sense And Respond ◽

Adhesion Complex

Cells interact with 3D fibrous platform topography via a nano-scaled focal adhesion complex, and more research is required on how osteoblasts sense and respond to random and aligned fibers through nano-sized focal adhesions and their downstream events. The present study assessed human primary osteoblast cells’ sensing and response to random and aligned medical-grade polycaprolactone (PCL) fibrous 3D scaffolds fabricated via the melt electrowriting (MEW) technique. Cells cultured on a tissue culture plate (TCP) were used as 2D controls. Compared to 2D TCP, 3D MEW fibrous substrates led to immature vinculin focal adhesion formation and significantly reduced nuclear localization of the mechanosensor-yes-associated protein (YAP). Notably, aligned MEW fibers induced elongated cell and nucleus shape and highly activated global DNA methylation of 5-methylcytosine, 5-hydroxymethylcytosine, and N-6 methylated deoxyadenosine compared to the random fibers. Furthermore, although osteogenic markers (osterix-OSX and bone sialoprotein-BSP) were significantly enhanced in PCL-R and PCL-A groups at seven days post-osteogenic differentiation, calcium deposits on all seeded samples did not show a difference after normalizing for DNA content after three weeks of osteogenic induction. Overall, our study linked 3D extracellular fiber alignment to nano-focal adhesion complex, nuclear mechanosensing, DNA epigenetics at an early point (24 h), and longer-term changes in osteoblast osteogenic differentiation.

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Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex

PLoS ONE ◽

10.1371/journal.pone.0079654 ◽

2013 ◽

Vol 8 (12) ◽

pp. e79654 ◽

Cited By ~ 25

Author(s):

Takeo Kawahara ◽

Naoe Hotta ◽

Yukiko Ozawa ◽

Seiichi Kato ◽

Keiko Kano ◽

...

Keyword(s):

Cell Adhesion ◽

Pancreatic Ductal Adenocarcinoma ◽

Focal Adhesion ◽

Ductal Adenocarcinoma ◽

Proteomic Profiling ◽

Focal Adhesion Complex ◽

Cell Adhesion And Migration ◽

And Migration ◽

Adhesion Complex

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Caspase-mediated Cleavage of Focal Adhesion Kinase pp125FAK and Disassembly of Focal Adhesions in Human Endothelial Cell Apoptosis

Journal of Experimental Medicine ◽

10.1084/jem.187.4.579 ◽

1998 ◽

Vol 187 (4) ◽

pp. 579-586 ◽

Cited By ~ 192

Author(s):

Bodo Levkau ◽

Barbara Herren ◽

Hidenori Koyama ◽

Russell Ross ◽

Elaine W. Raines

Keyword(s):

Extracellular Matrix ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Caspase 3 ◽

Umbilical Vein ◽

Focal Adhesions ◽

Focal Adhesion Complex ◽

Adhesion Sites ◽

Induced Apoptosis ◽

Adhesion Complex

Normal endothelial and epithelial cells undergo apoptosis when cell adhesion and spreading are prevented, implying a requirement for antiapoptotic signals from the extracellular matrix for cell survival. We investigated some of the molecular changes occurring in focal adhesions during growth factor deprivation–induced apoptosis in confluent monolayers of human umbilical vein endothelial cells. Among the first morphologic changes after initiation of the apoptotic process are membrane blebbing, loss of focal adhesion sites, and retraction from the matrix followed by detachment. We observe a specific proteolytic cleavage of focal adhesion kinase (pp125FAK), an important component of the focal adhesion complex, and identify pp125FAK as a novel substrate for caspase-3 and caspase-3–like apoptotic caspases. The initial cleavage precedes detachment, and coincides with loss of pp125FAK and paxillin from focal adhesion sites and their redistribution into the characteristic membrane blebs of apoptotically dying cells. Cleavage of pp125FAK differentially affects its association with signaling and cytoskeletal components of the focal adhesion complex; binding of paxillin, but not pp130Cas (Cas, Crk-associated substrate) and vinculin, to the COOH terminally truncated pp125FAK is abolished. Therefore, caspase-mediated cleavage of pp125FAK may be participating in the disassembly of the focal adhesion complex and actively interrupting survival signals from the extracellular matrix, thus propagating the cell death program.

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Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling

Gastroenterology ◽

10.1053/j.gastro.2003.10.078 ◽

2004 ◽

Vol 126 (1) ◽

pp. 8-18 ◽

Cited By ~ 81

Author(s):

Vijayalakshmi Thamilselvan ◽

Marc D. Basson

Keyword(s):

Colon Cancer ◽

Cell Adhesion ◽

Cancer Cell ◽

Focal Adhesion ◽

Colon Cancer Cell ◽

Focal Adhesion Complex ◽

Cancer Cell Adhesion ◽

Inside Out ◽

Adhesion Complex

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Molecular basis for Ras suppressor-1 recruitment to focal adhesions and stabilization of consensus adhesome complex

10.1101/2021.02.19.432017 ◽

2021 ◽

Author(s):

Koichi Fukuda ◽

Fan Lu ◽

Jun Qin

Keyword(s):

Cell Adhesion ◽

Focal Adhesion ◽

Specific Interaction ◽

Tumor Development ◽

Focal Adhesions ◽

Adaptor Protein ◽

Biological Data ◽

Structural Basis ◽

Lim Domain ◽

Insight Into

AbstractRas suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating fundamental cell adhesion processes and tumor development. Rsu-1 interacts with a zinc-finger type multi LIM domain-containing adaptor protein PINCH-1 involved in the integrin-mediated consensus adhesome but not with highly homologous isoform PINCH-2. However, the structural basis for such specific interaction and regulatory mechanism remains unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture with eight LRRs shielded by the N- and C-terminal capping modules. We show that a large conserved concave surface of the Rsu-1 LRR domain recognizes the PINCH-1 LIM5 domain, and that the C-terminal non-LIM region of PINCH-2 but not PINCH-1 sterically disfavors the Rsu-1 binding. We further show that Rsu-1 can be assembled, via PINCH-1-binding, into a tight hetero-pentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2 that constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Consistently, our mutagenesis and cell biological data consolidate the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading. Our results provide a crucial molecular insight into Rsu-1-mediated cell adhesion with implication on how it may regulate tumorigenic growth.

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Increased transcriptional response to mechanical strain in keloid fibroblasts due to increased focal adhesion complex formation

Journal of Cellular Physiology ◽

10.1002/jcp.20486 ◽

2005 ◽

Vol 206 (2) ◽

pp. 510-517 ◽

Cited By ~ 50

Author(s):

Zhen Wang ◽

Kenton D. Fong ◽

Toan-Thang Phan ◽

Ivor J. Lim ◽

Michael T. Longaker ◽

...

Keyword(s):

Complex Formation ◽

Focal Adhesion ◽

Mechanical Strain ◽

Transcriptional Response ◽

Focal Adhesion Complex ◽

Adhesion Complex ◽

Keloid Fibroblasts

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PHIP drives glioblastoma motility and invasion by regulating the focal adhesion complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1914505117 ◽

2020 ◽

Vol 117 (16) ◽

pp. 9064-9073

Author(s):

David de Semir ◽

Vladimir Bezrookove ◽

Mehdi Nosrati ◽

Kara R. Scanlon ◽

Eric Singer ◽

...

Keyword(s):

Focal Adhesion ◽

Pleckstrin Homology Domain ◽

Physical Interaction ◽

Glioblastoma Cells ◽

Focal Adhesion Complex ◽

Adhesion Proteins ◽

Focal Adhesion Proteins ◽

Tumor Cell Motility ◽

Adhesion Complex ◽

Force Transduction

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP’s role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.

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