Focal Adhesion Targeting: The Critical Determinant of FAK Regulation and Substrate Phosphorylation

The focal adhesion kinase (FAK) is discretely localized to focal adhesions via its C-terminal focal adhesion–targeting (FAT) sequence. FAK is regulated by integrin-dependent cell adhesion and can regulate tyrosine phosphorylation of downstream substrates, like paxillin. By the use of a mutational strategy, the regions of FAK that are required for cell adhesion–dependent regulation and for inducing tyrosine phosphorylation of paxillin were determined. The results show that the FAT sequence was the single region of FAK that was required for each function. Furthermore, the FAT sequence of FAK was replaced with a focal adhesion–targeting sequence from vinculin, and the resulting chimera exhibited cell adhesion–dependent tyrosine phosphorylation and could induce paxillin phosphorylation like wild-type FAK. These results suggest that subcellular localization is the major determinant of FAK function.

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The Cytoplasmic Tyrosines of Integrin Subunit β1 Are Involved in Focal Adhesion Kinase Activation

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5758-5765.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5758-5765 ◽

Cited By ~ 68

Author(s):

Krister Wennerberg ◽

Annika Armulik ◽

Takao Sakai ◽

Marjam Karlsson ◽

Reinhard Fässler ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Phosphorylation Site ◽

Focal Adhesions ◽

Cell Spreading ◽

Integrin Subunit ◽

Wild Type ◽

Directed Cell Migration ◽

Dependent Cell

ABSTRACT We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit β1 (Y783 and Y795) to phenylalanines markedly reduces the capability of β1A integrins to mediate directed cell migration. In this study, β1-dependent cell spreading was found to be delayed in GD25 cells expressing β1AY783/795F compared to that in wild-type GD25-β1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to β1-dependent adhesion in GD25-β1AY783/795F cells compared to that in wild-type GD25-β1A or mutants in which only a single tyrosine was altered (β1AY783F or β1AY795F). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via β1AY783/795F lies at the level of the initial autophosphorylation step. Indeed, β1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the β1AY783/795F cells, consistent with the impairment in FAK activation. In contrast, p130CAS overall tyrosine phosphorylation was unaffected by the β1 mutations. Despite the defect in β1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-β1AY783/795F cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit β1A are critical mediators of FAK activation and cell spreading in GD25 cells.

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Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

Molecular Biology of the Cell ◽

10.1091/mbc.5.9.977 ◽

1994 ◽

Vol 5 (9) ◽

pp. 977-988 ◽

Cited By ~ 28

Author(s):

S Kawaguchi ◽

J M Bergelson ◽

R W Finberg ◽

M E Hemler

Keyword(s):

Amino Acids ◽

Cell Adhesion ◽

Focal Adhesion ◽

Cho Cells ◽

Chinese Hamster ◽

Focal Adhesions ◽

Inverse Correlation ◽

Cytoplasmic Domain ◽

Negative Regulator ◽

Wild Type

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.

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Src SH2 Arginine 175 Is Required for Cell Motility: Specific Focal Adhesion Kinase Targeting and Focal Adhesion Assembly Function

Molecular and Cellular Biology ◽

10.1128/mcb.01147-05 ◽

2006 ◽

Vol 26 (12) ◽

pp. 4399-4409 ◽

Cited By ~ 33

Author(s):

Myeong Gu Yeo ◽

Michael A. Partridge ◽

Ellen J. Ezratty ◽

Qiong Shen ◽

Gregg G. Gundersen ◽

...

Keyword(s):

Cell Motility ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Src Kinase ◽

Focal Adhesions ◽

Sh2 Domain ◽

Wild Type ◽

Binding Function ◽

Generation Capacity ◽

Substrate Phosphorylation

ABSTRACT Src kinase is a crucial mediator of adhesion-related signaling and motility. Src binds to focal adhesion kinase (FAK) through its SH2 domain and subsequently activates it for phosphorylation of downstream substrates. In addition to this binding function, data suggested that the SH2 domain might also perform an important role in targeting Src to focal adhesions (FAs) to enable further substrate phosphorylations. To examine this, we engineered an R175L mutation in cSrc to prevent the interaction with FAK pY397. This constitutively open Src kinase mediated up-regulated substrate phosphorylation in SYF cells but was unable to promote malignant transformation. Significantly, SrcR175L cells also had a profound motility defect and an impaired FA generation capacity. Importantly, we were able to recapitulate wild-type motile behavior and FA formation by directing the kinase to FAs, clearly implicating the SH2 domain in recruitment to FAK and indicating that this targeting capacity, and not simply Src-FAK scaffolding, was critical for normal Src function.

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Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3489 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3489-3505 ◽

Cited By ~ 134

Author(s):

Michael D. Schaller ◽

Jeffrey D. Hildebrand ◽

J. Thomas Parsons

Keyword(s):

Subcellular Localization ◽

Focal Adhesion Kinase ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Tyrosine Kinases ◽

Focal Adhesions ◽

Src Kinases ◽

High Affinity Binding Site ◽

Src Homology ◽

Src Homology 2 Domain

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60c-srcor p59fynresults in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60c-srcor p59fynto induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60c-srcis hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60c-srcfrom a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60c-srcto focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

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Tyrosine phosphorylation within the SH3 domain regulates CAS subcellular localization, cell migration, and invasiveness

Molecular Biology of the Cell ◽

10.1091/mbc.e11-03-0207 ◽

2011 ◽

Vol 22 (22) ◽

pp. 4256-4267 ◽

Cited By ~ 33

Author(s):

Radoslav Janoštiak ◽

Ondřej Tolde ◽

Zuzana Brůhová ◽

Marian Novotný ◽

Steven K. Hanks ◽

...

Keyword(s):

Cell Migration ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Phosphorylation Site ◽

Biological Significance ◽

Focal Adhesions ◽

Sh3 Domain ◽

Transformed Cells ◽

Wild Type ◽

Human Carcinoma

Crk-associated substrate (CAS) is a major tyrosine-phosphorylated protein in cells transformed by v-crk and v-src oncogenes and plays an important role in invasiveness of Src-transformed cells. A novel phosphorylation site on CAS, Tyr-12 (Y12) within the ligand-binding hydrophobic pocket of the CAS SH3 domain, was identified and found to be enriched in Src-transformed cells and invasive human carcinoma cells. To study the biological significance of CAS Y12 phosphorylation, phosphomimicking Y12E and nonphosphorylatable Y12F mutants of CAS were studied. The phosphomimicking mutation decreased interaction of the CAS SH3 domain with focal adhesion kinase (FAK) and PTP-PEST and reduced tyrosine phosphorylation of FAK. Live-cell imaging showed that green fluorescent protein–tagged CAS Y12E mutant is, in contrast to wild-type or Y12F CAS, excluded from focal adhesions but retains its localization to podosome-type adhesions. Expression of CAS-Y12F in cas–/– mouse embryonic fibroblasts resulted in hyperphosphorylation of the CAS substrate domain, and this was associated with slower turnover of focal adhesions and decreased cell migration. Moreover, expression of CAS Y12F in Src-transformed cells greatly decreased invasiveness when compared to wild-type CAS expression. These findings reveal an important role of CAS Y12 phosphorylation in the regulation of focal adhesion assembly, cell migration, and invasiveness of Src-transformed cells.

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Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

The Journal of Cell Biology ◽

10.1083/jcb.143.3.861 ◽

1998 ◽

Vol 143 (3) ◽

pp. 861-873 ◽

Cited By ~ 99

Author(s):

Carlos O. Arregui ◽

Janne Balsamo ◽

Jack Lilien

Keyword(s):

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Tyrosine Phosphatase ◽

Focal Adhesions ◽

Dominant Negative ◽

Stress Fibers ◽

Dominant Negative Mutant ◽

Β1 Integrin ◽

Wild Type ◽

In Cells

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.

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Candida albicans Expresses a Focal Adhesion Kinase-Like Protein That Undergoes Increased Tyrosine Phosphorylation upon Yeast Cell Adhesion to Vitronectin and the EA.hy 926 Human Endothelial Cell Line

Infection and Immunity ◽

10.1128/iai.70.7.3804-3815.2002 ◽

2002 ◽

Vol 70 (7) ◽

pp. 3804-3815 ◽

Cited By ~ 11

Author(s):

Giorgio Santoni ◽

Roberta Lucciarini ◽

Consuelo Amantini ◽

Jordan Jacobelli ◽

Elisabetta Spreghini ◽

...

Keyword(s):

Endothelial Cells ◽

Candida Albicans ◽

Cell Adhesion ◽

Endothelial Cell ◽

Yeast Cell ◽

Focal Adhesion Kinase ◽

Cell Line ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Human Endothelial Cell Line

ABSTRACT The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of αvβ3 and αvβ5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.

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Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK

Molecular and Cellular Biology ◽

10.1128/mcb.13.2.785-791.1993 ◽

1993 ◽

Vol 13 (2) ◽

pp. 785-791

Author(s):

M D Schaller ◽

C A Borgman ◽

J T Parsons

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Focal Adhesion ◽

Protein Tyrosine Kinase ◽

Signal Transduction Pathway ◽

Focal Adhesions ◽

Cellular Adhesion ◽

Protein Tyrosine ◽

Intracellular Signals ◽

Embryo Cells

Integrins play a central role in cellular adhesion and anchorage of the cytoskeleton and participate in the generation of intracellular signals, including tyrosine phosphorylation. We have recently isolated a cDNA encoding a unique, focal adhesion-associated protein tyrosine kinase (FAK) that is a component of an integrin-mediated signal transduction pathway. Here we report the isolation of cDNAs encoding the C-terminal, noncatalytic domain of the FAK kinase, termed FRNK (FAK-related nonkinase). Both the FAK- and FRNK-encoded polypeptides, pp125FAK and p41/p43FRNK, are expressed in normal chicken embryo cells. pp125FAK and p41/p43FRNK were localized to focal adhesions, suggesting that pp125FAK is directed to the focal adhesions by sequences within its C-terminal domain. We also show that the fibronectin-dependent increase in tyrosine phosphorylation of pp125FAK is accompanied by a concomitant posttranslational modification of p41FRNK.

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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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Abstract 110: Thrombospondin Type-1 Domain-Containing Protein 1 (THSD1), an Intracranial Aneurysm Susceptibility Gene, Mediates Cell Adhesion in Human Umbilical Vein Endothelial Cells

Stroke ◽

10.1161/str.46.suppl_1.110 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Xiaoqian Fang ◽

Dong H Kim ◽

Teresa Santiago-Sim

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Focal Adhesion ◽

Umbilical Vein ◽

Adhesion Formation ◽

Wild Type ◽

Focal Adhesion Formation ◽

Umbilical Vein Endothelial Cells

Introduction: An intracranial aneurysm (IA) is a weak spot in cerebral blood vessel wall that can lead to its abnormal bulging. Previously, we reported that mutations in THSD1 , encoding thrombospondin type-1 domain-containing protein 1, are associated with IA in a subset of patients. THSD1 is a transmembrane molecule with a thrombospondin type-1 repeat (TSR). Proteins with TSR domain have been implicated in a variety of processes including regulation of matrix organization, cell adhesion and migration. We have shown that in mouse brain Thsd1 is expressed in endothelial cells. Hypothesis: THSD1 plays an important role in maintaining the integrity of the endothelium by promoting adhesion of endothelial cells to the underlying basement membrane. Methods: Human umbilical vein endothelial cells are used to investigate the role of THSD1 in vitro . THSD1 expression was knocked-down by RNA interference. Cell adhesion assay was done on collagen I-coated plates and focal adhesion formation was visualized using immunofluorescence by paxillin and phosphorylated focal adhesion kinase (pFAK) staining. THSD1 re-expression is accomplished by transfection with a pCR3.1-THSD1-encoding plasmid. Results: Knockdown of THSD1 caused striking change in cell morphology and size. Compared to control siRNA-treated cells that exhibited typical cobblestone morphology, THSD1 knockdown cells were narrow and elongated, and were significantly smaller ( p <0.01). Cell adherence to collagen I-coated plates was also attenuated in THSD1 knockdown cells ( p <0.01). Consistent with this finding is the observation that the number and size of focal adhesions, based on paxillin and pFAK staining, were significantly reduced after THSD1 knockdown ( p <0.01). These defects in cell adhesion and focal adhesion formation were rescued by re-expression of wild type THSD1 ( p <0.05). In contrast, initial studies indicate that expression of mutated versions of THSD1 as seen in human patients (L5F, R450*, E466G, P639L) could not restore cell adhesion and focal adhesion formation to wild type levels. Conclusions: Our studies provide evidence for a role of THSD1 and THSD1 mutations in endothelial cell adhesion and suggest a possible mechanism underlying THSD1 -mediated aneurysm disease.

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