Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

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BPGAP1 Interacts with Cortactin and Facilitates Its Translocation to Cell Periphery for Enhanced Cell Migration

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0141 ◽

2004 ◽

Vol 15 (6) ◽

pp. 2873-2883 ◽

Cited By ~ 26

Author(s):

Bee Leng Lua ◽

Boon Chuan Low

Keyword(s):

Cell Migration ◽

Control Cell ◽

Actin Binding ◽

Ionization Mass ◽

Cell Periphery ◽

Nucleotide Exchange ◽

Cortical Actin ◽

Cell Dynamics

Rho GTPases control cell dynamics during growth and development. They are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). Many GAPs exist with various protein modules, the functions of which largely remain unknown. We recently cloned and identified BPGAP1 as a novel RhoGAP that coordinately regulates pseudopodia and cell migration via the interplay of its BNIP-2 and Cdc42GAP homology, RhoGAP, and the proline-rich domains. To further elucidate the molecular mechanism underlying cell dynamics control by BPGAP1, we used protein precipitations and matrix-assisted laser desorption/ionization mass spectrometry and identified cortactin, a cortical actin binding protein as a novel partner of BPGAP1 both in vitro and in vivo. Progressive deletion studies confirmed that cortactin interacted directly and constitutively with the proline-rich motif 182-PPPRPPLP-189 of BPGAP1 via its Src homology 3 domain. Together, they colocalized to periphery and enhanced cell migration. Furthermore, substitution of prolines at 184 and 186 with alanines abolished their interaction. Consequently, this BPGAP1 mutant failed to facilitate translocation of cortactin to the periphery, and no enhanced cell migration was observed. These results provide the first evidence that a RhoGAP functionally interacts with cortactin and represents a novel determinant in the regulation of cell dynamics.

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Focal adhesion kinase confers pro-migratory and anti-apoptotic properties and is a potential therapeutic target in Ewing sarcoma

10.1101/604207 ◽

2019 ◽

Author(s):

Konrad Steinestel ◽

Esther-Pia Jansen ◽

Marcel Trautmann ◽

Uta Dirksen ◽

Jan Rehkämper ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Ewing Sarcoma ◽

Chorioallantoic Membrane ◽

Focal Adhesions ◽

Childhood And Adolescence ◽

Tissue Samples

ABSTRACTOncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1-ETS fusion oncogenes, most often EWSR1-FLI1 (E/F).E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F-dependent way involving Ezrin. Employing different EwS cell as in vitro models, we found that key malignant properties of E/F are mediated via substrate-independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho-dependent cell migration, and impaired caspase-3-mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor Y15 enhanced caspase-mediated apoptosis and EwS cell migration, independent from the respective EWSR1-ETS fusion type, mimicking an anoikis-like phenotype. Our findings were confirmed in vivo using an avian chorioallantoic membrane (CAM) model. Our results provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

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Regulation of cell migration and survival by focal adhesion targeting of Lasp-1

The Journal of Cell Biology ◽

10.1083/jcb.200311045 ◽

2004 ◽

Vol 165 (3) ◽

pp. 421-432 ◽

Cited By ~ 103

Author(s):

Yi Hsing Lin ◽

Zee-Yong Park ◽

Dayin Lin ◽

Anar A. Brahmbhatt ◽

Marie-Christine Rio ◽

...

Keyword(s):

Cell Migration ◽

Growth Factors ◽

Focal Adhesion ◽

Large Scale ◽

Actin Polymerization ◽

Focal Adhesions ◽

Leading Edge ◽

Actin Binding ◽

Cell Periphery ◽

Ecm Proteins

Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8–12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.

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Transition from actin-driven to water-driven cell migration dependson external hydraulic resistance

10.1101/275123 ◽

2018 ◽

Author(s):

Y. Li ◽

S.X. Sun

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Actin Polymerization ◽

Metastatic Cancer ◽

Focal Adhesions ◽

Force Balance ◽

Confined Geometries ◽

Two Phase ◽

Water Permeation

AbstractIn vivo, cells can reside in diverse physical and biochemical environments. For example, epithelial cells typically live in a two-dimensional (2D) environment while metastatic cancer cells can move through dense three-dimensional (3D) matrices. These distinct environments impose different kinds of mechanical forces on cells, and thus potentially can influence the mechanism of cell migration. For example, cell movement on 2D flat surfaces is mostly driven by forces from focal adhesion and actin polymerization, while in confined geometries, it can be driven by water permeation. In this work, we utilize a two-phase model of the cellular cytoplasm, where the mechanics of the cytosol and the F-actin network are treated on an equal footing. Using conservation laws and simple force balance considerations, we are able to describe the contribution of water flux, actin polymerization and flow, and focal adhesions to cell migration in both 2D surfaces and in confined spaces. The theory shows how cell migration can seamlessly transition from a focal adhesion- and actin-based mechanism on 2D surfaces to a water-based mechanism in confined geometries.

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Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats

Journal of Cell Science ◽

10.1242/jcs.112.24.4589 ◽

1999 ◽

Vol 112 (24) ◽

pp. 4589-4599 ◽

Cited By ~ 14

Author(s):

F. Li ◽

Y. Zhang ◽

C. Wu

Keyword(s):

Focal Adhesion ◽

Critical Role ◽

Focal Adhesions ◽

Subcellular Compartment ◽

Cell Matrix ◽

Integrin Binding Site ◽

Integrin Linked Kinase ◽

Cell Cell

Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function.

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Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

Experimental Biology and Medicine ◽

10.1177/153537020222700607 ◽

2002 ◽

Vol 227 (6) ◽

pp. 412-424 ◽

Cited By ~ 20

Author(s):

Imre L. Szabó ◽

Rama Pai ◽

Michael K. Jones ◽

George R. Ehring ◽

Hirofumi Kawanaka ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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Force transduction by Triton cytoskeletons

The Journal of Cell Biology ◽

10.1083/jcb.200110068 ◽

2002 ◽

Vol 156 (4) ◽

pp. 609-615 ◽

Cited By ~ 188

Author(s):

Yasuhiro Sawada ◽

Michael P. Sheetz

Keyword(s):

Focal Adhesion ◽

Tyrosine Phosphatase ◽

Actin Binding ◽

Two Dimensional Gel Electrophoresis ◽

Conformation Changes ◽

Cytoplasmic Proteins ◽

Ionic Mechanisms ◽

Triton X

Force-initiated signal transduction can occur either via membrane-based ionic mechanisms or through changes in cytoskeletal–matrix linkages. We report here the stretch-dependent binding of cytoplasmic proteins to Triton X-100 cytoskeletons of L-929 cells grown on collagen-coated silicone. Triton X-100–insoluble cytoskeletons were stretched by 10% and incubated with biotinylated cytoplasmic proteins. Analysis with two-dimensional gel electrophoresis showed stretch-dependent binding of more than 10 cytoplasmic protein spots. Bound cytoplasmic proteins were purified by a photocleavable biotin tag and stretch-dependent binding of paxillin, focal adhesion kinase, and p130Cas was found, whereas the binding of vinculin was unchanged and actin binding decreased with stretch. Paxillin binding upon stretch was morphologically and biochemically similar in vitro and in vivo, that is, enhanced in the periphery and inhibited by the tyrosine phosphatase inhibitor, phenylarsine oxide. Thus, we suggest that transduction of matrix forces occurs through force-dependent conformation changes in the integrated cytoskeleton.

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The Abl-related Gene Tyrosine Kinase Acts through p190RhoGAP to Inhibit Actomyosin Contractility and Regulate Focal Adhesion Dynamics upon Adhesion to Fibronectin

Molecular Biology of the Cell ◽

10.1091/mbc.e07-01-0075 ◽

2007 ◽

Vol 18 (10) ◽

pp. 3860-3872 ◽

Cited By ~ 55

Author(s):

Justin G. Peacock ◽

Ann L. Miller ◽

William D. Bradley ◽

Olga C. Rodriguez ◽

Donna J. Webb ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Actin Polymerization ◽

Focal Adhesions ◽

Fiber Formation ◽

Cell Periphery ◽

Related Gene ◽

Cell Contractility ◽

Fibroblast Migration ◽

Actomyosin Contractility

In migrating cells, actin polymerization promotes protrusion of the leading edge, whereas actomyosin contractility powers net cell body translocation. Although they promote F-actin–dependent protrusions of the cell periphery upon adhesion to fibronectin (FN), Abl family kinases inhibit cell migration on FN. We provide evidence here that the Abl-related gene (Arg/Abl2) kinase inhibits fibroblast migration by attenuating actomyosin contractility and regulating focal adhesion dynamics. arg−/− fibroblasts migrate at faster average speeds than wild-type (wt) cells, whereas Arg re-expression in these cells slows migration. Surprisingly, the faster migrating arg−/− fibroblasts have more prominent F-actin stress fibers and focal adhesions and exhibit increased actomyosin contractility relative to wt cells. Interestingly, Arg requires distinct functional domains to inhibit focal adhesions and actomyosin contractility. The kinase domain–containing Arg N-terminal half can act through the RhoA inhibitor p190RhoGAP to attenuate stress fiber formation and cell contractility. However, Arg requires both its kinase activity and its cytoskeleton-binding C-terminal half to fully inhibit focal adhesions. Although focal adhesions do not turn over efficiently in the trailing edge of arg−/− cells, the increased contractility of arg−/− cells tears the adhesions from the substrate, allowing for the faster migration observed in these cells. Together, our data strongly suggest that Arg inhibits cell migration by restricting actomyosin contractility and regulating its coupling to the substrate through focal adhesions.

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Isolation and characterization of a regulated form of actin depolymerizing factor

The Journal of Cell Biology ◽

10.1083/jcb.122.3.623 ◽

1993 ◽

Vol 122 (3) ◽

pp. 623-633 ◽

Cited By ~ 110

Author(s):

TE Morgan ◽

RO Lockerbie ◽

LS Minamide ◽

MD Browning ◽

JR Bamburg

Keyword(s):

Muscle Development ◽

Peptide Mapping ◽

Actin Binding ◽

Actin Assembly ◽

Actin Depolymerizing Factor ◽

Isolation And Characterization ◽

Relative Molecular Weight

Actin depolymerizing factor (ADF) is an 18.5-kD protein with pH-dependent reciprocal F-actin binding and severing/depolymerizing activities. We previously showed developing muscle down-regulates ADF (J. R. Bamburg and D. Bray. 1987. J. Cell Biol. 105: 2817-2825). To further study this process, we examined ADF expression in chick myocytes cultured in vitro. Surprisingly, ADF immunoreactivity increases during the first 7-10 d in culture. This increase is due to the presence of a new ADF species with higher relative molecular weight which reacts identically to brain ADF with antisera raised against either brain ADF or recombinant ADF. We have purified both ADF isoforms from myocytes and have shown by peptide mapping and partial sequence analysis that the new isoform is structurally related to ADF. Immunoprecipitation of both isoforms from extracts of cells prelabeled with [32P]orthophosphate showed that the new isoform is radiolabeled, predominantly on a serine residue, and hence is called pADF. pADF can be converted into a form which comigrates with ADF on 1-D and 2-D gels by treatment with alkaline phosphatase. pADF has been quantified in a number of cells and tissues where it is present from approximately 18% to 150% of the amount of unphosphorylated ADF. pADF, unlike ADF, does not bind to G-actin, or affect the rate or extent of actin assembly. Four ubiquitous protein kinases failed to phosphorylate ADF in vitro suggesting that ADF phosphorylation in vivo is catalyzed by a more specific kinase. We conclude that the ability to regulate ADF activity is important to muscle development since myocytes have both pre- and posttranslational mechanisms for regulating ADF activity. The latter mechanism is apparently a general one for cell regulation of ADF activity.

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EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells

The Journal of Cell Biology ◽

10.1083/jcb.200706206 ◽

2007 ◽

Vol 179 (6) ◽

pp. 1247-1259 ◽

Cited By ~ 155

Author(s):

Jacco van Rheenen ◽

Xiaoyan Song ◽

Wies van Roosmalen ◽

Michael Cammer ◽

Xiaoming Chen ◽

...

Keyword(s):

Actin Polymerization ◽

Carcinoma Cells ◽

Actin Binding ◽

Temporal Regulation ◽

Vitro Binding ◽

Epidermal Growth ◽

Membrane Bound ◽

Regulation Of Actin Cytoskeleton

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP2; however, this is mainly based on in vitro–binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP2 in living cells. We show that EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

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