Actopaxin, a New Focal Adhesion Protein That Binds Paxillin Ld Motifs and Actin and Regulates Cell Adhesion

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor– and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M.C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851–863). In this study, we report the cloning and initial characterization of a new paxillin LD motif–binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell–cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

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Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

10.1101/2021.04.01.438077 ◽

2021 ◽

Author(s):

Erik S Linklater ◽

Emily Duncan ◽

Ke Jun Han ◽

Algirdas Kaupinis ◽

Mindaugas Valius ◽

...

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Focal Adhesion ◽

Focal Adhesions ◽

Leading Edge ◽

Stress Fibers ◽

Actin Dynamics ◽

Migration And Invasion ◽

Matrix Remodeling ◽

Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin

Biochemical Journal ◽

10.1042/bj3180753 ◽

1996 ◽

Vol 318 (3) ◽

pp. 753-757 ◽

Cited By ~ 135

Author(s):

Nicholas P. J. BRINDLE ◽

Mark R. HOLT ◽

Joanna E DAVIES ◽

Caroline J PRICE ◽

David R. CRITCHLEY

Keyword(s):

Focal Adhesion ◽

Mammalian Cells ◽

Focal Adhesions ◽

In Vitro Transcription ◽

Translation System ◽

Rich Region ◽

Rich Domain ◽

Focal Adhesion Protein ◽

Proline Rich Region

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actin-filament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-1 (residues 839–850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

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Rab40–Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration

The Journal of Cell Biology ◽

10.1083/jcb.202008060 ◽

2021 ◽

Vol 220 (7) ◽

Author(s):

Erik S. Linklater ◽

Emily D. Duncan ◽

Ke-Jun Han ◽

Algirdas Kaupinis ◽

Mindaugas Valius ◽

...

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Focal Adhesion ◽

Focal Adhesions ◽

Leading Edge ◽

Stress Fibers ◽

Actin Dynamics ◽

Migration And Invasion ◽

Matrix Remodeling ◽

Cell Migration And Invasion

Rab40b is a SOCS box–containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here, we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b–Cullin5 binding decreases cell motility and invasive potential and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b–Cullin5-dependent localized ubiquitylation and degradation. Thus, we propose a model where Rab40b–Cullin5-dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.711 ◽

1995 ◽

Vol 15 (2) ◽

pp. 711-722 ◽

Cited By ~ 71

Author(s):

M Bergman ◽

V Joukov ◽

I Virtanen ◽

K Alitalo

Keyword(s):

Focal Adhesion ◽

Tyrosine Kinases ◽

Cell Attachment ◽

Focal Adhesions ◽

Inducible Promoter ◽

Sh2 Domain ◽

Sh3 Domains ◽

Focal Adhesion Protein ◽

In Cells

The C-terminal Src kinase p50csk phosphorylates Src family tyrosine kinases and down-regulates their activity in vitro. To gain insight into the cellular functions of this potentially antioncogenic enzyme, we have overexpressed the csk cDNA by using an inducible promoter in HeLa cells. Despite some differences in basal Src activity in the clones analyzed, Src activity was not significantly suppressed, while the amount of p50csk and Csk activity increased at least 10-fold during 3 days of induction. Immunofluorescence for the induced p50csk was localized in the cytoplasm and distinctly in focal adhesions, in which the amount of phosphotyrosine containing proteins was also increased. Point and deletion mutagenesis experiments showed that localization in focal adhesions was dependent on the SH2 and SH3 domains of Csk but not on its catalytic activity. Csk formed a complex with the focal adhesion protein paxillin in cells, and its SH2 domain was shown to interact with pp125FAK and paxillin in vitro. After Csk induction, the cells became spherical and more loosely attached to the culture substratum, and the alpha v beta 5 integrin complex (vitronectin receptor) of focal adhesions was redistributed to a novel type of structure consisting of punctate plaques on the ventral cell surface. These phenotypic changes occurred in several clones analyzed and were totally reversible when Csk was switched off, but they did not occur in cells overexpressing the catalytically inactive Csk R-222 mutant or luciferase. Our results thus show that a fraction of cellular Csk is targeted to focal adhesions via its SH2 and SH3 domains, probably interacting with tyrosyl-phosphorylated focal adhesion proteins. They also suggest that Csk is involved in the regulation of integrins controlling cell attachment and shape.

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Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

The Journal of Cell Biology ◽

10.1083/jcb.139.3.759 ◽

1997 ◽

Vol 139 (3) ◽

pp. 759-771 ◽

Cited By ~ 156

Author(s):

Claudio Brancolini ◽

Dean Lazarevic ◽

Joe Rodriguez ◽

Claudio Schneider

Keyword(s):

Cell Death ◽

Cell Adhesion ◽

Actin Cytoskeleton ◽

Caspase 3 ◽

Cell Types ◽

Cell Contacts ◽

Carboxy Terminal ◽

Cell Cell

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis. In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

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Paxillin: a new vinculin-binding protein present in focal adhesions.

The Journal of Cell Biology ◽

10.1083/jcb.111.3.1059 ◽

1990 ◽

Vol 111 (3) ◽

pp. 1059-1068 ◽

Cited By ~ 412

Author(s):

C E Turner ◽

J R Glenney ◽

K Burridge

Keyword(s):

Focal Adhesion ◽

Specific Interaction ◽

Focal Adhesions ◽

Adhesion Protein ◽

Rous Sarcoma ◽

Sds Page ◽

Cytoskeletal Component ◽

Sarcoma Virus ◽

Focal Adhesion Protein

The 68-kD protein (paxillin) is a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers in chicken embryo fibroblasts. It is also present in the focal adhesions of Madin-Darby bovine kidney (MDBK) epithelial cells but is absent, like talin, from the cell-cell adherens junctions of these cells. Paxillin purified from chicken gizzard smooth muscle migrates as a diffuse band on SDS-PAGE gels with a molecular mass of 65-70 kD. It is a protein of multiple isoforms with pIs ranging from 6.31 to 6.85. Using purified paxillin, we have demonstrated a specific interaction in vitro with another focal adhesion protein, vinculin. Cleavage of vinculin with Staphylococcus aureus V8 protease results in the generation of two fragments of approximately 85 and 27 kD. Unlike talin, which binds to the large vinculin fragment, paxillin was found to bind to the small vinculin fragment, which represents the rod domain of the molecule. Together with the previous observation that paxillin is a major substrate of pp60src in Rous sarcoma virus-transformed cells (Glenney, J. R., and L. Zokas. 1989. J. Cell Biol. 108:2401-2408), this interaction with vinculin suggests paxillin may be a key component in the control of focal adhesion organization.

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The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration

Molecular Biology of the Cell ◽

10.1091/mbc.e11-01-0015 ◽

2012 ◽

Vol 23 (4) ◽

pp. 591-601 ◽

Cited By ~ 39

Author(s):

Inbal Dahan ◽

Ahuv Yearim ◽

Yarin Touboul ◽

Shoshana Ravid

Keyword(s):

Tumor Suppressor ◽

Cell Polarity ◽

Focal Adhesion ◽

Cellular Localization ◽

Focal Adhesions ◽

Leading Edge ◽

Membrane Dynamics ◽

Migrating Cells

The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical–basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. In this study we show direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). We demonstrate that Lgl1 and NMII-A form a complex in vivo and provide data that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, we show that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly.

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EVL is a novel focal adhesion protein involved in the regulation of cytoskeletal dynamics and vascular permeability

Pulmonary Circulation ◽

10.1177/20458940211049002 ◽

2021 ◽

Vol 11 (4) ◽

pp. 204589402110490

Author(s):

Joseph B. Mascarenhas ◽

Amir A. Gaber ◽

Tania M. Larrinaga ◽

Rachel Mayfield ◽

Stefanie Novak ◽

...

Keyword(s):

Endothelial Cell ◽

Actin Cytoskeleton ◽

Focal Adhesion Kinase ◽

Vascular Permeability ◽

Focal Adhesion ◽

Regulatory Protein ◽

Focal Adhesions ◽

Adhesion Protein ◽

Cytoskeletal Responses ◽

Focal Adhesion Protein

Increases in lung vascular permeability is a cardinal feature of inflammatory disease and represents an imbalance in vascular contractile forces and barrier-restorative forces, with both forces highly dependent upon the actin cytoskeleton. The current study investigates the role of Ena-VASP-like (EVL), a member of the Ena-VASP family known to regulate the actin cytoskeleton, in regulating vascular permeability responses and lung endothelial cell barrier integrity. Utilizing changes in transendothelial electricial resistance (TEER) to measure endothelial cell barrier responses, we demonstrate that EVL expression regulates endothelial cell responses to both sphingosine-1-phospate (S1P), a vascular barrier-enhancing agonist, and to thrombin, a barrier-disrupting stimulus. Total internal reflection fluorescence demonstrates that EVL is present in endothelial cell focal adhesions and impacts focal adhesion size, distribution, and the number of focal adhesions generated in response to S1P and thrombin challenge, with the focal adhesion kinase (FAK) a key contributor in S1P-stimulated EVL-transduced endothelial cell but a limited role in thrombin-induced focal adhesion rearrangements. In summary, these data indicate that EVL is a focal adhesion protein intimately involved in regulation of cytoskeletal responses to endothelial cell barrier-altering stimuli. Keywords: cytoskeleton, vascular barrier, sphingosine-1-phosphate, thrombin, focal adhesion kinase (FAK), Ena-VASP like protein (EVL), cytoskeletal regulatory protein

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Disruption of the actin cytoskeleton after microinjection of proteolytic fragments of alpha-actinin.

The Journal of Cell Biology ◽

10.1083/jcb.114.3.481 ◽

1991 ◽

Vol 114 (3) ◽

pp. 481-491 ◽

Cited By ~ 87

Author(s):

F M Pavalko ◽

K Burridge

Keyword(s):

Actin Cytoskeleton ◽

Binding Site ◽

Focal Adhesions ◽

Cell Types ◽

Cytoplasmic Domain ◽

Stress Fibers ◽

Actin Binding ◽

Cell Periphery

Alpha-actinin can be proteolytically cleaved into major fragments of 27 and 53 kD using the enzyme thermolysin. The 27-kD fragment contains an actin-binding site and we have recently shown that the 53-kD fragment binds to the cytoplasmic domain of beta 1 integrin in vitro (Otey, C. A., F. M. Pavalko, and K. Burridge. 1990. J. Cell Biol. 111:721-729). We have explored the behavior of the isolated 27- and 53-kD fragments of alpha-actinin after their microinjection into living cells. Consistent with its containing a binding site for actin, the 27-kD fragment was detected along stress fibers within 10-20 min after injection into rat embryo fibroblasts (REF-52). The 53-kD fragment of alpha-actinin, however, concentrated in focal adhesions of REF-52 cells 10-20 min after injection. The association of this fragment with focal adhesions in vivo is consistent with its interaction in vitro with the cytoplasmic domain of the beta 1 subunit of integrin, which was also localized at these sites. When cells were injected with greater than 5 microM final concentration of either alpha-actinin fragment and cultured for 30-60 min, most stress fibers were disassembled. At this time, however, many of the focal adhesions, particularly those around the cell periphery, remained after most stress fibers had gone. By 2 h after injection only a few small focal adhesions persisted, yet the cells remained spread. Identical results were obtained with other cell types including primary chick fibroblasts, BSC-1, MDCK, and gerbil fibroma cells. Stress fibers and focal adhesions reformed if cells were allowed to recover for 18 h after injection. These data suggest that introduction of the monomeric 27-kD fragment of alpha-actinin into cells may disrupt the actin cytoskeleton by interfering with the function of endogenous, intact alpha-actinin molecules along stress fibers. The 53-kD fragment may interfere with endogenous alpha-actinin function at focal adhesions or by displacing some other component that binds to the rod domain of alpha-actinin and that is needed to maintain stress fiber organization.

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The extracellular-regulated protein kinase 5 (ERK5) enhances metastatic burden in triple-negative breast cancer through focal adhesion protein kinase (FAK)-mediated regulation of cell adhesion

Oncogene ◽

10.1038/s41388-021-01798-2 ◽

2021 ◽

Author(s):

Qiuping Xu ◽

Jingwei Zhang ◽

Brian A. Telfer ◽

Hao Zhang ◽

Nisha Ali ◽

...

Keyword(s):

Breast Cancer ◽

Protein Kinase ◽

Tumor Growth ◽

Focal Adhesion ◽

Triple Negative Breast Cancer ◽

Triple Negative ◽

Metastatic Breast ◽

Adhesion Protein ◽

Focal Adhesion Protein

AbstractThere is overwhelming clinical evidence that the extracellular-regulated protein kinase 5 (ERK5) is significantly dysregulated in human breast cancer. However, there is no definite understanding of the requirement of ERK5 in tumor growth and metastasis due to very limited characterization of the pathway in disease models. In this study, we report that a high level of ERK5 is a predictive marker of metastatic breast cancer. Mechanistically, our in vitro data revealed that ERK5 was critical for maintaining the invasive capability of triple-negative breast cancer (TNBC) cells through focal adhesion protein kinase (FAK) activation. Specifically, we found that phosphorylation of FAK at Tyr397 was controlled by a kinase-independent function of ERK5. Accordingly, silencing ERK5 in mammary tumor grafts impaired FAK phosphorylation at Tyr397 and suppressed TNBC cell metastasis to the lung without preventing tumor growth. Collectively, these results establish a functional relationship between ERK5 and FAK signaling in promoting malignancy. Thus, targeting the oncogenic ERK5-FAK axis represents a promising therapeutic strategy for breast cancer exhibiting aggressive clinical behavior.

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