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

1996 ◽  
Vol 318 (3) ◽  
pp. 753-757 ◽  
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.

scholarly journals Mammalian Septins Regulate Microtubule Stability through Interaction with the Microtubule-binding Protein MAP4

2005 ◽  
Vol 16 (10) ◽  
pp. 4648-4659 ◽  
Author(s):  
Brandon E. Kremer ◽  
Timothy Haystead ◽  
Ian G. Macara
Keyword(s):  
Mammalian Cells ◽  
Spectroscopic Analysis ◽  
Molecular Function ◽  
Rich Region ◽  
Intact Cells ◽  
Pronounced Increase ◽  
Binding Partner ◽  
Microtubule Stability ◽  
Proline Rich Region

Mammalian septins constitute a family of at least 12 GTP-binding proteins that can form hetero-oligomers and that are sometimes found in association with actin or microtubule filaments. However, their functions are not understood. Using RNA interference, we found that suppression of septin expression in HeLa cells caused a pronounced increase in microtubule stability. Mass spectroscopic analysis of proteins coprecipitating with Sept6 identified the microtubule-associated protein MAP4 as a septin binding partner. A small, proline-rich region in the C-terminal half of MAP4 bound directly to a Sept 2:6:7 heterotrimer, and to the Sept2 monomer. The trimer blocked the ability of this MAP4 fragment to bind and bundle microtubules in vitro. In intact cells, MAP4 was required for the stabilization of microtubules induced by septin depletion. Moreover, septin depletion increased the number of cells with abnormal nuclei, and this effect was blocked by gene silencing of MAP4. These data identify a novel molecular function for septins in mammalian cells: the modulation of microtubule dynamics through interaction with MAP4.

scholarly journals Variations in in vivo phosphorylation at the proline-rich domain of the microtubule-associated protein 2 (MAP2) during rat brain development

1995 ◽  
Vol 306 (2) ◽  
pp. 481-487 ◽  
Author(s):  
C Sánchez ◽  
J Díaz-Nido ◽  
J Avila
Keyword(s):  
Brain Development ◽  
Map Kinase ◽  
Rat Brain ◽  
Iron Chelation ◽  
Rich Region ◽  
Adult Rat ◽  
Rich Domain ◽  
Proline Rich Region

Microtubule-associated protein 2 (MAP2) is an in vitro substrate for MAP kinase. Part of the phosphorylation occurs at the C-terminal microtubule-binding domain of the molecule which contains a cluster of putative consensus sites for MAP kinase on a proline-rich region. A peptide with the sequence RTPGTPG-TPSY, located at this region of the molecule, is efficiently phosphorylated by MAP kinase in vitro. An antibody (972) raised against this non-phosphorylated peptide has been used to test for in vivo phosphorylation at the proline-rich domain of the MAP2 molecule. The reaction of purified MAP2 with antibody 972 diminishes after in vitro phosphorylation by MAP kinase and is enhanced after in vitro dephosphorylation by alkaline phosphatase. A fraction of brain MAP2 isolated by iron-chelation affinity chromatography appears to be phosphorylated in vivo at the site recognized by antibody 972. There is some variation in the phosphorylation of MAP2 at the proline-rich region throughout rat brain development. MAP2C is more highly phosphorylated in the developing rat brain, whereas high-molecular-mass MAP2 is more extensively phosphorylated in the adult rat brain.

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

2000 ◽  
Vol 151 (7) ◽  
pp. 1435-1448 ◽  
Author(s):  
Sotiris N. Nikolopoulos ◽  
Christopher E. Turner
Keyword(s):  
Cell Adhesion ◽  
Actin Cytoskeleton ◽  
Focal Adhesion ◽  
Substantial Reduction ◽  
Ectopic Expression ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Types ◽  
Focal Adhesion Protein

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.

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

1995 ◽  
Vol 15 (2) ◽  
pp. 711-722 ◽  
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.

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

1990 ◽  
Vol 111 (3) ◽  
pp. 1059-1068 ◽  
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.

scholarly journals 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 ◽  
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.

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

scholarly journals An efficient in vitro translation system from mammalian cells lacking the translational inhibition caused by eIF2 phosphorylation

RNA ◽  
2008 ◽  
Vol 14 (3) ◽  
pp. 593-602 ◽  
Author(s):  
V. V. Zeenko ◽  
C. Wang ◽  
M. Majumder ◽  
A. A. Komar ◽  
M. D. Snider ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
In Vitro Translation ◽  
Translation System ◽  
Translational Inhibition ◽  
Eif2 Phosphorylation

scholarly journals Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1192
Author(s):  
Hideyuki Nakanishi
Keyword(s):  
Adverse Effects ◽  
Mammalian Cells ◽  
Translational Regulation ◽  
Insertional Mutagenesis ◽  
In Vitro Transcription ◽  
Preclinical Studies ◽  
High Efficacy ◽  
Gene Circuits ◽  
Synthetic Mrna

Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

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

1999 ◽  
Vol 112 (24) ◽  
pp. 4589-4599 ◽  
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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