The Amino-terminal Domain of the B Subunit of Vacuolar H+-ATPase Contains a Filamentous Actin Binding Site

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

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Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Biochemical Journal ◽

10.1042/bj20120209 ◽

2012 ◽

Vol 444 (1) ◽

pp. 89-96 ◽

Cited By ~ 20

Author(s):

Keefe T. Chan ◽

David W. Roadcap ◽

Nicholas Holoweckyj ◽

James E. Bear

Keyword(s):

Binding Site ◽

Leading Edge ◽

Actin Binding ◽

Filamentous Actin ◽

Unique Region ◽

Equivalent Residue ◽

Filament Networks ◽

Actin Binding Site

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg30) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.

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The RhoGAP activity of OPHN1, a new F-actin-binding protein, is negatively controlled by its amino-terminal domain

Molecular and Cellular Neuroscience ◽

10.1016/s1044-7431(03)00078-2 ◽

2003 ◽

Vol 23 (4) ◽

pp. 574-586 ◽

Cited By ~ 67

Author(s):

F Fauchereau

Keyword(s):

Binding Protein ◽

Actin Binding ◽

Actin Binding Protein ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

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Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family.

The Journal of Cell Biology ◽

10.1083/jcb.126.6.1445 ◽

1994 ◽

Vol 126 (6) ◽

pp. 1445-1453 ◽

Cited By ~ 259

Author(s):

O Turunen ◽

T Wahlström ◽

A Vaheri

Keyword(s):

Binding Site ◽

Protein Family ◽

Actin Binding ◽

Capping Protein ◽

Terminal Domain ◽

Membrane Cytoskeleton ◽

Actin Capping Protein ◽

Binding Capability ◽

Actin Capping ◽

Actin Binding Site

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH-terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.

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Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

Molecular Biology of the Cell ◽

10.1091/mbc.6.8.1061 ◽

1995 ◽

Vol 6 (8) ◽

pp. 1061-1075 ◽

Cited By ~ 290

Author(s):

R Gary ◽

A Bretscher

Keyword(s):

Amino Acids ◽

Binding Site ◽

Sodium Dodecyl ◽

Actin Binding ◽

Terminal Domain ◽

Cell Extracts ◽

C Terminus ◽

Self Association ◽

Actin Binding Site

Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking.

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Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase

Nature ◽

10.1038/18050 ◽

1999 ◽

Vol 398 (6722) ◽

pp. 84-90 ◽

Cited By ~ 215

Author(s):

Wuyi Meng ◽

Sansana Sawasdikosol ◽

Steven J. Burakoff ◽

Michael J. Eck

Keyword(s):

Binding Site ◽

Amino Terminal ◽

Terminal Domain ◽

Amino Terminal Domain

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The N-Terminal Domain of MYO18A Has an ATP-Insensitive Actin-Binding Site†

Biochemistry ◽

10.1021/bi0475931 ◽

2005 ◽

Vol 44 (16) ◽

pp. 6190-6196 ◽

Cited By ~ 37

Author(s):

Yasushi Isogawa ◽

Takahide Kon ◽

Takeshi Inoue ◽

Reiko Ohkura ◽

Hisashi Yamakawa ◽

...

Keyword(s):

Binding Site ◽

Actin Binding ◽

Terminal Domain ◽

Actin Binding Site

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Analysis of the actin-binding domain of alpha-actinin by mutagenesis and demonstration that dystrophin contains a functionally homologous domain.

The Journal of Cell Biology ◽

10.1083/jcb.116.6.1369 ◽

1992 ◽

Vol 116 (6) ◽

pp. 1369-1380 ◽

Cited By ~ 120

Author(s):

L Hemmings ◽

P A Kuhlman ◽

D R Critchley

Keyword(s):

Smooth Muscle ◽

Binding Site ◽

Muscle Protein ◽

Actin Binding ◽

In Vitro Assays ◽

E Coli ◽

Cos Cells ◽

Terminal Domain ◽

Actin Binding Site

To define the actin-binding site within the NH2-terminal domain (residues 1-245) of chick smooth muscle alpha-actinin, we expressed a series of alpha-actinin deletion mutants in monkey Cos cells. Mutant alpha-actinins in which residues 2-19, 217-242, and 196-242 were deleted still retained the ability to target to actin filaments and filament ends, suggesting that the actin-binding site is located within residues 20-195. When a truncated alpha-actinin (residues 1-290) was expressed in Cos cells, the protein localized exclusively to filament ends. This activity was retained by a deletion mutant lacking residues 196-242, confirming that these are not essential for actin binding. The actin-binding site in alpha-actinin was further defined by expressing both wild-type and mutant actin-binding domains as fusion proteins in E. coli. Analysis of the ability of such proteins to bind to F-actin in vitro showed that the binding site was located between residues 108 and 189. Using both in vivo and in vitro assays, we have also shown that the sequence KTFT, which is conserved in several members of the alpha-actinin family of actin-binding proteins (residues 36-39 in the chick smooth muscle protein) is not essential for actin binding. Finally, we have established that the NH2-terminal domain of dystrophin is functionally as well as structurally homologous to that in alpha-actinin. Thus, a chimeric protein containing the NH2-terminal region of dystrophin (residues 1-233) fused to alpha-actinin residues 244-888 localized to actin-containing structures when expressed in Cos cells. Furthermore, an E. coli-expressed fusion protein containing dystrophin residues 1-233 was able to bind to F-actin in vitro.

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