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

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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Transgelin-1 (SM22α) interacts with actin stress fibers and podosomes in smooth muscle cells without using its actin binding site

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2018.09.176 ◽

2018 ◽

Vol 505 (3) ◽

pp. 879-884 ◽

Cited By ~ 9

Author(s):

Tsubasa S. Matsui ◽

Akihiro Ishikawa ◽

Shinji Deguchi

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Binding Site ◽

Muscle Cells ◽

Stress Fibers ◽

Actin Binding ◽

Actin Binding Site ◽

Actin Stress Fibers

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In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2

Bioscience Reports ◽

10.1042/bsr20080079 ◽

2008 ◽

Vol 28 (4) ◽

pp. 195-203 ◽

Cited By ~ 11

Author(s):

Mechthild M. Schroeter ◽

Brent Beall ◽

Hans W. Heid ◽

Joseph M. Chalovich

Keyword(s):

Smooth Muscle ◽

Muscle Protein ◽

Actin Binding ◽

Dependent Manner ◽

In Vitro Characterization ◽

Purification Scheme ◽

Synaptopodin 2 ◽

Muscle Myosin

An analysis of the primary structure of the actin-binding protein fesselin revealed it to be the avian homologue of mammalian synaptopodin 2 [Schroeter, Beall, Heid, and Chalovich (2008) Biochem. Biophys. Res. Commun. 371, 582–586]. We isolated two synaptopodin 2 isoforms from rabbit stomach that corresponded to known types of human synaptopodin 2. The purification scheme used was that developed for avian fesselin. These synaptopodin 2 forms shared several key functions with fesselin. Both avian fesselin and mammalian synaptopodin 2 bound to Ca2+–calmodulin, α-actinin and smooth-muscle myosin. In addition, both proteins stimulated the polymerization of actin in a Ca2+–calmodulin-dependent manner. Synaptopodin 2 has never before been shown to polymerize actin in the absence of α-actinin, to polymerize actin in a Ca2+–calmodulin-dependent manner, or to bind to Ca2+–calmodulin or myosin. These properties are consistent with the proposed function of synaptopodin 2 in organizing the cytoskeleton.

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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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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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Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

Journal of Cell Science ◽

10.1242/jcs.105.3.765 ◽

1993 ◽

Vol 105 (3) ◽

pp. 765-775 ◽

Cited By ~ 2

Author(s):

E. Friederich ◽

T.E. Kreis ◽

D. Louvard

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Binding Site ◽

Actin Filaments ◽

Living Cells ◽

Cytochalasin D ◽

Actin Binding ◽

Fast Growing ◽

Actin Binding Site

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

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A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0956 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1971-1984 ◽

Cited By ~ 31

Author(s):

Michael G. Clark ◽

Joseph Teply ◽

Brian K. Haarer ◽

Susan C. Viggiano ◽

David Sept ◽

...

Keyword(s):

Binding Site ◽

Binding Sites ◽

Actin Filaments ◽

Random Mutagenesis ◽

Site Directed Mutagenesis ◽

Actin Binding ◽

Interacting Protein ◽

Actin Binding Site

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

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