The structure of crystalline profilin–β-actin

Schutt, Clarence E.; Myslik, James C.; Rozycki, Michael D.; Goonesekere, Nalin C. W.; Lindberg, Uno

doi:10.1038/365810a0

Cited by 660 publications

(712 citation statements)

References 46 publications

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“…The three-dimensional structure of cytoplasmic (3-actin, in complex with profilin, was recently solved (Schutt et al, 1993). Although its primary sequence is generally similar to skeletal a-actin, cytoplasmic (3-actin displays several structural differences.…”

Section: Discussionmentioning

confidence: 99%

“…Major structural differences exist in their N-terminal region, and these differences are important because of the N-terminal role in binding myosin and other actinbinding proteins (Vandekerckhove, 1990). In addition, recent crystallographic studies reveal several differences in the subdomain structure of cytoplasmic (3-actin and a-skeletal actin (Schutt et al, 1993). Such structural comparisons might provide insights into the physiological significance of the isoforms caused by the small difference in primary sequences.…”

Section: Introductionmentioning

confidence: 99%

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Polarized distribution of actin isoforms in gastric parietal cells.

Yao

Chaponnier

Gabbiani

et al. 1995

MBoC

109

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The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic ,B-actin and -y-actin. Densitometry revealed a ratio for f3-actin/ y-actin that equaled 0.73 + 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the ,B-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of ,B-actin were also identified near the basolateral membrane. The y-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of ,B-actin, but not y-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of 13-actin/ y-actin in the immunoprecipitate (3/,y = 2.14 + 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that ,B-and 'y-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between 1B-actin and ezrin in gastric parietal cells.Finally, our results suggest that the ,B-and y-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion. INTRODUCTION essential role in a variety of cellular processes such as maintaining cell shape, cell motility, and cytokinesis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Polarized distribution of actin isoforms in gastric parietal cells.

Yao

Chaponnier

Gabbiani

et al. 1995

MBoC

109

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“…In mammals four profilin isoforms are expressed with slightly differing actin and poly-L-proline binding activities: the ubiquitous profilin 1, the neuronal profilin 2, and the testicular profilins 3 and 4. More than 50 different interaction partners of the pofilins have been identified that bind to its poly-L-proline binding site, which is spatially separated from its actin but may overlap with the PIP2 binding site [Schutt et al, 1993;Metzler et al, 1994; Chaudhary, 1998]. Many of the profilin binding proteins are involved in cell signaling, membrane trafficking, or synaptic scaffolding allowing the formation of a multitude of complexes that orchestrate different cellular activities and cytoskeletal rearrangements.…”

Section: Regulation Of Thymosin B4 Activitymentioning

confidence: 99%

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

Mannherz

Hannappel

2009

Cell Motil. Cytoskeleton

105

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The b-thymosins are N-terminally acetylated peptides of about 5 kDa molecular mass and composed of about 40-44 amino acid residues. The first member of the family, thymosin b4, was initially isolated from thymosin fraction 5, prepared in five steps from calf thymus. Thymosin b4 was supposed to be specifically produced and released by the thymic gland and to possess hormonal activities modulating the immune response. Various paracrine effects have indeed been reported for these peptides such as cardiac protection, angiogenesis, stimulation of wound healing, and hair growth. Besides these paracrine effects, it was noted that b-thymosins occur in high concentration in the cytoplasm of many eukaryotic cells and bind to the cytoskeletal component actin. Subsequently it became apparent from in vitro experiments that they preferentially bind to monomeric (G-)actin and stabilize it in its monomeric form. Due to this ability the b-thymosins are the main intracellular actin sequestering factor, i.e., they posses the ability to remove monomeric actin from the dynamic assembly and disassembly processes of the actin cytoskeleton that constantly occur in activated cells. In this review we will concentrate on the intracellular activity and localization of the b-thymosins, i.e., their modulating effect on the actin cytoskeleton. Cell Motil. Cytoskeleton 66: 839-851, 2009. '

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“…In the DNase I:actin and the tight-state profilin:actin crystals [2,4], the guanidinium group of R177 forms a salt cluster across the interdomain cleft involving the carboxyl group of D179 and the carbonyl oxygen of H73. Thus, the R177 can be perceived as a`clamp' over the nucleotide-binding cleft (Fig.…”

Section: Arginine 177 Is Important For Actin Stabilitymentioning

confidence: 99%

Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin

et al. 2000

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Actin ADP-ribosylated at arginine 177 is unable to hydrolyze ATP, and the R177 side chain is in a position similar to that of the catalytically essential lysine 71 in heat shock cognate protein Hsc70, another member of the actin-fold family of proteins. Therefore, actin residue R177 has been implicated in the mechanism of ATP hydrolysis. This paper compares wild-type b-actin with a mutant in which R177 has been replaced by aspartic acid. The mutant b-actin was expressed in Saccharomyces cerevisiae and purified by DNase I-affinity chromatography. The mutant protein exhibited a reduced thermal stability and an increased nucleotide exchange rate, suggesting a weakened interdomain connection. The ATPase activity of G-actin and the ATPase activity expressed during polymerization were unaffected by the R177D replacement, showing that this residue is not involved in catalysis. In the presence of polymerizing salts, ATP hydrolysis by both wild-type Mg-b-actin and the mutant protein preceded filament formation. With the mutant actin, the initial rate of ATP hydrolysis was as high as with wild-type actin, but polymer formation was slower, reached lower steady-state levels, and the polymers formed exhibited much lower viscosity. The critical concentration of polymerization (A cc ) of the mutant actin was increased 10-fold as compared to wild-type actin. Filaments formed from the R177D mutant b-actin bound phalloidin.

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The structure of crystalline profilin–β-actin

Cited by 660 publications

References 46 publications

Polarized distribution of actin isoforms in gastric parietal cells.

Polarized distribution of actin isoforms in gastric parietal cells.

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

Mutational analysis of arginine 177 in the nucleotide binding site of beta-actin

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