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Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

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Predicted sequence and domain structure of bovine  p205 (supervillin). (A) The amino acid sequence starting with the  first methionine of the translated ORF includes all eight peptides  obtained from purified p205 (double underline). The NH2-terminal half contains four putative nuclear targeting signals (gray boxes)  the longest of which resembles the nucleoplasmin targeting signal  (single underline). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site (black). Amino  acid positions are indicated by numbers in the left margin. These  sequence data are available from GenBank/EMBL/DDBJ under  accession number AF025996. (B) Schematic representation of the  domain structure showing the NH2-terminal region with putative  nuclear targeting regions (gray boxes) juxtaposed with potential  protein kinase A phosphorylation sites (asterisks). The COOH-terminal domain (cross-hatched) shows extensive similarity to villin and gelsolin, with three regions of especially high homology  that correspond to potential F-actin–binding sites (black boxes).  The potential tyrosine phosphorylation site is indicated (·).
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Figure 12: Predicted sequence and domain structure of bovine p205 (supervillin). (A) The amino acid sequence starting with the first methionine of the translated ORF includes all eight peptides obtained from purified p205 (double underline). The NH2-terminal half contains four putative nuclear targeting signals (gray boxes) the longest of which resembles the nucleoplasmin targeting signal (single underline). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site (black). Amino acid positions are indicated by numbers in the left margin. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF025996. (B) Schematic representation of the domain structure showing the NH2-terminal region with putative nuclear targeting regions (gray boxes) juxtaposed with potential protein kinase A phosphorylation sites (asterisks). The COOH-terminal domain (cross-hatched) shows extensive similarity to villin and gelsolin, with three regions of especially high homology that correspond to potential F-actin–binding sites (black boxes). The potential tyrosine phosphorylation site is indicated (·).

Mentions: The consensus DNA sequence encoded a protein of 1,792 amino acids (Fig. 12 A) that is absent from current protein databases. The predicted mol wt of 200,626 (isoelectric point ∼6.44) (8) is in good agreement with that predicted for p205 by SDS-PAGE (Fig. 1). Relative to an “average” protein (20), p205 is high in arginine (7.1% vs. 4.7%) and glutamine (9.2% vs. 6.2%) and low in tyrosine (2.3% vs. 3.5%) and cysteine (1.3% vs. 2.8%), variations that are consistent with its relative insensitivity to staining by silver (Fig. 3 A) (62). The deduced amino acid sequence includes all eight peptides obtained from purified p205 (Figs. 3 B and 12 A, double underlines). Since antibodies against two of these peptides immunoprecipitate the F-actin–binding protein of ∼205,000 D from SDS-solubilized neutrophil plasma membranes (Fig. 4), the sequence in Fig. 12 A undoubtedly encodes p205.


Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily.

Pestonjamasp KN, Pope RK, Wulfkuhle JD, Luna EJ - J. Cell Biol. (1997)

Predicted sequence and domain structure of bovine  p205 (supervillin). (A) The amino acid sequence starting with the  first methionine of the translated ORF includes all eight peptides  obtained from purified p205 (double underline). The NH2-terminal half contains four putative nuclear targeting signals (gray boxes)  the longest of which resembles the nucleoplasmin targeting signal  (single underline). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site (black). Amino  acid positions are indicated by numbers in the left margin. These  sequence data are available from GenBank/EMBL/DDBJ under  accession number AF025996. (B) Schematic representation of the  domain structure showing the NH2-terminal region with putative  nuclear targeting regions (gray boxes) juxtaposed with potential  protein kinase A phosphorylation sites (asterisks). The COOH-terminal domain (cross-hatched) shows extensive similarity to villin and gelsolin, with three regions of especially high homology  that correspond to potential F-actin–binding sites (black boxes).  The potential tyrosine phosphorylation site is indicated (·).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140202&req=5

Figure 12: Predicted sequence and domain structure of bovine p205 (supervillin). (A) The amino acid sequence starting with the first methionine of the translated ORF includes all eight peptides obtained from purified p205 (double underline). The NH2-terminal half contains four putative nuclear targeting signals (gray boxes) the longest of which resembles the nucleoplasmin targeting signal (single underline). The COOH-terminal half of the molecule contains a potential tyrosine phosphorylation site (black). Amino acid positions are indicated by numbers in the left margin. These sequence data are available from GenBank/EMBL/DDBJ under accession number AF025996. (B) Schematic representation of the domain structure showing the NH2-terminal region with putative nuclear targeting regions (gray boxes) juxtaposed with potential protein kinase A phosphorylation sites (asterisks). The COOH-terminal domain (cross-hatched) shows extensive similarity to villin and gelsolin, with three regions of especially high homology that correspond to potential F-actin–binding sites (black boxes). The potential tyrosine phosphorylation site is indicated (·).
Mentions: The consensus DNA sequence encoded a protein of 1,792 amino acids (Fig. 12 A) that is absent from current protein databases. The predicted mol wt of 200,626 (isoelectric point ∼6.44) (8) is in good agreement with that predicted for p205 by SDS-PAGE (Fig. 1). Relative to an “average” protein (20), p205 is high in arginine (7.1% vs. 4.7%) and glutamine (9.2% vs. 6.2%) and low in tyrosine (2.3% vs. 3.5%) and cysteine (1.3% vs. 2.8%), variations that are consistent with its relative insensitivity to staining by silver (Fig. 3 A) (62). The deduced amino acid sequence includes all eight peptides obtained from purified p205 (Figs. 3 B and 12 A, double underlines). Since antibodies against two of these peptides immunoprecipitate the F-actin–binding protein of ∼205,000 D from SDS-solubilized neutrophil plasma membranes (Fig. 4), the sequence in Fig. 12 A undoubtedly encodes p205.

Bottom Line: The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin.The NH2 terminus is novel, but contains four potential nuclear targeting signals.Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

View Article: PubMed Central - PubMed

Affiliation: Worcester Foundation for Biomedical Research, University of Massachusetts Medical Center, Shrewsbury, Massachusetts 01545, USA.

ABSTRACT
Actin-binding membrane proteins are involved in both adhesive interactions and motile processes. We report here the purification and initial characterization of p205, a 205-kD protein from bovine neutrophil plasma membranes that binds to the sides of actin filaments in blot overlays. p205 is a tightly bound peripheral membrane protein that cosediments with endogenous actin in sucrose gradients and immunoprecipitates. Amino acid sequences were obtained from SDS-PAGE-purified p205 and used to generate antipeptide antibodies, immunolocalization data, and cDNA sequence information. The intracellular localization of p205 in MDBK cells is a function of cell density and adherence state. In subconfluent cells, p205 is found in punctate spots along the plasma membrane and in the cytoplasm and nucleus; in adherent cells, p205 concentrates with E-cadherin at sites of lateral cell-cell contact. Upon EGTA-mediated cell dissociation, p205 is internalized with E-cadherin and F-actin as a component of adherens junctions "rings." At later times, p205 is observed in cytoplasmic punctae. The high abundance of p205 in neutrophils and suspension-grown HeLa cells, which lack adherens junctions, further suggests that this protein may play multiple roles during cell growth, adhesion, and motility. Molecular cloning of p205 cDNA reveals a bipartite structure. The COOH terminus exhibits a striking similarity to villin and gelsolin, particularly in regions known to bind F-actin. The NH2 terminus is novel, but contains four potential nuclear targeting signals. Because p205 is now the largest known member of the villin/gelsolin superfamily, we propose the name, "supervillin." We suggest that supervillin may be involved in actin filament assembly at adherens junctions and that it may play additional roles in other cellular compartments.

Show MeSH
Related in: MedlinePlus