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Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y - J. Cell Biol. (1997)

Bottom Line: A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain.We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin).These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

View Article: PubMed Central - PubMed

Affiliation: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

ABSTRACT
A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

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Mono Q column chromatography. (a) Absorbance at  280 nm (A280). (b) 125I-labeled F-actin blot overlay. An aliquot (3  μl) of each fraction was subjected to 125I-labeled F-actin blot  overlay. (c) Protein staining with Coomassie brilliant blue. An aliquot (10 μl) of each fraction was subjected to SDS-PAGE (8%  polyacrylamide gel), followed by protein staining with Coomassie  brilliant blue.
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Figure 1: Mono Q column chromatography. (a) Absorbance at 280 nm (A280). (b) 125I-labeled F-actin blot overlay. An aliquot (3 μl) of each fraction was subjected to 125I-labeled F-actin blot overlay. (c) Protein staining with Coomassie brilliant blue. An aliquot (10 μl) of each fraction was subjected to SDS-PAGE (8% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue.

Mentions: All the purification procedures were carried out at 0–4°C. Fetal brains from 60 mother rats (17-d gestation) were homogenized with a solution containing 10 mM Tris/Cl, pH 8.0, 2 mM EDTA, 5 mM EGTA, and a protease inhibitor cocktail (1 mM PMSF, 20 μg/ml of leupeptin, and 1 μg/ml of pepstatin A). The homogenate was mildly stirred for 1 h and centrifuged at 200,000 g for 1 h. The supernatant (600 ml, 2,070 mg of protein) was stored at −80°C until use. The supernatant was applied to a Q-Sepharose FF column (2.6 × 34 cm; Pharmacia Biotech, Inc., Piscataway, NJ) equilibrated with buffer A (20 mM Tris/Cl, pH 8.0, 0.5 mM EDTA, and 1 mM DTT). After the column was washed with 700 ml of buffer A, elution was performed with an 800-ml linear gradient of NaCl (0–0.5 M) in buffer A. 10-ml fractions were collected. l-Afadin appeared in fractions 56–77. These fractions (220 ml, 500 mg of protein) were collected, and 1.2 M (NH4)2SO4 was added to give a final concentration of 0.6 M. The sample was centrifuged at 200,000 g for 20 min, and the supernatant was applied to a phenyl-5PW column (2.15 × 15 cm; Tosoh, Tokyo, Japan) equilibrated with buffer A containing 0.6 M (NH4)2SO4. After the column was washed with 250 ml of the same buffer, elution was performed with a 240-ml linear gradient of (NH4)2SO4 (0.6–0 M) in buffer A. 6-ml fractions were collected. l-Afadin appeared in fractions 28–34. The active fractions (42 ml, 24 mg of protein) were collected and applied to a hydroxyapatite column (0.78 × 10 cm; Koken, Tokyo, Japan) equilibrated with buffer B (10 mM potassium phosphate, pH 7.8, and 1 mM DTT). After the column was washed with 30 ml of buffer B, elution was performed with a 150-ml linear gradient of potassium phosphate (10–500 mM), pH 7.8, in buffer B. 2.0-ml fractions were collected. l-Afadin appeared in fractions 39–41. The active fractions (6 ml, 0.86 mg of protein) were collected and diluted with an equal volume of buffer A, and half of the sample was applied to a Mono Q HR 5/5 column (Pharmacia Biotech, Inc.) equilibrated with buffer A. After the column was washed with 6 ml of buffer A, elution was performed with a 30-ml linear gradient of NaCl (0.15–0.4 M) in buffer A. 0.5-ml fractions were collected. The other half of the sample was subjected to Mono Q column chromatography in a similar manner. l-Afadin appeared in fractions 34–36 (see Fig. 1). The active fractions of the two Mono Q column chromatographies (3.0 ml, 0.2 mg of protein) were combined and stored at −80°C until use.


Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction.

Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, Itoh M, Mizoguchi A, Aoki T, Fujimoto T, Matsuda Y, Tsukita S, Takai Y - J. Cell Biol. (1997)

Mono Q column chromatography. (a) Absorbance at  280 nm (A280). (b) 125I-labeled F-actin blot overlay. An aliquot (3  μl) of each fraction was subjected to 125I-labeled F-actin blot  overlay. (c) Protein staining with Coomassie brilliant blue. An aliquot (10 μl) of each fraction was subjected to SDS-PAGE (8%  polyacrylamide gel), followed by protein staining with Coomassie  brilliant blue.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139800&req=5

Figure 1: Mono Q column chromatography. (a) Absorbance at 280 nm (A280). (b) 125I-labeled F-actin blot overlay. An aliquot (3 μl) of each fraction was subjected to 125I-labeled F-actin blot overlay. (c) Protein staining with Coomassie brilliant blue. An aliquot (10 μl) of each fraction was subjected to SDS-PAGE (8% polyacrylamide gel), followed by protein staining with Coomassie brilliant blue.
Mentions: All the purification procedures were carried out at 0–4°C. Fetal brains from 60 mother rats (17-d gestation) were homogenized with a solution containing 10 mM Tris/Cl, pH 8.0, 2 mM EDTA, 5 mM EGTA, and a protease inhibitor cocktail (1 mM PMSF, 20 μg/ml of leupeptin, and 1 μg/ml of pepstatin A). The homogenate was mildly stirred for 1 h and centrifuged at 200,000 g for 1 h. The supernatant (600 ml, 2,070 mg of protein) was stored at −80°C until use. The supernatant was applied to a Q-Sepharose FF column (2.6 × 34 cm; Pharmacia Biotech, Inc., Piscataway, NJ) equilibrated with buffer A (20 mM Tris/Cl, pH 8.0, 0.5 mM EDTA, and 1 mM DTT). After the column was washed with 700 ml of buffer A, elution was performed with an 800-ml linear gradient of NaCl (0–0.5 M) in buffer A. 10-ml fractions were collected. l-Afadin appeared in fractions 56–77. These fractions (220 ml, 500 mg of protein) were collected, and 1.2 M (NH4)2SO4 was added to give a final concentration of 0.6 M. The sample was centrifuged at 200,000 g for 20 min, and the supernatant was applied to a phenyl-5PW column (2.15 × 15 cm; Tosoh, Tokyo, Japan) equilibrated with buffer A containing 0.6 M (NH4)2SO4. After the column was washed with 250 ml of the same buffer, elution was performed with a 240-ml linear gradient of (NH4)2SO4 (0.6–0 M) in buffer A. 6-ml fractions were collected. l-Afadin appeared in fractions 28–34. The active fractions (42 ml, 24 mg of protein) were collected and applied to a hydroxyapatite column (0.78 × 10 cm; Koken, Tokyo, Japan) equilibrated with buffer B (10 mM potassium phosphate, pH 7.8, and 1 mM DTT). After the column was washed with 30 ml of buffer B, elution was performed with a 150-ml linear gradient of potassium phosphate (10–500 mM), pH 7.8, in buffer B. 2.0-ml fractions were collected. l-Afadin appeared in fractions 39–41. The active fractions (6 ml, 0.86 mg of protein) were collected and diluted with an equal volume of buffer A, and half of the sample was applied to a Mono Q HR 5/5 column (Pharmacia Biotech, Inc.) equilibrated with buffer A. After the column was washed with 6 ml of buffer A, elution was performed with a 30-ml linear gradient of NaCl (0.15–0.4 M) in buffer A. 0.5-ml fractions were collected. The other half of the sample was subjected to Mono Q column chromatography in a similar manner. l-Afadin appeared in fractions 34–36 (see Fig. 1). The active fractions of the two Mono Q column chromatographies (3.0 ml, 0.2 mg of protein) were combined and stored at −80°C until use.

Bottom Line: A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain.We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin).These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

View Article: PubMed Central - PubMed

Affiliation: Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., Kobe 651-22, Japan.

ABSTRACT
A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.

Show MeSH
Related in: MedlinePlus