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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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Biochemical properties of l-afadin. (a) Inhibition by  myosin S1 of the binding of  l-afadin to 125I-labeled F-actin.  The Mono Q sample of l-afadin  (0.1 μg of protein) was subjected  to SDS-PAGE (8% polyacrylamide gel), followed by the blot  overlay with 125I-labeled F-actin  pretreated with myosin S1 in the  presence or absence of ATP. (b)  Increase by l-afadin of the viscosity of F-actin. The viscosity of  F-actin was measured by low  shear viscometry. •, With l-afadin; ○, with α-actinin. (c) Binding of His6–l-afadin-C to F-actin.  The binding curve was generated by the cosedimentation of  His6–l-afadin-C with F-actin.
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Figure 5: Biochemical properties of l-afadin. (a) Inhibition by myosin S1 of the binding of l-afadin to 125I-labeled F-actin. The Mono Q sample of l-afadin (0.1 μg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by the blot overlay with 125I-labeled F-actin pretreated with myosin S1 in the presence or absence of ATP. (b) Increase by l-afadin of the viscosity of F-actin. The viscosity of F-actin was measured by low shear viscometry. •, With l-afadin; ○, with α-actinin. (c) Binding of His6–l-afadin-C to F-actin. The binding curve was generated by the cosedimentation of His6–l-afadin-C with F-actin.

Mentions: It was examined by competition experiments whether the Mono Q sample of l-afadin (including s-afadin) bound along the sides of F-actin or at its ends. The binding of 125I-labeled F-actin to l-afadin was completely inhibited by an excessive amount of myosin S1, a well-characterized protein that binds along the sides of F-actin (Rayment et al., 1993; Schröder et al., 1993) (Fig. 5 a). This inhibition was reversed by the addition of MgATP because MgATP dissociates the actin–myosin complex (Fraser et al., 1975). These results indicate that l-afadin binds along the sides of F-actin. The effect of the Mono Q sample of l-afadin on F-actin was examined using the falling ball method for low shear viscometry. This effect was compared to that of α-actinin, a well-characterized protein that shows F-actin–cross-linking activity (Burridge and Feramisco, 1981). l-Afadin increased the viscosity of F-actin in a dose-dependent manner, and the viscosity became maximal at approximately threefold (Fig. 5 b). α-Actinin also increased the viscosity, but the viscosity became unmeasurably high. This effect of the Mono Q sample of l-afadin on F-actin was further assessed by transmission electron microscopy of negatively stained specimens. l-Afadin hardly caused F-actin to associate into bundles and meshworks under conditions where α-actinin showed a marked F-actin–cross-linking activity (data not shown). This result was consistent with that of the low shear viscometry. The stoichiometry of the binding of l-afadin to actin and the affinity constant were examined using His6– l-afadin-C. The stoichiometry was calculated to be 1 His6– l-afadin-C molecule per ∼500 actin molecules (Fig. 5 c). The kilodalton value was calculated to be the order of 10−7. A similar result was obtained with GST–l-afadin-C (data not shown). The effect of the Mono Q sample of l-afadin on actin polymerization was finally examined using pyrene-conjugated actin. Pyrene-conjugated actin is known to increase its fluorescent intensity with the actin polymerization (Cooper and Pollard, 1982). l-Afadin did not affect the actin polymerization under the conditions where gelsolin stimulated it (data not shown).


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)

Biochemical properties of l-afadin. (a) Inhibition by  myosin S1 of the binding of  l-afadin to 125I-labeled F-actin.  The Mono Q sample of l-afadin  (0.1 μg of protein) was subjected  to SDS-PAGE (8% polyacrylamide gel), followed by the blot  overlay with 125I-labeled F-actin  pretreated with myosin S1 in the  presence or absence of ATP. (b)  Increase by l-afadin of the viscosity of F-actin. The viscosity of  F-actin was measured by low  shear viscometry. •, With l-afadin; ○, with α-actinin. (c) Binding of His6–l-afadin-C to F-actin.  The binding curve was generated by the cosedimentation of  His6–l-afadin-C with F-actin.
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Related In: Results  -  Collection

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

Figure 5: Biochemical properties of l-afadin. (a) Inhibition by myosin S1 of the binding of l-afadin to 125I-labeled F-actin. The Mono Q sample of l-afadin (0.1 μg of protein) was subjected to SDS-PAGE (8% polyacrylamide gel), followed by the blot overlay with 125I-labeled F-actin pretreated with myosin S1 in the presence or absence of ATP. (b) Increase by l-afadin of the viscosity of F-actin. The viscosity of F-actin was measured by low shear viscometry. •, With l-afadin; ○, with α-actinin. (c) Binding of His6–l-afadin-C to F-actin. The binding curve was generated by the cosedimentation of His6–l-afadin-C with F-actin.
Mentions: It was examined by competition experiments whether the Mono Q sample of l-afadin (including s-afadin) bound along the sides of F-actin or at its ends. The binding of 125I-labeled F-actin to l-afadin was completely inhibited by an excessive amount of myosin S1, a well-characterized protein that binds along the sides of F-actin (Rayment et al., 1993; Schröder et al., 1993) (Fig. 5 a). This inhibition was reversed by the addition of MgATP because MgATP dissociates the actin–myosin complex (Fraser et al., 1975). These results indicate that l-afadin binds along the sides of F-actin. The effect of the Mono Q sample of l-afadin on F-actin was examined using the falling ball method for low shear viscometry. This effect was compared to that of α-actinin, a well-characterized protein that shows F-actin–cross-linking activity (Burridge and Feramisco, 1981). l-Afadin increased the viscosity of F-actin in a dose-dependent manner, and the viscosity became maximal at approximately threefold (Fig. 5 b). α-Actinin also increased the viscosity, but the viscosity became unmeasurably high. This effect of the Mono Q sample of l-afadin on F-actin was further assessed by transmission electron microscopy of negatively stained specimens. l-Afadin hardly caused F-actin to associate into bundles and meshworks under conditions where α-actinin showed a marked F-actin–cross-linking activity (data not shown). This result was consistent with that of the low shear viscometry. The stoichiometry of the binding of l-afadin to actin and the affinity constant were examined using His6– l-afadin-C. The stoichiometry was calculated to be 1 His6– l-afadin-C molecule per ∼500 actin molecules (Fig. 5 c). The kilodalton value was calculated to be the order of 10−7. A similar result was obtained with GST–l-afadin-C (data not shown). The effect of the Mono Q sample of l-afadin on actin polymerization was finally examined using pyrene-conjugated actin. Pyrene-conjugated actin is known to increase its fluorescent intensity with the actin polymerization (Cooper and Pollard, 1982). l-Afadin did not affect the actin polymerization under the conditions where gelsolin stimulated it (data not shown).

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