title: Nuclear localization of the dystrophin-associated protein α-dystrobrevin through importin α2/β1 is critical for interaction with the nuclear lamina/maintenance of nuclear integrity.
abstract: Although α-dystrobrevin (DB) is assembled into the dystrophin-associated protein complex, which is central to cytoskeletal organization, it has also been found in the nucleus. Here we delineate the nuclear import pathway responsible for nuclear targeting of α-DB for the first time, together with the importance of nuclear α-DB in determining nuclear morphology. We map key residues of the nuclear localization signal of α-DB within the zinc finger domain (ZZ) using various truncated versions of the protein, and site-directed mutagenesis. Pulldown, immunoprecipitation, and AlphaScreen assays showed that the importin (IMP) α2/β1 heterodimer interacts with high affinity with the ZZ domain of α-DB. In vitro nuclear import assays using antibodies to specific importins, as well as in vivo studies using siRNA or a dominant negative importin construct, confirmed the key role of IMPα2/β1 in α-DB nuclear translocation. Knockdown of α-DB expression perturbed cell cycle progression in C2C12 myoblasts, with decreased accumulation of cells in S phase and, significantly, altered localization of lamins A/C, B1, and B2 with accompanying gross nuclear morphology defects. Because α-DB interacts specifically with lamin B1 in vivo and in vitro, nuclear α-DB would appear to play a key role in nuclear shape maintenance through association with the nuclear lamina.
authors: *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico.
authors: Areli
authors: A
authors: Aguilar
authors: *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico.
authors: Kylie M
authors: KM
authors: Wagstaff
authors: *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico.
authors: Rocío
authors: R
authors: Suárez-Sánchez
authors: *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico.
authors: Samuel
authors: S
authors: Zinker
authors: *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico bcisnero@cinvestav.mx david.jans@monash.edu.
authors: David A
authors: DA
authors: Jans
authors: *Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México City, Mexico; Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia; and Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México City, Mexico bcisnero@cinvestav.mx david.jans@monash.edu.
authors: Bulmaro
authors: B
authors: Cisneros
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation; D002478:Cells, Cultured; D016189:Dystrophin / Q000378:metabolism*; D049029:Dystrophin-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D034881:Nuclear Lamina / Q000378:metabolism*; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D028901:alpha Karyopherins; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D016189:Dystrophin; D049029:Dystrophin-Associated Proteins; C490631:Kpna2 protein, mouse; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D012333:RNA, Messenger; D028901:alpha Karyopherins; D028961:beta Karyopherins; C098885:dystrobrevin
keywords: lamin B1; nuclear envelope; nuclear import; nuclear morphology