Nucleocytoplasmic shuttling of the Duchenne muscular dystrophy gene product dystrophin Dp71d is dependent on the importin α/β and CRM1 nuclear transporters and microtubule motor dynein.
Even though the Duchenne muscular dystrophy (DMD) gene product Dystrophin Dp71d is involved in various key cellular processes through its role as a scaffold for structural and signalling proteins at the plasma membrane as well as the nuclear envelope, its subcellular trafficking is poorly understood. Here we map the nuclear import and export signals of Dp71d by truncation and point mutant analysis, showing for the first time that Dp71d shuttles between the nucleus and cytoplasm mediated by the conventional nuclear transporters, importin (IMP) α/β and the exportin CRM1. Binding was confirmed in cells using pull-downs, while in vitro binding assays showed direct, high affinity (apparent dissociation coefficient of c. 0.25nM) binding of Dp71d to IMPα/β. Interestingly, treatment of cells with the microtubule depolymerizing reagent nocodazole or the dynein inhibitor EHNA both decreased Dp71d nuclear localization, implying that Dp71d nuclear import may be facilitated by microtubules and the motor protein dynein. The role of Dp71d in the nucleus appears to relate in part to interaction with the nuclear envelope protein emerin, and maintenance of the integrity of the nuclear architecture. The clear implication is that Dp71d's previously unrecognised nuclear transport properties likely contribute to various, important physiological roles.
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico; Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México D.F, Mexico.
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Suárez-Sánchez
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico.
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Aguilar
Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
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Wagstaff
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico.
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Velez
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico.
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Azuara-Medina
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico.
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Gomez
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico.
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Vásquez-Limeta
Laboratorio de Medicina Genómica, Departamento de Genética, Instituto Nacional de Rehabilitación, México D.F, Mexico.
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O
Hernández-Hernández
Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia.
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Lieu
Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC, Australia. Electronic address: david.jans@monash.edu.
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Jans
Departamento de Genética y Biología Molecular, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), México D.F, Mexico. Electronic address: bcisnero@cinvestav.mx.
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Cisneros
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D017931:DNA Primers; D004398:Dyneins / Q000378:metabolism*; D016189:Dystrophin / Q000235:genetics*; D028884:Karyopherins / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics*; D021381:Protein Transport; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D028901:alpha Karyopherins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*; D000097606:Exportin 1 Protein
D017931:DNA Primers; D004398:Dyneins; D016189:Dystrophin; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D028901:alpha Karyopherins; D028961:beta Karyopherins; D000097606:Exportin 1 Protein; C085339:apo-dystrophin 1
Dynein; Dystrophin Dp71; Emerin; Exportin Crm1; Importins; ZZ domain