Bpag1 localization to actin filaments and to the nucleus is regulated by its N-terminus.
Plakins are a family of giant cytoskeleton binding proteins. One member of this group is bullous pemphigoid antigen 1 (Bpag1)/dystonin, which has neuronal and muscle isoforms that consist of actin-binding and microtubule-binding domains at either end separated by a plakin domain and several spectrin repeats. The better-characterized epithelial isoform has only the plakin domain in common with the neuronal and muscle isoforms. Here, we have analyzed the localization of muscle/neuronal (Bpag1a/b) isoforms and the epithelial (Bpag1e) isoform within C2C12 myoblast cells. Although an antibody specific to Bpag1a/b isoform 2 detected protein co-aligning actin stress fibers, this same antibody and two Bpag1e antibodies predominantly detected protein in the nuclei. A Bpag1a/b isoform 2 N-terminal fusion protein containing the plakin domain also localized to actin stress fibers and to nuclei. Within the plakin domain, we characterized a functional nuclear localization signal, which was responsible for localization of the fusion protein to the nucleus. Bpag1a/b isoform 1 N-terminal fusion proteins differed in their interaction with the actin cytoskeleton and with their ability to localize to the nucleus, suggesting that Bpag1 isoforms with different N-termini have differing roles. These results show the importance of N-terminal domains in dictating the localization and function of Bpag1 isoforms. We provide the first indication that Bpag1 is not strictly a cytoplasmic/membrane protein but that it can also localize to the nucleus.
Ottawa Health Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6, and Center for Neuromuscular Disease, and Departments of Cellular and Molecular Medicine, and Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5.
Kevin G
KG
Young
Madeline
M
Pool
Rashmi
R
Kothary
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens / Q000378:metabolism*; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003094:Collagen / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D000071821:Dystonin; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D032446:Myoblasts / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D024041:Non-Fibrillar Collagens; D019913:Nuclear Localization Signals / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022502:Stress Fibers / Q000378:metabolism; D000097805:Collagen Type XVII
D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000097805:Collagen Type XVII; C095667:Dst protein, mouse