Cytolinker cross-talk: periplakin N-terminus interacts with plectin to regulate keratin organisation and epithelial migration.
Periplakin is a cytoskeletal linker protein that participates in the assembly of epidermal cell cornified envelope and regulates keratin organisation in simple epithelial cells. We have generated a stably transfected MCF-7 subclone expressing HA-tagged periplakin N-terminus to identify molecular interactions of periplakin. Co-immunoprecipitation with anti-HA antibodies and mass spectrometry identified a 500-kDa periplakin-interacting protein as plectin, another plakin family member. Plectin-periplakin interaction was confirmed by immunoblotting of complexes immunoprecipitated by either anti-HA or anti-plectin antibodies. Transient transfections of periplakin deletion constructs indicated that first 133 amino acid residues of the N-terminus are sufficient for co-localisation with plectin at MCF-7 cell borders. Immunofluorescence analysis demonstrated that periplakin and plectin isoforms 1, 1f and 1k co-localise at cell borders of MCF-7 epithelia and that plectin-1f and 1k co-localise with periplakin in suprabasal epidermis. Ablation of plectin by siRNA in HaCaT keratinocytes resulted in aggregation of periplakin to small clusters. Scratch-wounded MCF-7 epithelia expressing periplakin N-terminus showed accelerated keratin re-organisation that was inhibited by siRNA knock-down of plectin. Finally, ablation of either periplakin or plectin, or both proteins simultaneously, impaired migration of MCF-7 epithelial sheets. Thus, we have identified a novel functional co-localisation between two plakin cytolinker proteins.
Centre for Stem Cell Research and Regenerative Medicine, School of Biological and Biomedical Sciences, University of Durham, DH1 3LE, Durham, UK.
Veronika
V
Boczonadi
Lorna
L
McInroy
Arto
A
Määttä
D045744:Cell Line, Tumor; D002465:Cell Movement; D002999:Clone Cells; D003599:Cytoskeleton / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D007633:Keratins / Q000378:metabolism*; D051179:Plakins / Q000737:chemistry* / Q000378:metabolism*; D051190:Plectin / Q000378:metabolism*; D011485:Protein Binding; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
C491674:PPL protein, human; D051179:Plakins; D051190:Plectin; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D007633:Keratins