A hemidesmosomal transmembrane collagenous molecule, the 180-kDa bullous pemphigoid antigen (BPA II), is phosphorylated with 12-O-tetradecanoylphorbol-13-acetate in a human squamous cell carcinoma cell line (DJM-1).
We have previously shown that the 180-kD bullous pemphigoid antigen (BPAII), which is a transmembrane collagenous protein of hemidesmosomes, is distributed at adhesion sites on glass coverslips on the basal membrane forming a concentric ring, or arch pattern, in a human squamous cell carcinoma cell line (DJM-1), when studied by immunofluorescence microscopy using monoclonal antibodies to BPA II. This concentric ring/arch pattern of "footsteps" of BPA II has been shown to be collapsed in association with a transient activation of protein kinase C by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In the present study, therefore, the effects of TPA on the phosphorylation of BPA II was examined. DJM-1 cells, which were metabolically labelled with [32Pi], were lysed and the extracts were subjected to immunoprecipitation with anti-BPAII and anti-230 kDa bullous pemphigoid antigen (BPAI) monoclonal antibodies. The results showed that only BPA II, but not BPA I, was phosphorylated at serine residues before TPA treatment. After TPA treatment phosphorylation was prominently increased so as to generate a 190 kDa-phosphorylated peptide. This 190-kDa peptide was reacted with anti-BPA II monoclonal antibodies by immunoblotting, and it was not detected when cells were pretreated with a specific protein kinase C inhibitor (H7) before TPA treatment, suggesting that the 190 kDa peptide is phosphorylated BPAII with TPA. Prolonged treatment with TPA abolished both of 180- and 190-kDa BPA II from Triton X-100-soluble fractions. These findings suggest that the BPA II, but not BPA I, is a substrate of protein kinase C, and the generation of 190-kDa-phosphorylated BPA II has a key role in the TPA-induced collapse of the assembly of BPA II on the basal plasma membrane, probably, at hemidesmosomes.
Kitajima
Y
Y
Department of Dermatology, Gifu University School of Medicine, Gifu City, Japan.
Owada
M K
MK
Fujisawa
Y
Y
Seishima
M
M
Yaoita
H
H
Hirako
Y
Y
Owaribe
K
K
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D001324:Autoantigens / Q000378:metabolism*; D001345:Autoradiography; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell; D002352:Carrier Proteins; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D003094:Collagen; D003315:Cornea / Q000166:cytology; D003598:Cytoskeletal Proteins; D003896:Desmosomes / Q000737:chemistry* / Q000276:immunology; D000071821:Dystonin; D004847:Epithelial Cells; D004848:Epithelium / Q000276:immunology; D006801:Humans; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000276:immunology*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests; D012878:Skin Neoplasms; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D000097805:Collagen Type XVII
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001324:Autoantigens; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010761:Phosphorus Radioisotopes; D013755:Tetradecanoylphorbol Acetate; D000097805:Collagen Type XVII; C486268:DST protein, human