Transient translocation of hemidesmosomal bullous pemphigoid antigen 1 from cytosol to membrane fractions by 12-O-tetradecanoylphorbol-13-acetate treatment and Ca2+-switch in a human carcinoma cell line

We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca 2+-switch from low (0.07 mM) to normal (1.87 mM) concentration in culture m edium., which were also linked to activation of protein kinase C (PKC), lea d to phosphorylation of 180 kDa-bullous pemphigoid antigen (BPAG) 2, but no t of 230 kDa-BPAG1, and possibly to its disassembly from hemidesmosomes in a human squamous cell carcinoma cell line (DJM-1). In this study. we examin ed the effects of TPA and Ca2+-switch on intracellular localization of BPAG 1 by immuno-blotting and immuno-fluorescence microscopy with monoclonal ant ibodies to the antigen after sub-cellular fractionation. In DJM-1 cells cul tured in low Call medium, BPAG1 was detected as phosphate buffered saline-s oluble (cytosolic), Triton X-100 soluble (roughly membrane-associated) and Triton X-100 insoluble (cytoskeleton-bound) forms, whereas in normal Ca2+-g rown cells only as cytosolic and cytoskeleton-bound forms. In normal Ca2+-c ultured cells, TPA (50 nM) caused a complete translocation of BPAG1 from cy tosol to membrane fractions within 10 min, that was inhibited by pretreatme nt with H7 (a selective PKC inhibitor) at 40 muM. After 30 min and 4 h of T PA-treatment, BPAG I was exclusively detected in cytoskeleton fractions. Mo rphologically, immuno-fluorescence microscopy showed that treatment caused a marked reduction of BPAG1 from the cytoplasm and generated a linear patte rn at cell-cell contacts. suggesting translocation of BPAG I from the cytos ol to the plasma membrane. In contrast, the Ca2+-switch from low to normal caused a prominent increase of BPAG1. both in cytosolic and membrane-associ ated forms after 4 h. that was inhibited both with H7 and cycloheximide (an inhibitor of protein synthesis) at 70 muM. suggesting a role for PKC and B PAG1 synthesis in these Ca2+-induced effects. These results suggest that TP A and Ca2+-switch induced BPAG1 translocation to membrane fractions possibl y mediated by PKC-activation. Furthermore, whereas TPA affects the redistri bution of BPAG1 among their pools without inducing their synthesis, Ca2+-sw itch induces both membrane translocation and synthesis of BPAG1, suggesting involvement of signaling other than PKC pathways in control of BPAG1 synth esis. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.