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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.

Abstract：

:We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+-switch from low (0.07 mM) to normal (1.87 mM) concentration in culture medium, which were also linked to activation of protein kinase C (PKC), lead to phosphorylation of 180 kDa-bullous pemphigoid antigen (BPAG) 2, but not 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 examined the effects of TPA and Ca2+-switch on intracellular localization of BPAG1 by immuno-blotting and immuno-fluorescence microscopy with monoclonal antibodies to the antigen after sub-cellular fractionation. In DJM-1 cells cultured in low Ca2+ medium, BPAG1 was detected as phosphate buffered saline-soluble (cytosolic), Triton X-100 soluble (roughly membrane-associated) and Triton X-100 insoluble (cytoskeleton-bound) forms, whereas in normal Ca2+-grown cells only as cytosolic and cytoskeleton-bound forms. In normal Ca2+-cultured cells, TPA (50 nM) caused a complete translocation of BPAG1 from cytosol to membrane fractions within 10 min, that was inhibited by pretreatment with H7 (a selective PKC inhibitor) at 40 microM. After 30 min and 4 h of TPA-treatment, BPAG1 was exclusively detected in cytoskeleton fractions. Morphologically, immuno-fluorescence microscopy showed that treatment caused a marked reduction of BPAG1 from the cytoplasm and generated a linear pattern at cell-cell contacts, suggesting translocation of BPAG1 from the cytosol to the plasma membrane. In contrast, the Ca2+-switch from low to normal caused a prominent increase of BPAG1, both in cytosolic and membrane-associated forms after 4 h, that was inhibited both with H7 and cycloheximide (an inhibitor of protein synthesis) at 70 microM, suggesting a role for PKC and BPAG1 synthesis in these Ca2+-induced effects. These results suggest that TPA and Ca2+-switch induced BPAG1 translocation to membrane fractions possibly mediated by PKC-activation. Furthermore, whereas TPA affects the redistribution of BPAG1 among their pools without inducing their synthesis, Ca2+-switch induces both membrane translocation and synthesis of BPAG1, suggesting involvement of signaling other than PKC pathways in control of BPAG1 synthesis.

journal_name

J Dermatol Sci

journal_title

Journal of dermatological science

authors

Matsuoka Y,Yamada T,Seishima M,Hirako Y,Owaribe K,Kitajima Y

doi

10.1016/s0923-1811(01)00137-2

subject

Has Abstract

pub_date

2001-11-01 00:00:00

pages

206-14

issue

eissn

0923-1811

issn

1873-569X

pii

S0923181101001372

journal_volume

pub_type

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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.