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SILKWORM 30 K PROTEIN INHIBITS ECDYSONE‐INDUCED APOPTOSIS BY BLOCKING THE BINDING OF ULTRASPIRACLE TO ECDYSONE RECEPTOR‐ B 1 IN CULTURED B m5 CELLS
Author(s) -
Kim Mi Young,
Song Hwa Young,
Kim Jin Hee,
Kim Bo Yong,
Park Sun Whan,
Sung Dong Kyung,
Park Hun Hee,
Jeon Soung Hoo,
Chung In Sik,
Lee Bong Hee
Publication year - 2012
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.21050
Subject(s) - transfection , apoptosis , microbiology and biotechnology , ecdysone receptor , receptor , chemistry , biology , cell culture , biochemistry , nuclear receptor , transcription factor , gene , genetics
This study investigates the mechanism through which increased 30K protein inhibits ecdysone‐induced apoptosis in the B m5 silkworm ovarian cell line. Treatment of Bm5 cells with 20‐hydroxyecdysone (20 E ) after transfection with the p IZT / V 5‐ H is control vector triggered apoptosis, but 20 E treatment did not trigger apoptosis in B m5 cells transfected with the p IZT /30 K / V 5‐ H is vector. To confirm its inhibitory effect on apoptosis, 30 K protein was first purified from Escherichia coli transformed with a 30 K expression vector and used to generate specific antibodies in mice. Anti‐30 K antiserum was used to confirm synthesis of the 30 K protein in p IZT /30 K / V 5‐ H is‐transfected B m5 cells and to detect 30 K protein binding to the ecdysone receptor‐ B 1 ( E c R ‐ B 1). Anti‐30 K antiserum was used to immunoprecipitate protein complexes containing 30 K from B m5 cells transfected with p IZT /30 K / V 5‐ H is vector and treated with 20 E . We observed that 30 K proteins bound primarily to the E c R ‐ B 1 and not to ultraspiracle ( USP ). Reciprocal immunoprecipitation of E c R ‐ B 1‐containing complexes from B m5 cells transfected with control p IZT / V 5‐ H is vector and treated with 20 E showed that E c R ‐ B 1 bound to USP in the absence of 30 K but did not bind to USP in p IZT /30 K / V 5‐ H is‐transfected B m5 cells. These results demonstrate that 30 K proteins block USP binding to E c R ‐ B 1 through formation of a 30 K / E c R ‐ B 1 complex, resulting in inhibition of 20 E ‐induced B m5 cell apoptosis.