| Co-Investigator(Kenkyū-buntansha) |
MUTO Yoshinori Gifu University, School of Medicine, Professor, 医学部, 教授 (80190859)
YOSHIOKA Takashi Gifu University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (20311699)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
We previously reported the interaction of the RING-finger protein, RNF8, a binding partner of UBE2E2 (a ubiquitin-conjugating enzyme), with retinoid X receptor (RXR). Using the deletion mutants of RNF8 and RXR, the important domains in these proteins were examined by two-hybrid assay or in vitro pull-down assay. Wild-type RNF8 were distributed in nucleus, whereas an N-terminal deletion mutant (deltaN) and the RING-disrupted mutant (dominant negative) of RNF8 were observed in cytosole. Although RNF8 did not seem to act as an E3 for RXR ubiquitylation, it enhanced RXR transcriptional activity independently of a selective ligand, 9-cis retinoic acid. This transactivation effects were abolished with deltaN and dominant negative mutants of RNF8.
Furthermore, in collaboration with Dr.Timothy Thomson's group in Spain, following observations were made. Two-hybrid screening using UBC13 as a bait, we cloned the variant E2, UEV, and several RING-finger proteins including RNF8, KIAA00675, CHFR, KF1, and ZNRF2. UEV is known to form multi-ubiquitin chains via K63 but not ordinary K48. Autoubiquitylation of RNF8 was examined using wild-type ubiquitin (UbWT), and 3 types of K to R mutants of ubiquitin (UbK48,63R; UbK29,63R; UbK29,48R). In vitro ubiquitylation experiments in combination with the expressions of dominant negative UBC13 mutant (UBC13-C87A) and UbK29,48R, revealed ubiquitylation of RNF8 via K63 but not K48.
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