| Co-Investigator(Kenkyū-buntansha) |
KISHIDA Michiko Faculty of Medicine, HIROSHIMA UNIVERSITY, Teaching Associate, 医学部, 教務員 (40274089)
ANDO Tomoko Faculty of Medicine, HIROSHIMA UNIVERSITY, Research Associate, 医学部, 助手 (20294548)
KISHIDA Shosei Faculty of Medicine, HIROSHIMA UNIVERSITY, Research Associate, 医学部, 助手 (50274064)
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| Research Abstract |
In this study, we tried to clarify the functions of Rat signaling pathway which acts downstream of Ras.
(1) Identification of downstream molecules of Ral and their characterization. We identified POB1 as a novel RalBP1-binding protein. PCB1 bound to Grb2, was tyrosine-phosphorylated, and formed a complex with EGF receptor in response to EGF.The N-terminal region of POB1 has an EH domain. We purified Epsin and Eps15 as EH domain binding proteins from bovine brain. Both proteins formed a complex with AP-2 and clathrin, and were known to be important for the regulation of receptor-mediated endocytosis.
(2) Functional analyses. Ral, RalBP1, and POB1 regulated the endocytosis of the receptors for insulin and EGF but not for transferrin, while Epsin was involved in the receptor-mediated endocytosis of these three agonists. Therefore, the signaling complex of Ral/RalBP1/POB1 transmitted the signal from a ligand such as insulin and EGF to Eps 15 and Epsin, thereby induces the ligand-dependent endocytosis.
(3) Phosphorylation of RalBP1, POB1, Epsin, and Eps15 in mitotic phase. The regulation of assembly of the complex of these proteins was examined. RalBP1, POB1, Epsin, and Eps15 formed a complex with a-adaptin of AP-2 in CHO cells, but the formation was reduced in mitotic phase. All of RalBP1, POB1, Epsin, and Eps15 were phosphorylated in mitotic phase. POB1 and Epsin were phosphorylated by p34cdc2 kinase in vitro. Their phosphorylation sites (411S of POB1 and 357S of Epsin) were determined. Phosphorylated Epsin and EpsinS357D formed a complex with a-adaptin less efficiently than wild-type Epsin. Although the EH domain of POB1 bound directly to Epsin, phosphorylation of Epsin inhibited the binding. Furthermore, EpsinS357D but not EpsinS357A lost the effect of Epsin on the insulin-dependent endocytosis. These results suggest that phosphorylation of Epsin in mitotic phase inhibits receptor-mediated endocytosis by disassembly of its complex with POB1 and α-adaptin.
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