KISHIDA Michiko Faculty of Medicine, Teaching Associate, 医学部, 教務員 (40274089)
ANDO Tomoko Faculty of Medicine, Research Associate, 医学部, 助手 (20294548)
KISHIDA Shosei Faculty of Medicine, Research Associate, 医学部, 助手 (50274064)
|Budget Amount *help
¥13,400,000 (Direct Cost : ¥13,400,000)
Fiscal Year 1998 : ¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1997 : ¥10,100,000 (Direct Cost : ¥10,100,000)
We analyzed the novel signaling pathway of Ras and its physiological functions in this project. Especially, the mode of activation of Ral by RaIGDS, a Ras effector protein, and the functions of the Ral signaling pathway were intensively examined.
(1)Mode of activation of Ral by Ras
So far, we had clarified that Ras bound to the plasma membrane through its lipid modification induces the translocation of RaIGDS from the cytosol to the plasma membrane. Extending these observations, we found that the lipid modifications of both Ras and Ral are necessary for Ras to activate Ral through RaIGDS.We generated the Ras/Rap1 chimera to determine the relationship between subcellular localization of Ras and the activation of Ral. Since Rap1 is localized to the perinuclear region through its lipid modification, Ras/Rap1 chimera did not activate Ral. These results indicate that once Ras is activated, RaIGDS is translocated to the plasma membrane where Ral is present, resulting in the activation of Ral.
(2) Downstream molecules of Ral and their functions
We identified POB1 as a novel protein that binds to RalBP1, a putative effector protein of Ral. POB1 bound to Grb2, was tyrosine phosphoiylated in response to EGF, and formed a complex with EGF receptor. Further, we identified Epsin as a binding protein of POB1. Since Both POB1 and Epsin have the common domains that are found in the proteins regulating endocytosis, we examined whether Ral, RalBP1, POB1, and Epsin are involved in the regulation of endocytosis. The mutants of these proteins inhibited the internalization of EGF and insulin. These results indicate that the Ral signaling pathway regulates ligand-dependent receptor-mediated endocytosis.