2005 Fiscal Year Final Research Report Summary
Single-molecule analysis of the spatio-temporal properties of cell signaling processes by small GTPases.
| Project/Area Number |
15310092
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Nanomaterials/Nanobioscience
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| Research Institution | Osaka University |
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Principal Investigator |
SAKO Yasushi Osaka University, Graduate School of Frontier Biosciences, Associate Professor, 生命機能研究科, 助教授 (20215700)
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| Project Period (FY) |
2003 – 2005
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| Keywords | Ras / Single molecule detection / Raf / GFP / GTP / EGF |
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| Research Abstract |
A small GTPase H-Ras (Ras) is an important molecular switch controlling various intracellular signaling processes. Many extracellular stimulations, such as carried by epidermal growth factor (EGF), activate Ras by exchanging gunanine nucleotide bound to Ras from GDP to GTP. Ras in GTP binding form is recognized by cytoplasmic effecter proteins to transduce signals inside cells. However, details of interactions between Ras and its effectors are largely unknown because the interactions are weak and unstable. We have developed single-molecule imaging technique that allows kinetic analysis of molecular interactions even in living cells. In this research project, we have applied this technique to study kinetics of interaction between Ras and c-Raf1 (Raf) one of the effecters of Ras in living cells.
The results can be summarized as follows :
1)The interaction between Ras and Raf includes kinetic intermediate before dissociation.
2)Both two of the binding domain of Raf to Ras, RBD and CRD are required for the formation of the kinetic intermediate.
3)At the initial state of binding, both RBD and CRD of Raf bind to Ras. Thus, binding of CRD to Ras (after binding of RBD) is not the rate limiting step of the intermediate formation.
4)Phosphorylations of Raf in therine/threonine residues affect the reaction rate of both intermediate formation and dissociation steps.
5)Evident accumulations of Raf in the plasma membrane for more than 60 minutes are maintained as a dynamic equilibrium. Individual molecules of Raf are continuously circulating between the plasma membrane and the cytoplasm.
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[Journal Article] Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration.2004
Author(s)
Murai, T., Miyazaki, Y., Nshinakamura, H., Sugahara, K.N., Miyauchi, T., Sako, Y., Yanagida, T., Miyasaka, M.
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Journal Title
J.Biol.Chem. 279
Pages: 4541-4550
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Single- and multiple-molecule dynamics of the signaling from H-Ras to c-Raf1 visualized on the plasma membrane of living cells.2003
Author(s)
Hibino, K., Watanabe, T., Kozuka, J., Iwane, A.H., Okada, T., Kataoka, T., Yanagida, T., Sako, Y.
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Journal Title
Chem.Phys.Chem. 4
Pages: 748-753
Description
「研究成果報告書概要(欧文)」より
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