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Visualization of an interaction between Ras and Ras‐binding domain in living cells by bimolecular fluorescence complementation.
Author(s) -
Tsutsumi Kaori,
Tsuda Masumi,
Kawaguchi Hideaki,
Ohba Yusuke
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.645.19
Subject(s) - endosome , bimolecular fluorescence complementation , microbiology and biotechnology , golgi apparatus , chemistry , effector , vesicle , complementation , pi3k/akt/mtor pathway , epidermal growth factor , intracellular , signal transduction , biology , biochemistry , receptor , cell , membrane , mutant , gene
Recent accumulating evidences converge on the importance of spatiotemporal regulation to determine the specificity and diversity of intracellular signalings. Here, we constructed a system to analyze the protein‐protein interaction between Ras and its effectors in living cells by using bimolecular fluorescence complementation (BiFC) system, in which the dynamic interactions could be observed as an increase in fluorescent intensity. We used H‐Ras and Ras‐binding domains (RBDs) of Raf, PI3K, and RalGDS, and found that the binding of Ras to the effector molecules was specific in a manner dependent on subcellular localization. Ras and Raf bound predominantly at the plasma membrane followed by at Golgi apparatus by epidermal growth factor (EGF) stimulation, as reported previously. On the other hand, the interaction between H‐Ras and PI3K‐RBD upon EGF stimulation was observed at the endosome‐like vesicles, on which early endosome autoantigen 1 (EEA1), a marker for endosome, were localized. This result indicates that PI3K activated by Ras may play an important role in the production of phosphatidylinositol triphosphate (PIP3) at the endosome, wherein PIP3 is highly enriched. Since we used only RBD but not functional domains of Raf, PI3K and RalGDS in the present study, it will be thereby necessary to clarify the mechanism how RBDs dictate the selectivity of localization of H‐Ras‐PI3K binding.