RRC ID 33216
Author Hibino K, Shibata T, Yanagida T, Sako Y.
Title Activation kinetics of RAF protein in the ternary complex of RAF, RAS-GTP, and kinase on the plasma membrane of living cells: single-molecule imaging analysis.
Journal J Biol Chem
Abstract RAS is an important cell signaling molecule, regulating the activities of various effector proteins, including the kinase c-RAF (RAF). Despite the critical function of RAS signaling, the activation kinetics have not been analyzed experimentally in living cells for any of the RAS effectors. Here, we analyzed the kinetics of RAF activation on the plasma membrane in living HeLa cells after stimulation with EGF to activate RAS. RAF is recruited by the active form of RAS (RAS-GTP) from the cytoplasm to the plasma membrane through two RAS-binding sites (the RAS-binding domain and the cysteine-rich domain (CRD)) and is activated by its phosphorylation by still undetermined kinases on the plasma membrane. Using single-molecule imaging, we measured the dissociation time courses of GFP-tagged molecules of wild type RAF and fragments or mutants of RAF containing one or two of the three functional domains (the RAS-binding domain, the CRD, and the catalytic domain) to determine their interaction with membrane components. Each molecule showed a unique dissociation time course, indicating that both its interaction with RAS-GTP and its phosphorylation by the kinases are rate-limiting steps in RAF activation. Based on our experimental results, we propose a kinetic model for the activation of RAF. The model suggests the importance of the interaction between RAS-GTP and CRD for the effective activation of RAF, which is triggered by rapid RAS-GTP-induced conformational changes in RAF and the subsequent presentation of RAF to the kinase. The model also suggests necessary properties of the kinases that activate RAF.
Volume 286(42)
Pages 36460-8
Published 2011-10-21
DOI 10.1074/jbc.M111.262675
PII M111.262675
PMID 21862573
PMC PMC3196144
MeSH Cell Membrane / enzymology* Enzyme Activation / physiology HeLa Cells Humans Kinetics Microscopy, Fluorescence Models, Biological* Multienzyme Complexes / genetics Multienzyme Complexes / metabolism* Mutation Proto-Oncogene Proteins c-raf / genetics Proto-Oncogene Proteins c-raf / metabolism* ras Proteins / genetics ras Proteins / metabolism*
IF 4.106
Times Cited 23
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material GL348 (RDB13255) GL207 (RDB13256) GL107 (RDB13257).