Concentration of Ras in HeLa cells
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| Value | 0.4 µM Range: Table - link µM |
|---|---|
| Organism | Mammalian tissue culture cell |
| Reference | Fujioka A, Terai K, Itoh RE, Aoki K, Nakamura T, Kuroda S, Nishida E, Matsuda M., Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes. J Biol Chem. 2006 Mar 31 281(13):8917-26PubMed ID16418172 |
| Method | FRET (Fluorescence Resonance Energy Transfer)probe. In FRET, complex formation between two molecules such as DNA-Protein or protein-protein is tracked by attaching a fluorescing molecules, a photon donor and a photon acceptor to each biomolecule of interest. When the donor and acceptor are dissociated the donor's fluorescence is mainly observed and when they are brought to close proximity (1-10 nm) the acceptor's fluorescence is observed. Similarly conformational changes of a protein can be observed when donor and acceptor molecules are attached to two sites on it through genetic engineering. Green Fluorescent Protein (GFP) variants, CFP (C=Cyan) and YFP (Y=Yellow) are commonly used as the donor and acceptor. Researchers aimed at collecting missing parameters of simulation kinetic models using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, they developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, they developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. |
| Comments | In silico (performed on computer) kinetic simulation models of the RAS/ERK MAPK pathway, while describing it very nicely, have produced astonishingly different parameters. The aim of this study was to collect and evaluate the parameters for the development of a kinetic simulation model for Ras/ERK MAPK cascades. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study. For Ras conc. in HeLa cell of 1.6 µM see table link. See BNID 103954 |
| Entered by | Cellina Cohen-Saidon |
| ID | 100853 |