Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes.
To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we 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, we 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. 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.
Fujioka
Aki
A
Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan.
Terai
Kenta
K
Itoh
Reina E
RE
Aoki
Kazuhiro
K
Nakamura
Takeshi
T
Kuroda
Shinya
S
Nishida
Eisuke
E
Matsuda
Michiyuki
M
D000818:Animals; D001693:Biological Transport, Active; D019556:COS Cells; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D002522:Chlorocebus aethiops; D002999:Clone Cells; D003198:Computer Simulation; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis* / Q000378:metabolism; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000378:metabolism; D008954:Models, Biological; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry*; D010766:Phosphorylation; D019908:Proto-Oncogene Proteins c-raf / Q000032:analysis* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018631:ras Proteins / Q000032:analysis* / Q000378:metabolism
D005456:Fluorescent Dyes; D015335:Molecular Probes; D034741:RNA, Small Interfering; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins