Dynamics of the Ras/ERK MAPK Cascade as Monitored by Fluorescent Probes

Abstract

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 Ras⅐Raf, Raf⅐MEK, and MEK⅐ERK 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.The Ras/ERK 2 MAPK cascade has been highly conserved throughout evolution and plays a pivotal role in many aspects of cellular events, including proliferation, differentiation, and survival (reviewed in Refs. 1-5). The principal components of this Ras/ERK MAPK cascade include H-Ras, N-Ras, K-Ras, c-Raf, B-Raf, A-Raf, MEK1, MEK2, ERK1, and ERK2. In addition to these, a number of proteins regulate this signaling pathway either positively or negatively: A few examples include phosphatases and scaffold proteins that have been shown to play critical roles in the spatiotemporal regulation of ERK MAPK (reviewed in Refs. 6 -9). Furthermore, there are many positive and negative feedback loops that modulate the activity of each signaling component, rendering this signal transduction cascade ever more complicated.To comprehend the Ras/ERK MAPK cascade, many research groups have attempted to reconstruct this cascade in silico (10 -17). These systems analyses are used to propose mechanisms to explain the ultrasensitivity of ERK to the input signal (18), the stable response of the Ras/ERK signaling cascade to a wide range of epidermal growth factor (EGF) concentrations (15), and the different responses of PC12 pheochromocytoma cells upon stimulation by EGF or nerve growth factor (17). Each kinetic simulation model reported previously recapitulates the stimulus-induced ERK activation very nicely. Nevertheless, the parameters used therein are sometimes astonishingly different from each other. One apparent reason for this discrepancy is that many studies set parameters to fit experimental data using different algorithms (11-13). Another reason...

ERK Nuclear Translocation Is Dimerization-independent but Controlled by the Rate of Phosphorylation

“…We have not detected any differences in aggregation state between ERK1-WT and ERK1-⌬4, using both FCS and emFRET measurements in live cells. Our experimental conditions were optimal because all ERK1 molecules expressed were tagged with GFP, and the concentration of GFP-ERKs was in the recently determined range of endogenous ERK expression (39). Our results demonstrate that ERK1-⌬4 activation is less efficient than that of WT ERK, possibly due to abnormal formation of the scaffold complex (24,25).…”

“…when the nucleus and cytoplasm displayed equal intensity in the resting condition. In this case, the GFP-ERK1 concentration was 0.82 Ϯ 0.4 M in the cytoplasm and 0.77 Ϯ 0.2 M in the nucleus, values still within endogenous expression levels (39).…”

“…7B). Furthermore, it has been demonstrated that at peak stimulation only 5% of MEK molecules are active, whereas up to 60% of ERK molecules are active (39). Hence, rapidly inactivated ERK molecules are trapped instantly by the large pool of inactive MEK inside the cytoplasm, and inefficient activation of ERK1-⌬4 would then retard further release from MEK.…”

Multiple Decisive Phosphorylation Sites for the Negative Feedback Regulation of SOS1 via ERK*

“…Each kinetic simulation model reported previously recapitulates the stimulus-induced ERK activation fairly well. Nevertheless, the parameters used therein are sometimes astonishingly different from each other (14). One apparent reason for this discrepancy is that these studies often use different algorithms to fit the parameters to the experimental data (15)(16)(17).…”

“…To collect and evaluate the parameters in living cells, we previously adopted fluorescent protein technologies and developed a kinetic simulation model based on the experimentally validated parameters (14). This simulation model contained only four signaling molecules, Ras, Raf, MEK, and ERK, but still successfully reproduced the essential features of the Ras-ERK MAPK pathway and demonstrated the usefulness of the parameters collected in living cells.…”