Abstract
Shoc2/SUR-8 positively regulates Ras/ERK MAP kinase signaling by serving as a scaffold for Ras and Raf. Here, we examined the role of Shoc2 in the spatio-temporal regulation of Ras by using a fluorescence resonance energy transfer (FRET)-based biosensor, together with computational modeling. In epidermal growth factor-stimulated HeLa cells, RNA-mediated Shoc2 knockdown reduced the phosphorylation of MEK and ERK with half-maximal inhibition, but not the activation of Ras. For the live monitoring of Ras binding to Raf, we utilized a FRET biosensor wherein Ras and the Ras-binding domain of Raf were connected tandemly and sandwiched with acceptor and donor fluorescent proteins for the FRET measurement. With this biosensor, we found that Shoc2 was required for the rapid interaction of Ras with Raf upon epidermal growth factor stimulation. To decipher the molecular mechanisms underlying the kinetics, we developed two computational models that might account for the action of Shoc2 in the Ras-ERK signaling. One of these models, the Shoc2 accelerator model, provided a reasonable explanation of the experimental observations. In this Shoc2 accelerator model, Shoc2 accelerated both the association and dissociation of Ras-Raf interaction. We propose that Shoc2 regulates the spatio-temporal patterns of the Ras-ERK signaling pathway primarily by accelerating the Ras-Raf interaction.
Highlights
In addition to the aforementioned components, scaffold proteins, which bind two or more of the components and thereby provide platforms for signaling, have been shown to regulate the Ras-ERK signaling cascade in many ways [10, 11]
It has been reported that Erbin, a member of the leucine-rich repeat and PDZ domain family, inhibits the interaction of Shoc2 with Ras and Raf and thereby inhibits the Ras/ERK MAP kinase cascade, suggesting that the binding of Ras and Raf to the Shoc2 scaffold protein is subject to regulation [18]
In the HeLa cells used in our experiments, Shoc2 knockdown was found to affect the level of phospho-MEK and phosphoERK upon stimulation, at a low concentration of
Summary
In addition to the aforementioned components, scaffold proteins, which bind two or more of the components and thereby provide platforms for signaling, have been shown to regulate the Ras-ERK signaling cascade in many ways [10, 11]. In activation of the MAP kinase signaling cascade as previously Shoc2-depleted cells the FRET/TFP ratio was increased slowly reported [33].
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