An optogenetic approach to study how protein interaction domains shape signaling dynamics

Abstract

Cell signaling provides the link between inputs from a cell’s environment, and the appropriate output by intracellular components. These pathways are often rich in complexity, with various combinations of signaling modules as well as the dynamics of activation dictating specific cellular responses. Attempts to study these pathways using inputs such as growth factors renders analysis difficult, as there is a fan-like activation of various modules downstream of receptors capable of regulating each other’s activity. Optogenetic tools allow for the disentanglement of these pathways, as they are capable of directly stimulating a specific module with precise temporal control.1, 2 These tools have been used to characterize how specific dynamic patterns can encode information.1 A topic that remains poorly understood, however, is how the relative strength of interactions between components of a pathway impacts signaling. This paper investigates this topic by pairing optogenetic control of the Ras/ERK pathway with mutations in the DRS and FRS binding domains of ERK. We find that the mutations investigated lead to a variety of changes to signal transduction, with destruction of the CD domain of the DRS via removal of charge (D316N-D319N) leading to an absence of ERK nuclear translocation. We show that phosphorylation of ERK in each of these mutants occurs at a high level, suggesting that the differences in dynamics are not due simply to an inability to activate ERK. We investigated dimerization as a mechanism of providing a stable interaction with MEK that should be deficient in doing so (D319N and D316N-D319N), and found that inhibiting dimerization using DEL-22379 did not significantly affect the level of phosphorylation. However, dimerization inhibition was able to restore nuclear translocation in D316N-D319N, suggesting that the mutation leads to sequestering via dimerization. Future work needs to be performed to validate the effects of dimerization inhibition using dimerization defective mutants, investigate the impact of altering the phosphorylation-activated nuclear import site of ERK, and study mutations in other proteins within the Ras/ERK modules perhaps those implicated in cancer. This study provides a glimpse at how binding domains impact cell signaling, as well as a framework for studying mutations both within ERK and upstream components.