Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A

Molecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA). By arranging the sequence for Kemptide in tandem, we designed a farnesylated peptide whose localization can dramatically change upon phosphorylation by PKA.

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Fig: Basic Design and characterization of the Synthetic Farnesyl-Electrostatic Switch.(A) Plasma membrane targeting of farnesylated proteins relies on the presence of positively charged residues near the C-terminus. The farnesyl-electrostatic switch relies on the regulation of this sequence through phosphorylation. Yellow cylinders indicate a fluorescent protein for visualization. (B) Design of a farnesyl-electrostatic switch for PKA activity (FES-PKA) that consists of positive charges, two phosphate acceptors, and a CAAX tail which undergoes multi-step modifications including farnesylation. (C) Time-profile shows the quantification of the fluorescent signal in response to PKA activation and reversal with PKA inhibition. Representative micrographs of cells are shown on top in the following states: pre-treatment, post-activation, and post-inhibition. Fluorescence intensity of FES-PKA at the endomembranes at a given time point (F) was divided by that of time 0 (F0). The resulting value (F/F0) was then normalized to that of time 0, and used as an indicator of relative redistribution of FES-PKA in (C) and (D). All data points represent an average signal intensity collected from 30 cells over 3 independent experiments (10 cells each), and error bars represent standard error of mean. Scale bar represents 10 µm. (D)Bar chart represents the signal change pre-treatment (at 5 min), post-treatment with FSK + IBMX (at 15 min), and washout with H89 (at 25 min), respectively. * represents statistical significance: p < 0.05.

After testing a different valence number of Kemptide as well as modulating the linker sequence connecting them, we identified an efficient peptide switch that exhibited dynamic translocation between plasma membranes and internal endomembranes in a PKA activity dependent manner. Due to the modular design and small size, our PKA switch can have versatile utility in future studies as a platform for visualizing and perturbing signal transduction pathways, as well as for performing synthetic operations in cells.

Kim, A.K., Wu, H.D. & Inoue, T. Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch. Sci Rep 11, 16421 (2021). https://doi.org/10.1038/s41598-021-95840-8

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