Abstract
Herein, we report the development of a photocleavable analog of AP20187, a cell-permeable molecule used to dimerize FK506-binding protein (FKBP) fusion proteins and initiate biological signaling cascades and gene expression or disrupt protein-protein interactions. We demonstrate that this reagent permits the unique ability to rapidly and specifically antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g. endosome dispersion) and to release the trap upon photocleavage to follow the cell's return to homeostasis. In addition, this photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways, offering the ability to correlate the duration of a signaling event and a cellular response.
Highlights
The ability to rapidly turn on and off an acute antagonist is helpful to understand the initiation of a cellular program
We used native PAGE to follow the formation of the LC8 and FK506binding protein (FKBP)-LC8 molecular trap (LC8TRAP) complex induced by the addition of PhAP [6]
We have developed a photocleavable analog of AP, demonstrated that PhAP can induce formation of the LC8LC8TRAP complex, and shown that photocleavage of PhAP within this molecular trap leads to dissociation and rapid reversal of endosome dispersion
Summary
The ability to rapidly turn on and off an acute antagonist is helpful to understand the initiation of a cellular program. We report the development of a photocleavable analog of AP20187, a cell-permeable molecule used to dimerize FK506binding protein (FKBP) fusion proteins and initiate biological signaling cascades and gene expression or disrupt protein-protein interactions We demonstrate that this reagent permits the unique ability to rapidly and antagonize a molecular interaction in vitro and follow a biological process due to this acute antagonism (e.g. endosome dispersion) and to release the trap upon photocleavage to follow the cell’s return to homeostasis. This photocleavable AP20187 analog can be used in other systems where the dimerization of FKBP has been used to initiate signaling pathways, offering the ability to correlate the duration of a signaling event and a cellular response.
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