Small GTPases are important signaling proteins that regulate a variety of cellular processes such as cell morphogenesis, division and migration. The function of these proteins is dependent on a GTP-GDP switch that modulates effector binding. Given the involvement of these enzymes in human disease, numerous efforts have been undertaken to directly modulate the activity and function of small GTPases. However, most current approaches are hampered by associated challenges in targeting small GTPases with small molecules as well as complex cellular feedback mechanisms that are not fully understood. The Stains lab has previously developed a split-protein platform to control effector binding of the small GTPase termed Cdc42 <i>in vitro</i>. Here we propose to expand this strategy to gate the activity of small GTPases in living cells. We demonstrate that split-Cdc42 constructs are able of activating downstream effectors in a tunable and temporally controlled fashion. In addition, due to the high degree of structural homology among small GTPases, we hypothesize that our fragmentation site could be readily applied across the entire superfamily. As a proof-of-concept, we have generated split-KRas constructs and demonstrated their ability to temporally control signaling in a dose-dependent manner. In the long term, we anticipate our methodology could allow for reprogramming cellular function and investigating the fundamental roles of small GTPases in cellular signaling.

Development of a general method to control the activity of small GTPases in living cells

ACS Spring 2022

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