Fluorescent Ubiquitination-based Trbl Turnover Indicator (FUTTI): A tool to detect activity of the Tribbles pseudokinase in vivo

The Fluorescent Ubiquitination-based Trbl Turnover Indicator (FUTTI) is a novel tool designed to detect the activity of the Tribbles pseudokinase in living organisms. Tribbles proteins, which are known to target proteins for degradation, bind to a specific degron motif in transcription factor C/EBP. This leads to the recruitment of a ubiquitin ligase and the subsequent degradation of C/EBP.

To exploit this mechanism, researchers fused the degron motif to a fluorescent protein (either GFP or RFP) under the control of a constitutive promoter. The resulting transgenic flies, known as the FUTTI sensor, exhibited reduced fluorescence levels when Drosophila Tribbles (Trbl) levels were high, and increased fluorescence when Trbl activity was reduced.

Conversely, mutations in the Trbl conserved pseudokinase substrate binding pocket, as well as in a divergent PEST-like motif in the C-terminal tail region, were found to disrupt Trbl activity in the FUTTI assay. These findings suggest that FUTTI can be a valuable tool for identifying sites of Trbl activity in living organisms and for determining the essential features of the protein necessary for its function.