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

Abstract

Targeted protein turnover of the transcription factor C/EBP is enhanced by the Tribbles (Trib) pseudokinase family of adaptor proteins, which bind to a conserved degron motif in C/EBP substrate and recruit a cognate COP1 E3 ligase to ubiquitinate C/EBP for degradation. We identified a conserved degron in the Drosophila C/EBP ortholog Slbo and fused this motif to a fluorescent protein (GFP or RFP) under the control of a constitutive promoter. Transgenic flies expressing this fluorescent protein (FP)::degron transgene, which we call the Fluorescent Ubiquitination-based Trbl Turnover Indicator (or FUTTI) sensor, showed reduced levels of fluorescence in response to increased levels of Drosophila Tribbles (Trbl) and conversely, increased levels when Trbl activity was reduced. Mutations in the Trbl conserved pseudokinase substrate binding pocket and in a divergent PEST-like motif in the C-terminal tail region disrupted Trbl activity in the FUTTI assay. These data indicate that FUTTI will be a valuable tool to identify sites of Trbl activity in vivo and the critical features of the protein required for function.