Structure-Function Analysis of the Tribbles Pseudokinase

Abstract

Members of the Tribbles (Trb) family of pseudokinases have roles in development, cell cycle control, insulin signaling, and the immune response and have been linked to many human diseases. The structure of Drosophila Tribbles includes a conserved kinase-like core that binds targets and an N-terminal region that contains a PEST domain, and a Carboxy-terminal tail that binds the E3 ligase, constitutive photomorphogenic I (COP1, Murphy et al., 2015; Jamieson et al., 2018). The bound COP1 ubiquitin-ligase ubiquitinates the target protein, leading to degradation of the target by the proteolytic machinery (Murphy. et al., 2015; Jamieson et al., 2018). The C-terminal tail of Tribbles is responsible for recruiting ubiquitination complexes that target proteins such as String (Stg) and Slow border cells (Slbo) for proteolytic degradation. While Drosophila Tribbles (Trbl) retains this conserved function, it lacks a C-terminal COP1 binding site found in most Trb family members that have been shown necessary for target protein destruction. To uncover the cryptic binding site, we misexpressed Tribbles and mutant variants in Drosophila tissue, including the wing and egg chamber to determine function. Multiple sequence alignments of closely and distantly related Tribbles proteins in arthropods determined that the divergence from a COP1 binding site in fly Tribble is a recent evolutionary event. Motif scans led to the discovery of a PEST domain in the C-terminal tail and site-directed mutagenesis of the PEST domain revealed its importance for Trbl function. Genetic interaction with the Cullin 3 proteome adaptor protein has shed light on a possible mechanism of Trbl tail function.