Insulin stimulation induces dynamic interaction between 14-3-3 and the C-terminal tail domain of Kinesin-2 subunit KIF3B in vitro

Abstract

Heterotrimeric kinesin[ndash]2 mediates diverse intracellular transport processes, yet the mechanisms governing its cargo specificity and regulation remain poorly understood. In this study, we report the regulation of interaction between the kinesin[ndash]2 subunit KIF3B and a candidate adaptor protein, 14[ndash]3[ndash]3[epsilon];. Using splitAPEX2 proximity labelling and fluorescence lifetime imaging microscopy (FLIM) assays in HEK293T cells, we demonstrate that 14[ndash]3[ndash]3[epsilon]; interacts with KIF3B within discrete subcellular compartments. This interaction is significantly enhanced upon insulin stimulation, indicating regulation by signalling pathways. Time resolved FLIM analysis further reveals that the site of interaction is dynamic, suggesting spatial and temporal control of motora[ndash]daptor association. Finally, the interactions are mapped to two serine residues in the C[ndash]terminal tail region of KIF3B that are highly conserved across species. Together, these findings uncover a conditional and reversible interaction between KIF3B and 14[ndash]3[ndash]3[epsilon], providing insight into how kinesin[ndash]2 may achieve context specific cargo engagement and transport within cells.