Polo-like kinase phosphorylation of the orphan kinesin KIN-G negatively regulates centrin arm biogenesis in Trypanosoma brucei

Abstract

The unicellular parasite Trypanosoma brucei assembles a motile flagellum that is required for locomotion, cell division plane placement, and cell-cell communication. Inheritance of the flagellum during the cell cycle relies on the faithful duplication/segregation of multiple flagellum-associated cytoskeletal structures, including a centrin-marked, bar-shaped structure termed centrin arm, which also determines the site for Golgi assembly. Biogenesis of the centrin arm requires the Polo-like kinase homolog TbPLK and the orphan kinesin KIN-G, but the mechanistic role of TbPLK in centrin arm biogenesis remains elusive. Here we report that TbPLK phosphorylates KIN-G, disrupts its microtubule-binding activity, and negatively regulates its function. TbPLK phosphorylates KIN-G in vitro at multiple residues, two of which are in vivo TbPLK phosphosites, including the Thr301 residue within one of the microtubule-binding motifs of the kinesin motor domain. Phosphorylation of Thr301 by TbPLK inhibits the microtubule-binding activity of KIN-G in vitro, and expression of a Thr301 phospho-mimic mutant in T. brucei disrupts centrin arm integrity, Golgi duplication, flagellum attachment zone elongation, flagellum positioning, and cell division plane placement. In wild-type T. brucei cells, Thr301 phosphorylation occurs on a small portion of the KIN-G population, suggesting that KIN-G undergoes phosphorylation/dephosphorylation cycles to regulate its activity. Together, these findings uncover a negative role of TbPLK-mediated phosphorylation of KIN-G in regulating centrin arm biogenesis in trypanosomes.