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

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 biogenesis site for Golgi. 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 in the procyclic form of T. brucei. TbPLK phosphorylates KIN-G in vitro at multiple residues, some of which are phosphorylated in vivo in T. brucei, 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, thereby impairing Golgi biogenesis, flagellum attachment zone elongation, flagellum positioning, and cell division plane placement. Therefore, TbPLK negatively regulates KIN-G activity by phosphorylating Thr301, and dephosphorylation of Thr301 is required for KIN-G to fulfill its cellular function in promoting centrin arm biogenesis.