Polo-like kinases (Plks) play multiple roles in mitosis and cytokinesis in eukaryotes and are characterized by the C-terminal Polo-box domain (PBD), which is implicated in binding to Plk substrates, targeting Plk and regulating Plk activity. The Plk homolog in Trypanosoma brucei (TbPLK) possesses a similar architecture, but it lacks the crucial residues involved in substrate binding and regulates cytokinesis but not mitosis. Little is known about the regulation of TbPLK and the role of the PBD in TbPLK localization and function. Here, we addressed the requirement of the kinase activity and the PBD for TbPLK localization and function through coupling RNAi of endogenous TbPLK with ectopic expression of TbPLK mutants. We demonstrate that the kinase activity and phosphorylation of two threonine residues, Thr198 and Thr202, in the activation loop (T-loop) of the kinase domain are essential for TbPLK function but not for TbPLK localization. Deletion of the PBD abolishes TbPLK localization, but the PBD itself is not correctly targeted, indicating that TbPLK localization requires both the PBD and the kinase domain. Surprisingly, the kinase domain of TbPLK, but not the PBD, binds to its substrates TbCentrin2 and p110, suggesting that TbPLK might interact with its substrate through different mechanisms. Finally, the PBD interacts with the kinase domain of TbPLK and inhibits its activity, and this inhibition is relieved when Thr198 is phosphorylated. Together, these results suggest an essential role of T-loop phosphorylation in TbPLK activation and crucial roles of the PBD in regulating TbPLK activity and localization.

SummaryMicrotubules are a vital part of the cytoskeleton of eukaryotic cells and are involved in various cellular processes. The cytoskeleton of Trypanosoma brucei is characterized by an array of subpellicular microtubules and is essential for maintenance of cell shape and polarity, but little is known about the regulation of the assembly and organization of the subpellicular microtubule corset. Here, we report that the orphan kinesin TbKIN-D regulates the organization of subpellicular microtubules and is required for maintaining cell morphology. TbKIN-D possesses in vitro ATPase activity, associates with cytoskeletal microtubules and is distributed throughout the cytoskeleton at all cell cycle stages. RNAi of TbKIN-D disrupts the organization of the subpellicular microtubule corset and distorts cell morphology, resulting in round cells with an elongated posterior filled with newly assembled microtubules. Depletion of TbKIN-D also abolishes the segregation of organelles and cytoskeletal structures, suggesting that cellular morphogenesis is essential for proper organelle segregation. Moreover, TbKIN-D deficiency impairs the attachment of the new flagellum without compromising the formation of the flagellum attachment zone. Finally, we identified TbKIN-C, a kinetoplastid-specific kinesin known to regulate subpellicular microtubules and cell morphogenesis in T. brucei, as a partner of TbKIN-D. Further, we demonstrate that interaction between TbKIN-C and TbKIN-D requires the coiled-coil motifs in the C-termini of both proteins. Altogether, our results suggest that TbKIN-D cooperates with TbKIN-C to maintain cell morphology by regulating the organization of the subpellicular microtubule corset.

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Tobacco BY-2 suspension cells were used to study the chemical damage and its associated mechanisms caused by Cu2+. Treatment with 100 micromol/L Cu2+ generated a large amount of H2O2 and thiobarbituric acid-reactive substances (TBARS) in cells. Using phospholipase D (PLD) specific inhibitor (1-butanol) or phosphatidic acid (PA), we demonstrated that PLD plays an important role in the generation of H2O2 and TBARS. Semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme activity assays with wild type and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-overexpressing BY-2 cells revealed that PLD and PA are the key factors leading to NADPH oxidase activation, which is responsible for H2O2 and TBARS production induced by Cu2+. Moreover, the content of ascorbic acid (AsA), an effective antioxidant, was sharply reduced in BY-2 cells exposed to excessive Cu2+. Furthermore, a significant downregulation of the enzymes of AsA biosynthesis and the antioxidant system was found. This evidence suggests that excessive Cu2+-elevated reactive oxygen species (ROS) production is caused by upregulated PLD that elevates the activity of NADPH oxidase and its collapsed antioxidant systems that scavenges ROS.

The chromosomal passenger complex (CPC) in animals, consisting of Aurora B kinase and three evolutionarily conserved proteins, plays crucial roles in mitosis and cytokinesis. However, Trypanosoma brucei expresses an unusual CPC consisting of an Aurora-like kinase, TbAUK1, and two kinetoplastid-specific proteins, TbCPC1 and TbCPC2. Despite their essential functions, little is known about the regulation of TbAUK1 and the roles of TbCPC1 and TbCPC2. Here, we investigate the effect of post-translational modification on the activity and spatiotemporal control of TbAUK1, and demonstrate that phosphorylation of two conserved threonine residues in the activation loop of the kinase domain contributes to TbAUK1 activation and function. TbAUK1 is SUMOylated in vivo, and mutation of the SUMO-conjugation site compromises TbAUK1 function. Degradation of TbAUK1 requires two destruction boxes and is mediated by the anaphase-promoting complex/cyclosome (APC/C), whereas degradation of TbCPC1 and TbCPC2 is not dependent on the predicted destruction boxes and is APC/C-independent. Moreover, we determine the domains in CPC subunits that mediate the pairwise interactions, and show that disruption of the interaction impairs the localization of TbAUK1 and TbCPC2 but not TbCPC1. Our results demonstrate the requirement of post-translational modifications for TbAUK1 function and a crucial role of TbCPC1 in mediating TbAUK1 localization.