Structure-function relationship of the Polo-like kinase in Trypanosoma brucei

Abstract

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.

Fig. 1.

The kinase activity is essential for TbPLK function but not for localization. (A) In vitro kinase assay of wild-type TbPLK and the K70R mutant. GW843286, an inhibitor of human PLK1, was used to inhibit TbPLK. (B) Localization of TbPLK-3HA and TbPLK-K70R-3HA. Arrows point to wild-type TbPLK and K70R mutant fluorescence. (C,D) RNAi of TbPLK by targeting the 3′-UTR of TbPLK, and overexpression (OE) of wild-type TbPLK (C) or the TbPLK-K70R mutant (D) in the TbPLK-UTR RNAi cell line or the 29-13 cell line. Growth curves (left) are shown with western blot (top right) detecting overexpressed TbPLK-3HA or TbPLK-K70R-3HA and RT-PCR (bottom right) monitoring endogenous TbPLK mRNA level in the RNAi/OE double transfectant. (E) Quantitation of cells with different numbers of nuclei (N) and kinetoplasts (K) in TbPLK OE cell line and TbPLK-UTR RNAi/TbPLK-K70R OE cell line upon induction for 3 days. Data are presented as the mean percent ± s.d. of ~200 cells counted from three independent experiments. (F) Effect on basal body duplication/segregation in control and TbPLK-UTR RNAi/TbPLK-K70R OE cell lines. (G) Effect on Golgi duplication/segregation in control and TbPLK-UTR RNAi/TbPLK-K70R OE cell lines. BB, basal body; N, nucleus; K, kinetoplast; G, Golgi.

Fig. 2.

Phosphorylation in the T-loop is essential for TbPLK function but not localization. (A) Alignment of the T-loop of Plks from T. brucei, human and Xenopus. The two threonine residues, Thr198 and Thr202, in TbPLK were each mutated to alanine and aspartic acid. (B) In vitro kinase assay of wild-type TbPLK and T198A, T198D, T202A and T202D mutants using TbCentrin2 as the substrate. (C) Localization of wild-type TbPLK and T198A/D and T202A/D mutants. Arrows indicate where the wild-type and mutant TbPLK proteins are localized. (D) Overexpression of T198A, T198D, T202A or T202D mutants of TbPLK in the TbPLK-UTR RNAi cell line or in the 29-13 cell line. Growth curves (left) are shown with western blot (top right) detecting overexpressed 3HA-tagged TbPLK mutants and RT-PCR (bottom right) monitoring the endogenous TbPLK mRNA level. (E) Effect on Golgi duplication/segregation in control, TbPLK-UTR RNAi/TbPLK-T198A OE, and TbPLK-UTR RNAi/TbPLK-T202A OE cell lines. G, Golgi; N, nucleus; K, kinetoplast.

Fig. 3.

Localization and function of TbPLK bearing various mutations in the PBD. (A) In vitro kinase assay of wild-type TbPLK and W557F, F561A, H710A and K712A mutants using TbCentrin2 as the substrate. (B) Localization of W557F, F561A, H710A and K712 mutants. Arrows indicate where TbPLK mutants are localized. (C) Overexpression of W557F, F561A, H710A or K712A mutants in the TbPLK-UTR RNAi cell line or the 29-13 cell line. Growth curves (left) are shown with western blot (top right) detecting overexpressed 3HA-tagged TbPLK mutants and RT-PCR (bottom right) monitoring endogenous TbPLK mRNA levels.

Fig. 4.

Localization of TbPLK in HeLa cells and of human PLK1 in trypanosome cells. (A) Localization of EYFP-tagged trypanosome TbPLK in HeLa cells. (B) Localization of 3HA-tagged human PLK1 (hPlk1) in trypanosomes. N, nucleus; K, kinetoplast.

Fig. 5.

The PBD is required but is not sufficient for TbPLK targeting. (A) The TbPLK truncation mutants overexpressed in trypanosomes. The putative nuclear localization signal (NLS) sequence is shown with the basic residues highlighted in red. (B) Levels of overexpressed truncation mutants detected by western blot with anti-HA antibody. (C) Effect of overexpression of the truncation mutants on cell growth. (D) Localization of the truncation mutants in trypanosomes. The arrow points to the anterior tip of the new flagellum attachment zone (FAZ) where TbPLK is localized, and the arrowhead indicates the anterior tip of the cell where overexpressed TbPLK was also localized.

Fig. 6.

Interaction of the KD of TbPLK with its substrates. (A) In vitro GST pull-down to test interactions between TbCentrin2 and TbPLK, TbPLK-K70R, the KD and the PBD. (B) In vitro GST pull-down to test interactions between p110 and the KD and the PBD of TbPLK. (C) In vitro GST pull-down to test the interactions between the KD and PBD of TbPLK and phosphorylated and dephosphorylated TbCentrin2. Trypanosome cells expressing TbCentrin2-3HA were lysed and incubated with or without λPPase prior to pull-down assays. The bait proteins were stained with Coomassie Brilliant Blue (CBB) as presented beneath the immunoblot (IB).

Fig. 7.

Interactions between the KD and the PBD and their effect on TbPLK activity. (A) Yeast two-hybrid assays to test the interaction between the KD and the PBD of TbPLK. (B) In vitro GST pull-down to test the interaction between the KD and the PBD of TbPLK. The bait proteins were stained with Coomassie Brilliant Blue (CBB) as presented beneath the immunoblot (IB). (C) Effect of PBD binding on the kinase activity of wild-type TbPLK, TbPLK-T198D and the KD of TbPLK. To the right is shown a quantitation of the kinase assay from three independent experiments. Error bars indicate s.d.