MCAK is phosphorylated by AuroraB at several sites within the N-terminus and the neck domain for localisation and activity reasons (Lan et al., 2004; Zhang et al., 2007). Little is known about a possible regulation of TbKif13-1 in T. brucei. One issue of this thesis was to analyse whether TbKif13-1 was phosphorylated by the mitotic Aurora-like kinase TbAuk1 and whether this phosphorylation inhibited TbKif13-1´s depolymerisation activity.
In a first step, mycTbAuk1 was immunoprecipitated from a transgenic 449 cell line ectopically expressing mycTbAuk1 in an active state, being able to phosphorylate TbHistoneH3His6 (Figure 32). The N-terminus of TbHistoneH3 lacks phosphorylation sites corresponding to mammalian S10 and S28, which are phosphorylated by AuroraB. LC/MS/MS revealed that TbHistoneH3 is phosphorylated by TbAuk1 at T116 within the C-terminal domain (Jetton et al., 2009).
The published kinase-dead mutants mycTbAuk1 K58R and mycTbAuk1 T184A were chosen as negative controls. However, both mutants were able to phosphorylate TbHistoneH3His6 to some extend (Figure 32; Hu et al., 2014; Li and Wang, 2006). There was no TbHistoneH3His6 phosphorylation by the product of the mock IP, in which the protein G-sepharose was applied to wild type 449 cell lysate without antibody. This control excludes the possibility that contaminations of the cell lysate were responsible for the phosphorylation.
The control IP product from wild type 449 cell lysate also showed slight TbHistoneH3His6 phosphorylation, but to an even lesser extend than the kinase-dead controls (Figure 32). This
Discussion
| 83 indicates that the myc-antibody bound protein G-sepharose purified a protein from Trypanosomes´ cell lysate capable of phosphorylating TbHistoneH3His6 to a small degree.
The, in comparison, slightly increased TbHistoneH3His6 phosphorylation by the kinase-dead
mycTbAuk1 K58R and mycTbAuk1 T184A mutants suggests the possibility of additional co-immunoprecipitation of a protein able to oligomerise with mycTbAuk1. Yeast-two-hybrid assays showed that TbAuk1 does not dimerise with itself but it does with TbTlk1 (Li et al., 2007). TbTlk1 was already found co-immunoprecipitated with TbAuk1 from T. brucei cell lysates and it was shown to phosphorylate TbHistoneH3 in vitro (Li et al., 2007). Westernblot analysis or mass spectrometry of the immunoprecipitated products could determine whether TbTlk1 is contained in the IP products and whether other possible kinases were immunoprecipitated.
In a next step, the product from mycTbAuk1 IP was shown to phosphorylate His6TbKif13-1 (Figure 33). This occurred independent of microtubule addition. At first sight it suggests that TbAuk1, in contrast to AuroraB kinase, would not need tubulin for in vitro activity (Rosasco-Nitcher et al., 2008). However, Westernblot analysis showed tubulin to a minor degree also in samples where microtubules were not added (Figure S 32). This could be the result of a spillover during gel loading. Another possibility is that it confirms studies where PTP mediated purification of TbAuk1 from T. brucei cell lysates also isolated tubulin, which could be due to TbAuk1´s association to spindle microtubules (Li et al., 2008a; Tu et al., 2006).
Hence, as previously supposed for AuroraB kinase, TbAuk1 could have an increased activity towards other microtubule-bound proteins like TbKif13-1, compared to non-bound proteins (Noujaim et al., 2014).
mycTbAuk1 K58R and mycTbAuk1 T184A resulted in the same phosphorylation effects, while wild type 449 IP product did not phosphorylate His6TbKif13-1 and tubulin. One explanation could be the possibility that the published kinase-dead mutants are not entirely devoid of kinase activity. Another explanation could be the previously mentioned possibility of co-purification of other kinases, capable to oligomerise with mycTbAuk1. In addition to TbTlk1´s ability to phosphorylate TbHistoneH3, there is nothing known about its ability to phosphorylate TbAuk1 or TbKif13-1 (Li et al., 2007). MCAK´s motor domain phosphorylation by Cdk1 (cyclin-dependent kinase) diminishes its depolymerisation activity and contributes to spindle formation and chromosome positioning (Sanhaji et al., 2010). Cdk1 also phosphorylates unpolymerised tubulin (Fourest-Lieuvin et al., 2006). For the T. brucei Cdk1
| 84 homologue TbCrk3 (cdc2-related kinase) no interaction with tubulin, TbKif13-1 or TbAuk1 has yet been identified (Hammarton et al., 2003; Tu and Wang, 2004).
Furthermore, MCAK is also phosphorylated at five residues within the C-terminal domain by Plk1, stimulating its depolymerisation activity during early phases of mitosis (Shao et al., 2015; Zhang et al., 2011). Such an stimulating effect of TbPlk to TbKif13-1 could explain the absent inhibition of His6TbKif13-1´s depolymerisation activity by the mycTbAuk1 IP product (Figure 34). However, for TbPlk an interaction with TbAuk1 or TbKif13-1 has not yet been shown. TbPlk is excluded from the nucleus throughout the cell cycle and does not play a role during mitosis, but during cytokinesis (Hammarton et al., 2007; Kumar and Wang, 2006).
TbPlk, together with its substrates, is necessary for basal body segregation, Golgi and bilobe biogenesis and flagellum inheritance (de Graffenried et al., 2008; Hammarton et al., 2007;
Hu et al., 2015; Ikeda and de Graffenried, 2012; McAllaster et al., 2015). Also for mammalian Plk1 there is nothing known about a AuroraB kinase phosphorylation.
Other possible explanations could be that the used mycTbAuk1 concentration in comparison to His6TbKif13-1 was insufficient or that TbAuk1 simply does not inhibit TbKif13-1´s depolymerisation activity.
To conclude, data from this thesis suggest a TbAuk1 mediated phosphorylation of TbKif13-1 that does not inhibit its microtubule depolymerisation activity. However, mass spectrometry analysis of the immunoprecipitated products could figure out a possible co-immunoprecipitation of other kinases. This will provide more information about the detected TbKif13-1 phosphorylation and the possible involvement of TbAuk1.
Material and methods
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