Fig. 4. TLK1 interacts with AUK1 in vivo and is phosphorylated by AUK1 in vitro. (A) In vitro GST pulldown assays. (B) Yeast two-hybrid assays. (C) Co-immunoprecipitation of TLK1 with AUK1 from T. brucei cells. TLK1-HA and TLK2-HA were each expressed in T. brucei cells harboring endogenous AUK1 tagged with a PTP epitope. Cells were lysed in IP lysis buffer and immunoprecipitation was performed with a pAb against HA, followed by immunoblotting with a mAb against HA or a mAb against protein C that recognizes the PTP epitope. Reciprocally, immunoprecipitation was carried out with a mAb against protein C to bring down AUK1-PTP and then blotted with a pAb against HA and a mAb against protein C, respectively. Note that no slower-migrating protein band was observed in the TLK1-HA lane because the cells were lysed in IP lysis buffer and incubated on ice for 30 minutes before boiling in SDS sample solution. The incubation might have dephosphorylated the phosphorylated protein. (D,E) AUK1 phosphorylates TLK1 (D) and TLK2 (E) in vitro. The wild-type and kinase-dead (KD) mutants of AUK1, TLK1 and TLK2 were expressed as GST-fusion proteins in E. coli, purified and mixed in various combinations for kinase assays. (F) In vitro phosphorylation of histone H3 by the purified wild-type and kinase-dead mutant (KD) TLK1, TLK2 and AUK1 recombinant proteins.