Identification and characterization of a cell division-regulating kinase AKB1 (associated kinase of Trypanosoma brucei 14-3-3) through proteomics study of the Tb14-3-3 binding proteins

Masahiro Inoue, Kenta Okamoto, Haruki Uemura, Kouichi Yasuda, Yoshihiko Motohara, Kouichi Morita, Makoto Hiromura, E. Premkumar Reddy, Toshihide Fukuma, Nobuo Horikoshi

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

We used a proteomics approach to identify the binding partners of Trypanosoma brucei 14-3-3 (Tb14-3-3) which led to the identification of a novel kinase, AKB1. The binding between these two proteins was mediated by an amphipathic groove structure in Tb14- 3-3 and 1-438 amino acid sequence of AKB1. Recombinant AKB1 but not its ATP-binding-deficient mutant (DFG to NFG) possessed an auto-phosphorylation activity as well as a kinase activity towards a peptide substrate in vitro. However, the autophosphorylation was not required for the binding of AKB1 to Tb14-3-3. Interestingly, the kinase activity of AKB1 was inhibited by calcium, and the kinase was found to utilize GTP, and dATP in addition to ATP as phospho-donors. AKB1 formed homodimers through a leucine-zipper structure. Either knockdown of AKB1 or overexpression of AKB1, but not kinase-dead AKB1 mutant, deregulated cytokinesis and cell division, suggesting that kinase activity of AKB1 is crucial for its function. Furthermore, we showed that AKB1 exists in a detergent insoluble fraction. Laser confocal microscopy revealed that the majority of AKB1 is co-localized with α-tubulin. Taken together, these findings suggest that AKB1 might regulate cytokinesis and cell division by phosphorylating cytoskeleton-associated proteins.

Original languageEnglish (US)
Pages (from-to)49-60
Number of pages12
JournalJournal of Biochemistry
Volume158
Issue number1
DOIs
StatePublished - 2015

Keywords

  • 14-3-3
  • Kinase
  • Proteomics
  • Trypanosoma brucei

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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