title: Phosphorylation-dependent protein interaction with Trypanosoma brucei 14-3-3 proteins that display atypical target recognition.
abstract: BACKGROUND
  The 14-3-3 proteins are structurally conserved throughout eukaryotes and participate in protein kinase signaling. All 14-3-3 proteins are known to bind to evolutionally conserved phosphoserine-containing motifs (modes 1 and/or 2) with high affinity. In Trypanosoma brucei, 14-3-3I and II play pivotal roles in motility, cytokinesis and the cell cycle. However, none of the T. brucei 14-3-3 binding proteins have previously been documented.
  
  
  RESULTS
  Initially we showed that T. brucei 14-3-3 proteins exhibit far lower affinity to those peptides containing RSxpSxP (mode 1) and RxY/FxpSxP (mode 2) (where x is any amino acid residue and pS is phosphoserine) than human 14-3-3 proteins, demonstrating the atypical target recognition by T. brucei 14-3-3 proteins. We found that the putative T. brucei protein phosphatase 2C (PP2c) binds to T. brucei 14-3-3 proteins utilizing its mode 3 motif (-pS/pTx(1-2)-COOH, where x is not Pro). We constructed eight chimeric PP2c proteins replacing its authentic mode 3 motif with potential mode 3 sequences found in Trypanosoma brucei genome database, and tested their binding. As a result, T. brucei 14-3-3 proteins interacted with three out of eight chimeric proteins including two with high affinity. Importantly, T. brucei 14-3-3 proteins co-immunoprecipitated with an uncharacterized full-length protein containing identified high-affinity mode 3 motif, suggesting that both proteins form a complex in vivo. In addition, a synthetic peptide derived from this mode 3 motif binds to T. brucei 14-3-3 proteins with high affinity.
  
  
  CONCLUSIONS
  Because of the atypical target recognition of T. brucei 14-3-3 proteins, no 14-3-3-binding proteins have been successfully identified in T. brucei until now whereas over 200 human 14-3-3-binding proteins have been identified. This report describes the first discovery of the T. brucei 14-3-3-binding proteins and their binding motifs. The high-affinity phosphopeptide will be a powerful tool to identify novel T. brucei 14-3-3-binding proteins.
authors.affiliation: Division of Eukaryotic Microbiology, Department of Infectious Medicine, Kurume University School of Medicine, Kurume, Japan. inouedna@med.kurume-u.ac.jp
authors.forename: Masahiro
authors.initials: M
authors.lastname: Inoue
authors.forename: Kouichi
authors.initials: K
authors.lastname: Yasuda
authors.forename: Haruki
authors.initials: H
authors.lastname: Uemura
authors.forename: Natsumi
authors.initials: N
authors.lastname: Yasaka
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Inoue
authors.forename: Yoshitatsu
authors.initials: Y
authors.lastname: Sei
authors.forename: Nobuo
authors.initials: N
authors.lastname: Horikoshi
authors.forename: Toshihide
authors.initials: T
authors.lastname: Fukuma
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002453:Cell Cycle; D002465:Cell Movement; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D010748:Phosphopeptides / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D000911:Antibodies, Monoclonal; D010748:Phosphopeptides; D011993:Recombinant Fusion Proteins; D019908:Proto-Oncogene Proteins c-raf