Phosphorylation-dependent protein interaction with Trypanosoma brucei 14-3-3 proteins that display atypical target recognition

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. Methodology/Principal Findings: 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. Conclusion/Significance: 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.