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.

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.

Figure 1. Interactions of T. brucei 14-3-3 proteins with c-Raf and the 14-3-3 binding phosphopeptide.

(A) Interaction of GST-I, -II and τ with human c-Raf. HeLa cell lysates were subjected to GST pull-down assay using the indicated proteins bound to glutathione beads. Bound proteins were separated with SDS-PAGE, transferred to a PVDF membrane and detected with α-human c-Raf-1 antibodies (WB). The membrane was stained with coomassie brilliant blue (CBB) to visualize GST-14-3-3 proteins used in the assays. (B) Surface plasmon resonance measurements. Surface plasmon resonance analysis of interaction of c-Raf-derived phosphopeptide pSRaf259 (biotin-MAGGGRQRSTpSTPN) (mode 1 peptide) to GST- and/or MBP-14-3-3 fusion proteins. Surface plasmon resonance analysis of a mode 2 peptide (biotin-MAGGGRLYHpSLP) to GST-14-3-3 fusion proteins. Sensorgram patterns are shown in each injected protein. (C) Peptide pull-down assay. T. brucei PCF cell lysates (5×10⁸ cells/pull-down) were subjected to peptide pull-down assay followed by Western blotting (WB) with α-I or –II antibodies. WB of total cell lysates (1×10⁷ cells) using α-I and -II detected bands of 28 kDa and 30 kDa, respectively.

Figure 2. Interaction of T. brucei PP2c with T. brucei and human 14-3-3 proteins.

(A) and (B) Mammalian expression vectors pCR3 N-V5-PP2c, –PP2c S744A or -C-terminal mutants of PP2c were transiently transfected to HEK293T cells. The cell lysates were subjected to immunoprecipitation with α-V5 Ab followed by far-Western blot analyses (Far-WB) using indicated probes (upper panel). Immunoprecipitated proteins were reprobed with α-V5 Ab (lower panel). (C) Modes 1-3 phosphopeptides summarized in Table 1 have far less affinity to T. brucei 14-3-3 than human 14-3-3ζ proteins. Indicated biotinylated phosphopeptides were mixed with streptavidine and spotted to nitrocellulose filters and dried. The filters were then incubated with indicated GST-14-3-3 proteins and detected with α-GST antibodies. (D) Tet-inducible T. brucei expression vector, pLew82 T7bsr-N-V5-PP2c was transfected and clones were isolated with blasticidin selection. Tet-uninduced (Tet−) or –induced (Tet +) cell lysates of the clone were subjected to immunoprecipitation followed by Western blotting using a mixture of α-I and II Ab (lower panel), and horseradish peroxidase (HRP)-labeled α-V5 Ab (upper panel).

Figure 3. p31-SAP binds to T. brucei 14-3-3 proteins in vivo (PCF cells).

(A) C-terminal chimeric PP2c proteins were produced by transfection of HEK293T cells and purified by immunoprecipitation with α-V5 Ab. Immunoprecipitated proteins were analyzed by Far-WB with GST-II + I as a probe (upper panel). Immunoprecipitated V5-tagged proteins were visualized with α-V5 Ab (lower panel). [*PP2c, **PP2c W (-)] (B) Full-length V5-tagged p31-SAP or –SAP (S286A)-expressing cell lines under the control of tetracycline (Tet) were established. Cell lysates of clones with (+) or without (-) Tet-induction were subjected to immunoprecipitation with α-V5 Ab followed by Western blotting (WB) with a mixture of α-14-3-3I and II Abs (upper panel). Western blotting with HRP-labeled α-V5 monoclonal Ab (lower panel, WB: α-V5) serves as the immunoprecipitation control. Associated 14-3-3 proteins with V5-tagged p31-SAP, and IgG heavy chain (H) are indicated with arrows (upper panel) and precipitated total V5-p31-SAP or –SAP (S286A) proteins are indicated (lower panel).

Figure 4. Identification of the high-affinity mode 3 phosphopeptide derived from p31-SAP.

Indicated biotinylated mode 3 phosphopeptides (1 and 2) as indicated were mixed with streptavidine and spotted to nitrocellulose filters and dried. The filters were then incubated with the indicated GST-14-3-3 proteins and detected with α-GST antibodies.