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14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides.

Madeira F, Tinti M, Murugesan G, Berrett E, Stafford M, Toth R, Cole C, MacKintosh C, Barton GJ - Bioinformatics (2015)

Bottom Line: ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from -6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60.The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome.Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful.

View Article: PubMed Central - PubMed

Affiliation: Division of Computational Biology.

No MeSH data available.


Related in: MedlinePlus

Regulated binding of 14-3-3 proteins to FAM122-GFP proteins. (A) HEK293 cells growing in media containing 10% (v/v) serum were transfected to express FAM122A-GFP, FAM122B-GFP and FAM122C-GFP proteins (the latter as 152 and 196 residue isoforms, respectively, excluding the GFP). GFP-tagged proteins isolated from cell lysates (120 mg) with GFP-Trap® were tested for their ability to bind directly to 14-3-3s in Far-Western assays (overlay) and by coimmunoprecipitation of endogenous 14-3-3s (14-3-3s) using the K19 pan-14-3-3 antibody. Anti-GFP signals show levels of the tagged proteins in the immunoprecipitates. (B) FAM122A-GFP bound to GFPTrap® was dephosphorylated with lambda phosphatase or not when the phosphatase was inhibited with EDTA. The immunoprecipitates were washed and FAM122A-GFP analyzed for its ability to bind directly to 14-3-3s (overlay) and for retention of co-purified endogenous 14-3-3 proteins (14-3-3s). Cells had been grown in standard medium (serum), with 100 nM calyculin A (a protein phosphatase inhibitor) added for approximately 5 min before lysis, as indicated. (C) Wild-type FAM122A-GFP and the indicated serine/threonine-to-alanine mutant proteins were isolated from transfected cells and tested for direct binding to 14-3-3s and for co-immunoprecipitating endogenous 14-3-3s. (D) HEK293 cells were transfected to express Ser37Ala/Ser62Ala- FAM122A-GFP and Ser37Ala/Thr64Ala-FAM122A-GFP, as indicated. Cells were serum starved for 10 h, then stimulated with serum (10% (v/v) for 30 min) or forskolin (20 µM for 30 min) with or without H89 pre-treatment (30 µM for 30 min), as indicated. Proteins immunoprecipitated from lysates were tested for 14-3-3 binding (overlay) and coimmunoprecipitation of endogenous 14-3-3 (14-3-3)
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btv133-F2: Regulated binding of 14-3-3 proteins to FAM122-GFP proteins. (A) HEK293 cells growing in media containing 10% (v/v) serum were transfected to express FAM122A-GFP, FAM122B-GFP and FAM122C-GFP proteins (the latter as 152 and 196 residue isoforms, respectively, excluding the GFP). GFP-tagged proteins isolated from cell lysates (120 mg) with GFP-Trap® were tested for their ability to bind directly to 14-3-3s in Far-Western assays (overlay) and by coimmunoprecipitation of endogenous 14-3-3s (14-3-3s) using the K19 pan-14-3-3 antibody. Anti-GFP signals show levels of the tagged proteins in the immunoprecipitates. (B) FAM122A-GFP bound to GFPTrap® was dephosphorylated with lambda phosphatase or not when the phosphatase was inhibited with EDTA. The immunoprecipitates were washed and FAM122A-GFP analyzed for its ability to bind directly to 14-3-3s (overlay) and for retention of co-purified endogenous 14-3-3 proteins (14-3-3s). Cells had been grown in standard medium (serum), with 100 nM calyculin A (a protein phosphatase inhibitor) added for approximately 5 min before lysis, as indicated. (C) Wild-type FAM122A-GFP and the indicated serine/threonine-to-alanine mutant proteins were isolated from transfected cells and tested for direct binding to 14-3-3s and for co-immunoprecipitating endogenous 14-3-3s. (D) HEK293 cells were transfected to express Ser37Ala/Ser62Ala- FAM122A-GFP and Ser37Ala/Thr64Ala-FAM122A-GFP, as indicated. Cells were serum starved for 10 h, then stimulated with serum (10% (v/v) for 30 min) or forskolin (20 µM for 30 min) with or without H89 pre-treatment (30 µM for 30 min), as indicated. Proteins immunoprecipitated from lysates were tested for 14-3-3 binding (overlay) and coimmunoprecipitation of endogenous 14-3-3 (14-3-3)

Mentions: Consistent with the 14-3-3-Pred results (Table 2), all three FAM122 family members displayed phosphorylation-dependent binding to 14-3-3 proteins when isolated from transfected cells (Fig. 2A). The binding of 14-3-3 to FAM122A was abolished by its dephosphorylation (Fig. 2B) and by substitution of Ser37 of FAM122A with alanine (Fig. 2C). Although phosphoSer62 and phosphoThr64 of FAM122A also had relatively high 14-3-3-Pred scores (0.614 and 1.076, respectively), mutation of these residues did not affect 14-3-3 binding to FAM122A isolated from cells cultured in standard serum-containing medium (Fig. 2C). However, in the absence of Ser37, stimulating cells with the adenylate cyclase activator forskolin caused a marked increase in 14-3-3 binding to FAM122A, which was abolished when Ser62 was also mutated to alanine and when cells were pre-treated with H89, which is a non-specific cAMP-dependent protein kinase (PKA) inhibitor (Fig. 2D). These data indicate that a 14-3-3 dimer binds to both phosphoSer37 and phosphoSer62 on FAM122A, the latter likely phosphorylated by PKA. Similar experiments showed that 14-3-3 binds to phosphoSer25 and forskolin-regulated phosphoSer50 of FAM122B and to phosphoSer29 (ILRRVNSAPLI) of FAM122C. Thus, this is an example of a 2R-ohnologue family for which protein members share a conserved 14-3-3-binding ‘lynchpin’. In fact, half of the top 20 candidate proteins (10/20) belong to 2R-ohnologue families.Fig. 2.


14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides.

Madeira F, Tinti M, Murugesan G, Berrett E, Stafford M, Toth R, Cole C, MacKintosh C, Barton GJ - Bioinformatics (2015)

Regulated binding of 14-3-3 proteins to FAM122-GFP proteins. (A) HEK293 cells growing in media containing 10% (v/v) serum were transfected to express FAM122A-GFP, FAM122B-GFP and FAM122C-GFP proteins (the latter as 152 and 196 residue isoforms, respectively, excluding the GFP). GFP-tagged proteins isolated from cell lysates (120 mg) with GFP-Trap® were tested for their ability to bind directly to 14-3-3s in Far-Western assays (overlay) and by coimmunoprecipitation of endogenous 14-3-3s (14-3-3s) using the K19 pan-14-3-3 antibody. Anti-GFP signals show levels of the tagged proteins in the immunoprecipitates. (B) FAM122A-GFP bound to GFPTrap® was dephosphorylated with lambda phosphatase or not when the phosphatase was inhibited with EDTA. The immunoprecipitates were washed and FAM122A-GFP analyzed for its ability to bind directly to 14-3-3s (overlay) and for retention of co-purified endogenous 14-3-3 proteins (14-3-3s). Cells had been grown in standard medium (serum), with 100 nM calyculin A (a protein phosphatase inhibitor) added for approximately 5 min before lysis, as indicated. (C) Wild-type FAM122A-GFP and the indicated serine/threonine-to-alanine mutant proteins were isolated from transfected cells and tested for direct binding to 14-3-3s and for co-immunoprecipitating endogenous 14-3-3s. (D) HEK293 cells were transfected to express Ser37Ala/Ser62Ala- FAM122A-GFP and Ser37Ala/Thr64Ala-FAM122A-GFP, as indicated. Cells were serum starved for 10 h, then stimulated with serum (10% (v/v) for 30 min) or forskolin (20 µM for 30 min) with or without H89 pre-treatment (30 µM for 30 min), as indicated. Proteins immunoprecipitated from lysates were tested for 14-3-3 binding (overlay) and coimmunoprecipitation of endogenous 14-3-3 (14-3-3)
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btv133-F2: Regulated binding of 14-3-3 proteins to FAM122-GFP proteins. (A) HEK293 cells growing in media containing 10% (v/v) serum were transfected to express FAM122A-GFP, FAM122B-GFP and FAM122C-GFP proteins (the latter as 152 and 196 residue isoforms, respectively, excluding the GFP). GFP-tagged proteins isolated from cell lysates (120 mg) with GFP-Trap® were tested for their ability to bind directly to 14-3-3s in Far-Western assays (overlay) and by coimmunoprecipitation of endogenous 14-3-3s (14-3-3s) using the K19 pan-14-3-3 antibody. Anti-GFP signals show levels of the tagged proteins in the immunoprecipitates. (B) FAM122A-GFP bound to GFPTrap® was dephosphorylated with lambda phosphatase or not when the phosphatase was inhibited with EDTA. The immunoprecipitates were washed and FAM122A-GFP analyzed for its ability to bind directly to 14-3-3s (overlay) and for retention of co-purified endogenous 14-3-3 proteins (14-3-3s). Cells had been grown in standard medium (serum), with 100 nM calyculin A (a protein phosphatase inhibitor) added for approximately 5 min before lysis, as indicated. (C) Wild-type FAM122A-GFP and the indicated serine/threonine-to-alanine mutant proteins were isolated from transfected cells and tested for direct binding to 14-3-3s and for co-immunoprecipitating endogenous 14-3-3s. (D) HEK293 cells were transfected to express Ser37Ala/Ser62Ala- FAM122A-GFP and Ser37Ala/Thr64Ala-FAM122A-GFP, as indicated. Cells were serum starved for 10 h, then stimulated with serum (10% (v/v) for 30 min) or forskolin (20 µM for 30 min) with or without H89 pre-treatment (30 µM for 30 min), as indicated. Proteins immunoprecipitated from lysates were tested for 14-3-3 binding (overlay) and coimmunoprecipitation of endogenous 14-3-3 (14-3-3)
Mentions: Consistent with the 14-3-3-Pred results (Table 2), all three FAM122 family members displayed phosphorylation-dependent binding to 14-3-3 proteins when isolated from transfected cells (Fig. 2A). The binding of 14-3-3 to FAM122A was abolished by its dephosphorylation (Fig. 2B) and by substitution of Ser37 of FAM122A with alanine (Fig. 2C). Although phosphoSer62 and phosphoThr64 of FAM122A also had relatively high 14-3-3-Pred scores (0.614 and 1.076, respectively), mutation of these residues did not affect 14-3-3 binding to FAM122A isolated from cells cultured in standard serum-containing medium (Fig. 2C). However, in the absence of Ser37, stimulating cells with the adenylate cyclase activator forskolin caused a marked increase in 14-3-3 binding to FAM122A, which was abolished when Ser62 was also mutated to alanine and when cells were pre-treated with H89, which is a non-specific cAMP-dependent protein kinase (PKA) inhibitor (Fig. 2D). These data indicate that a 14-3-3 dimer binds to both phosphoSer37 and phosphoSer62 on FAM122A, the latter likely phosphorylated by PKA. Similar experiments showed that 14-3-3 binds to phosphoSer25 and forskolin-regulated phosphoSer50 of FAM122B and to phosphoSer29 (ILRRVNSAPLI) of FAM122C. Thus, this is an example of a 2R-ohnologue family for which protein members share a conserved 14-3-3-binding ‘lynchpin’. In fact, half of the top 20 candidate proteins (10/20) belong to 2R-ohnologue families.Fig. 2.

Bottom Line: ANN, position-specific scoring matrix and SVM methods showed best performance for a motif window spanning from -6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60.The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome.Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful.

View Article: PubMed Central - PubMed

Affiliation: Division of Computational Biology.

No MeSH data available.


Related in: MedlinePlus