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Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1.

Yun SM, Moulaei T, Lim D, Bang JK, Park JE, Shenoy SR, Liu F, Kang YH, Liao C, Soung NK, Lee S, Yoon DY, Lim Y, Lee DH, Otaka A, Appella E, McMahon JB, Nicklaus MC, Burke TR, Yaffe MB, Wlodawer A, Lee KS - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction.Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death.The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

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Minimal p-T78 peptides specifically bind to Plk1 with a high affinity. (a) HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) were mixed before incubating with the bead-bound T78 peptides or synthetic optimal peptide (MQSpTPL) as indicated. Precipitates were immunoblotted with anti-Flag antibody. Because of the distinct binding nature of Plk4 PBD, Plk4 was not tested. Numbers indicate the fraction of Plk2 over Plk1 bound to the peptide. Arrows indicate Flag-Plk1, 2, 3 proteins. (b) Mitotic HeLa lysates were incubated with the indicated bead-bound peptides. Co-precipitating proteins were analyzed by silver staining. Arrows, Plk1 precipitated with p-T78 peptides. (c) Soluble control GST, GST-PBD, or GST-PBD(H538A K540M) was incubated with the indicated T78 peptides immobilized to the beads. Bound proteins were immunoblotted with anti-GST antibody. (d) Isothermal titration calorimetry for the p-T78 peptides was performed using purified Plk1 PBD. Representative calorimetric isotherms for the binding of two 5-mers (PLHSpT and LHSpTA) to the PBD are shown. The solid lines represent fits to the data. The overall H (kcal/mol) is easily observed as the difference between the pre- and the post-binding baselines extrapolated along the y-axis. (e) Mitotic HeLa lysates were pre-incubated with bead-bound GST-PBD for 1.5 h prior to the addition of the indicated peptides. After another 1.5 h incubation, GST-PBD-binding proteins were precipitated and analyzed as in (a). Detection of GST-PBD in the anti-Cdc25C blot is the result of the previous anti-GST immunoblotting. Numbers indicate relative efficiency of p-Cdc25C pull-down by GST-PBD. (f) Mitotic HeLa lysates were treated with the indicated peptide prior to immunoprecipitation with either control IgG or anti-Plk1 antibody. Immunoprecipitates were blotted with the indicated antibodies. Asterisk, a cross-reacting protein.
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Figure 2: Minimal p-T78 peptides specifically bind to Plk1 with a high affinity. (a) HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) were mixed before incubating with the bead-bound T78 peptides or synthetic optimal peptide (MQSpTPL) as indicated. Precipitates were immunoblotted with anti-Flag antibody. Because of the distinct binding nature of Plk4 PBD, Plk4 was not tested. Numbers indicate the fraction of Plk2 over Plk1 bound to the peptide. Arrows indicate Flag-Plk1, 2, 3 proteins. (b) Mitotic HeLa lysates were incubated with the indicated bead-bound peptides. Co-precipitating proteins were analyzed by silver staining. Arrows, Plk1 precipitated with p-T78 peptides. (c) Soluble control GST, GST-PBD, or GST-PBD(H538A K540M) was incubated with the indicated T78 peptides immobilized to the beads. Bound proteins were immunoblotted with anti-GST antibody. (d) Isothermal titration calorimetry for the p-T78 peptides was performed using purified Plk1 PBD. Representative calorimetric isotherms for the binding of two 5-mers (PLHSpT and LHSpTA) to the PBD are shown. The solid lines represent fits to the data. The overall H (kcal/mol) is easily observed as the difference between the pre- and the post-binding baselines extrapolated along the y-axis. (e) Mitotic HeLa lysates were pre-incubated with bead-bound GST-PBD for 1.5 h prior to the addition of the indicated peptides. After another 1.5 h incubation, GST-PBD-binding proteins were precipitated and analyzed as in (a). Detection of GST-PBD in the anti-Cdc25C blot is the result of the previous anti-GST immunoblotting. Numbers indicate relative efficiency of p-Cdc25C pull-down by GST-PBD. (f) Mitotic HeLa lysates were treated with the indicated peptide prior to immunoprecipitation with either control IgG or anti-Plk1 antibody. Immunoprecipitates were blotted with the indicated antibodies. Asterisk, a cross-reacting protein.

Mentions: Next, we tested the specificity of minimal p-T78 peptides against Plk1 PBD. The results showed that, similar to the initial 14-mer peptide, minimal p-T78 peptides precipitated Plk1 from cellular lysates containing similar levels of Plk1, Plk2, and Plk3 (Fig. 2a and Supplementary Fig. 1b). In contrast, the 6-mer optimal MQSpTPL peptide precipitated Plk2 with ~27% efficiency of Plk1 precipitation (Fig. 2a), suggesting that it possesses a significantly lower Plk1 specificity than PLHSpT. Consistently, MQSpTPL, but not the p-T78 peptides, also precipitated Plk2 from the lysates expressing Plk2 alone (Supplementary Fig. 1c). Remarkably, although much shorter than the initial 14-mer peptide, a minimal p-T78 peptide, PLHSpT, exhibited an undiminished Plk1 specificity and precipitated Plk1 as the only major binding protein from total cellular lysates (Fig. 2b). Another 5-mer, LHSpTA, displayed a similar but somewhat reduced level of Plk1 affinity (Fig. 2b). These results suggest that elements critical for Plk1-binding affinity and specificity reside within these minimal sequences. Furthermore, PLHSpT efficiently bound to GST-fused PBD (GST-PBD), but not to the respective GST-PBD(H538A, K540A) phosphate pincer mutant (Fig. 2c), indicating that an intact phosphoepitope-binding module is required for the interaction.


Structural and functional analyses of minimal phosphopeptides targeting the polo-box domain of polo-like kinase 1.

Yun SM, Moulaei T, Lim D, Bang JK, Park JE, Shenoy SR, Liu F, Kang YH, Liao C, Soung NK, Lee S, Yoon DY, Lim Y, Lee DH, Otaka A, Appella E, McMahon JB, Nicklaus MC, Burke TR, Yaffe MB, Wlodawer A, Lee KS - Nat. Struct. Mol. Biol. (2009)

Minimal p-T78 peptides specifically bind to Plk1 with a high affinity. (a) HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) were mixed before incubating with the bead-bound T78 peptides or synthetic optimal peptide (MQSpTPL) as indicated. Precipitates were immunoblotted with anti-Flag antibody. Because of the distinct binding nature of Plk4 PBD, Plk4 was not tested. Numbers indicate the fraction of Plk2 over Plk1 bound to the peptide. Arrows indicate Flag-Plk1, 2, 3 proteins. (b) Mitotic HeLa lysates were incubated with the indicated bead-bound peptides. Co-precipitating proteins were analyzed by silver staining. Arrows, Plk1 precipitated with p-T78 peptides. (c) Soluble control GST, GST-PBD, or GST-PBD(H538A K540M) was incubated with the indicated T78 peptides immobilized to the beads. Bound proteins were immunoblotted with anti-GST antibody. (d) Isothermal titration calorimetry for the p-T78 peptides was performed using purified Plk1 PBD. Representative calorimetric isotherms for the binding of two 5-mers (PLHSpT and LHSpTA) to the PBD are shown. The solid lines represent fits to the data. The overall H (kcal/mol) is easily observed as the difference between the pre- and the post-binding baselines extrapolated along the y-axis. (e) Mitotic HeLa lysates were pre-incubated with bead-bound GST-PBD for 1.5 h prior to the addition of the indicated peptides. After another 1.5 h incubation, GST-PBD-binding proteins were precipitated and analyzed as in (a). Detection of GST-PBD in the anti-Cdc25C blot is the result of the previous anti-GST immunoblotting. Numbers indicate relative efficiency of p-Cdc25C pull-down by GST-PBD. (f) Mitotic HeLa lysates were treated with the indicated peptide prior to immunoprecipitation with either control IgG or anti-Plk1 antibody. Immunoprecipitates were blotted with the indicated antibodies. Asterisk, a cross-reacting protein.
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Figure 2: Minimal p-T78 peptides specifically bind to Plk1 with a high affinity. (a) HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) were mixed before incubating with the bead-bound T78 peptides or synthetic optimal peptide (MQSpTPL) as indicated. Precipitates were immunoblotted with anti-Flag antibody. Because of the distinct binding nature of Plk4 PBD, Plk4 was not tested. Numbers indicate the fraction of Plk2 over Plk1 bound to the peptide. Arrows indicate Flag-Plk1, 2, 3 proteins. (b) Mitotic HeLa lysates were incubated with the indicated bead-bound peptides. Co-precipitating proteins were analyzed by silver staining. Arrows, Plk1 precipitated with p-T78 peptides. (c) Soluble control GST, GST-PBD, or GST-PBD(H538A K540M) was incubated with the indicated T78 peptides immobilized to the beads. Bound proteins were immunoblotted with anti-GST antibody. (d) Isothermal titration calorimetry for the p-T78 peptides was performed using purified Plk1 PBD. Representative calorimetric isotherms for the binding of two 5-mers (PLHSpT and LHSpTA) to the PBD are shown. The solid lines represent fits to the data. The overall H (kcal/mol) is easily observed as the difference between the pre- and the post-binding baselines extrapolated along the y-axis. (e) Mitotic HeLa lysates were pre-incubated with bead-bound GST-PBD for 1.5 h prior to the addition of the indicated peptides. After another 1.5 h incubation, GST-PBD-binding proteins were precipitated and analyzed as in (a). Detection of GST-PBD in the anti-Cdc25C blot is the result of the previous anti-GST immunoblotting. Numbers indicate relative efficiency of p-Cdc25C pull-down by GST-PBD. (f) Mitotic HeLa lysates were treated with the indicated peptide prior to immunoprecipitation with either control IgG or anti-Plk1 antibody. Immunoprecipitates were blotted with the indicated antibodies. Asterisk, a cross-reacting protein.
Mentions: Next, we tested the specificity of minimal p-T78 peptides against Plk1 PBD. The results showed that, similar to the initial 14-mer peptide, minimal p-T78 peptides precipitated Plk1 from cellular lysates containing similar levels of Plk1, Plk2, and Plk3 (Fig. 2a and Supplementary Fig. 1b). In contrast, the 6-mer optimal MQSpTPL peptide precipitated Plk2 with ~27% efficiency of Plk1 precipitation (Fig. 2a), suggesting that it possesses a significantly lower Plk1 specificity than PLHSpT. Consistently, MQSpTPL, but not the p-T78 peptides, also precipitated Plk2 from the lysates expressing Plk2 alone (Supplementary Fig. 1c). Remarkably, although much shorter than the initial 14-mer peptide, a minimal p-T78 peptide, PLHSpT, exhibited an undiminished Plk1 specificity and precipitated Plk1 as the only major binding protein from total cellular lysates (Fig. 2b). Another 5-mer, LHSpTA, displayed a similar but somewhat reduced level of Plk1 affinity (Fig. 2b). These results suggest that elements critical for Plk1-binding affinity and specificity reside within these minimal sequences. Furthermore, PLHSpT efficiently bound to GST-fused PBD (GST-PBD), but not to the respective GST-PBD(H538A, K540A) phosphate pincer mutant (Fig. 2c), indicating that an intact phosphoepitope-binding module is required for the interaction.

Bottom Line: Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction.Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death.The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.

ABSTRACT
Polo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

Show MeSH
Related in: MedlinePlus