Minimization of PBIP1 p-T78 peptide that binds to Plk1. (a–c) Various lengths of N-terminal Cys-(CH₂)₆-fused T78 peptides were cross-linked to the beads (a) and then tested for their ability to bind to Plk1. The Ser residue (blue) in (a, right) indicates the invariable S77 residue critical for PBD binding. For comparison, a shortened form of the synthetic peptide optimized for Plk1 PBD binding (MQSpTPL)13 was included. (d) A 6-mer T78 peptide (LHSpTAI) analogous to the synthetic optimal peptide (MQSpTPL) was tested for Plk1 binding. Numbers indicate the efficiency of Plk1 precipitation by each peptide relative to the Plk1 signal in the input.

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.

The nature of PBD binding and specificity. (a) Superposition of the phosphopeptide-binding pockets of PBD^PL, PBD^PP, PBD^S+G, and PBD^S. PBD is drawn in grey. PLHSpT is in green and its associated glycerol molecule is in yellow. PPHSpT is drawn in cyan. The glycerol molecule (two half-occupancy conformations at the Ser-1 position) of PBD^S+G is drawn in magenta. The two sulfate anions of PBD^S+G and PBD^S are drawn with the sulfur atoms in black and oxygen atoms in red. The differences in the exact positions of sulfate and phosphate groups could be due to the fact that the sulfate is a free anion, whereas the phosphate is covalently linked to the phosphopeptide. PDB ID for PBD^PL, 3HIK; PDB ID for PBD^PP, 3C5L; PDB ID for PBD^S+G and PBD^S, 3HIH. (b) The PBD residues involved in binding of PLHSpT are labeled and shown in cyan. All water molecules that form an interface between the phosphopeptide and PBD are drawn in red mesh. (c) Superposition of PLHSpT (green), PPHSpT (cyan), MQSpTPL (magenta), and PMQSpTPL (grey). (d,e) The mixture of HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R) was subjected to pull-down assays as in Fig. 2a with the indicated 5-mer wild-type (PLHSpT) and mutants cross-linked to the beads. The respective non-phospho-T78 peptide (PLHST) was used as a control. The numbers at the top of the blot indicate the relative efficiency of Plk2 precipitation, whereas the numbers at the bottom denote the relative efficiency of Plk1 precipitation. (f) Illustration depicting the nature of the interactions between the SpT-containing peptides and the Plk1 PBD. Alignment of minimal p-T78 peptides (PLHST and LHSTA) and synthetic optimal peptides (PMQSTPL and MQSTPL) showed that, in addition to the critical SpT motif, the N-terminal Pro-4 and Met-3 residues are important to stabilize the interactions by docking into a hydrophobic core surrounded by the Trp414, Phe535 and Arg516 residues in Plk1 PBD. The His-2 residue is important for Plk1 specificity since substitution of Gln for His enhances Plk2 binding. The Ala+1 or Pro+1 residue is central for guiding a priming kinase to phosphorylate the Thr residue.

A 5-mer p-T78 mimetic peptide (PLHS-Pmab) induces mitotic arrest by specifically inhibiting Plk1 localization. (a) Illustration of a nonhydrolyzable p-Thr derivative, Pmab (Top), used for the synthesis of mimetic peptides. The indicated, bead-immobilized, peptides were incubated with mitotic HeLa lysates in the presence of phosphatase inhibitors, and then analyzed as in Fig. 1a. T78, C-(CH₂)₆-PLHST; p-T78, C-(CH₂)₆-PLHSpT; Pmab, C-(CH₂)₆-PLHS-Pmab; Pmab(S77A), C-(CH₂)₆-PLHA-Pmab. (b) The same peptides used in (a) were incubated with the mixture of HeLa lysates expressing the kinase-inactive Flag-Plk1(K82M), Flag-Plk2(K108M), or Flag-Plk3(K52R). Bead-associated proteins were analyzed as in Fig. 2a. The membrane was stained with Coomassie (CBB). (c) To quantitatively determine the efficiency of PBD-binding inhibition by the indicated peptides, an ELISA-based inhibition assay was performed in the presence of various amounts of the indicated competitor peptides (see Methods for details). (d,e) HeLa cells arrested in S phase by thymidine treatment were released into fresh medium. Two hours after release, all the cells (~150 cells) in a single grid were microinjected with a mixture containing 2.5 mM of the indicated peptide and 30 ng/μl of pEGFP-C1 vector (to visualize the injected cells), and then further incubated. Cells were photographed 12 h after releasing from the S phase block (d). To monitor cell cycle progression, the percentages of mitotic cells were quantified at the indicated time points (e). Bars, standard deviation. (f) Cells microinjected similarly as in (d) were fixed and immunostained with anti-Plk1 antibody. Images were acquired from EGFP-positive cells. Asterisks, centrosome-localized Plk1 signals; arrowed brackets, kinetochore-localized Plk1 signals; arrows, misaligned chromosomes. (g) Fluorescence intensities for centrosome-localized (n > 20 centrosomes) or kinetochore-localized (n > 45 kinetochores, average of 6 kinetochores per cell) anti-Plk1 signals in (f) were quantified as described in the Methods. Bars (red), the averages of relative fluorescence intensities.