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Genes Cells
2024 Nov 11;2911:1002-1011. doi: 10.1111/gtc.13159.
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Development of luminescent probes for real-time detection of the CDK/PP2A balance during the cell cycle.
Hino H
,
Takaki K
,
Kobe M
,
Mochida S
.
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From a biochemical viewpoint, the cell cycle is controlled by the phosphorylation of cyclin-dependent kinase (CDK) substrates, and the phosphorylation level is determined by the enzymatic balance between CDK and protein phosphatase 2A (PP2A). However, the conventional techniques for analyzing protein phosphorylation using radioisotopes and antibodies involve many operational steps and take days before obtaining results, making them difficult to apply to high-throughput screening and real-time observations. In this study, we developed luminescent probes with a light intensity that changes depending on its phosphorylation state. We modified the Nano-lantern probe (Renilla luciferase-based Ca2+ probe) by introducing a CDK-substrate peptide and a phosphopeptide-binding domain into the luciferase. Our initial trial resulted in new probes that could report the CDK/PP2A balance in a purified system. Further modifications of these probes (replacing the phospho-Ser with phospho-Thr and randomly replacing its surrounding amino acids) improved the dynamic range by up to four-fold, making them practical for use in the Xenopus egg extracts system, where many physiological events can be reproduced. Taken together, our new probes enabled the monitoring of the CDK/PP2A balance in real time, and are applicable to high-throughput systems; the new probes thus appear promising for use in substrate and drug screening.
FIGURE 1
Design of luminescent probes for cell cycle-dependent phosphorylation. (a) Schematic representation of the luminescent probe. The conformational change shown is conceptual. CTZ, coelenterazine; RLuc(C), carboxy-terminal fragment of Renilla luciferase; RLuc(N), amino-terminal fragment of Renilla luciferase; S/TP-pep, phospho-peptide of the CDK1:cyclin B substrate; WW, WW domain of Xenopus Pin1. (b) S/TP-pep sequences derived from CDK substrate proteins (Xenopus Fizzy, LaminA/B2, PP1gamma, and Cdc6) employed in the initial screening. Letters in red indicate CDK-phosphorylation sites. Underlines indicate the S/T-P motif, a minimal CDK target motif. (c, d) The ratio of the luminescent intensity of the phosphorylated/hypo-phosphorylated probes are shown as the dynamic range. Probes without (c) or with (d) the WW domain. Data are shown as the average values from three independent experiments with the SD.
FIGURE 2
Dephosphorylation of probes in Xenopus egg extracts. (a) The dephosphorylation kinetics of 32P-phosphorylated probes in Xenopus egg extracts were analyzed by autoradiography at the indicated timepoints after Ca2+
FIGURE 3
Changes in the dynamic range of the probes due to modifications of CDK-targeted peptide sequences. (a) Alignment of the original and modified S50 sequences. S and T in red followed by P are CDK-targeted Ser and Thr, respectively. Hyphens indicate gaps. RS and LE at each end (black underline) are restriction enzyme sequences. Gray boxes indicate the sequences derived from the PP1 peptide (Figure 1b). The effects of Gly insertions were assessed in Figure S4. (b) The luminescence of the probes in (a) and the corresponding non-phosphorylatable mutants (Ser/Thr to Ala) were measured. Each probe with Ser, Thr, or Ala at the CDK site is indicated by blue, orange, or gray bars, respectively. Data are shown as the average values from three independent experiments with the SD.
FIGURE 4
Screening for larger phospho-dependent changes in probe luminescence. (a) Schematic representation of the screening. Blue and yellow boxes indicate regions randomly substituted in the T50-NC sequence. (b) The dynamic range of the top 35 clones from the N-terminal screening. (c) The dynamic range of the top 35 clones from the C-terminal screening. Asterisks indicate clones containing an in-frame stop codon in the modified region. (d) Peptide sequences of clones found in this screening (N-random: 7G6/- [#1 in (b)], C-random: -/5A12 [#2 in (c)], 7G6/5A12: combined) and their phosphorylation assay results. Orange, blue, and gray bars show the Thr, Ser, and Ala substitutions at the phosphorylation site, respectively. Data are shown as the average values from three independent experiments with the SD. (e) The probe proteins used in (d) were separated by SDS-PAGE (10% acrylamide gel containing 15 μM Phos-tag), and detected by western blotting using anti-GFP antibody (for Venus). P and hypo-P indicate the phosphorylated and hypo-phosphorylated state, respectively.
FIGURE 5
Phosphorylation and dephosphorylation speeds of the probes by CDK2:cyclin A and PP2A:B55. (a) The S50, T50-NCP, and T50(7G6/5A12) probes were mixed with CDK2:cyclin A at time 0. Data are presented with the maximum phosphorylated state set to 100%. (b) The phosphorylation speed calculated from (a). (c) The S50, T50-NCP, and T50(7G6/5A12) probes were phosphorylated by CDK2:cyclin A, then mixed with p27Kip1 and PP2A:B55 at time 0 to start dephosphorylation. Data were standardized using a sample with CDK and without PP2A:B55 as a 100% control sample. (d) The dephosphorylation speed calculated from (c). (e) The phosphorylation assay of S50, T50, and other probes obtained in the screening (1F6/-, 1E1/-, -/1E10, and -/1F7) was performed as described in (a). (f) The phosphorylation rate calculated from (e). (g) The dephosphorylation assay of S50, T50-NCP, and other probes obtained in the screening (1C4/-, -/1B7, 1H5/-, and -/1G10) was performed as described in (c). (h) The dephosphorylation speed calculated from (g). In (a), (c), (e), and (g), representative data of three independent analyses are shown. In (b), (d), (f), and (h), the average values from three independent experiments are shown with the S.D.
FIGURE 6
Real-time monitoring of cell cycle progression in Xenopus egg extracts using the T50(7G6/5A12) probe. Xenopus interphase egg extracts were mixed with the T50(7G6/5A12) probe and cyclin B1ΔN172 (CycB1-ΔN), then divided into two tubes; one was used for real-time measurements of the luminescence (a), and aliquots of the other were fractionated at the indicated timepoints and analyzed by western blotting using anti-Apc3 antibody (b). P and hypo-P indicate the phosphorylated and hypo-phosphorylated state of Apc3, respectively. At 75 min (indicated by dashed line), p27Kip1 was added to the extracts to induce mitotic exit.
