1. A Quantitative Model for Ordered Cdk Substrate Dephosphorylation during Mitotic Exit 
Céline Bouchoux, Frank Uhlmann 
Cell 
Volume 147, Issue 4, Pages (November 2011)
DOI: /j.cell
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3. Figure 1 Ordered Cdk Substrate Dephosphorylation during Mitotic Exit
(A) Western blot analysis of phosphorylation-dependent mobility shifts and levels of the indicated Cdk substrates and cyclins during synchronous cell-cycle progression at 16°C. Red-, blue-, and green-shaded boxes indicate the dephosphorylation time windows of early, intermediate, and late substrates, respectively.
(B) Clb2 was immunoprecipitated at the indicated times, and its associated kinase activity against histone H1 was measured. Cdc14 release from the nucleolus was scored by indirect immunofluorescence. The data in (B), (C), and (E) are representative of all cultures and stem from the Fin1-Pk strain.
(C) FACS analysis of DNA content.
(D) Mitotic progression was monitored by scoring elongated anaphase spindles, confirming reproducible cell-cycle synchrony between the individual cultures.
(E) Examples of cells during the time course, stained for DNA with 4′,6-diamidino-2-phenylindole (DAPI) and an anti-tubulin antibody. Scale bar, 7 μm.
See also Figure S1.
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4. Figure 2
Ordered Substrate Dephosphorylation in Response to Ectopic Cdc14 Expression
(A) MET3-CDC20 cells were arrested in mitosis by Cdc20 depletion at 25°C, and GAL1 promoter-driven Cdc14-myc expression was induced. Aliquots of the cultures were taken for western blot analysis, measurement of Clb2/Cdk activity against histone H1, and Cdc14-myc activity against para-nitrophenyl phosphate (pNPP). FACS analysis of DNA content and immunofluorescence staining of tubulin and septin (Cdc11) confirmed that cells remained in a metaphase-like state during the course of the experiment. Scale bar, 7 μm.
(B) As (A), but Cdc14 induction was terminated in half of the culture by glucose addition after 30 min.
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5. Figure 3
Persistent Cdk Activity Prevents Late but Not Early Substrate Dephosphorylation
(A) Western blot analysis to follow electrophoretic mobility and levels of the indicated proteins during cell-cycle progression in presence of indestructible Clb2Δdb at 16°C.
(B) Clb2 was immunoprecipitated, and its associated kinase activity against histone H1 was measured. The analysis was performed in parallel and is comparable to that shown in Figure 1. Cdc14 release from the nucleolus was scored by indirect immunofluorescence. The data in (B), (C), and (E) are representative and stem from the Fin1-Pk strain.
(C) FACS analysis of DNA content.
(D) Mitotic progression was monitored by scoring elongated anaphase spindles, confirming reproducible cell-cycle synchrony between the individual cultures.
(E) Examples of cells during the time course, stained with DAPI and an anti-tubulin antibody. Scale bar, 7 μm.
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6. Figure 4 Reconstitution of In Vitro Clb2/Cdk Substrate Phosphorylation
(A) Purified Cdk substrates, Clb2/Cdk and Cdc14 (3 μg each), were separated on 12% SDS-PAGE and stained with Coomassie blue.
(B) Time course of Clb2/Cdk phosphorylation of purified Fin1, Ask1, Sli15, and Orc6. A total of 1.66 μM of each substrate was incubated with 33 nM of Clb2/Cdc28 and 100 μM ATP, including 0.5 μM of γ-33P-ATP, for 1 hr at 30°C. Aliquots of the phosphorylation reaction were retrieved at the indicated times and stopped by addition of SDS-PAGE loading buffer. Proteins were resolved by SDS-PAGE, the gel was fixed, dried, and phosphate incorporation was quantified by phosphoimager analysis.
See also Figure S2.
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7. Figure 5 Kinetic Parameters for Clb2/Cdk Substrate Phosphorylation
The indicated concentrations of purified recombinant Fin1, Ask1, Sli15, and Orc6 were incubated with 2 nM Clb2/Cdk for 90 s at 30°C in the presence of 100 μM ATP, including 0.5 μM of γ-33P-ATP. The reactions were resolved by SDS-PAGE, and phosphate incorporation was quantified by phosphoimager analysis. The mean and standard deviation from three independent experiments are presented. The phosphate incorporation data were fitted using a nonlinear least-square regression to the Michaelis-Menten equation. kcat, KM, and kcat/KM are listed with their standard errors and the coefficient of determination R2 of the fit.
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8. Figure 6
Greater Catalytic Efficiency of Cdc14 for Its Early Substrates
Fully phosphorylated Fin1, Ask1, Sli15, and Orc6 were prepared as in Figure 4. A total of 640 nM of each substrate was subjected to dephosphorylation by addition of 6.4 nM (Fin1 and Sli15) or 64 nM (Ask1 and Orc6) Cdc14. Aliquots of the reaction were taken in 15 s intervals and resolved by SDS-PAGE. The remaining phosphosubstrate concentration at each time was quantified by phosphoimager analysis, from which substrate dephosphorylation was inferred. Mean and standard deviation from three independent experiments are presented.
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9. Figure 7
Late Substrate Dephosphorylation Is Impeded by Persisting Clb2/Cdk Activity and Substrate Competition
(A) Cdc14 dephosphorylates early, but not late, substrates in the presence of persisting Clb2/Cdk activity. A total of 500 nM Fin1, Sli15, and Orc6 was phosphorylated by 50 nM Clb2/Cdk for 5 min at 30°C in the presence of 100 μM ATP, including 0.5 μM of γ-33P-ATP. Further phosphorylation was (left), or was not (right), terminated by addition of 15 mM EDTA. A total of 53, 133, or 330 nM of Cdc14 was now added (t = 0), and aliquots of the reaction were taken every 15 s to assess the percentage of substrate dephosphorylation by SDS-PAGE followed by phosphoimager analysis. Mean and standard deviation from two independent experiments are shown.
(B) The presence of early substrates delays late substrate dephosphorylation, but not vice versa. Fin1, Sli15, and Orc6 were Clb2/Cdk phosphorylated as in Figure 4. The phosphosubstrates were then incubated at a concentration of 640 nM either individually with 128 nM Cdc14 (left), or the three substrates were mixed together in a single reaction, and dephosphorylation occurred in competition (right). Aliquots were taken every 15 s, and substrate dephosphorylation was quantified. Mean and standard deviation from three independent experiments are presented.
See also Figure S4.
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10. Figure S1
Ordered Substrate Dephosphorylation Detected by Cdk Phosphorylation-Specific Antibodies, Related to Figure 1
Substrate dephosphorylation timing in Figure 1 was evaluated by their electrophoretic gel mobility shifts. To confirm that Cdk substrate dephosphorylation indeed occurs with sequential timing during mitotic exit, we analyzed the phosphorylation status of three representative Cdk substrates using Cdk phosphosite-specific antibodies. Cells carrying Fin1, Sli15, or Orc6, respectively, fused to a Pk epitope tag were arrested in metaphase and released into synchronous progression through anaphase and mitotic exit by depletion and readdition of Cdc20 under control of the MET3 promoter at 23°C. Cell-cycle progression was halted in the next G1 by pheromone α factor addition. Fin1, Sli15 and Orc6 were immunopurified from cell extracts at the indicated times and their phosphorylation status was analyzed by western blotting. Several Cdk phosphosite-specific antibodies have been developed that each show some context dependence. We found that antibodies 2325 (Fin1 and Sli15) and 2321 (Orc6, both Cell Signaling) gave reproducible Cdk phosphorylation-specific signals. Substrate detection using an α-Pk antibody served as the loading control in each case. Synchrony of cell-cycle progression was monitored by scoring anaphase (disappearance of mononucleated cells) and cytokinesis (disappearance of large budded cells). This analysis confirms the early (Fin1), intermediate (Sli15) and late (Orc6) dephosphorylation timing of these three substrates during mitotic exit.
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11. Figure S2
Specific and Near-Complete In Vitro Phosphorylation of Recombinant Substrates by Clb2/Cdk, Related to Figure 4
(A) 1.66 μM of recombinant, purified His6-Ask1, His6-Fin1, His6-Sli15 or Orc6-His6 were incubated with 33 nM of Clb2/Cdk in kinase buffer containing 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2, 150 mM NaCl with or without 100 μM ATP. After 1 hr at 30°C, kinase reactions were stopped by addition of 15 mM EDTA, and proteins were separated by 12% SDS-PAGE. The gel was stained with Coomassie Blue. The near complete mobility shift of the Cdk substrates suggests that substrate phosphorylation proceeded close to completion.
(B) The stained bands corresponding to the phosphorylated substrates were excised and subjected to trypsin digestion followed by mass spectrometric analysis. A second aliquot of the phosphorylation reactions was processed for mass spectrometric analysis without prior gel separation. The combined peptide coverage of the substrates was for Fin1 78%, Ask1 92%, Sli15 58% and Orc6 71%. Identified phosphorylation sites are depicted by asterisks along a graphic representation of the four substrates. Green bars represent the position of Cdk phosphorylation consensus motifs. Red asterisks correspond to phosphorylation at these sites, while brown asterisks mark phosphorylation at sites different from Cdk consensus sites. Cdk consensus phosphorylation sites for which we did not obtain information because of incomplete peptide coverage are indicated by ”N.” This analysis confirms that the Cdk substrates were phosphorylated specifically and efficiently under the conditions of our in vitro phosphorylation reactions.
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12. Figure S3
Supporting Analysis to Determine kcat/KM of Cdc14 for the Four Cdk Substrates from Their Dephosphorylation Progress Curves, Related to Figure 6
(A) Exponential fit of the dephosphorylation progress curves to an integrated form of the Michaelis-Menten equation. A nonlinear least-square fit of the experimental progress curves to an integrated exponential form of the Michaelis-Menten equation is shown. Even prolonged incubation with high concentrations of Cdc14 resulted in residual persistence of a small fraction of the phosphosubstrate that appeared refractory to dephosphorylation (not shown). This might have been due to denaturation of the substrate in the reaction. To reflect this, the actual phosphosubstrate concentration at the beginning of the timecourse (Sp0) was corrected downward. This allowed a good fit to exponential progress curves, indicated by a coefficient of determination R2 close to 1, and determination of kcat/KM from the exponent (Sp0 and kcat/KM are given together with their 95% confidence intervals). The individual values of kcat and KM are not accessible from this analysis.
(B) Supporting analysis to constrain kcat/KM by kinetic simulations of the dephosphorylation progress curves. Cdk phosphosubstrate dephosphorylation by Cdc14 involves reversible association of the phosphosubstrate and phosphatase, characterized by the on and off rates k1 and k-1, respectively. This is followed by transfer of the phosphate from the substrate onto the Cdc14 catalytic cysteine residue, at a rate described by k2. The phosphoenzyme intermediate is hydrolyzed by water to release phosphate and regenerate the free enzyme at a rate k3, previously determined in pre-steady-state burst analyses using synthetic substrates to 20 ± 5 s-1 (Wang et al., 2004). Because of the fast hydrolysis of the phosphoenzyme intermediate, and because of the large excess of water in our assays and the intracellular environment, reactions two and three were considered irreversible for the purpose of our analysis. Empirical kinetic simulations generated good fits to the experimental progress curves for a wide range of rate constants, i.e., the individual rate constants k1, k-1 and k2 were not well constrained by the experiment. Only in the case of Fin1, k2 was relatively well constrained to between 8 and 12 s-1. However, the characteristic kcat/KM ratio, that takes into account up to and including the first irreversible step in the kinetic mechanism kcat/KM = k1·k2/(k2+k-1), was well constrained by the experiment over a very wide range of possible rate constants. We performed 40 kinetic simulations, fixing each of the individual rate constants over a wide range of values, covering any biologically relevant numbers. Despite the wide possible range of individual rate constants, kcat/KM remained well constrained within a narrow margin for each of the substrates. The rate constants that gave rise to the kinetic simulations shown in Figure 6 are, Fin1: k1 = 1.27·107 M-1 s-1, k-1 = 5.65 s-1, k2 = 9.75 s-1; Ask1: k1 = 1.50·108 M-1 s-1, k-1 = 9740 s-1, k2 = s-1; Sli15: k1 = 1.47·108 M-1 s-1, k-1 = 547 s-1, k2 = 9.82 s-1; Orc6: k1 = 3.50·108 M-1 s-1, k-1 = 12.8·103 s-1, k2 = 9.27 s-1.
(C) In order to derive kcat/KM from the kinetic simulations, k1·k2 was plotted as a function of k2+k-1 from all 120 simulations for each substrate in (B) that covered the parameter space for the three rate constants. kcat/KM = k1·k2/(k2+k-1) is the slope of the linear relationship between the two terms. A coefficient of determination R2 close to 1 suggests that a linear function correctly describes the relationship between k1·k2 and k2+k-1 and that the ratio k1·k2/(k2+k-1) is well constrained by the kinetic simulations. kcat/KM and its 95% confidence intervals from this analysis are shown.
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13. Figure S4
Phosphorylation-Dependent and -Independent Cdc14 Substrate Interactions, Related to Figure 7
(A) GST-Cdc14 shows comparable phosphatase activity to His6-Cdc14. The indicated amounts of purified His6-Cdc14, GST-Cdc14, or their phosphatase-inactive counterparts His6-Cdc14(C283S) and GST-Cdc14(C283S) carrying a mutation in the active site cysteine residue, were incubated in 200 μl phosphatase buffer with 100 mM para-nitrophenyl phosphate (pNPP) at 30°C. The reaction was stopped after 15 min and the absorbance at 410 nM determined. Having confirmed that GST-Cdc14 is active as a phosphatase, and therefore likely adopts its native conformation, we used GST-Cdc14(C283S), for the following interaction studies. We chose the catalytically inactive form to prevent protein dephosphorylation during the binding reaction.
(B) Phosphorylation-dependent and -independent substrate interaction with GST-Cdc14(C283S). Thirty picomoles Fin1, Ask1, Sli15 or Orc6 were incubated at a concentration of 1.33 μM in kinase buffer with 50 nM Clb2/Cdc28 at 30°C, with or without 100 μM ATP for 1 hr. The kinase reactions were stopped by EDTA addition and diluted to 1 μM substrate concentration in phosphatase buffer. The phosphorylated or nonphosphorylated substrates were then incubated at this concentration with 3 picomoles of GST or GST-Cdc14(C283S) immobilized on a small volume of glutathione sepharose beads. After 5 min incubation, the beads were thoroughly washed in phosphatase buffer, and bound protein eluted at 65°C in SDS-PAGE loading buffer. Input and bound proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. The stained gels were scanned and band intensities were quantified (ImageQuant, GE Healthcare). The relative binding, normalized to that of phosphorylated Fin1, in three independent experiments is shown (the mean and standard error are presented). The interactions seen in this assay were strongest for substrates for which Cdc14 displayed a high catalytic efficiency (Fin1 and Sli15) and weaker for substrates with a lower catalytic efficiency (Orc6 and Ask1). In case of Orc6, the degree of specific binding is more difficult to estimate because of reproducible background binding of the nonphosphorylated protein also to GST. Cdc14 interacted with its substrates more strongly if they were Cdk phosphorylated, although phosphorylation-independent interaction was also apparent, in particular in the case of the high catalytic efficiency substrate Fin1. These results are consistent with the possibility that substrate affinity contributes to the high catalytic efficiency of Cdc14 for its early substrates and that this affinity is in part achieved by a phosphorylation-independent interaction. However, this semiquantitative pull-down assay does not allow absolute affinity measurements.
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