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Graphical abstract
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Figure 1. Apc1-300L dephosphorylation in PP2A-B55-activated Xenopus egg extracts
(A) Schematic depicting the mutual regulation between CDK and PP2A-B55. Downstream targets remain underexplored.
(B) Circular plots (xiVIEW) show intra- and inter-protein cross-links in rAPC/C complexes, comparing unphosphorylated (top) to hyper-phosphorylated states (bottom). Gray lines represent all Apc subunit cross-links; Apc1-300L cross-links are highlighted in red.
(C) Phosphatase assay of Cdc20 (T79) and Apc1-300L in mitosis. After phosphorylation with CDK-Cyclin A and [γ-32P]-ATP, substrates were added to CSF extracts with DMSO or 2 μM okadaic acid. Samples were analyzed by SDS-PAGE and autoradiography.
(D) Quantification of (C), normalizing 32P levels to initial intensities, with 0 min set to 1.0. Data represent the average of three experiments ± SEM.
(E) Phosphatase assay of Cdc20 (T79) and Apc1-300L in PP2A-B55-activated extracts. CSF extracts underwent immunodepletion using either beads (MockΔ) or Gwl antibody-bound beads (GwlΔ) at 23°C, and then either buffer or 5 μM pEnsa was added. Analysis was performed as in (C).
(F) Quantification of (E), with data from three (Cdc20) or four (Apc1-300L) experiments ± SEM and initial intensities set to 1.0.
(G) Short-interval Apc1-300L dephosphorylation by PP2A-B55, following the procedure in (E) and quantified as in (F).
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Figure 2. Apc1-300L dephosphorylation by PP2A-B55, not PP1, in Xenopus egg extracts
(A) Multiple alignment of Apc1-300L sequences from various species (Hs, Homo sapiens; Pt, Pan troglodytes; Mm, Mus musculus; Xl, Xenopus laevis; Xt, Xenopus tropicalis), with the CDK phosphorylation site highlighted in yellow and orange. Conserved amino acids are marked with asterisks, while similar ones are denoted by colons and dots.
(B) Investigation of endogenous Apc1-300L phosphorylation status in PP2A-B55 activated CSF extracts. Beads (MockΔ) or Gwl antibody-bound beads (GwlΔ) were mixed with CSF extracts at 23°C for 20 min. Before recovering the immunodepleted extracts, either buffer, 5 μM pEnsa or 2 μM okadaic acid was introduced. Samples taken at the indicated time points were analyzed by SDS-PAGE and immunoblotting.
(C) Comparison of Apc1-300L dephosphorylation by PP2A-B55 against PP1, using methods from (B) with added NIPP1 (12 μM) and pEnsa (5 μM) and then p27 to induce mitotic exit.
(D) In vitro phosphatase assay using phosphorylated Apc1-300L and purified PP2A-B55. Post CDK-Cyclin A phosphorylation, Apc1-300L was incubated with mock or PP2A-B55, with/without 10 μM pEnsa, and analyzed by SDS-PAGE and immunoblotting.
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Figure 3. PP2A-B55 activation during mitosis deactivates APC/C via Apc1-300L
(A) Cyclin B degradation was evaluated following premature PP2A-B55 activation or with 5 μM pEnsa (see
Figure 2
B for details). Samples post immunodepletion using XErp1 antibody-bound (XErp1Δ) or unbound (MockΔ) beads were analyzed by SDS-PAGE and immunoblotting.
(B) Premature PP2A-B55 activation affects APC/C substrate degradation. 35S-labeled Nek2A, Cyclin B, and Cyclin BΔ67 (stable control) tested in mock- or Gwl-depleted extracts with or without 5 μM pEnsa were analyzed by SDS-PAGE and autoradiography at the indicated times.
(C) Quantitative analysis of (B), with data means (±SEM) from three experiments. Intensities at 0 min were set to 1.0.
(D) rAPC/C destruction assay showing Apc1-300L’s involvement in PP2A-B55-mediated APC/C deactivation. Top: experimental schematic. 35S-labeled substrate quantification is shown (data from three experiments ± SEM). Refer to
Figures S3
A and S3B.
(E) In vitro ubiquitylation assay in the PP2A-B55-activated context. APC/C activity and Cdc20 levels were quantified post immunoprecipitation (procedure in
Figure 2
B), relative to CSF intensity, averaged over three trials (±SEM). See also
Figure S3
E.
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Figure 4. Impact of Apc1-300L dephosphorylation on APC/C-Cdc20 interaction
(A) Schematic of the dissociation assay.
(B) Analysis of Cdc20 dissociation from APC/C in Xenopus egg extracts. FLAG-Cdc20-5A was introduced into Cdc20-depleted extracts with MG132, and the APC/C-Cdc20 complex’s dissociation was tested under various conditions (mock or Gwl depletion). Samples were analyzed via SDS-PAGE and immunoblotting.
(C) Dynamic reassociation of endogenous Cdc20 with APC/C in Xenopus egg extracts, following the approach in (B).
(D) Assays for de novo Cdc20 loading. Activation of PP2A-B55 lessens Cdc20’s APC/C binding. After mock and Gwl depletion, extracts were supplemented with FLAG-Cdc20-5A and Apc3-bound Dynabeads, and the retrieved APC/C-Cdc20 complex was examined using SDS-PAGE and immunoblotting.
(E) Model overview: CDK1-PP2A-B55 interplay and its role in cell cycle oscillation through Apc1-loop300. During interphase, inactive CDK1 allows PP2A-B55 to dephosphorylate Apc1-300L, preventing Cdc20 binding. Entering mitosis, activated CDK1 phosphorylates Apc1-300L and subsequently displaces of Apc1-300L, enabling the APC/C-Cdc20 complex formation and Cyclin B destruction. CDK1 also inhibits PP2A-B55 through the Gwl-Ensa/Arpp19 pathway, preserving this active state. Cdc20’s interaction with the APC/C is dynamic. If PP2A-B55 activates prematurely (e.g., GwlΔ scenario), then it disrupts Cdc20’s association, even if CDK is active. This effect can be counteracted by pEnsa (as indicated by GwlΔ+pEnsa), pinpointing PP2A-B55 as the principal regulator of Apc1-300L and Cdc20 dynamics. The APC/C functions as a pivotal substrate, with its activities fine-tuned by CDK and PP2A-B55, ensuring cell cycle oscillation.
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Figure S1. Key residues crucial for Apc1-300L binding to the APC/C, related to Figure 2.
(A) Schematic diagram of Apc1-300L constructs (residues 294-399). Conserved CDK phosphorylation consensus sites are shown as 'S', and their respective alanine substitutions by 'A'.
(B) Binding assay of purified Apc1-300L fragments to the APC/C complex in anaphase extract.
Apc1-300L constructs were immobilized on FLAG-M2 agarose beads and then incubated with
interphase extract supplemented with non-degradable purified Cyclin B∆167 at 23˚C for 1.5
hours, to mimic mitotic entry to anaphase. Proteins bound to the beads were eluted, and a
portion of each eluate was treated with CIP. Both eluted and CIP-treated samples were analyzed by SDS-PAGE and immunoblotted with specific antibodies.
(C) Quantification of binding efficiency (B). Apc2 levels relative to FLAG-Apc1-300L are
presented. Intensities were normalized to those obtained with wild type Apc1-300L. Data are
the mean ± SEM from three independent experiments.
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Figure S2. Analysis of Apc1-300L dephosphorylation in the presence of phosphatase
inhibitors, related to Figure 2.
(A) Apc1-300L, post phosphorylation with purified CDK-Cyclin A and [γ-32P]-ATP, was introduced to anaphase extracts containing phosphatase inhibitors: 4 µM okadaic acid, 64 µM B56i,
12 µM NIPP1, or 5 µM pEnsa. Subsequently, 0.35 µM purified p27 was added to facilitate
mitotic exit. The samples were then analyzed by SDS-PAGE and autoradiography.
(B) Substrate validation for phosphatase assay. Phosphorylation of wild type Apc1-300L and its
derivatives (7T or 7A) following incubation with purified CDK-Cyclin A and [γ-32P]-ATP, confirming that CDK-Cyclin A phosphorylates seven conserved CDK sites as indicated in Figure S1A.
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Figure S3. Impact of PP2A-B55 activation on APC/C activity, related to Figure 3.
(A) APC/C-mediated substrate destruction assay using rAPC/C. This experiment underscores
the role of Apc1-300L in the deactivation of APC/C by PP2A-B55. The rAPC/C samples were
incubated with CSF extract depleted of APC/C and supplemented with 150 µM MG132 at 23˚C
to allow phosphorylation. These phosphorylated rAPC/C samples were subsequently introduced into APC/C-depleted anaphase extracts, either mock-depleted or Gwl-depleted, for
dephosphorylation at 23˚C for 40 min. 35S-labelled substrates—Nek2A, Cyclin B, and a version
of Cyclin B lacking the N-terminal 67 residues (Δ67, stable control)—were added to start the
destruction assay at 23˚C. Samples from the extracts were processed through SDS-PAGE and
visualized using autoradiography.
(B) Quantification of (A). The relative levels of 35S-labelled Nek2A were quantified and averaged
across three experiments (± SEM). The intensity at 0 min was arbitrarily set to 1.0. (C) Initial rAPC/C input analysis used in (A): Samples were collected immediately after adding
rAPC/C to the indicated anaphase extracts, and analyzed by SDS-PAGE and immunoblotting
with the indicated antibodies (* denotes an irrelevant lane).
(D) Co-immunoprecipitation of Cdc20 with APC/C in Gwl-depleted CSF extract. APC/C was
purified from CSF extract at the indicated times after Gwl depletion using anti-Apc3
antibody-conjugated beads, washed, and a portion was subjected to CIP treatment. All samples were eluted with SDS-sample buffer, analyzed by SDS-PAGE, and immunoblotting with
the indicated antibodies.
(E) An in vitro ubiquitylation assay for APC/C in a PP2A-B55 activated context. Endogenous
APC/C was purified from CSF extracts at various times post Gwl depletion by immunoprecipitation and followed by ubiquitylation assay using methylated ubiquitin and FLAG-Ub-K0-Cyclin
B as the substrate at 23˚C. Samples taken at the indicated times were examined using
SDS-PAGE and immunoblotting.
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Figure S4. Dynamic interaction between APC/C and its co-activators, related to Figure 4.
(A) Schematic of the experimental design to assess Cdc20 dissociation dynamics from rAPC/C
in Xenopus egg extracts.
(B) The wild type rAPC/C and its variant lacking Apc1-300L were phosphorylated in CSF
extracts devoid of Cdc20 and APC/C and supplemented with 150 µM MG132 at 23˚C.
FLAG-Cdc20-5A was then added to form the rAPC/C-Cdc20 complexes. These complexes
were subjected to a dissociation assay in APC/C- and Gwl-depleted extracts. The
rAPC/C-Cdc20 complexes were purified at the indicated times, analyzed by SDS-PAGE and
immunoblotting.(C) Quantification of (B). The intensities of Cdc20 at 0 min were arbitrarily set to 1.0.
(D) Dynamics of Cdc20 and Cdh1 dissociation from APC/C in Xenopus egg extracts. 35S-labelled
Cdc20-5A or FLAG-Cdh1-4A were added to Cdc20-depleted CSF extract containing MG132 at
23˚C. The subsequent APC/C-Cdc20 or APC/C-Cdh1 complex was purified and subjected to
dissociation assay at 23˚C in Cdc20- and APC/C-depleted interphase extract or XB-CSF buffer.
APC/C-Cdc20 complex was purified at the indicated times. Samples were processed through
SDS-PAGE and visualized using autoradiography.
(E) Quantification of (D): The intensities at 0 min were arbitrarily set to 1.0.
(F) Dynamic association of FLAG-Cdc20-5A with APC/C in Xenopus egg extracts. Inactivation of
PP2A-B55 increases Cdc20 loading to the APC/C by “reopening the Apc1-300L gate”.
FLAG-Cdc20-5A with either buffer or pEnsa, were added to Cdc20-depleted CSF extracts with
either mock or Gwl depletion. Immunoprecipitated APC/C-Cdc20 complex was examined using
SDS-PAGE and immunoblotting.
(G) De novo Cdc20 loading assays on Apc1-300L-deleted rAPC/C. The interaction of Cdc20 with
the Apc1-300L-deleted rAPC/C complex remains stable despite PP2A-B55 activation. After
depleting endogenous APC/C from the specified extracts, they were supplemented with the
purified rAPC/C complex, introduced to FLAG-Cdc20-5A, and the resultant rAPC/C-Cdc20
complexes were examined by SDS-PAGE and immunoblotting.
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