Shevah-Sitry D et al. (2022), Role of phosphorylation of Cdc20 in the regulat...

XB-ART-59271

Proc Natl Acad Sci U S A 2022 Aug 30;11935:e2210367119. doi: 10.1073/pnas.2210367119.

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Role of phosphorylation of Cdc20 in the regulation of the action of APC/C in mitosis.

Shevah-Sitry D , Miniowitz-Shemtov S , Teichner A , Kaisari S , Hershko A .

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The ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for the control of mitosis, and its activity is subject to tight regulation. In early mitosis, APC/C is inhibited by the mitotic checkpoint system, but subsequently it regains activity and promotes metaphase-anaphase transition by targeting cyclin B and securin for degradation. The phosphorylation of APC/C by the mitotic protein kinase Cdk1-cyclin B facilitates its interaction with its coactivator Cdc20, while the phosphorylation of Cdc20 inhibits its binding to APC/C. This raises the question of how Cdc20 binds to APC/C under conditions of high Cdk1 activity. It seemed possible that the opposing action of protein phosphatases produces a fraction of unphosphorylated Cdc20 that binds to APC/C. We found, however, that while inhibitors of protein phosphatases PP2A and PP1 increased the overall phosphorylation of Cdc20 in anaphase extracts from Xenopus eggs, they did not decrease the levels of Cdc20 bound to APC/C. Searching for alternative mechanisms, we found that following the binding of Cdc20 to APC/C, it became significantly protected against phosphorylation by Cdk1. Protection was mainly at threonine sites at the N-terminal region of Cdc20, known to affect its interaction with APC/C. A model is proposed according to which a pool of unphosphorylated Cdc20, originating from initial stages of mitosis or from phosphatase action, combines with phosphorylated APC/C and thus becomes stabilized against further phosphorylation, possibly by steric hindrance of Cdk1 action. This pool of APCCdc20 appears to be required for the regulation of APC/C activity at different stages of mitosis.

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Species referenced: Xenopus laevis
Genes referenced: btg3 cdc20 cdc27 cdk1 cdkn1b gopc lpp myc npy4r ptpa
GO keywords: mitotic cell cycle [+]

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	Figure 1. Influence of phosphatase inhibitors on the binding of Cdc20 to APC/C in anaphase extracts from Xenopus eggs. (A) Effects of okadaic acid and calyculin A on the phosphorylation state of Cdc27 and Cdc20 in anaphase extracts. Interphase or anaphase extracts from Xenopus eggs were prepared as described under Materials and Methods. Anaphase extracts were incubated at 23 °C for 30 min with okadaic acid (Calbiochem #MBS459620) or calyculin A (Cell Signaling #CST-9902S) (1 mM, each), as indicated. DMSO solvent was at a final concentration of 0.5% (v/v) in all incubations. Where indicated, the sample was treated with lambda phosphatase (see Materials and Methods). Samples of 0.1 mL of extracts were subjected to SDS-PAGE and were immunoblotted as indicated. Numbers on the right indicate the position of molecular mass marker proteins. Ana., anaphase; Ok. ac, okadaic acid; Cal. A., calyculin A; lPP, lambda phosphatase. (B) Effects of phosphatase inhibitors on the binding to APC/C of total and T79-phosphorylated Cdc20. Anaphase extracts from Xenopus eggs were incubated with or without phosphatase inhibitors as in A, and then were subjected to immunoprecipitation with anti-Cdc27 antibodies or with nonimmune IgG, as indicated. Immunoprecipitation was carried out as described under Materials and Methods, except that beads were washed four times with buffer A that contained 0.3 M NaCl and 0.1% (vol/vol) NP-40 and then twice with buffer A. Where indicated, samples were treated with lambda phosphatase (“lPP”), as described under Materials and Methods. Samples of immunoprecipitates derived from 5 mL extracts (lanes 4–12) or 0.1 mL of unfractionated extracts (lanes 1–3) were subjected to immunoblotting with antibody directed against Xenopus Cdc20 (Upper) or against its T79-phosphorylated derivative (Center). (Lower) Immunoblot form Cdc27 bait in same immunoprecipitates. Asterisks, cross-reacting material; arrows, position of pT79-Cdc20. (C) Quantitation of data similar to B. Data from three experiments similar to B were quantified. APC/C-bound total Cdc20 or pT79-Cdc20 were expressed as the percentage of each in unfractionated extracts.
	Figure 2. Differences in effects of Cdk1 on free and APC/C-bound Cdc20. (A) Effects of Cdk1-catalyzed phosphorylation of free or APC/C-bound Cdc20 on its association with APC/C. (Upper) Free myc-Cdc20 (50 nM) was phosphorylated in a reaction mixture that contained in a volume of 50 mL: 40 mM Tris-HCl (pH 7.6), 3 mM MgCl2, 1 mM ATP, 10 mM phosphocreatine, 0.1 mg/mL creatine phosphokinase, 2 mg/mL BSA and 40 units/mL Cdk1-cyclin B (referred to as “Cdk1”). Following incubation at 23 °C for the time periods indicated, the binding of myc-Cdc20 to immunopurified APC/C (on 4 mL anti-Cdc27 beads) was assayed following washing of beads as described under Materials and Methods. (Lower) myc-Cdc20 pre-bound to immunopurified APC/C (see Materials and Methods) was incubated with Cdk1 under conditions similar to those described above and then beads were washed and the amount myc-Cdc20 that remained associated with APC/C was estimated by immunoblotting. All samples had been subjected to treatment with lambda phosphatase. “No APC/C,” nonspecific absorption of myc-Cdc20 to anti-Cdc27 beads without APC; “No Cdk1,” samples incubated without Cdk1 for 30 min. (B) Quantitation of data from A. Background values (“No APC/C”) were subtracted and myc-Cdc20 bound to APC/C were expressed as the percentage of the control sample incubated without Cdk1. (C) Effects of phosphorylation by Cdk1 of free or APC/C-bound myc-Cdc20 on the activity of APC/C. Quantitation of APC/C activity data from SI Appendix, Figs. S1 A and B. Background values if ubiquitylated 125I-cyclin obtained without added myc-Cdc20 (lanes 2 in SI Appendix, Figs. S1 A and B, “No Cdc20”) were subtracted and results were expressed as the percentage of sample incubated without Cdk1 for 30 min (lane 3 in SI Appendix, Fig. S1A and lane 4 in SI Appendix, Fig. S1B). (D) Phos-tag-SDS-PAGE analysis of patterns of phosphorylation by Cdk1 of free and APC/C-bound myc-Cdc20. The experiment was similar to that described in A, except free myc-Cdc20 was not applied to APC/C, samples were not treated with lambda phosphatase and were subjected to Phos-tag-SDS-PAGE (see Materials and Methods).
	Figure 3. Protection of sites affecting the binding of Cdc20 to APC/C against phosphorylation by Cdk1. (A) Protection against Cdk1 action of a mutant of Cdc20 (5AV) that can be phosphorylated only on three T residues at its N-terminal region. Free or APC/C-bound forms of the indicated wild-type or mutant myc-Cdc20s were incubated (at 23 °C for 30 min) with or without Cdk1, as described for Fig. 2A. Myc-Cdc20 bound to APC/C was detected by immunoblotting and was expressed as the percentage of the control incubated without Cdk1. In separate samples of immunoprecipitates, not treated by lambda phosphatase, the activity of APC/C was determined as described for SI Appendix, Figs. S1 A and B and results were expressed as described for Fig. 2C. (B) Patterns of phosphorylation of free and APC/C-bound 5AV mutant of Cdc20, analyzed by Phos-tag-SDS-PAGE. Free of APC/C-bound mutant of myc-Cdc20 were subjected to phosphorylation by Cdk1, as described in Fig. 2 A and D. Samples were resolved by Phos-tag-SDS-PAGE and were immunoblotted with Myc-tag. Phosphorylated free myc-Cdc20 5AV was applied at one-half (lane 3, “0.5”) or at equal amount (lane 4, “1.0”) to that of wild-type myc-Cdc20 (lane 5).
	Figure 4. Possible roles of Cdc20 phosphorylation in the regulation of APC/C activity in mitosis. See the Discussion. (A) Inhibition of APC/C in active mitotic checkpoint. (B) Release of APC/C from mitotic checkpoint inhibition. Phosphorylation of Cdc20 refers to sites known to affect its interaction with APC/C, such as the three T residues at its N-terminal region. APC/C-P, Cdc20-P, phosphorylated forms of APC/C or Cdc20.
	Fig. S1. Effects of phosphorylation of free or APC/C-bound myc-Cdc20 on the activity of APC/C. (A) Time-course of the effects of Cdk1-catalyzed phosphorylation of free myc-Cdc20 on its stimulation of APC/C activity. The experiment was similar to that described for Fig. 2A, upper panel, except that samples were not subjected to treatment with lambda phosphatase. The activity of APC/C in the ubiquitylation of 125I-cyclin B was determined as described under Materials and Methods, in samples of 1 l of APC/C beads. “No Cdc20”, activity of endogenous APC/C without added myc-Cdc20. Numbers on the right indicate the position of molecular mass marker proteins. (B) Time-course of the effects of Cdk1-catalyzed phosphorylation of APC/C-bound Cdc20 on APC/C activity. The experiment was similar to that described under Fig. 2A, lower panel, but without lambda phosphatase treatment of samples. APC/C activity was assayed as described under Materials and Methods, in samples of 1 l of anti-Cdc27 beads containing either APC/C without myc-Cdc20 (lanes 2, 3) or APC/C to which myc-Cdc20 had been bound (lanes 4-7).
	Fig. S2. (A) Effects of okadaic acid and calyculin A on the activity of APC/CmycCdc20 incubated with Cdk1. Immunopurified APC/C to which myc-Cdc20 has been bound (“APC/C-myc-Cdc20”) was incubated under conditions as in Fig. 2A, with additions as indicated. Okadaic acid and calyculin A were supplemented at 0.5 M. Following incubation at 23ºC for 30 min, beads were washed and APC/C activity was determined as described under Materials and Methods. (B) Preincubation of APC/C from HeLa cells with Cdc20 reduces the extent of the inhibition of its action by Cdk1. Phosphorylated APC/C was immunopurified from extracts of mitotic HeLa cells as described under Materials and Methods. Left panel, “Cdc20 + Cdk1, then APC/C”: Free myc-Cdc20 was first incubated with Cdk1 and ATP, under conditions similar to those described in Fig. 2A, except that the reaction volume was 10 l, and incubations were carried out at 18 ºC. Cdk1 action was terminated by at the times indicated by the addition of 3 M p27kip1, and then samples were mixed with HeLa cell APC/C (on 1 l of anti-Cdc27 beads), at 1,400 rpm for 15 min. Finally, ubiquitylation mixture containing 125I-cyclin B (see Materials and Methods) was added and incubation was continued at 18 ºC for 30 min, with shaking at 1,400 rpm. Right panel, “Cdc20 + APC/C, then Cdk1”: Experimental conditions were similar to those described for left panel, except that the order of incubations was reversed, so that myc-Cdc20 was first mixed with APC/C from HeLa cells for 15 min, then Cdk1 was added for the time periods indicated, followed by 125I-cyclin B ubiquitylation mixture. Results were expressed as in Fig. 2C.
	Fig. S3. Effects of Cdk1-catalyzed phosphorylation of different free phosphorylation site mutants of Cdc20 on their action to stimulate the activity of APC/C. Wild type (“WT”) myc-Cdc20 or its mutants as indicated, were incubated (23 ºC,30 min) with or without Cdk1, under conditions similar to those described in Fig. 2A, and then were added to immunopurified APC/C on anti-Cdc27 beads, as described under Materials and Methods. The activity of APC/C on ubiquitylation of 125I-cyclin B (see Materials and Methods) was estimated in samples of 1 l APC/C beads. The activity of APC/C following treatment of Cdc20 constructs with Cdk1 was expressed as the percentage of respective controls incubated without Cdk1.

References [+] :

Alfieri, Molecular basis of APC/C regulation by the spindle assembly checkpoint. 2016, Pubmed