Fujimitsu K , Yamano H .

MR/M010899/1 UK Research and Innovation | Medical Research Council (MRC), 205150/Z/16/Z Wellcome Trust (WT), BB/N008383/1 BBSRC, MR/M010899/1 Medical Research Council , 205150/Z/16/Z Wellcome Trust , Wellcome Trust

	Figure 1. Cdk‐dependent phosphorylation of Apc1‐loop500 ensures PP2A‐B56γ recruitment Multiple alignment of a predicted Apc1‐loop500 domain of vertebrate APC/Cs. The sequences corresponding to residues 515–584 in Xenopus tropicalis Apc1 are shown. A putative B56 binding region is coloured in green. Hs, Homo sapiens human; Pt, Pan troglodytes chimpanzee; Mm, Mus musculus mouse; Gg, Gallus gallus chicken; Xt, Xenopus tropicalis frog.Schematic diagrams of Apc1‐loop500 constructs. Apc1‐loop500 (residues 515–584) was fused with maltose binding protein (MBP) at N‐terminus and 6xHis at C‐terminus. Conserved Cdk phosphorylation sites (SP/TP) are shown as S or T, respectively. The putative B56 binding region is shown in green. The 11‐residue [LSPVPELRDST] deletion (∆11) and alanine substitution mutations to three Cdk phosphorylation sites (3A) or to B56 binding motif (L557A/V560A) are shown.Binding assay using MBP‐fused Apc1‐loop500 fragments and B56γ. Apc1‐loop500 WT or its derivatives (∆11 or 3A) were incubated with the 35S‐labelled Flag‐B56γ in interphase extract (Inter) or anaphase extract (Ana) supplemented with CycB∆167 at 23°C for 1 h. The bound proteins were recovered by amylose beads, separated by SDS–PAGE and detected by autoradiography or Coomassie brilliant blue (CBB) staining. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.Cdk‐dependent in vitro kinase assay of Apc1‐loop500. MBP‐fused WT or 3A Apc1‐loop500 fragment was incubated with Cdk2‐cyclin A in the presence of [γ‐32P]‐ATP at 23°C for 10 min, separated by SDS–PAGE and detected by autoradiography.Cdk can promote Apc1‐loop500 and B56 interaction. MBP‐fused WT or 3A Apc1‐loop500 fragment was incubated in the presence or absence of Cdk2‐cyclin A at 30°C for 60 min. MBP‐fused peptides (−/+ kinase) were isolated and incubated with purified Flag‐B56γ at 23°C for 30 min. The bound proteins were recovered by amylose beads and analysed by SDS–PAGE and immunoblotting with indicated antibodies.Quantification of (E) Bound B56γ to WT Apc1‐loop500 control (–Cdk) was arbitrarily set to 1.0. Error bars, SEM from three independent experiments.
	Figure EV1. Effects of mutations at Cdk sites of Apc1‐loop500 towards B56γ binding and the specificity of Apc1‐loop500 to B subunits Schematic diagrams of Apc1‐loop500 constructs used (B). Same as Fig 1B, Apc1‐loop500 (residues 515–584) was fused with maltose binding protein (MBP) at N‐terminus and 6xHis at C‐terminus. Conserved Cdk phosphorylation sites (SP/TP) are shown as S or T, respectively. The putative B56 binding region is shown in green. The alanine substitution mutations to three Cdk phosphorylation sites (3A) or to single point mutation S558A are shown.Binding assay using MBP‐fused Apc1‐loop500 fragments and B56γ using Apc1‐loop500 WT or its derivatives (3A or S558A) in A. The bound proteins were analysed as in Fig 1C. The bar graph is quantification of bound B56γ. The intensities of MBP control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.Specific binding of Apc1‐loop500 to B56γ. Apc1‐loop500 WT or its derivatives (∆11 or L557A/V560A) were incubated with the 35S‐labelled Flag‐B56γ, Flag‐B55δ or Flag‐PR70 in anaphase extract supplemented with CycB∆167 at 23°C for 1 h. The bound proteins were analysed as in Fig 1C. The bar graph is quantification of bound B subunits. The intensities of MBP control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.
	Figure 2. B56 binding to Apc1‐loop500 is important for the formation of the active APC/C‐Cdc20 complex Cdc20 binding assay in Xenopus egg extracts. The purified recombinant wild‐type (WT) or B56 binding site mutant APC/C (1‐L557A/V560A) was incubated with APC/C‐depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23°C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS–PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho‐site‐specific antibody that binds only when both S314 and S318 are phosphorylated.Quantification of (A). The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.B56 binding site mutant APC/C (1‐L557A/V560A) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT APC/C or B56 binding motif mutant APC/C (1‐L557A/V560A) was incubated with ∆APC anaphase extract. The recovered APC/C‐Cdc20 complex was subjected to ubiquitylation assay using 35S‐labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS–PAGE and autoradiography. Cdc20 bound to the APC/C is presented in Fig EV2B.Quantification of (C). Error bars, SEM from three independent experiments.
	Figure EV2. Mutant apo‐APC/C complexes deficient in B56‐APC/C interactions show less activity than WT APC/C (left panel) The purified recombinant wild‐type (WT) or B56 binding site mutant APC/C (1‐∆11) was incubated with APC/C‐depleted (∆APC) interphase extract (Inter) or ∆APC anaphase extract supplemented with CycB∆167 (Ana) at 23°C for indicated times. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS–PAGE and immunoblotting with indicated antibodies. pApc1 (pS314/pS318) is a phospho‐site‐specific antibody. (right panel) Quantification of bound Cdc20. The intensities of WT control in interphase were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.(left panel) Mutant apo‐APC/C carrying mutations in Apc1 (1‐L557A/V560A) binds Cdc20 at a lower level than WT APC/C. The recovered APC/C‐Cdc20 complex used in Fig 2C was analysed by SDS–PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.(left panel) B56 binding site mutant APC/C in Apc1‐loop500 (1‐∆11) is less active in ubiquitylation assay than WT APC/C. The purified recombinant WT or 1‐∆11 APC/C was incubated with ∆APC anaphase extract. The recovered APC/C‐Cdc20 complex was subjected to ubiquitylation assay using 35S‐labelled cyclin B as a substrate. Samples were taken at indicated time points and analysed by SDS–PAGE and autoradiography. (right panel) Quantification of ubiquitylation assays. Error bars, SEM from three independent experiments.(left panel) The recovered APC/C‐Cdc20 complex used in (C) was analysed by SDS–PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.
	Figure EV3. Cyclin destruction assays using cell‐free Xenopus egg extracts (left panel) The B56 binding site mutant APC/C (1‐L557A/V560A) is less active than WT APC/C in cyclin destruction assays in an anaphase‐specific manner, whereas both activities are similar in interphase cyclin destruction assays. The purified recombinant WT APC/C or Apc1 mutant APC/C (1‐L557A/V560A) was incubated with APC/C‐depleted (∆APC) extract supplemented with CycB∆167 (anaphase, upper panel) or with Cdh1 (interphase, lower panel) at 23°C. 35S‐labelled cyclin B and a version of cyclin B lacking the N‐terminal 67 residues (Δ67, stable control) were used as substrates. Samples taken at indicated time points after addition of substrates were analysed by SDS–PAGE and autoradiography. (right panel) Quantification of cyclin destruction assays. Error bars, SEM from three independent experiments.(left panel) The activity of the B56 binding site mutant APC/C (1‐∆11) is less active than WT APC/C in anaphase cyclin destruction assay. The activity of recombinant APC/C (1‐∆11) was examined as in (A) (right panel) Quantification of cyclin destruction assays. Error bars, SEM from three independent experiments.
	Figure EV4. Deletion of Apc1‐loop300 activates the APC/C even in interphase extractIn order to perform cyclin destruction assay, the purified recombinant WT APC/C or Apc1‐loop300‐deleted APC/C (1‐∆L300) was incubated with ∆APC interphase extract in the presence (upper panel) or absence (lower panel) of Cdh1 at 23°C. 35S‐labelled cyclin B and a version of cyclin B lacking the N‐terminal 67 residues (Δ67, stable control) were used as substrates. Samples taken at indicated time points after addition of substrates were analysed by SDS–PAGE and autoradiography. 1‐∆L300 APC/C is activated by the endogenous Cdc20 and initiates cyclin destruction in the absence of Cdh1 in interphase (lanes 19–24, lower panel).
	Figure 3. Apc1‐loop500 regulates APC/C activation independently of phosphorylation of Apc1‐loop300 (left panel) Cdc20 binding to APC/C loop domain mutants in interphase extract. The purified recombinant WT or Apc1 mutant APC/Cs (1‐∆L300, 1‐∆L300/∆11 or 1‐∆L300/L557A/V560A) were incubated with ∆APC interphase extract at 23°C for 1 h. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS–PAGE and immunoblotting with indicated antibodies. (right panel) Quantification of bound Cdc20. The intensities of 1‐∆L300 control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.(left panel) Cdc20 binding to APC/C loop domain mutants in anaphase extracts. Same as (A) but ∆APC anaphase extract was used and incubated at 23°C for 50 min in anaphase extract. (right panel) Quantification of bound Cdc20. The intensities of 1‐∆L300 control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.
	Figure 4. PP2A‐B56 bound to Apc1‐loop500 promotes APC/C‐Cdc20 complex formation through dephosphorylation of N‐terminal Cdc20 (left panel) Cdk‐dependent in vitro kinase assay of Apc1‐loop300. 3xFlag‐tagged WT or 7T Apc1‐loop300 fragment was incubated with Cdk2‐cyclin A in the presence of [γ‐32P]‐ATP at 30°C for 30 min, and separated by SDS–PAGE and detected by autoradiography. (right panel) 32P‐phosphorylated WT or 7T Apc1‐loop300 fragment was incubated in anaphase extract, and removal of radioactivity was analysed by SDS–PAGE followed by autoradiography.Quantification of removal of radioactivity in (A). Error bars, SEM from three independent experiments.Cdc20‐5A efficiently binds to the B56 binding site mutant APC/C. The purified recombinant WT APC/C or Apc1‐loop500 mutant APC/C (1‐L557A/V560A) was incubated with WT Cdc20 or non‐phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23°C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS–PAGE and immunoblotting with indicated antibodies.Quantification of (C). The intensities of WT APC/C control were arbitrarily set to 1.0. Error bars, SEM from three independent experiments.The APC/C and Cdc20 immunoprecipitated from CSF extract were incubated in the presence of a range of concentrations or absence of PP2A‐B56γ at 23°C. Samples were taken at the indicated time points and analysed by SDS–PAGE and immunoblotting with antibodies, including phospho‐site‐specific antibodies for pCdc20 (pT79) and pApc1 (pS314/pS318).A model for Apc1‐loop500‐mediated Cdc20‐APC/C complex formation in mitosis. Apc1‐loop500 is phosphorylated by Cdk1 and binds to PP2A‐B56 in anaphase. PP2A‐B56 dephosphorylates inhibitory phosphorylation sites in N‐Cdc20 and promotes the formation of active APC/C‐Cdc20 complex. Apc1‐loop500 may control phosphorylation of other APC/C subunits and possibly interacting proteins although the mechanisms involved remain elusive. The indicated numbers on the schematic view of the APC/C (the back view is rotated by 180° around the vertical axis from the front view) represent APC/C subunits.
	Figure EV5. Cdc20‐5A efficiently binds to the B56 binding site mutant APC/CThe purified recombinant WT APC/C or B56 binding site mutant APC/C (1‐∆11) was incubated with WT Cdc20 or non‐phosphorylatable Cdc20 mutant (5A) in ∆APC∆Cdc20 anaphase extract at 23°C for 55 min. The APC/C was recovered with Apc3 monoclonal antibody (AF3.1) beads, and the bound proteins were analysed by SDS–PAGE and immunoblotting with indicated antibodies.

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