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Co-activator independent differences in how the metaphase and anaphase APC/C recognise the same substrate.

Matsusaka T, Enquist-Newman M, Morgan DO, Pines J - Biol Open (2014)

Bottom Line: We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis.Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1.Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.

View Article: PubMed Central - PubMed

Affiliation: The Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

No MeSH data available.


The N-terminus of Cdc20 can substitute for a D-box when cyclin B1 is targeted to the APC/C by Cks1.(A,B) The anaphase APC/C can degrade a D-box mutant of cyclin B1 targeted to it by Cks1. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus (RL-V, black, n = 35), cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, red, n = 24), cyclin B1 M21A/R42A/L45A triple mutant venus-cks1 (MRL-V-C, blue, n = 20), or cyclin B1-venus (grey, n = 36) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (C,D) Cks1 cannot be substituted by a C-terminal IR motif. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, black, n = 24) or cyclin B1 R42A/L45A double mutant-venus-IR (RL-V-IR, red, n = 29) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) The N-terminus of Cdc20 can promote degradation of cyclin B1 lacking its destruction box in metaphase if it is targeted to the APC/C by Cks1. The N-terminal 151 residues of Cdc20 were fused to cyclin B1 lacking its D-box, designated Cdc20 (1–151)-R42A/L45A double mutant cyclin B1, with or without Cks1 at the C-terminus. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus (20-B1RL-V, black, n = 24) or Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (20-B1RL-V-C, red, n = 31) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (G,H) The N-terminus of Cdc20 requires its C-box and KILR motif to promote degradation. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 wt, black, n = 31), Cdc20 (1–151) KEN cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KEN, green, n = 14), Cdc20 (1–151) C-box cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 C-box, red, n = 14), or Cdc20 (1–151) KILR cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KILR, blue, n = 14) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (I,J) The non-phosphorylatable mutant N-terminus of Cdh1 can also promote degradation of cyclin B1 lacking its D-box. HeLa cells were injected with plasmids encoding Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20, black, n = 31), or a construct in which the N-terminus of Cdc20 was replaced with the N-terminus of non-phosphorylatable Cdh1, Cdh1 (1–155) Ala-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala, red, n = 16), or the N-terminus of non-phosphorylatable Cdh1 with the KILR motif of Cdc20, Cdh1 (1–155) Ala+KILR-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala+KILR, blue, n = 27). Constructs were analysed as in Fig. 1. Data are from 3 independent experiments. (K) Sequence alignment of Cdc20 and Cdh1 around C-box and KILR motif. The residues marked in blue (KNEL) in Cdh1 were mutated to those found in Cdc20 (KILR).
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f04: The N-terminus of Cdc20 can substitute for a D-box when cyclin B1 is targeted to the APC/C by Cks1.(A,B) The anaphase APC/C can degrade a D-box mutant of cyclin B1 targeted to it by Cks1. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus (RL-V, black, n = 35), cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, red, n = 24), cyclin B1 M21A/R42A/L45A triple mutant venus-cks1 (MRL-V-C, blue, n = 20), or cyclin B1-venus (grey, n = 36) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (C,D) Cks1 cannot be substituted by a C-terminal IR motif. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, black, n = 24) or cyclin B1 R42A/L45A double mutant-venus-IR (RL-V-IR, red, n = 29) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) The N-terminus of Cdc20 can promote degradation of cyclin B1 lacking its destruction box in metaphase if it is targeted to the APC/C by Cks1. The N-terminal 151 residues of Cdc20 were fused to cyclin B1 lacking its D-box, designated Cdc20 (1–151)-R42A/L45A double mutant cyclin B1, with or without Cks1 at the C-terminus. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus (20-B1RL-V, black, n = 24) or Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (20-B1RL-V-C, red, n = 31) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (G,H) The N-terminus of Cdc20 requires its C-box and KILR motif to promote degradation. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 wt, black, n = 31), Cdc20 (1–151) KEN cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KEN, green, n = 14), Cdc20 (1–151) C-box cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 C-box, red, n = 14), or Cdc20 (1–151) KILR cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KILR, blue, n = 14) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (I,J) The non-phosphorylatable mutant N-terminus of Cdh1 can also promote degradation of cyclin B1 lacking its D-box. HeLa cells were injected with plasmids encoding Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20, black, n = 31), or a construct in which the N-terminus of Cdc20 was replaced with the N-terminus of non-phosphorylatable Cdh1, Cdh1 (1–155) Ala-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala, red, n = 16), or the N-terminus of non-phosphorylatable Cdh1 with the KILR motif of Cdc20, Cdh1 (1–155) Ala+KILR-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala+KILR, blue, n = 27). Constructs were analysed as in Fig. 1. Data are from 3 independent experiments. (K) Sequence alignment of Cdc20 and Cdh1 around C-box and KILR motif. The residues marked in blue (KNEL) in Cdh1 were mutated to those found in Cdc20 (KILR).

Mentions: The cyclin B1-Cdk1 complex has previously been shown to bind to the APC/C through its partner Cks1 protein, and this improves the efficiency of its destruction (van Zon et al., 2010). To test whether the difference in D-box residues recognised by metaphase and anaphase APC/Cs was due to a difference in their ability to bind cyclin B1, we recruited wild type or a R42A/L45A double mutant of cyclin B1 directly to the APC/C by fusing its carboxyl terminus to the Cks1 protein (Di Fiore and Pines, 2010; Wolthuis et al., 2008). This revealed a marked difference between metaphase and anaphase cells: recruiting the double mutant to the metaphase APC/C had no effect, but recruiting it to the anaphase APC/C allowed it to be degraded (Fig. 4A,B). These experiments further underlined the importance of M21 to anaphase recognition because mutating M21 partially stabilised this construct in anaphase (Fig. 4A,B). Note that we could not substitute Cks1 with a C-terminal IR motif (Fig. 4C,D); therefore it may be important where and how the C-terminus of cyclin B1 binds to the APC/C.


Co-activator independent differences in how the metaphase and anaphase APC/C recognise the same substrate.

Matsusaka T, Enquist-Newman M, Morgan DO, Pines J - Biol Open (2014)

The N-terminus of Cdc20 can substitute for a D-box when cyclin B1 is targeted to the APC/C by Cks1.(A,B) The anaphase APC/C can degrade a D-box mutant of cyclin B1 targeted to it by Cks1. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus (RL-V, black, n = 35), cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, red, n = 24), cyclin B1 M21A/R42A/L45A triple mutant venus-cks1 (MRL-V-C, blue, n = 20), or cyclin B1-venus (grey, n = 36) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (C,D) Cks1 cannot be substituted by a C-terminal IR motif. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, black, n = 24) or cyclin B1 R42A/L45A double mutant-venus-IR (RL-V-IR, red, n = 29) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) The N-terminus of Cdc20 can promote degradation of cyclin B1 lacking its destruction box in metaphase if it is targeted to the APC/C by Cks1. The N-terminal 151 residues of Cdc20 were fused to cyclin B1 lacking its D-box, designated Cdc20 (1–151)-R42A/L45A double mutant cyclin B1, with or without Cks1 at the C-terminus. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus (20-B1RL-V, black, n = 24) or Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (20-B1RL-V-C, red, n = 31) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (G,H) The N-terminus of Cdc20 requires its C-box and KILR motif to promote degradation. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 wt, black, n = 31), Cdc20 (1–151) KEN cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KEN, green, n = 14), Cdc20 (1–151) C-box cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 C-box, red, n = 14), or Cdc20 (1–151) KILR cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KILR, blue, n = 14) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (I,J) The non-phosphorylatable mutant N-terminus of Cdh1 can also promote degradation of cyclin B1 lacking its D-box. HeLa cells were injected with plasmids encoding Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20, black, n = 31), or a construct in which the N-terminus of Cdc20 was replaced with the N-terminus of non-phosphorylatable Cdh1, Cdh1 (1–155) Ala-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala, red, n = 16), or the N-terminus of non-phosphorylatable Cdh1 with the KILR motif of Cdc20, Cdh1 (1–155) Ala+KILR-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala+KILR, blue, n = 27). Constructs were analysed as in Fig. 1. Data are from 3 independent experiments. (K) Sequence alignment of Cdc20 and Cdh1 around C-box and KILR motif. The residues marked in blue (KNEL) in Cdh1 were mutated to those found in Cdc20 (KILR).
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f04: The N-terminus of Cdc20 can substitute for a D-box when cyclin B1 is targeted to the APC/C by Cks1.(A,B) The anaphase APC/C can degrade a D-box mutant of cyclin B1 targeted to it by Cks1. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus (RL-V, black, n = 35), cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, red, n = 24), cyclin B1 M21A/R42A/L45A triple mutant venus-cks1 (MRL-V-C, blue, n = 20), or cyclin B1-venus (grey, n = 36) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (C,D) Cks1 cannot be substituted by a C-terminal IR motif. HeLa cells were injected with cyclin B1 R42A/L45A double mutant-venus-cks1 (RL-V-C, black, n = 24) or cyclin B1 R42A/L45A double mutant-venus-IR (RL-V-IR, red, n = 29) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) The N-terminus of Cdc20 can promote degradation of cyclin B1 lacking its destruction box in metaphase if it is targeted to the APC/C by Cks1. The N-terminal 151 residues of Cdc20 were fused to cyclin B1 lacking its D-box, designated Cdc20 (1–151)-R42A/L45A double mutant cyclin B1, with or without Cks1 at the C-terminus. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus (20-B1RL-V, black, n = 24) or Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (20-B1RL-V-C, red, n = 31) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (G,H) The N-terminus of Cdc20 requires its C-box and KILR motif to promote degradation. HeLa cells were injected with Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 wt, black, n = 31), Cdc20 (1–151) KEN cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KEN, green, n = 14), Cdc20 (1–151) C-box cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 C-box, red, n = 14), or Cdc20 (1–151) KILR cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20 KILR, blue, n = 14) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (I,J) The non-phosphorylatable mutant N-terminus of Cdh1 can also promote degradation of cyclin B1 lacking its D-box. HeLa cells were injected with plasmids encoding Cdc20 (1–151) cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdc20, black, n = 31), or a construct in which the N-terminus of Cdc20 was replaced with the N-terminus of non-phosphorylatable Cdh1, Cdh1 (1–155) Ala-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala, red, n = 16), or the N-terminus of non-phosphorylatable Cdh1 with the KILR motif of Cdc20, Cdh1 (1–155) Ala+KILR-cyclin B1 R42A/L45A double mutant-venus-cks1 (Cdh1 Ala+KILR, blue, n = 27). Constructs were analysed as in Fig. 1. Data are from 3 independent experiments. (K) Sequence alignment of Cdc20 and Cdh1 around C-box and KILR motif. The residues marked in blue (KNEL) in Cdh1 were mutated to those found in Cdc20 (KILR).
Mentions: The cyclin B1-Cdk1 complex has previously been shown to bind to the APC/C through its partner Cks1 protein, and this improves the efficiency of its destruction (van Zon et al., 2010). To test whether the difference in D-box residues recognised by metaphase and anaphase APC/Cs was due to a difference in their ability to bind cyclin B1, we recruited wild type or a R42A/L45A double mutant of cyclin B1 directly to the APC/C by fusing its carboxyl terminus to the Cks1 protein (Di Fiore and Pines, 2010; Wolthuis et al., 2008). This revealed a marked difference between metaphase and anaphase cells: recruiting the double mutant to the metaphase APC/C had no effect, but recruiting it to the anaphase APC/C allowed it to be degraded (Fig. 4A,B). These experiments further underlined the importance of M21 to anaphase recognition because mutating M21 partially stabilised this construct in anaphase (Fig. 4A,B). Note that we could not substitute Cks1 with a C-terminal IR motif (Fig. 4C,D); therefore it may be important where and how the C-terminus of cyclin B1 binds to the APC/C.

Bottom Line: We have addressed this question by determining whether the same substrate, cyclin B1, is recognised in the same way by the APC/C at different times in mitosis.Unexpectedly, we find that distinct but overlapping motifs in cyclin B1 are recognised by the APC/C in metaphase compared with anaphase, and this does not depend on the exchange of Cdc20 for Cdh1.Thus, changes in APC/C substrate specificity in mitosis can potentially be conferred by altering interaction sites in addition to exchanging Cdc20 for Cdh1.

View Article: PubMed Central - PubMed

Affiliation: The Gurdon Institute, Tennis Court Road, Cambridge CB2 1QN, UK Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.

No MeSH data available.