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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.


Related in: MedlinePlus

Residues N-terminal to the canonical D-box of cyclin B1 are required for its ubiquitylation and degradation in anaphase.(A) In vitro ubiquitylation reactions using purified budding yeast APC/C-Cdh1 and the indicated in vitro translated human cyclin B1 mutants. Data are representative of results from two independent experiments. (B) The data from (A) were quantified and the amount of ubiquitylated cyclin B1 was plotted as a function of time. (C,D) HeLa cells were injected with cyclin B1-Venus (grey, n = 36) or cyclin B1 M21A-Venus (red, n = 20) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) Cdh1+/+ (red), or Cdh1−/− (blue) mouse embryonic fibroblasts were transfected with cyclin B1-Venus constructs and analysed as in Fig. 1. Error bars indicate mean ± SD of 41 and 50, cells, for panels E and F, respectively.
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f03: Residues N-terminal to the canonical D-box of cyclin B1 are required for its ubiquitylation and degradation in anaphase.(A) In vitro ubiquitylation reactions using purified budding yeast APC/C-Cdh1 and the indicated in vitro translated human cyclin B1 mutants. Data are representative of results from two independent experiments. (B) The data from (A) were quantified and the amount of ubiquitylated cyclin B1 was plotted as a function of time. (C,D) HeLa cells were injected with cyclin B1-Venus (grey, n = 36) or cyclin B1 M21A-Venus (red, n = 20) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) Cdh1+/+ (red), or Cdh1−/− (blue) mouse embryonic fibroblasts were transfected with cyclin B1-Venus constructs and analysed as in Fig. 1. Error bars indicate mean ± SD of 41 and 50, cells, for panels E and F, respectively.

Mentions: To narrow down the region responsible for promoting anaphase recognition by the APC/C we used an in vitro ubiquitylation system (Enquist-Newman et al., 2008) and found that the Δ11–41 mutant could hardly be ubiquitylated (Fig. 3A,B). We made a series of smaller internal deletions and found that deleting residues 21 to 25 greatly reduced ubiquitylation by APC/C in vitro (Fig. 3A,B). Moreover, this mutant was also stabilised in anaphase in vivo (supplementary material Fig. S1A,B). To refine this analysis we constructed point mutations in each of the residues from positions 21 to 25 and identified M21 as the critical residue for anaphase degradation (Fig. 3C,D). (Positions 22, 23, 24 are A–G–A, and mutating K25 to alanine did not affect degradation, data not shown.) Cells expressing the M21A mutant arrested in anaphase and it was notable that M21 was not required for degradation in metaphase (Fig. 3C).


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)

Residues N-terminal to the canonical D-box of cyclin B1 are required for its ubiquitylation and degradation in anaphase.(A) In vitro ubiquitylation reactions using purified budding yeast APC/C-Cdh1 and the indicated in vitro translated human cyclin B1 mutants. Data are representative of results from two independent experiments. (B) The data from (A) were quantified and the amount of ubiquitylated cyclin B1 was plotted as a function of time. (C,D) HeLa cells were injected with cyclin B1-Venus (grey, n = 36) or cyclin B1 M21A-Venus (red, n = 20) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) Cdh1+/+ (red), or Cdh1−/− (blue) mouse embryonic fibroblasts were transfected with cyclin B1-Venus constructs and analysed as in Fig. 1. Error bars indicate mean ± SD of 41 and 50, cells, for panels E and F, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197439&req=5

f03: Residues N-terminal to the canonical D-box of cyclin B1 are required for its ubiquitylation and degradation in anaphase.(A) In vitro ubiquitylation reactions using purified budding yeast APC/C-Cdh1 and the indicated in vitro translated human cyclin B1 mutants. Data are representative of results from two independent experiments. (B) The data from (A) were quantified and the amount of ubiquitylated cyclin B1 was plotted as a function of time. (C,D) HeLa cells were injected with cyclin B1-Venus (grey, n = 36) or cyclin B1 M21A-Venus (red, n = 20) constructs and analysed as in Fig. 1. Data are from 3 independent experiments. (E,F) Cdh1+/+ (red), or Cdh1−/− (blue) mouse embryonic fibroblasts were transfected with cyclin B1-Venus constructs and analysed as in Fig. 1. Error bars indicate mean ± SD of 41 and 50, cells, for panels E and F, respectively.
Mentions: To narrow down the region responsible for promoting anaphase recognition by the APC/C we used an in vitro ubiquitylation system (Enquist-Newman et al., 2008) and found that the Δ11–41 mutant could hardly be ubiquitylated (Fig. 3A,B). We made a series of smaller internal deletions and found that deleting residues 21 to 25 greatly reduced ubiquitylation by APC/C in vitro (Fig. 3A,B). Moreover, this mutant was also stabilised in anaphase in vivo (supplementary material Fig. S1A,B). To refine this analysis we constructed point mutations in each of the residues from positions 21 to 25 and identified M21 as the critical residue for anaphase degradation (Fig. 3C,D). (Positions 22, 23, 24 are A–G–A, and mutating K25 to alanine did not affect degradation, data not shown.) Cells expressing the M21A mutant arrested in anaphase and it was notable that M21 was not required for degradation in metaphase (Fig. 3C).

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.


Related in: MedlinePlus