Limits...
MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C.

Voets E, Wolthuis R - Biol Open (2015)

Bottom Line: However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active.Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20.Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AvL), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Cyclin B1 associates with the mitotic APC/C, independent of Cdc20.(A) Cyclin B1 recruitment to the APC/C does not involve direct Cdc20 binding, different from cyclin A2. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a rabbit anti-Cdc20 and aliquots of the immunoprecipitates were analysed by western blotting. Asynch refers to an asynchronous, non-transfected lysate. (B) Genetic ablation of CDC20 results in a mitotic arrest due to cyclin B1 stabilisation. Cdc20(Δ/Δ); RERT(+/Cre) mouse embryonic fibroblasts (MEFs) were asynchronously grown or thymidine synchronised for 9 hours and treated with 1 µM of 4-hydroxytamoxifen (4-OHT) to induce Cre activity, resulting in the excision of exon 2 of CDC20 (Cdc20(Δ) allele). Cells arrested in mitosis by the 4-OHT treatment were obtained by mitotic shake-off. Extracts of asynchronous or 4-OHT treated cells were subjected to western blot analysis. The dashed line indicates where two lanes have been placed next to each other. (C) Cyclin B1 binds the mitotic APC/C regardless of the presence of Cdc20. Cdc20(Δ/Δ); RERT(+/Cre) MEFs were either asynchronously grown or synchronised in mitosis using thymidine and nocodazole or 4-OHT treatment. Enrichment of cells arrested in mitosis was obtained by mitotic shake-off. MEF lysates were subjected to immunoprecipitation using a mouse anti-cyclin B1. Aliquots of the immunoprecipitates were analysed by western blotting. The asterisk (*) indicates the remaining APC3 signal after reprobing the blot with anti-APC4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400591&req=5

f04: Cyclin B1 associates with the mitotic APC/C, independent of Cdc20.(A) Cyclin B1 recruitment to the APC/C does not involve direct Cdc20 binding, different from cyclin A2. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a rabbit anti-Cdc20 and aliquots of the immunoprecipitates were analysed by western blotting. Asynch refers to an asynchronous, non-transfected lysate. (B) Genetic ablation of CDC20 results in a mitotic arrest due to cyclin B1 stabilisation. Cdc20(Δ/Δ); RERT(+/Cre) mouse embryonic fibroblasts (MEFs) were asynchronously grown or thymidine synchronised for 9 hours and treated with 1 µM of 4-hydroxytamoxifen (4-OHT) to induce Cre activity, resulting in the excision of exon 2 of CDC20 (Cdc20(Δ) allele). Cells arrested in mitosis by the 4-OHT treatment were obtained by mitotic shake-off. Extracts of asynchronous or 4-OHT treated cells were subjected to western blot analysis. The dashed line indicates where two lanes have been placed next to each other. (C) Cyclin B1 binds the mitotic APC/C regardless of the presence of Cdc20. Cdc20(Δ/Δ); RERT(+/Cre) MEFs were either asynchronously grown or synchronised in mitosis using thymidine and nocodazole or 4-OHT treatment. Enrichment of cells arrested in mitosis was obtained by mitotic shake-off. MEF lysates were subjected to immunoprecipitation using a mouse anti-cyclin B1. Aliquots of the immunoprecipitates were analysed by western blotting. The asterisk (*) indicates the remaining APC3 signal after reprobing the blot with anti-APC4.

Mentions: Does the binding of cyclin B1 to the mitotic APC/C depend on Cdc20? Previously, in CDC20 RNAi experiments, we found that CDC20 depletion by RNAi did not affect binding (van Zon et al., 2010). In line with that observation, the cyclin B1 D-box, which is known to bind Cdc20 with low affinity, was also not required. These results were questioned, however, in another study (Izawa and Pines, 2011). Here, we aimed to more thoroughly investigate this matter, and particularly to further address the role of MASTL and Cdc20 in directing cyclin B1 to the APC/C. Immunoprecipitation of Cdc20 specifically pulls down the APC/C in extracts from cells arrested in mitosis, but cyclin B1 is hardly detectable under these conditions (Fig. 4A). These immunoprecipitations were carried out under non-saturating conditions of the Cdc20 antibody, resulting in the isolation of mostly mitotic checkpoint complexes (MCCs) bound to the APC/C in mitosis. We showed before that only a fraction of the cellular pool of APC/C is found in complex with cyclin B1 (van Zon et al., 2010). We then used tamoxifen-inducible CDC20 knockout cells (Manchado et al., 2010) to test the contribution of Cdc20 to the observed interaction between cyclin B1 and the APC/C more thoroughly. After genetic ablation of CDC20, these cells arrested in mitosis and could be collected by mitotic shake-off, for immunopreciptation experiments. Importantly, genetic ablation of CDC20 did not prevent cyclin B1-APC/C complex formation in prometaphase, showing that Cdc20 is dispensable for cyclin B1 recruitment to the mitotic APC/C (Fig. 4B,C). Cyclin B1 can be recruited by phosphorylated APC/C, in a D-box–independent manner, and MASTL supports this. Recently, it was shown that this interaction between cyclin B and the mitotic APC/C plays a role in residual cyclin B1 degradation, observed after inhibiting the APC/C-Cdc20 interaction together with the Cdc20–D-box interaction, when combining two distinct APC/C inhibitors (Sackton et al., 2014).


MASTL promotes cyclin B1 destruction by enforcing Cdc20-independent binding of cyclin B1 to the APC/C.

Voets E, Wolthuis R - Biol Open (2015)

Cyclin B1 associates with the mitotic APC/C, independent of Cdc20.(A) Cyclin B1 recruitment to the APC/C does not involve direct Cdc20 binding, different from cyclin A2. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a rabbit anti-Cdc20 and aliquots of the immunoprecipitates were analysed by western blotting. Asynch refers to an asynchronous, non-transfected lysate. (B) Genetic ablation of CDC20 results in a mitotic arrest due to cyclin B1 stabilisation. Cdc20(Δ/Δ); RERT(+/Cre) mouse embryonic fibroblasts (MEFs) were asynchronously grown or thymidine synchronised for 9 hours and treated with 1 µM of 4-hydroxytamoxifen (4-OHT) to induce Cre activity, resulting in the excision of exon 2 of CDC20 (Cdc20(Δ) allele). Cells arrested in mitosis by the 4-OHT treatment were obtained by mitotic shake-off. Extracts of asynchronous or 4-OHT treated cells were subjected to western blot analysis. The dashed line indicates where two lanes have been placed next to each other. (C) Cyclin B1 binds the mitotic APC/C regardless of the presence of Cdc20. Cdc20(Δ/Δ); RERT(+/Cre) MEFs were either asynchronously grown or synchronised in mitosis using thymidine and nocodazole or 4-OHT treatment. Enrichment of cells arrested in mitosis was obtained by mitotic shake-off. MEF lysates were subjected to immunoprecipitation using a mouse anti-cyclin B1. Aliquots of the immunoprecipitates were analysed by western blotting. The asterisk (*) indicates the remaining APC3 signal after reprobing the blot with anti-APC4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400591&req=5

f04: Cyclin B1 associates with the mitotic APC/C, independent of Cdc20.(A) Cyclin B1 recruitment to the APC/C does not involve direct Cdc20 binding, different from cyclin A2. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 µM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a rabbit anti-Cdc20 and aliquots of the immunoprecipitates were analysed by western blotting. Asynch refers to an asynchronous, non-transfected lysate. (B) Genetic ablation of CDC20 results in a mitotic arrest due to cyclin B1 stabilisation. Cdc20(Δ/Δ); RERT(+/Cre) mouse embryonic fibroblasts (MEFs) were asynchronously grown or thymidine synchronised for 9 hours and treated with 1 µM of 4-hydroxytamoxifen (4-OHT) to induce Cre activity, resulting in the excision of exon 2 of CDC20 (Cdc20(Δ) allele). Cells arrested in mitosis by the 4-OHT treatment were obtained by mitotic shake-off. Extracts of asynchronous or 4-OHT treated cells were subjected to western blot analysis. The dashed line indicates where two lanes have been placed next to each other. (C) Cyclin B1 binds the mitotic APC/C regardless of the presence of Cdc20. Cdc20(Δ/Δ); RERT(+/Cre) MEFs were either asynchronously grown or synchronised in mitosis using thymidine and nocodazole or 4-OHT treatment. Enrichment of cells arrested in mitosis was obtained by mitotic shake-off. MEF lysates were subjected to immunoprecipitation using a mouse anti-cyclin B1. Aliquots of the immunoprecipitates were analysed by western blotting. The asterisk (*) indicates the remaining APC3 signal after reprobing the blot with anti-APC4.
Mentions: Does the binding of cyclin B1 to the mitotic APC/C depend on Cdc20? Previously, in CDC20 RNAi experiments, we found that CDC20 depletion by RNAi did not affect binding (van Zon et al., 2010). In line with that observation, the cyclin B1 D-box, which is known to bind Cdc20 with low affinity, was also not required. These results were questioned, however, in another study (Izawa and Pines, 2011). Here, we aimed to more thoroughly investigate this matter, and particularly to further address the role of MASTL and Cdc20 in directing cyclin B1 to the APC/C. Immunoprecipitation of Cdc20 specifically pulls down the APC/C in extracts from cells arrested in mitosis, but cyclin B1 is hardly detectable under these conditions (Fig. 4A). These immunoprecipitations were carried out under non-saturating conditions of the Cdc20 antibody, resulting in the isolation of mostly mitotic checkpoint complexes (MCCs) bound to the APC/C in mitosis. We showed before that only a fraction of the cellular pool of APC/C is found in complex with cyclin B1 (van Zon et al., 2010). We then used tamoxifen-inducible CDC20 knockout cells (Manchado et al., 2010) to test the contribution of Cdc20 to the observed interaction between cyclin B1 and the APC/C more thoroughly. After genetic ablation of CDC20, these cells arrested in mitosis and could be collected by mitotic shake-off, for immunopreciptation experiments. Importantly, genetic ablation of CDC20 did not prevent cyclin B1-APC/C complex formation in prometaphase, showing that Cdc20 is dispensable for cyclin B1 recruitment to the mitotic APC/C (Fig. 4B,C). Cyclin B1 can be recruited by phosphorylated APC/C, in a D-box–independent manner, and MASTL supports this. Recently, it was shown that this interaction between cyclin B and the mitotic APC/C plays a role in residual cyclin B1 degradation, observed after inhibiting the APC/C-Cdc20 interaction together with the Cdc20–D-box interaction, when combining two distinct APC/C inhibitors (Sackton et al., 2014).

Bottom Line: However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active.Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20.Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology I (B5) and Division of Molecular Carcinogenesis (B7), The Netherlands Cancer Institute (NKI-AvL), Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus