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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

MASTL depletion significantly impairs cyclin B1 recruitment to the mitotic APC/C.(A) Cyclin B1 binds to the APC/C specifically in mitosis. HeLa cells were synchronised in G2 and M phase by a thymidine block, followed by nocodazole or taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off, and treated for another 2 hours with 5 µM MG132 where indicated. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4. Aliquots of the immunoprecipitates were analysed by western blotting. (B) MASTL silencing impairs the recruitment of cyclin A2 and cyclin B1 to the mitotic APC/C. 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 goat anti-APC4 and aliquots of the immunoprecipitates were analysed by western blotting. Note that equal amounts of APC4 are immunoprecipitated in the mitotic fractions, whereas cyclins A2 and B1 bound to the APC/C are hardly detectable in the MASTL RNAi extracts. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin (right-hand panel). (C) Direct binding of cyclin B1 to the APC/C may depend on extensive APC3 phosphorylation in mitosis. HeLa cells treated as in (B), were subjected to immunoprecipitation using a mouse anti-cyclin B1. G1 phase cells were harvested 90 minutes after treatment with 10 µM of RO-3306. Aliquots of the immunoprecipitates were analysed by western blotting. For cyclin A2 blots in A,B, * denotes an aspecific band that is only observed upon immunoprecipitation.
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f03: MASTL depletion significantly impairs cyclin B1 recruitment to the mitotic APC/C.(A) Cyclin B1 binds to the APC/C specifically in mitosis. HeLa cells were synchronised in G2 and M phase by a thymidine block, followed by nocodazole or taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off, and treated for another 2 hours with 5 µM MG132 where indicated. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4. Aliquots of the immunoprecipitates were analysed by western blotting. (B) MASTL silencing impairs the recruitment of cyclin A2 and cyclin B1 to the mitotic APC/C. 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 goat anti-APC4 and aliquots of the immunoprecipitates were analysed by western blotting. Note that equal amounts of APC4 are immunoprecipitated in the mitotic fractions, whereas cyclins A2 and B1 bound to the APC/C are hardly detectable in the MASTL RNAi extracts. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin (right-hand panel). (C) Direct binding of cyclin B1 to the APC/C may depend on extensive APC3 phosphorylation in mitosis. HeLa cells treated as in (B), were subjected to immunoprecipitation using a mouse anti-cyclin B1. G1 phase cells were harvested 90 minutes after treatment with 10 µM of RO-3306. Aliquots of the immunoprecipitates were analysed by western blotting. For cyclin A2 blots in A,B, * denotes an aspecific band that is only observed upon immunoprecipitation.

Mentions: We then reasoned that stabilisation of cyclin B1 may depend on the recruitment of cyclin B1 by the mitotic APC/C. Previously, we reported that cyclin B1, bound to Cdk1 and Cks, is retained at the APC/C in prometaphase, in a manner strictly dependent on APC3 (van Zon et al., 2010). Indeed, when we performed immunoprecipitation experiments using an APC4 antibody, to pull-down the APC/C from mitotic HeLa cell extracts and analysed interacting proteins on western blots, we found that cyclin B1, like cyclin A2, specifically bound to APC/CCdc20 in prometaphase (Fig. 3A). The binding of cyclins A2 and B1 to the APC/C was specific for mitosis, and occurred regardless of the strength of the spindle checkpoint (Fig. 3A; compare nocodazole- with taxol-arrested cells) (Collin et al., 2013). In strong contrast, the APC/CCdc20 substrate Nek2A bound the APC/C already in G2 phase, in line with previous findings and our own recent results (Hames et al., 2001; Boekhout and Wolthuis, 2015) (Fig. 3A). Interestingly, the amounts of APC/C-bound cyclin B1 or Nek2A did not correlate with the amounts of Cdc20 bound to the APC/C, indicating that Cdc20 may not be required for their binding (Fig. 3A; compare fourth and fifth lanes).


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

Voets E, Wolthuis R - Biol Open (2015)

MASTL depletion significantly impairs cyclin B1 recruitment to the mitotic APC/C.(A) Cyclin B1 binds to the APC/C specifically in mitosis. HeLa cells were synchronised in G2 and M phase by a thymidine block, followed by nocodazole or taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off, and treated for another 2 hours with 5 µM MG132 where indicated. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4. Aliquots of the immunoprecipitates were analysed by western blotting. (B) MASTL silencing impairs the recruitment of cyclin A2 and cyclin B1 to the mitotic APC/C. 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 goat anti-APC4 and aliquots of the immunoprecipitates were analysed by western blotting. Note that equal amounts of APC4 are immunoprecipitated in the mitotic fractions, whereas cyclins A2 and B1 bound to the APC/C are hardly detectable in the MASTL RNAi extracts. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin (right-hand panel). (C) Direct binding of cyclin B1 to the APC/C may depend on extensive APC3 phosphorylation in mitosis. HeLa cells treated as in (B), were subjected to immunoprecipitation using a mouse anti-cyclin B1. G1 phase cells were harvested 90 minutes after treatment with 10 µM of RO-3306. Aliquots of the immunoprecipitates were analysed by western blotting. For cyclin A2 blots in A,B, * denotes an aspecific band that is only observed upon immunoprecipitation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400591&req=5

f03: MASTL depletion significantly impairs cyclin B1 recruitment to the mitotic APC/C.(A) Cyclin B1 binds to the APC/C specifically in mitosis. HeLa cells were synchronised in G2 and M phase by a thymidine block, followed by nocodazole or taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off, and treated for another 2 hours with 5 µM MG132 where indicated. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4. Aliquots of the immunoprecipitates were analysed by western blotting. (B) MASTL silencing impairs the recruitment of cyclin A2 and cyclin B1 to the mitotic APC/C. 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 goat anti-APC4 and aliquots of the immunoprecipitates were analysed by western blotting. Note that equal amounts of APC4 are immunoprecipitated in the mitotic fractions, whereas cyclins A2 and B1 bound to the APC/C are hardly detectable in the MASTL RNAi extracts. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin (right-hand panel). (C) Direct binding of cyclin B1 to the APC/C may depend on extensive APC3 phosphorylation in mitosis. HeLa cells treated as in (B), were subjected to immunoprecipitation using a mouse anti-cyclin B1. G1 phase cells were harvested 90 minutes after treatment with 10 µM of RO-3306. Aliquots of the immunoprecipitates were analysed by western blotting. For cyclin A2 blots in A,B, * denotes an aspecific band that is only observed upon immunoprecipitation.
Mentions: We then reasoned that stabilisation of cyclin B1 may depend on the recruitment of cyclin B1 by the mitotic APC/C. Previously, we reported that cyclin B1, bound to Cdk1 and Cks, is retained at the APC/C in prometaphase, in a manner strictly dependent on APC3 (van Zon et al., 2010). Indeed, when we performed immunoprecipitation experiments using an APC4 antibody, to pull-down the APC/C from mitotic HeLa cell extracts and analysed interacting proteins on western blots, we found that cyclin B1, like cyclin A2, specifically bound to APC/CCdc20 in prometaphase (Fig. 3A). The binding of cyclins A2 and B1 to the APC/C was specific for mitosis, and occurred regardless of the strength of the spindle checkpoint (Fig. 3A; compare nocodazole- with taxol-arrested cells) (Collin et al., 2013). In strong contrast, the APC/CCdc20 substrate Nek2A bound the APC/C already in G2 phase, in line with previous findings and our own recent results (Hames et al., 2001; Boekhout and Wolthuis, 2015) (Fig. 3A). Interestingly, the amounts of APC/C-bound cyclin B1 or Nek2A did not correlate with the amounts of Cdc20 bound to the APC/C, indicating that Cdc20 may not be required for their binding (Fig. 3A; compare fourth and fifth lanes).

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