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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-mediated PP2A inhibition in mitosis is essential to direct cyclin B1 destruction by the APC/C.(A) Forced mitotic exit by either Aurora B or Cdk1 inhibition stabilises cyclin B1 in MASTL RNAi cells. HeLa cells were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 (Cdk1 inhibitor) or 1 µM of AZD1152 (Aurora B inhibitor) for 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. (B) APC/C phosphorylation in mitosis critically depends on cyclin B1-Cdk1 activity. HeLa cells transfected with indicated siRNA pools were synchronised in mitosis by a thymidine block followed by subsequent taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis either 24 hours (PLK1 RNAi) or 48 hours after the siRNA transfection and blotted for the proteins as indicated. (C) Enforcing mitotic exit in absence of MASTL severely impairs the proteolysis of cyclin B1. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1, geminin, and securin signals were quantified for each condition and corrected for a background band, which was used as loading control (n = 3; mean±s.e.m.). (D) MASTL depletion selectively stabilises cyclin B1 in G1 phase, independent of general APC/C activity. HeLa cells transfected with indicated siRNAs were synchronised in M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin. The intensity of the cyclin B1, securin, and geminin signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.).
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f02: MASTL-mediated PP2A inhibition in mitosis is essential to direct cyclin B1 destruction by the APC/C.(A) Forced mitotic exit by either Aurora B or Cdk1 inhibition stabilises cyclin B1 in MASTL RNAi cells. HeLa cells were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 (Cdk1 inhibitor) or 1 µM of AZD1152 (Aurora B inhibitor) for 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. (B) APC/C phosphorylation in mitosis critically depends on cyclin B1-Cdk1 activity. HeLa cells transfected with indicated siRNA pools were synchronised in mitosis by a thymidine block followed by subsequent taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis either 24 hours (PLK1 RNAi) or 48 hours after the siRNA transfection and blotted for the proteins as indicated. (C) Enforcing mitotic exit in absence of MASTL severely impairs the proteolysis of cyclin B1. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1, geminin, and securin signals were quantified for each condition and corrected for a background band, which was used as loading control (n = 3; mean±s.e.m.). (D) MASTL depletion selectively stabilises cyclin B1 in G1 phase, independent of general APC/C activity. HeLa cells transfected with indicated siRNAs were synchronised in M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin. The intensity of the cyclin B1, securin, and geminin signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.).

Mentions: To further determine whether the destruction of APC/C substrates related to impaired APC/C phosphorylation, HeLa cells were monitored by western blot analysis of their extracts taken at different time points after inducing exit from mitosis. Therefore, a pure fraction of mitotic cells, arrested in taxol, was collected by mitotic shake-off and analysed, or treated with a Cdk1 inhibitor (RO-3306) or an Aurora B inhibitor (AZD1152), which induce spindle checkpoint override and force mitotic exit (Fig. 2A). MASTL RNAi prevented the efficient destruction of the APC/CCdc20 substrate cyclin B1 upon mitotic exit, while securin destruction was less affected. Suppression of MASTL, CDC2, CCNB1, or a combination of CKS1B and CKS2, impaired APC/C phosphorylation in mitosis, detected by the incomplete phospho-shift of APC3 on western blot in fractions of purified mitotic cells (Fig. 2B). Cyclin B1-Cdk1, in a manner dependent on the Cdk1 subunit Cks, phosphorylates the APC/C subunit APC3 in mitosis (Kraft et al., 2003; Patra and Dunphy, 1998). Interestingly, in MASTL-depleted cells, cyclin B1 did not disappear for at least two hours after mitotic exit [Fig. 2C; western blot and left graph; 40%±0.07% cyclin B1 remained 120 minutes after mitotic exit (siMASTL) compared to 13%±0.04% (siCTRL)]. Securin and geminin levels followed a similar trend, but were clearly not stabilised as efficiently as cyclin B1 (Fig. 2C; securin levels after 120 minutes: 13%±0.02% in siMASTL compared to 7%±0.01% in siCTRL; geminin levels after 120 minutes: 13%±0.02% in siMASTL compared to 9%±0.04% in siCTRL). We conclude that either the APC/C is not properly activated by Cdc20 when MASTL-depleted cells enter mitosis, or the destruction of cyclin B1 is more dependent on MASTL than that of other Cdc20 substrates.


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-mediated PP2A inhibition in mitosis is essential to direct cyclin B1 destruction by the APC/C.(A) Forced mitotic exit by either Aurora B or Cdk1 inhibition stabilises cyclin B1 in MASTL RNAi cells. HeLa cells were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 (Cdk1 inhibitor) or 1 µM of AZD1152 (Aurora B inhibitor) for 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. (B) APC/C phosphorylation in mitosis critically depends on cyclin B1-Cdk1 activity. HeLa cells transfected with indicated siRNA pools were synchronised in mitosis by a thymidine block followed by subsequent taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis either 24 hours (PLK1 RNAi) or 48 hours after the siRNA transfection and blotted for the proteins as indicated. (C) Enforcing mitotic exit in absence of MASTL severely impairs the proteolysis of cyclin B1. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1, geminin, and securin signals were quantified for each condition and corrected for a background band, which was used as loading control (n = 3; mean±s.e.m.). (D) MASTL depletion selectively stabilises cyclin B1 in G1 phase, independent of general APC/C activity. HeLa cells transfected with indicated siRNAs were synchronised in M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin. The intensity of the cyclin B1, securin, and geminin signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.).
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f02: MASTL-mediated PP2A inhibition in mitosis is essential to direct cyclin B1 destruction by the APC/C.(A) Forced mitotic exit by either Aurora B or Cdk1 inhibition stabilises cyclin B1 in MASTL RNAi cells. HeLa cells were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 (Cdk1 inhibitor) or 1 µM of AZD1152 (Aurora B inhibitor) for 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. (B) APC/C phosphorylation in mitosis critically depends on cyclin B1-Cdk1 activity. HeLa cells transfected with indicated siRNA pools were synchronised in mitosis by a thymidine block followed by subsequent taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis either 24 hours (PLK1 RNAi) or 48 hours after the siRNA transfection and blotted for the proteins as indicated. (C) Enforcing mitotic exit in absence of MASTL severely impairs the proteolysis of cyclin B1. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1, geminin, and securin signals were quantified for each condition and corrected for a background band, which was used as loading control (n = 3; mean±s.e.m.). (D) MASTL depletion selectively stabilises cyclin B1 in G1 phase, independent of general APC/C activity. HeLa cells transfected with indicated siRNAs were synchronised in M phase by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 µM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin. The intensity of the cyclin B1, securin, and geminin signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.).
Mentions: To further determine whether the destruction of APC/C substrates related to impaired APC/C phosphorylation, HeLa cells were monitored by western blot analysis of their extracts taken at different time points after inducing exit from mitosis. Therefore, a pure fraction of mitotic cells, arrested in taxol, was collected by mitotic shake-off and analysed, or treated with a Cdk1 inhibitor (RO-3306) or an Aurora B inhibitor (AZD1152), which induce spindle checkpoint override and force mitotic exit (Fig. 2A). MASTL RNAi prevented the efficient destruction of the APC/CCdc20 substrate cyclin B1 upon mitotic exit, while securin destruction was less affected. Suppression of MASTL, CDC2, CCNB1, or a combination of CKS1B and CKS2, impaired APC/C phosphorylation in mitosis, detected by the incomplete phospho-shift of APC3 on western blot in fractions of purified mitotic cells (Fig. 2B). Cyclin B1-Cdk1, in a manner dependent on the Cdk1 subunit Cks, phosphorylates the APC/C subunit APC3 in mitosis (Kraft et al., 2003; Patra and Dunphy, 1998). Interestingly, in MASTL-depleted cells, cyclin B1 did not disappear for at least two hours after mitotic exit [Fig. 2C; western blot and left graph; 40%±0.07% cyclin B1 remained 120 minutes after mitotic exit (siMASTL) compared to 13%±0.04% (siCTRL)]. Securin and geminin levels followed a similar trend, but were clearly not stabilised as efficiently as cyclin B1 (Fig. 2C; securin levels after 120 minutes: 13%±0.02% in siMASTL compared to 7%±0.01% in siCTRL; geminin levels after 120 minutes: 13%±0.02% in siMASTL compared to 9%±0.04% in siCTRL). We conclude that either the APC/C is not properly activated by Cdc20 when MASTL-depleted cells enter mitosis, or the destruction of cyclin B1 is more dependent on MASTL than that of other Cdc20 substrates.

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