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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 counteracts the mitotic phosphatase PP2A.(A) Depletion of MASTL reduces mitotic phosphorylation events. HeLa cells transfected with a control RNAi (siCTRL) or with a siRNA pool targeting MASTL (siMASTL) were either asynchronously grown or synchronised in mitosis by a thymidine block and subsequent nocodazole treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. A quantification of the phosphorylated threonine signal (p-Threonine) demonstrates that MASTL depletion lowers the abundance of mitotic phosphorylations. The intensity of the p-Threonine signal was quantified for the entire lane, as well as for individual bands on the gel. (B) MASTL contributes to efficient Cdk1 substrate phosphorylation. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and subsequently blocked in mitosis by nocodazole treatment. Mitotic cells were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. The intensity of the phosphorylated serine Cdks substrate antibody signal (p-Serine CDKs substrate) was quantified for each individual lane [n = 3; mean±standard error of the mean (s.e.m.)]. (C) Silencing of the phosphatase PP2A by individual siRNAs. Asynchronously growing HeLa cells were transfected with either a pool of four individual siRNAs targeting human PPP2CA (siPPP2CA pool) or each of the siRNAs separately and processed for western blotting 48 hours after the transfection. Extracts were blotted for the proteins as indicated. The intensity of the PP2A-Cα and PP2A-A signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.). (D) MASTL counteracts the activity of PP2A in mitosis. 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 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1 and p-Serine signal was quantified for each individual lane and corrected for the Actin loading control (n = 3; mean±s.e.m.). Note that the APC3 phosphorylation is restored upon co-depletion of MASTL and PPP2CA.
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f01: MASTL counteracts the mitotic phosphatase PP2A.(A) Depletion of MASTL reduces mitotic phosphorylation events. HeLa cells transfected with a control RNAi (siCTRL) or with a siRNA pool targeting MASTL (siMASTL) were either asynchronously grown or synchronised in mitosis by a thymidine block and subsequent nocodazole treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. A quantification of the phosphorylated threonine signal (p-Threonine) demonstrates that MASTL depletion lowers the abundance of mitotic phosphorylations. The intensity of the p-Threonine signal was quantified for the entire lane, as well as for individual bands on the gel. (B) MASTL contributes to efficient Cdk1 substrate phosphorylation. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and subsequently blocked in mitosis by nocodazole treatment. Mitotic cells were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. The intensity of the phosphorylated serine Cdks substrate antibody signal (p-Serine CDKs substrate) was quantified for each individual lane [n = 3; mean±standard error of the mean (s.e.m.)]. (C) Silencing of the phosphatase PP2A by individual siRNAs. Asynchronously growing HeLa cells were transfected with either a pool of four individual siRNAs targeting human PPP2CA (siPPP2CA pool) or each of the siRNAs separately and processed for western blotting 48 hours after the transfection. Extracts were blotted for the proteins as indicated. The intensity of the PP2A-Cα and PP2A-A signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.). (D) MASTL counteracts the activity of PP2A in mitosis. 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 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1 and p-Serine signal was quantified for each individual lane and corrected for the Actin loading control (n = 3; mean±s.e.m.). Note that the APC3 phosphorylation is restored upon co-depletion of MASTL and PPP2CA.

Mentions: To reveal the role of MASTL in phosphorylation events in mitosis, we silenced MASTL by RNAi and then collected a pure fraction of mitotic cells (Fig. 1A, left panel). First, we analysed the status of threonine phosphorylation of mitotic epitopes by western blots. Phospho-threonine epitopes were, on average, two-fold reduced in MASTL-depleted mitotic cells as compared to control mitotic cells (Fig. 1A, second panel; the table shows relative lane density is 0.55 in siMASTL cells compared to siCTRL). The depletion of MASTL reduced the overall spectrum of cyclin B1-Cdk1 substrates, as shown by the quantification of individual bands recognised by the phospho-threonine antibody (Fig. 1A, right table). MASTL depletion led to a similar reduction of total mitotic phospho-serine epitopes (Fig. 1B). Further quantification of the phospho-serine signal showed that while MASTL depletion reduced mitotic phosphorylations (61%±0.02% of siCTRL), phospho-serine levels in G2 phase were maintained, so these occurred mostly independently of MASTL [8%±0.02% (siMASTL) or 10%±0.03% (siCTRL)] (Fig. 1B, right graph).


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 counteracts the mitotic phosphatase PP2A.(A) Depletion of MASTL reduces mitotic phosphorylation events. HeLa cells transfected with a control RNAi (siCTRL) or with a siRNA pool targeting MASTL (siMASTL) were either asynchronously grown or synchronised in mitosis by a thymidine block and subsequent nocodazole treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. A quantification of the phosphorylated threonine signal (p-Threonine) demonstrates that MASTL depletion lowers the abundance of mitotic phosphorylations. The intensity of the p-Threonine signal was quantified for the entire lane, as well as for individual bands on the gel. (B) MASTL contributes to efficient Cdk1 substrate phosphorylation. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and subsequently blocked in mitosis by nocodazole treatment. Mitotic cells were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. The intensity of the phosphorylated serine Cdks substrate antibody signal (p-Serine CDKs substrate) was quantified for each individual lane [n = 3; mean±standard error of the mean (s.e.m.)]. (C) Silencing of the phosphatase PP2A by individual siRNAs. Asynchronously growing HeLa cells were transfected with either a pool of four individual siRNAs targeting human PPP2CA (siPPP2CA pool) or each of the siRNAs separately and processed for western blotting 48 hours after the transfection. Extracts were blotted for the proteins as indicated. The intensity of the PP2A-Cα and PP2A-A signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.). (D) MASTL counteracts the activity of PP2A in mitosis. 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 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1 and p-Serine signal was quantified for each individual lane and corrected for the Actin loading control (n = 3; mean±s.e.m.). Note that the APC3 phosphorylation is restored upon co-depletion of MASTL and PPP2CA.
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Related In: Results  -  Collection

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f01: MASTL counteracts the mitotic phosphatase PP2A.(A) Depletion of MASTL reduces mitotic phosphorylation events. HeLa cells transfected with a control RNAi (siCTRL) or with a siRNA pool targeting MASTL (siMASTL) were either asynchronously grown or synchronised in mitosis by a thymidine block and subsequent nocodazole treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. A quantification of the phosphorylated threonine signal (p-Threonine) demonstrates that MASTL depletion lowers the abundance of mitotic phosphorylations. The intensity of the p-Threonine signal was quantified for the entire lane, as well as for individual bands on the gel. (B) MASTL contributes to efficient Cdk1 substrate phosphorylation. HeLa cells transfected with indicated siRNAs were synchronised in G2 and M phase by thymidine synchronisation and subsequently blocked in mitosis by nocodazole treatment. Mitotic cells were collected by mitotic shake-off. Extracts were processed for western blot analysis and blotted for the proteins as indicated. The intensity of the phosphorylated serine Cdks substrate antibody signal (p-Serine CDKs substrate) was quantified for each individual lane [n = 3; mean±standard error of the mean (s.e.m.)]. (C) Silencing of the phosphatase PP2A by individual siRNAs. Asynchronously growing HeLa cells were transfected with either a pool of four individual siRNAs targeting human PPP2CA (siPPP2CA pool) or each of the siRNAs separately and processed for western blotting 48 hours after the transfection. Extracts were blotted for the proteins as indicated. The intensity of the PP2A-Cα and PP2A-A signals were quantified for each condition and corrected for the Actin loading control (n = 3; mean±s.e.m.). (D) MASTL counteracts the activity of PP2A in mitosis. 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 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1 and p-Serine signal was quantified for each individual lane and corrected for the Actin loading control (n = 3; mean±s.e.m.). Note that the APC3 phosphorylation is restored upon co-depletion of MASTL and PPP2CA.
Mentions: To reveal the role of MASTL in phosphorylation events in mitosis, we silenced MASTL by RNAi and then collected a pure fraction of mitotic cells (Fig. 1A, left panel). First, we analysed the status of threonine phosphorylation of mitotic epitopes by western blots. Phospho-threonine epitopes were, on average, two-fold reduced in MASTL-depleted mitotic cells as compared to control mitotic cells (Fig. 1A, second panel; the table shows relative lane density is 0.55 in siMASTL cells compared to siCTRL). The depletion of MASTL reduced the overall spectrum of cyclin B1-Cdk1 substrates, as shown by the quantification of individual bands recognised by the phospho-threonine antibody (Fig. 1A, right table). MASTL depletion led to a similar reduction of total mitotic phospho-serine epitopes (Fig. 1B). Further quantification of the phospho-serine signal showed that while MASTL depletion reduced mitotic phosphorylations (61%±0.02% of siCTRL), phospho-serine levels in G2 phase were maintained, so these occurred mostly independently of MASTL [8%±0.02% (siMASTL) or 10%±0.03% (siCTRL)] (Fig. 1B, right graph).

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