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A specific form of phospho protein phosphatase 2 regulates anaphase-promoting complex/cyclosome association with spindle poles.

Torres JZ, Ban KH, Jackson PK - Mol. Biol. Cell (2010)

Bottom Line: Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity.Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle.Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. torres@chem.ucla.edu

ABSTRACT
In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

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Dynamic association of the APC/C with mitotic microtubule asters is regulated by a kinase-phosphatase equilibrium. (A) Inhibition of Cdk activity and an OA-sensitive phosphatase in vivo perturbs the association of APC/C with microtubules. Mitotic HeLa cells were treated with control vehicle, the Cdk inhibitor roscovitine (Rosco; 1 h), or the phosphatase inhibitor okadaic acid (OA; 30 min) before extract preparation. Mitotic microtubule copelleting reactions (as in Figure 2) were performed in the presence of control vehicle, Rosco, or OA. Supernatant and pellet fractions were immunoblotted with antibodies against APC/C subunits/coactivators (Cdc27, Apc8, Cdc20, and Cdh1) or Eg5 to detect microtubule association. Cdc27 phosphorylation status, association with microtubules, and tubulin polymerization was monitored, as described in Figure 2B. For A and B, arrows indicate phosphorylated and dephosphorylated forms of Cdc27. (B) Phosphorylation and dephosphorylation of APC/C in vitro perturbs APC/C microtubule association. Mitotic HeLa cells were treated with control vehicle, Rosco, or OA as described in A before extract preparation. Extracts from Rosco-treated cells were incubated with cyclin B/Cdk1 for 20 min at 30°C. Conversely, extracts from OA-treated cells were incubated with lambda phosphatase (λPPase) for 20 min at 30°C. All extracts were subjected to mitotic microtubule copelleting reactions and the status of Cdc27 phosphorylation, its association with microtubules, and tubulin polymerization was detected as described in Figure 2B.
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Figure 3: Dynamic association of the APC/C with mitotic microtubule asters is regulated by a kinase-phosphatase equilibrium. (A) Inhibition of Cdk activity and an OA-sensitive phosphatase in vivo perturbs the association of APC/C with microtubules. Mitotic HeLa cells were treated with control vehicle, the Cdk inhibitor roscovitine (Rosco; 1 h), or the phosphatase inhibitor okadaic acid (OA; 30 min) before extract preparation. Mitotic microtubule copelleting reactions (as in Figure 2) were performed in the presence of control vehicle, Rosco, or OA. Supernatant and pellet fractions were immunoblotted with antibodies against APC/C subunits/coactivators (Cdc27, Apc8, Cdc20, and Cdh1) or Eg5 to detect microtubule association. Cdc27 phosphorylation status, association with microtubules, and tubulin polymerization was monitored, as described in Figure 2B. For A and B, arrows indicate phosphorylated and dephosphorylated forms of Cdc27. (B) Phosphorylation and dephosphorylation of APC/C in vitro perturbs APC/C microtubule association. Mitotic HeLa cells were treated with control vehicle, Rosco, or OA as described in A before extract preparation. Extracts from Rosco-treated cells were incubated with cyclin B/Cdk1 for 20 min at 30°C. Conversely, extracts from OA-treated cells were incubated with lambda phosphatase (λPPase) for 20 min at 30°C. All extracts were subjected to mitotic microtubule copelleting reactions and the status of Cdc27 phosphorylation, its association with microtubules, and tubulin polymerization was detected as described in Figure 2B.

Mentions: The displacement of Cdc27 from spindle poles upon acute OA treatment indicated that this association was likely dynamic, which suggests it could be counteracted by a mitotic kinase. Cyclin B/Cdk1 is the predominant mitotic kinase that regulates APC/C activity and phosphorylates several APC/C subunits in vitro, including Cdc27 (Kraft et al., 2003). Thus, we analyzed whether cyclin B/Cdk1 could directly regulate APC/C-microtubule association. First, we inactivated Cdks with the small molecule inhibitor Rosco. Mitotic HeLa cells were treated in vivo with Rosco before extract preparation and in vitro microtubule aster polymerization reactions were performed in the continued presence of Rosco. Inhibition of Cdk activity led to an accumulation of hypophosphorylated Cdc27 with an increased binding to microtubules (Figure 3A). This is in agreement with previous in vitro results showing that cyclin B/Cdk1 phosphorylates Cdc27 in mitosis and that phosphorylated Cdc27 localizes to centrosomes and not the mitotic microtubule spindle (Kraft et al., 2003). Another APC/C subunit, Apc8 and the substrate adaptor proteins Cdc20 and Cdh1 also increased in microtubule binding upon Rosco treatment (Figure 3A).


A specific form of phospho protein phosphatase 2 regulates anaphase-promoting complex/cyclosome association with spindle poles.

Torres JZ, Ban KH, Jackson PK - Mol. Biol. Cell (2010)

Dynamic association of the APC/C with mitotic microtubule asters is regulated by a kinase-phosphatase equilibrium. (A) Inhibition of Cdk activity and an OA-sensitive phosphatase in vivo perturbs the association of APC/C with microtubules. Mitotic HeLa cells were treated with control vehicle, the Cdk inhibitor roscovitine (Rosco; 1 h), or the phosphatase inhibitor okadaic acid (OA; 30 min) before extract preparation. Mitotic microtubule copelleting reactions (as in Figure 2) were performed in the presence of control vehicle, Rosco, or OA. Supernatant and pellet fractions were immunoblotted with antibodies against APC/C subunits/coactivators (Cdc27, Apc8, Cdc20, and Cdh1) or Eg5 to detect microtubule association. Cdc27 phosphorylation status, association with microtubules, and tubulin polymerization was monitored, as described in Figure 2B. For A and B, arrows indicate phosphorylated and dephosphorylated forms of Cdc27. (B) Phosphorylation and dephosphorylation of APC/C in vitro perturbs APC/C microtubule association. Mitotic HeLa cells were treated with control vehicle, Rosco, or OA as described in A before extract preparation. Extracts from Rosco-treated cells were incubated with cyclin B/Cdk1 for 20 min at 30°C. Conversely, extracts from OA-treated cells were incubated with lambda phosphatase (λPPase) for 20 min at 30°C. All extracts were subjected to mitotic microtubule copelleting reactions and the status of Cdc27 phosphorylation, its association with microtubules, and tubulin polymerization was detected as described in Figure 2B.
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Figure 3: Dynamic association of the APC/C with mitotic microtubule asters is regulated by a kinase-phosphatase equilibrium. (A) Inhibition of Cdk activity and an OA-sensitive phosphatase in vivo perturbs the association of APC/C with microtubules. Mitotic HeLa cells were treated with control vehicle, the Cdk inhibitor roscovitine (Rosco; 1 h), or the phosphatase inhibitor okadaic acid (OA; 30 min) before extract preparation. Mitotic microtubule copelleting reactions (as in Figure 2) were performed in the presence of control vehicle, Rosco, or OA. Supernatant and pellet fractions were immunoblotted with antibodies against APC/C subunits/coactivators (Cdc27, Apc8, Cdc20, and Cdh1) or Eg5 to detect microtubule association. Cdc27 phosphorylation status, association with microtubules, and tubulin polymerization was monitored, as described in Figure 2B. For A and B, arrows indicate phosphorylated and dephosphorylated forms of Cdc27. (B) Phosphorylation and dephosphorylation of APC/C in vitro perturbs APC/C microtubule association. Mitotic HeLa cells were treated with control vehicle, Rosco, or OA as described in A before extract preparation. Extracts from Rosco-treated cells were incubated with cyclin B/Cdk1 for 20 min at 30°C. Conversely, extracts from OA-treated cells were incubated with lambda phosphatase (λPPase) for 20 min at 30°C. All extracts were subjected to mitotic microtubule copelleting reactions and the status of Cdc27 phosphorylation, its association with microtubules, and tubulin polymerization was detected as described in Figure 2B.
Mentions: The displacement of Cdc27 from spindle poles upon acute OA treatment indicated that this association was likely dynamic, which suggests it could be counteracted by a mitotic kinase. Cyclin B/Cdk1 is the predominant mitotic kinase that regulates APC/C activity and phosphorylates several APC/C subunits in vitro, including Cdc27 (Kraft et al., 2003). Thus, we analyzed whether cyclin B/Cdk1 could directly regulate APC/C-microtubule association. First, we inactivated Cdks with the small molecule inhibitor Rosco. Mitotic HeLa cells were treated in vivo with Rosco before extract preparation and in vitro microtubule aster polymerization reactions were performed in the continued presence of Rosco. Inhibition of Cdk activity led to an accumulation of hypophosphorylated Cdc27 with an increased binding to microtubules (Figure 3A). This is in agreement with previous in vitro results showing that cyclin B/Cdk1 phosphorylates Cdc27 in mitosis and that phosphorylated Cdc27 localizes to centrosomes and not the mitotic microtubule spindle (Kraft et al., 2003). Another APC/C subunit, Apc8 and the substrate adaptor proteins Cdc20 and Cdh1 also increased in microtubule binding upon Rosco treatment (Figure 3A).

Bottom Line: Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity.Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle.Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA. torres@chem.ucla.edu

ABSTRACT
In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

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