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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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Association of Cdc27 with spindle poles requires the PPP2 subunits CA, R1A, and R2B. (A) Dephosphorylated APC/C preferentially associates with microtubule asters in vitro. Mitotic HeLa cell extracts were used for in vitro microtubule polymerization reactions in the presence or absence of taxol. Polymerized microtubules were subjected to centrifugation, and samples from the supernatant (S) and pelleted (P) microtubules were analyzed by immunoblot. Immunoblotting was used to detect the association of the APC/C subunits Cdc27, Apc8, Cdc20, Cdh1, and positive (mitotic kinesin Eg5) and negative (CycD) controls with microtubules. The upper and lower arrows indicate phosphorylated and dephosphorylated forms of Cdc27, respectively. Tubulin polymerization was confirmed by staining the membrane with Coomassie blue. (B) Depletion of CA, R1A, or R2B by siRNA leads to delocalization of Cdc27 from spindle poles. RNAi-treated cells (as in Figure 1) or cells treated acutely with 175 nM OA for 13 min were fixed and stained with Hoechst 33342 and anti-α-tubulin and anti-Cdc27 antibodies to visualize DNA, the mitotic spindle, and Cdc27, respectively. Bar, 5 μm. (C) Quantitation of mean intensity of Cdc27 at spindle poles. A 2-μm square was drawn around each of 20 spindle poles from control, CA, R1A, or R2B siRNA-treated cells or cells treated acutely with OA, and the mean fluorescence intensity of Cdc27 spindle pole staining was quantified and presented as arbitrary units (AU). (D) Spindle associated Cdc27 is reduced in mitotic cells depleted of CA, R1A, or R2B or treated acutely with OA. Line intensity measurements taken along axis intersecting the two poles (marked by arrows in A) were quantified and graphed. X-axis, distance in micrometers from the center of the two poles. Y-axis, fluorescence intensities along the axis in arbitrary units (AU).
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Figure 2: Association of Cdc27 with spindle poles requires the PPP2 subunits CA, R1A, and R2B. (A) Dephosphorylated APC/C preferentially associates with microtubule asters in vitro. Mitotic HeLa cell extracts were used for in vitro microtubule polymerization reactions in the presence or absence of taxol. Polymerized microtubules were subjected to centrifugation, and samples from the supernatant (S) and pelleted (P) microtubules were analyzed by immunoblot. Immunoblotting was used to detect the association of the APC/C subunits Cdc27, Apc8, Cdc20, Cdh1, and positive (mitotic kinesin Eg5) and negative (CycD) controls with microtubules. The upper and lower arrows indicate phosphorylated and dephosphorylated forms of Cdc27, respectively. Tubulin polymerization was confirmed by staining the membrane with Coomassie blue. (B) Depletion of CA, R1A, or R2B by siRNA leads to delocalization of Cdc27 from spindle poles. RNAi-treated cells (as in Figure 1) or cells treated acutely with 175 nM OA for 13 min were fixed and stained with Hoechst 33342 and anti-α-tubulin and anti-Cdc27 antibodies to visualize DNA, the mitotic spindle, and Cdc27, respectively. Bar, 5 μm. (C) Quantitation of mean intensity of Cdc27 at spindle poles. A 2-μm square was drawn around each of 20 spindle poles from control, CA, R1A, or R2B siRNA-treated cells or cells treated acutely with OA, and the mean fluorescence intensity of Cdc27 spindle pole staining was quantified and presented as arbitrary units (AU). (D) Spindle associated Cdc27 is reduced in mitotic cells depleted of CA, R1A, or R2B or treated acutely with OA. Line intensity measurements taken along axis intersecting the two poles (marked by arrows in A) were quantified and graphed. X-axis, distance in micrometers from the center of the two poles. Y-axis, fluorescence intensities along the axis in arbitrary units (AU).

Mentions: Most APC/C subunits exist in a hypophosphorylated state during G1/S and several subunits are phosphorylated in mitosis by the activities of cyclin B/Cdk1 and Plk1 (Kraft et al., 2003; Pines, 2006). Previous immunofluorescence data indicated that phosphorylation might regulate APC/C subcellular localization, with phospho-APC/C (Cdc20 subset) associating with centrosomes and hypophosphorylated APC/C (Cdh1 subset) associating with the spindle, spindle poles, kinetochores, and chromosome arms (Tugendreich et al., 1995; Topper et al., 2002; Kraft et al., 2003). Thus, we hypothesized that the ability of the APC/C to associate with spindle poles was regulated through phosphorylation. To test this hypothesis, we used a mitotic microtubule aster copelleting assay (Mack and Compton, 2001) to monitor APC/C binding to the mitotic spindle. Mitotic HeLa cell extracts were prepared and treated with the microtubule-stabilizing drug, taxol, to induce microtubule polymerization. Polymerization of microtubule asters was monitored by fluorescence microscopy using anti-α-tubulin antibodies. Polymerized microtubules and their associated proteins were then sedimented through a sucrose cushion. Protein samples from the supernatant (S) and the pelleted microtubule-binding proteins (P) were probed with antibodies specific for Eg5, a kinesin necessary for proper mitotic spindle assembly, and cyclin D, a protein that does not bind the mitotic spindle (Sawin et al., 1992; Figure 2A). Eg5 kinesin associated with the taxol-stabilized microtubule pellet, whereas cyclin D remained in the supernatant (Figure 2A). Neither protein pelleted in the absence of taxol, indicating minimal nonspecific pelleting (Figure 2A).


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)

Association of Cdc27 with spindle poles requires the PPP2 subunits CA, R1A, and R2B. (A) Dephosphorylated APC/C preferentially associates with microtubule asters in vitro. Mitotic HeLa cell extracts were used for in vitro microtubule polymerization reactions in the presence or absence of taxol. Polymerized microtubules were subjected to centrifugation, and samples from the supernatant (S) and pelleted (P) microtubules were analyzed by immunoblot. Immunoblotting was used to detect the association of the APC/C subunits Cdc27, Apc8, Cdc20, Cdh1, and positive (mitotic kinesin Eg5) and negative (CycD) controls with microtubules. The upper and lower arrows indicate phosphorylated and dephosphorylated forms of Cdc27, respectively. Tubulin polymerization was confirmed by staining the membrane with Coomassie blue. (B) Depletion of CA, R1A, or R2B by siRNA leads to delocalization of Cdc27 from spindle poles. RNAi-treated cells (as in Figure 1) or cells treated acutely with 175 nM OA for 13 min were fixed and stained with Hoechst 33342 and anti-α-tubulin and anti-Cdc27 antibodies to visualize DNA, the mitotic spindle, and Cdc27, respectively. Bar, 5 μm. (C) Quantitation of mean intensity of Cdc27 at spindle poles. A 2-μm square was drawn around each of 20 spindle poles from control, CA, R1A, or R2B siRNA-treated cells or cells treated acutely with OA, and the mean fluorescence intensity of Cdc27 spindle pole staining was quantified and presented as arbitrary units (AU). (D) Spindle associated Cdc27 is reduced in mitotic cells depleted of CA, R1A, or R2B or treated acutely with OA. Line intensity measurements taken along axis intersecting the two poles (marked by arrows in A) were quantified and graphed. X-axis, distance in micrometers from the center of the two poles. Y-axis, fluorescence intensities along the axis in arbitrary units (AU).
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Figure 2: Association of Cdc27 with spindle poles requires the PPP2 subunits CA, R1A, and R2B. (A) Dephosphorylated APC/C preferentially associates with microtubule asters in vitro. Mitotic HeLa cell extracts were used for in vitro microtubule polymerization reactions in the presence or absence of taxol. Polymerized microtubules were subjected to centrifugation, and samples from the supernatant (S) and pelleted (P) microtubules were analyzed by immunoblot. Immunoblotting was used to detect the association of the APC/C subunits Cdc27, Apc8, Cdc20, Cdh1, and positive (mitotic kinesin Eg5) and negative (CycD) controls with microtubules. The upper and lower arrows indicate phosphorylated and dephosphorylated forms of Cdc27, respectively. Tubulin polymerization was confirmed by staining the membrane with Coomassie blue. (B) Depletion of CA, R1A, or R2B by siRNA leads to delocalization of Cdc27 from spindle poles. RNAi-treated cells (as in Figure 1) or cells treated acutely with 175 nM OA for 13 min were fixed and stained with Hoechst 33342 and anti-α-tubulin and anti-Cdc27 antibodies to visualize DNA, the mitotic spindle, and Cdc27, respectively. Bar, 5 μm. (C) Quantitation of mean intensity of Cdc27 at spindle poles. A 2-μm square was drawn around each of 20 spindle poles from control, CA, R1A, or R2B siRNA-treated cells or cells treated acutely with OA, and the mean fluorescence intensity of Cdc27 spindle pole staining was quantified and presented as arbitrary units (AU). (D) Spindle associated Cdc27 is reduced in mitotic cells depleted of CA, R1A, or R2B or treated acutely with OA. Line intensity measurements taken along axis intersecting the two poles (marked by arrows in A) were quantified and graphed. X-axis, distance in micrometers from the center of the two poles. Y-axis, fluorescence intensities along the axis in arbitrary units (AU).
Mentions: Most APC/C subunits exist in a hypophosphorylated state during G1/S and several subunits are phosphorylated in mitosis by the activities of cyclin B/Cdk1 and Plk1 (Kraft et al., 2003; Pines, 2006). Previous immunofluorescence data indicated that phosphorylation might regulate APC/C subcellular localization, with phospho-APC/C (Cdc20 subset) associating with centrosomes and hypophosphorylated APC/C (Cdh1 subset) associating with the spindle, spindle poles, kinetochores, and chromosome arms (Tugendreich et al., 1995; Topper et al., 2002; Kraft et al., 2003). Thus, we hypothesized that the ability of the APC/C to associate with spindle poles was regulated through phosphorylation. To test this hypothesis, we used a mitotic microtubule aster copelleting assay (Mack and Compton, 2001) to monitor APC/C binding to the mitotic spindle. Mitotic HeLa cell extracts were prepared and treated with the microtubule-stabilizing drug, taxol, to induce microtubule polymerization. Polymerization of microtubule asters was monitored by fluorescence microscopy using anti-α-tubulin antibodies. Polymerized microtubules and their associated proteins were then sedimented through a sucrose cushion. Protein samples from the supernatant (S) and the pelleted microtubule-binding proteins (P) were probed with antibodies specific for Eg5, a kinesin necessary for proper mitotic spindle assembly, and cyclin D, a protein that does not bind the mitotic spindle (Sawin et al., 1992; Figure 2A). Eg5 kinesin associated with the taxol-stabilized microtubule pellet, whereas cyclin D remained in the supernatant (Figure 2A). Neither protein pelleted in the absence of taxol, indicating minimal nonspecific pelleting (Figure 2A).

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.

Show MeSH
Related in: MedlinePlus