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Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720

Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720
Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720
Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.
cyclin-dependent kinase (CDK), cell cycle, embryo, mitosis, oocyte, phosphatase, anaphase-promoting complex, fertilization, oocyte maturation
0021-9258
14705-14712
Chang, HY
10ef9357-7f7b-432f-86d3-33c03f348980
Jennings, PC
fba9ff1d-c590-4715-b2d9-174bcb4c1678
Stewart, J
dec10e19-25fb-49bd-996b-d6237f5e1c87
Verrills, NM
625f908a-86b1-494a-abe1-178e200d80d8
Jones, KT
73e8e2b5-cd67-4691-b1a9-4e7bc9066af4
Chang, HY
10ef9357-7f7b-432f-86d3-33c03f348980
Jennings, PC
fba9ff1d-c590-4715-b2d9-174bcb4c1678
Stewart, J
dec10e19-25fb-49bd-996b-d6237f5e1c87
Verrills, NM
625f908a-86b1-494a-abe1-178e200d80d8
Jones, KT
73e8e2b5-cd67-4691-b1a9-4e7bc9066af4

Chang, HY, Jennings, PC, Stewart, J, Verrills, NM and Jones, KT (2011) Essential role of protein phosphatase 2A in metaphase II arrest and activation of mouse eggs shown by okadaic acid, dominant negative protein phosphatase 2A, and FTY720. The Journal of Biological Chemistry, 286 (16), 14705-14712. (doi:10.1074/jbc.M110.193227). (PMID:21383018)

Record type: Article

Abstract

Vertebrate eggs arrest at second meiotic metaphase. The fertilizing sperm causes meiotic exit through Ca(2+)-mediated activation of the anaphase-promoting complex/cyclosome (APC/C). Although the loss in activity of the M-phase kinase CDK1 is known to be an essential downstream event of this process, the contribution of phosphatases to arrest and meiotic resumption is less apparent, especially in mammals. Therefore, we explored the role of protein phosphatase 2A (PP2A) in mouse eggs using pharmacological inhibition and activation as well as a functionally dominant-negative catalytic PP2A subunit (dn-PP2Ac-L199P) coupled with live cell imaging. We observed that PP2A inhibition using okadaic acid induced events normally observed at fertilization: degradation of the APC/C substrates cyclin B1 and securin resulting from loss of the APC/C inhibitor Emi2. Although sister chromatids separated, chromatin remained condensed, and polar body extrusion was blocked as a result of a rapid spindle disruption, which could be ameliorated by non-degradable cyclin B1, suggesting that spindle integrity was affected by CDK1 loss. Similar cell cycle effects to okadaic acid were also observed using dominant-negative PP2Ac. Preincubation of eggs with the PP2A activator FTY720 could block many of the actions of okadaic acid, including Emi2, cyclin B1, and securin degradation and sister chromatid separation. Therefore, in conclusion, we used okadaic acid, dn-PP2Ac-L199P, and FTY720 on mouse eggs to demonstrate that PP2A is needed to for both continued metaphase arrest and successful exit from meiosis.

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More information

e-pub ahead of print date: 7 March 2011
Published date: 22 April 2011
Keywords: cyclin-dependent kinase (CDK), cell cycle, embryo, mitosis, oocyte, phosphatase, anaphase-promoting complex, fertilization, oocyte maturation
Organisations: Centre for Biological Sciences

Identifiers

Local EPrints ID: 340340
URI: http://eprints.soton.ac.uk/id/eprint/340340
ISSN: 0021-9258
PURE UUID: 286f5f6f-66b1-43d2-bf11-f0b5c125c8dc
ORCID for KT Jones: ORCID iD orcid.org/0000-0002-0294-0851

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Date deposited: 19 Jun 2012 13:36
Last modified: 14 Mar 2024 11:23

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Contributors

Author: HY Chang
Author: PC Jennings
Author: J Stewart
Author: NM Verrills
Author: KT Jones ORCID iD

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