Related Experiment Videos
Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus
D F Colgan1, K G Murthy, W Zhao
1Department of Biological Sciences, Columbia University, New York 10027, USA.
Abstract:
We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.
Insights
Poly(A) polymerase (PAP) activity is repressed by phosphorylation during M-phase. Full repression requires phosphorylation of both consensus and non-consensus cyclin-dependent kinase sites on PAP.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin-dependent kinases (CDKs) regulate cell cycle progression.
- Maturation promoting factor (MPF), a CDK/cyclin complex, controls entry into M-phase.
- Poly(A) polymerase (PAP) activity is regulated during the cell cycle.
Purpose of the Study:
- To investigate the role of non-consensus CDK sites in MPF-mediated repression of PAP activity.
- To determine the phosphorylation kinetics of consensus and non-consensus CDK sites on PAP.
- To elucidate the mechanism by which PAP activity is repressed during M-phase.
Main Methods:
- Site-directed mutagenesis of PAP C-terminal regulatory domain.
- In vitro phosphorylation assays using purified MPF and PAP.
- Analysis of PAP phosphorylation during Xenopus oocyte meiotic maturation.
Main Results:
- MPF phosphorylates both consensus and non-consensus CDK sites on PAP.
- Full repression of PAP activity requires phosphorylation of all identified CDK sites.
- Consensus sites are phosphorylated at lower MPF concentrations than non-consensus sites.
- Differential phosphorylation kinetics of consensus and non-consensus sites observed during Xenopus oocyte maturation.
Conclusions:
- PAP's repression is linked to late M-phase through differential phosphorylation of multiple CDK sites.
- The distinct sensitivities of CDK sites to MPF provide a mechanism for precise cell cycle regulation.
- This mechanism may represent a general strategy for timing CDK-dependent events in the cell cycle.