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A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in
I Ferby1, M Blazquez, A Palmer
1European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
Abstract:
The activation of maturation-promoting factor (MPF) is required for G(2)/M progression in eukaryotic cells. Xenopus oocytes are arrested in G(2) and are induced to enter M phase of meiosis by progesterone stimulation. This process is known as meiotic maturation and requires the translation of specific maternal mRNAs stored in the oocytes. We have used an expression cloning strategy to functionally identify proteins involved in G(2)/M progression in Xenopus oocytes. Here we report the cloning of two novel cDNAs that when expressed in oocytes induce meiotic maturation efficiently. The two cDNAs encode proteins of 33 kD that are 88% identical and have no significant homologies to other sequences in databases. These proteins, which we refer to as p33(ringo) (rapid inducer of G(2)/M progression in oocytes), induce very rapid MPF activation in cycloheximide-treated oocytes. Conversely, ablation of endogenous p33(ringo) mRNAs using antisense oligonucleotides inhibits progesterone-induced maturation, suggesting that synthesis of p33(ringo) is required for this process. We also show that p33(ringo) binds to and activates the kinase activity of p34(cdc2) but does not associate with p34(cdc2)/cyclin B complexes. Our results identify a novel p34(cdc2) binding and activating protein that regulates the G(2)/M transition during oocyte maturation.
Insights
Researchers identified p33(ringo), a novel protein essential for cell cycle progression. P33(ringo) rapidly activates maturation-promoting factor (MPF) and is crucial for meiotic maturation in Xenopus oocytes.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Maturation-promoting factor (MPF) activation is critical for G(2)/M phase transition in eukaryotic cells.
- Xenopus oocytes arrest in G(2) and require progesterone to initiate meiotic maturation, a process dependent on maternal mRNA translation.
Purpose of the Study:
- To identify novel proteins regulating G(2)/M progression in Xenopus oocytes using expression cloning.
- To characterize the function of newly identified proteins in meiotic maturation.
Main Methods:
- Expression cloning strategy to isolate functional cDNAs.
- Oocyte injection and stimulation assays.
- Antisense oligonucleotide experiments to inhibit endogenous gene expression.
- Biochemical assays to study protein-protein interactions and kinase activity.
Main Results:
- Two novel cDNAs encoding 33 kDa proteins, named p33(ringo), were cloned. These proteins are 88% identical and lack significant homology to known sequences.
- Overexpression of p33(ringo) in oocytes induced rapid MPF activation and meiotic maturation, even in the presence of cycloheximide.
- Antisense-mediated depletion of p33(ringo) mRNA blocked progesterone-induced maturation, indicating its necessity.
- P33(ringo) was shown to bind and activate p34(cdc2) kinase activity, but not in association with p34(cdc2)/cyclin B complexes.
Conclusions:
- P33(ringo) is a novel protein that plays a critical role in regulating the G(2)/M transition during oocyte maturation.
- P33(ringo) acts as a direct activator of p34(cdc2) kinase, independent of cyclin B association.
- The findings reveal a new mechanism for controlling MPF activation and cell cycle progression.