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Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates
Abstract:
The p34cdc2 protein kinase is a conserved regulator of the eukaryotic cell cycle. Here we show that residues Thr14 and Tyr15 of mouse p34cdc2 become phosphorylated as mouse fibroblasts proceed through the cell cycle. We have mutated these residues and measured protein kinase activity of the p34cdc2 variants in a Xenopus egg extract. Phosphorylation of residues 14 and 15, which lie within the presumptive ATP-binding region of p34cdc2, normally restrains the protein kinase until it is specifically dephosphorylated and activated at the G2/M transition. Regulation by dephosphorylation of Tyr15 is conserved from fission yeast to mammals, while an extra level of regulation of mammalian p34cdc2 involves Thr14 dephosphorylation. In the absence of phosphorylation on these two residues, the kinase still requires cyclin B protein for its activation. Inhibition of DNA synthesis inhibits activation of wild-type p34cdc2 in the Xenopus system, but a mutant which cannot be phosphorylated at residues 14 and 15 escapes this inhibition, suggesting that these phosphorylation events form part of the pathway linking completion of DNA replication to initiation of mitosis.
Insights
Mouse p34cdc2 protein kinase regulation involves phosphorylation at Thr14 and Tyr15. This restrains kinase activity until the G2/M transition, linking DNA replication to mitosis initiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The p34cdc2 protein kinase is a key regulator of the eukaryotic cell cycle.
- Understanding its precise regulatory mechanisms is crucial for cell cycle control.
Purpose of the Study:
- To investigate the role of Thr14 and Tyr15 phosphorylation in regulating mouse p34cdc2 protein kinase activity.
- To elucidate the conserved and novel regulatory mechanisms in mammalian cell cycle control.
Main Methods:
- Site-directed mutagenesis of mouse p34cdc2 at Thr14 and Tyr15.
- Assay of protein kinase activity in Xenopus egg extracts.
- Analysis of cell cycle-dependent phosphorylation patterns.
Main Results:
- Phosphorylation of Thr14 and Tyr15 restrains p34cdc2 kinase activity until the G2/M transition.
- Dephosphorylation of Tyr15 is conserved, while Thr14 dephosphorylation adds mammalian regulation.
- A mutant lacking Thr14/Tyr15 phosphorylation escapes DNA synthesis inhibition, indicating a link to cell cycle progression.
Conclusions:
- Phosphorylation of Thr14 and Tyr15 on p34cdc2 is essential for linking DNA replication completion to mitosis initiation.
- Mammalian p34cdc2 regulation involves both conserved (Tyr15) and novel (Thr14) dephosphorylation events.
- Cyclin B remains necessary for p34cdc2 activation, even in the absence of Thr14/Tyr15 phosphorylation.