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Sequential dephosphorylation of p34(cdc2) on Thr-14 and Tyr-15 at the prophase/metaphase transition
1Centre National de la Recherche Scientifique, Station Biologique, BP 74, 29682 Roscoff cedex, France. borgne@sb-roscoff.fr
Abstract:
The G2-M transition of the cell cycle is triggered by the p34(cdc2)/cyclin B kinase. During the prophase/metaphase transition, the inactive, Thr-14/Tyr-15 phosphorylated form of p34(cdc2) (TP-YP) is modified to an active, Thr-14/Tyr-15 dephosphorylated form (T-Y) by the cdc25 dual-specificity phosphatase. Using highly synchronized starfish oocytes as a cellular model, we show that dephosphorylation in vivo and in vitro occurs in two steps: Thr-14 dephosphorylation precedes Tyr-15 dephosphorylation. The transient intermediate form (T-YP), which can be obtained in vitro by treatment of TP-YP by protein phosphatase 2A, displays low but significant kinase activity. These results raise the possibility that the intermediate form T-YP may be involved in the autocatalytic amplification of the p34(cdc2)/cyclin B complex through phosphorylation/activation of the cdc25 phosphatase and phosphorylation/inactivation of the wee1 kinase.
Insights
The cell cycle
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The G2-M transition is regulated by p34(cdc2)/cyclin B kinase.
- Activation involves dephosphorylation of p34(cdc2) by cdc25 phosphatase.
- Inactive p34(cdc2) is phosphorylated at Thr-14 and Tyr-15 (TP-YP).
Purpose of the Study:
- To investigate the stepwise dephosphorylation mechanism of p34(cdc2).
- To characterize the intermediate dephosphorylation forms of p34(cdc2).
- To explore the role of intermediate forms in cell cycle regulation.
Main Methods:
- Utilized synchronized starfish oocytes as a cellular model.
- Performed in vivo and in vitro dephosphorylation assays.
- Employed protein phosphatase 2A for generating intermediate forms.
Main Results:
- p34(cdc2) dephosphorylation occurs in two distinct steps: Thr-14 first, then Tyr-15.
- A transient intermediate form (T-YP) with low kinase activity was identified.
- This intermediate may play a role in the positive feedback loop of cell cycle activation.
Conclusions:
- The stepwise dephosphorylation of p34(cdc2) is crucial for G2-M transition.
- The intermediate T-YP form could be involved in amplifying p34(cdc2)/cyclin B activity.
- Findings suggest a novel regulatory mechanism in cell cycle control.