Related Experiment Videos
Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase
Abstract:
Exit from metaphase of the cell cycle requires inactivation of MPF, a stoichiometric complex between the cdc2 catalytic and the cyclin B regulatory subunits, as well as that of cyclin A-cdc2 kinase. Inactivation of both complexes depends on proteolytic degradation of the cyclin subunit, yet cyclin proteolysis is not sufficient to inactivate the H1 kinase activity of cdc2. Genetic evidence strongly suggests that type 1 phosphatase plays a key role in the metaphase-anaphase transition of the cell cycle. Here we report that inhibition of both type 1 and type 2A phosphatases by okadaic acid allows cyclin degradation to occur, but prevents cdc2 kinase inactivation. Complete inhibition of type 2A phosphatase alone is not sufficient to prevent cdc2 kinase inactivation following cyclin proteolysis. We show further that residue 161 of cdc2 is phosphorylated in active cyclin A or cyclin B complexes at metaphase, whilst unassociated cdc2 is not phosphorylated. Proteolysis of cyclin releases a free cdc2 subunit, which subsequently undergoes dephosphorylation and then migrates more slowly than its Thr161 phosphorylated counterpart in Laemmli gels. Removal of phosphothreonine 161 requires cyclin proteolysis. However, it does not occur even after cyclin proteolysis, when both type 1 and type 2A phosphatases are inhibited. We conclude that both cyclin degradation and dephosphorylation of Thr161 on cdc2, catalysed at least in part by type 1 phosphatase, are required to inactivate either cyclin B- or cyclin A-cdc2 kinases and thus for cells to exit from M phase.
Insights
Cell cycle exit requires cyclin degradation and cdc2 dephosphorylation. Type 1 phosphatase is crucial for inactivating cyclin B-cdc2 and cyclin A-cdc2 kinases by removing Thr161 phosphorylation, enabling metaphase-anaphase transition.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell cycle progression, particularly exit from metaphase, depends on the inactivation of key kinases like MPF (maturation-promoting factor).
- MPF inactivation involves the degradation of cyclin subunits (cyclin B and cyclin A) complexed with cdc2.
- While cyclin degradation is necessary, it's insufficient to fully inactivate cdc2 kinase activity, suggesting other regulatory mechanisms are involved.
Purpose of the Study:
- To investigate the role of phosphatases, specifically type 1 and type 2A, in the inactivation of cdc2 kinase during cell cycle exit.
- To determine the precise events required for cdc2 kinase inactivation following cyclin proteolysis.
- To elucidate the contribution of Thr161 phosphorylation/dephosphorylation of cdc2 to cell cycle regulation.
Main Methods:
- Utilizing okadaic acid to inhibit type 1 and type 2A phosphatases in cell cycle studies.
- Monitoring cyclin degradation and cdc2 kinase activity.
- Analyzing the phosphorylation status of cdc2 at residue 161 using gel electrophoresis.
Main Results:
- Inhibition of both type 1 and type 2A phosphatases by okadaic acid allowed cyclin degradation but blocked cdc2 kinase inactivation.
- Type 2A phosphatase inhibition alone did not prevent cdc2 kinase inactivation after cyclin proteolysis.
- Phosphorylation of cdc2 at Thr161 was observed in active metaphase complexes and was removed upon cyclin proteolysis, but only when phosphatases were active.
Conclusions:
- Both cyclin degradation and the dephosphorylation of cdc2 at Thr161 are essential for the inactivation of cyclin B-cdc2 and cyclin A-cdc2 kinases.
- Type 1 phosphatase plays a significant role in catalyzing the dephosphorylation of Thr161, a critical step for exiting M phase.
- These coordinated events ensure the proper transition from metaphase to anaphase in the cell cycle.