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An inhibitor of p34cdc2/cyclin B that regulates the G2/M transition in Xenopus extracts
1Harvard Medical School, Department of Cell Biology, Boston, MA 02115, USA.
Abstract:
The activity of maturation-promoting factor (MPF), a protein kinase complex composed of p34cdc2 and cyclin B, is undetectable during interphase but rises abruptly at the G2/M transition to induce mitosis. After the synthesis of cyclin B, the suppression of MPF activity before mitosis has been attributed to the phosphorylation of p34cdc2 on sites (threonine-14 and tyrosine-15) that inhibit its catalytic activity. We previously showed that the activity of the mitotic p34cdc2/cyclin B complex is rapidly suppressed when added to interphase Xenopus extracts that lack endogenous cyclin B. Here we show that a mutant of p34cdc2 that cannot be inhibited by phosphorylation (threonine-14-->alanine, tyrosine-15-->phenylalanine) is also susceptible to inactivation, demonstrating that inhibitory mechanisms independent of threonine-14 and tyrosine-15 phosphorylation must exist. We have partially characterized this inhibitory pathway as one involving a reversible binding inhibitor of p34cdc2/cyclin B that is tightly associated with cell membranes. Kinetic analysis suggests that this inhibitor, in conjunction with the kinases that mediate the inhibitory phosphorylations on p34cdc2, maintains the interphase state in Xenopus; it may play an important role in the exact timing of the G2/M transition.
Insights
Maturation-promoting factor (MPF) activity is suppressed in Xenopus interphase extracts. This suppression involves mechanisms beyond inhibitory phosphorylation, including a membrane-associated inhibitor crucial for maintaining interphase.
Area of Science:
- Cell Cycle Regulation
- Molecular Biology
- Biochemistry
Background:
- Maturation-promoting factor (MPF), a complex of p34cdc2 and cyclin B, drives mitosis.
- MPF activity is low during interphase and rises sharply at the G2/M transition.
- Inhibitory phosphorylation of p34cdc2 at threonine-14 and tyrosine-15 was thought to suppress MPF activity before mitosis.
Purpose of the Study:
- To investigate mechanisms regulating MPF activity during the cell cycle.
- To determine if inhibitory pathways independent of threonine-14 and tyrosine-15 phosphorylation exist.
- To characterize the role of a novel inhibitor in maintaining interphase.
Main Methods:
- Utilized Xenopus interphase extracts.
- Employed a mutant p34cdc2 resistant to inhibitory phosphorylation (T14A/Y15F).
- Performed kinetic analysis of MPF inactivation and inhibitor binding.
Main Results:
- A non-phosphorylatable p34cdc2 mutant was still inactivated in interphase extracts.
- This indicates inhibitory mechanisms independent of T14/Y15 phosphorylation.
- A reversible, membrane-associated inhibitor of p34cdc2/cyclin B was identified.
Conclusions:
- Inhibitory phosphorylation and a novel inhibitor cooperate to maintain the interphase state in Xenopus.
- This regulatory system is critical for precise G2/M transition timing.
- The findings reveal a complex, multi-layered control of MPF activity.