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Direct activation of cdc2 with phosphatase: identification of p13suc1-sensitive and insensitive steps
C Jessus1, B Ducommun, D Beach
1Cold Spring Harbor Laboratory, NY 11724.
Abstract:
In Xenopus oocytes, activation of MPF during prophase-metaphase transition is associated with the tyrosine dephosphorylation of the cdc2 protein. In vivo and in cell-free extracts kinase activation can be inhibited by excess p13suc1, a subunit of the protein kinase. Here we have demonstrated that affinity-purified cdc2 from Xenopus prophase oocytes may be activated in vitro by exposure to potato acid phosphatase. In vitro, excess p13 does not inhibit tyrosine dephosphorylation of prophase cdc2, but nonetheless binds and prevents the activation of the enzyme. By contrast, fully activated enzyme from metaphase Xenopus eggs is insensitive to excess p13. These observations define a p13-sensitive state in the activation of fully active cdc2 that follows tyrosine dephosphorylation.
Insights
Maturation promoting factor (MPF) activation in Xenopus oocytes involves cdc2 dephosphorylation. A p13-sensitive state precedes full cdc2 activation, which is inhibited by p13suc1 binding after tyrosine dephosphorylation.
Area of Science:
- Cell Biology
- Molecular Biology
- Developmental Biology
Background:
- Maturation promoting factor (MPF) activation during the prophase-metaphase transition in Xenopus oocytes is linked to the tyrosine dephosphorylation of the cdc2 protein.
- Kinase activation in vivo and in cell-free extracts can be inhibited by excess p13suc1, a protein kinase subunit.
Purpose of the Study:
- To investigate the role of p13suc1 in the activation of cdc2.
- To define the p13-sensitive state in cdc2 activation following tyrosine dephosphorylation.
Main Methods:
- In vitro activation of affinity-purified cdc2 from Xenopus prophase oocytes using potato acid phosphatase.
- Assessing the effect of excess p13suc1 on cdc2 activation and tyrosine dephosphorylation in vitro.
Main Results:
- Xenopus prophase cdc2 can be activated in vitro by potato acid phosphatase.
- Excess p13suc1 does not inhibit tyrosine dephosphorylation of prophase cdc2 but prevents its activation by binding.
- Fully activated metaphase Xenopus egg cdc2 is insensitive to excess p13suc1.
Conclusions:
- A p13-sensitive state exists in cdc2 activation that occurs after tyrosine dephosphorylation.
- p13suc1 binding to cdc2 defines a specific regulatory step in the activation process.