Dephosphorylation of cdc25-C by a type-2A protein phosphatase: Specific regulation during the cell cycle in Xenopus egg extracts

Paul R. Clarke (Lead / Corresponding author), Ingrid Hoffmann, Giulio Draetta, Eric Karsenti

Research output: Contribution to journalArticle

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Abstract

We have examined the roles of type-1 (PP-1) and type-2A (PP-2A) protein-serine/threonine phosphatases in the mechanism of activation of p34cdc2/cyclin B protein kinase in Xenopus egg extracts. p34cdc2/cyclin B is prematurely activated in the extracts by inhibition of PP2A by okadaic acid but not by specific inhibition of PP-1 by inhibitor-2. Activation of the kinase can be blocked by addition of the purified catalytic subunit of PP-2A at a twofold excess over the activity in the extract. The catalytic subunit of PP-1 can also block kinase activation, but very high levels of activity are required. Activation of p34cdc2 /cyclin B protein kinase requires dephosphorylation of p34cdc2 on Tyr 15. This reaction is catalysed by cdc25C phosphatase that is itself activated by phosphorylation. We show that, in interphase extracts, inhibition of PP-2A by okadaic acid completely blocks cdc25-C dephosphorylation, whereas inhibition of PP-1 by specific inhibitors has no effect. This indicates that a type2A protein phosphatase negatively regulates p34cdc2 /cyclin B protein kinase activation primarily by maintaining cdc25-C phosphatase in a dephosphorylated, low activity state. In extracts containing active p34cdc2 /cyclin B protein kinase, dephosphorylation of cdc25-C is inhibited, whereas the activity of PP-2A (and PP-1) towards other substrates is unaffected. We propose that this specific inhibition of cdc25-C dephosphorylation is part of a positive feedback loop that also involves direct phosphorylation and activation of cdc25-C by p34cdc2 /cyclin B. Dephosphorylation of cdc25-C is also inhibited when cyclin A-dependent protein kinase is active, and this may explain the potentiation of p34cdc2 /cyclin B protein kinase activation by cyclin A. In extracts supplemented with nuclei, the block on p34cdc2 / cyclin B activation by unreplicated DNA is abolished when PP-2A is inhibited or when stably phosphorylated cdc25-C is added, but not when PP-1 is specifically inhibited. This suggests that unreplicated DNA inhibits p34cdc2 /cyclin B activation by maintaining cdc25C in a low activity, dephosphorylated state, probably by keeping the activity of a type-2A protein phosphatase towards cdc25-C at a high level.

Original languageEnglish
Pages (from-to)397-411
Number of pages15
JournalMolecular Biology of the Cell
Volume4
Issue number4
DOIs
Publication statusPublished - 1 Apr 1993

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