Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts.
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 PP-2A 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 Tyr15. This reaction is catalysed by cdc25-C 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 type-2A 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 cdc25-C in a low activity, dephosphorylated state, probably by keeping the activity of a type-2A protein phosphatase towards cdc25-C at a high level.
Cell Biology Program, European Molecular Biology Laboratory, Heidelberg, Germany.
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PR
Clarke
I
I
Hoffmann
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G
Draetta
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E
Karsenti
D000818:Animals; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D016213:Cyclins / Q000378:metabolism; D004261:DNA Replication; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D010063:Ovum / Q000166:cytology / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D014982:Xenopus laevis; D020687:cdc25 Phosphatases
D016213:Cyclins; D011506:Proteins; D014443:Tyrosine; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases; D020687:cdc25 Phosphatases