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) protei n-serine/threonine phosphatases in the mechanism of activation of p34c dc2/cyclin B protein kinase in Xenopus egg extracts. p34cdc2/cyclin B is prematurely activated in the extracts by inhibition of PP-2A by oka daic acid but not by specific inhibition of PP-1 by inhibitor-2. Activ ation of the kinase can be blocked by addition of the purified catalyt ic subunit of PP-2A at a twofold excess over the activity in the extra ct. The catalytic subunit of PP-1 can also block kinase activation, bu t very high levels of activity are required. Activation of p34cdc2/cyc lin B protein kinase requires dephosphorylation of p34cdc2 on Tyr15. T his reaction is catalysed by cdc25-C phosphatase that is itself activa ted by phosphorylation. We show that, in interphase extracts, inhibiti on of PP-2A by okadaic add completely blocks cdc25-C dephosphorylation , whereas inhibition of PP-1 by specific inhibitors has no effect. Thi s indicates that a type-2A protein phosphatase negatively regulates p3 4cdc2/cyclin B protein kinase activation primarily by maintaining cdc2 5-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) towa rds other substrates is unaffected. We propose that this specific inhi bition of cdc25-C dephosphorylation is part of a positive feedback loo p that also involves direct phosphorylation and activation of cdc25-C by p34cdc2/cyclin B. Dephosphorylation of cdc25-C is also inhibited wh en cyclin A-dependent protein kinase is active, and this may explain t he potentiation of p34cdc/cyclin B protein kinase activation by cyclin A. In extracts supplemented with nuclei, the block on p34cdc/cyclin B activation by unreplicated DNA is abolished when PP-2A is inhibited o r when stably phosphorylated cdc25-C is added, but not when PP-1 is sp ecifically inhibited. This suggests that unreplicated DNA inhibits p34 cdc/cyclin B activation by maintaining cdc25-C in a low activity, deph osphorylated state, probably by keeping the activity of a type-2A prot ein phosphatase towards cdc25-C at a high level.