THE XENOPUS CHK1 PROTEIN-KINASE MEDIATES A CAFFEINE-SENSITIVE PATHWAYOF CHECKPOINT CONTROL IN CELL-FREE-EXTRACTS

We have analyzed the role of the protein kinase Chk1 in checkpoint con trol by using cell-free extracts from Xenopus eggs. Recombinant Xenopu s Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on mu ltiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 protei ns. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc 25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the actio n of a distinct Ser-287-specific kinase in addition to Xchk1.Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which atte nuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensit ive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.