title: The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts.
abstract: We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. 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. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action 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 attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.
authors.affiliation: Division of Biology, 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: A
authors.initials: A
authors.lastname: Kumagai
authors.forename: Z
authors.initials: Z
authors.lastname: Guo
authors.forename: K H
authors.initials: KH
authors.lastname: Emami
authors.forename: S X
authors.initials: SX
authors.lastname: Wang
authors.forename: W G
authors.initials: WG
authors.lastname: Dunphy
mesh_terms: D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts; D002474:Cell-Free System; D000071877:Checkpoint Kinase 1; D003001:Cloning, Molecular; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D010063:Ovum; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014446:Tyrosine 3-Monooxygenase; D014981:Xenopus; D029867:Xenopus Proteins; D020687:cdc25 Phosphatases
chemical_list: D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D011506:Proteins; D029867:Xenopus Proteins; D002110:Caffeine; D012694:Serine; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D010749:Phosphoprotein Phosphatases; D020687:cdc25 Phosphatases