Positive regulation of Wee1 by Chk1 and 14-3-3 proteins.
Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.
Lee
J
J
Division of Biology 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Kumagai
A
A
Dunphy
W G
WG
D048948:14-3-3 Proteins; D000818:Animals; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D066298:In Vitro Techniques; D009154:Mutation; D009687:Nuclear Proteins; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D048948:14-3-3 Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D014446:Tyrosine 3-Monooxygenase; D011494:Protein Kinases; C530005:WEE1 protein, Xenopus; D011505:Protein-Tyrosine Kinases; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D016203:CDC2 Protein Kinase