Positive regulation of Wee1 by Chk1 and 14-3-3 proteins

• Published in: Molecular biology of the cell Vol. 12; no. 3; pp. 551 - 563
• Main Authors: Lee, J, Kumagai, A, Dunphy, W G
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.03.2001
• Subjects: 14-3-3 Proteins; Animals; Base Sequence; CDC2 Protein Kinase - metabolism; Cell Cycle; Cell Cycle Proteins; Cell Line; Cell Nucleus - metabolism; Checkpoint Kinase 1; DNA Primers - genetics; DNA Replication; Escherichia coli - genetics; Female; In Vitro Techniques; Mutation; Nuclear Proteins; Oocytes - metabolism; Protein Binding; Protein Kinases - genetics; Protein Kinases - metabolism; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spodoptera; Tyrosine 3-Monooxygenase - genetics; Tyrosine 3-Monooxygenase - metabolism; Xenopus; Xenopus Proteins
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.12.3.551

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.