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

Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphoryla ting Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the ce ll cycle. In frog egg extracts, it has been established previously that Xen opus Wee1 (Xwee1) is present in a hypophosphorylated, active form during in terphase and undergoes down-regulation by extensive phosphorylation at M-ph ase. We report that Xwee1 is also regulated by association with 14-3-3 prot eins. 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 intra nuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphoryl ates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these tw o enzymes are involved in a common pathway in the DNA replication checkpoin t 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.