The T104A/T150A double mutant was no more resistant to inactivation than the T150A single mutant (Fig. of M-phase entry. These results also show that multisite phosphorylation cooperatively inactivates Wee1A and cooperatively promotes Wee1A proteolysis. Wee1 was first identified through genetic studies of cell size control and cell cycle progression in (30, 31, 38). Subsequent work established Wee1 as a critical regulator of the G2/M transition in diverse organisms and cell types (1, 3, 7). Wee1 functions by phosphorylating cyclin-Cdk complexes at a conserved tyrosine residue (Tyr 15 in Cdc2/Cdk1), thereby inactivating the kinase (8, 17, 20, 33). Wee1’s activity is opposed by Cdc25A, -B, and -C, a small family of conserved phosphatases that dephosphorylate two inhibitory sites (Thr 14 and Tyr 15) in Cdk1 (1, 3, 4, 6, 7, 9, 13). In late G2 phase, the balance between Wee1-mediated Cdk1 phosphorylation and UAA crosslinker 2 Cdc25-mediated dephosphorylation shifts UAA crosslinker 2 in favor of dephosphorylation, bringing about activation of cyclin-Cdk1 complexes and entry into mitosis. Fungi and invertebrates possess a single Wee1 gene; vertebrates possess two, an embryonic Wee1 gene expressed predominantly in mature oocytes, testes, and UAA crosslinker 2 early embryos, and a somatic gene expressed later in development (16, 28, 32, 45). (The nomenclature of the somatic and embryonic Wee1 genes is described in Materials and Methods.) Although the biochemical function of Wee1 is relatively simpleits only known substrates are itself and the cyclin-Cdk complexesWee1’s regulation is complicated and incompletely understood. The levels, activity, and localization of Wee1 are all regulated, and multiple mechanisms contribute to each of these aspects of Wee1 control. Wee1 levels are regulated transcriptionally (46), translationally (5, 25, 27), and by ubiquitylation and proteolysis (21). The degradation of Wee1 in M phase or late G2 phase has been reported to be triggered by three SCF-type ubiquitin ligases: SCFTome-1, SCF-TrCP1, and SCF-TrCP2 (2, 45). Recognition of embryonic Wee1 by Tome-1 depends upon the phosphorylation of Ser 38 (2), an SP site that is present in embryonic Wee1 (Wee1A) but not in human or mouse embryonic Wee1 (Fig. ?(Fig.1A).1A). Recognition of somatic UAA crosslinker 2 Wee1 by -TrCPs depends upon the phosphorylation of two residues conserved in somatic Wee1 proteins but absent from embryonic Wee1 proteins: Ser 53 (a putative Plk1 phosphorylation site) and Ser 123 (a putative Cdk1 phosphorylation site) (45). Open in a separate window FIG. 1. OP11-Wee1A is resistant to mitotic inactivation. (A) Sequence alignment of the Wee1A and mutated in OP11-Wee1A are shown. (B) In vitro kinase activity of wild-type (WT) Wee1A and OP11-Wee1A. Bead-bound Wee1A proteins were incubated with interphase egg extracts or extracts treated with 200 nM Mouse monoclonal to FLT4 90-cyclin B (M-phase extracts). The Wee1A beads were then washed and incubated with a complex of kinase-minus T161A-Cdk1 and 65-cyclin B. The phosphorylation of Cdk1 was assessed by antiphosphotyrosine immunoblotting. Equal loading of substrate was verified by Cdk1 immunoblotting. (C) Autophosphorylation of wild-type Wee1A (WT), OP11-Wee1A (OP), and kinase-minus Wee1A (KM). Bead-bound Wee1A proteins were treated with interphase extracts or M-phase extracts as in panel B. The electrophoretic mobility of Wee1A was assessed by blotting with Flag antibody and the autophosphorylation of Wee1A assessed by antiphosphotyrosine immunoblotting. (D) Interphase extracts cause a shift in both wild-type Wee1A and OP11-Wee1A. Wee1A proteins were detected by Coomassie staining. In addition, the specific activity of the Wee1 protein is regulated posttranslationally. During mitosis, the N-terminal noncatalytic domain of Wee1 becomes hyperphosphorylated, UAA crosslinker 2 causing Wee1 to shift to a higher apparent molecular weight on sodium dodecyl sulfate (SDS) gels (19, 22, 34, 41). This hyperphosphorylation is accompanied by a marked decrease.