Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Abstract

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G 2 and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.The tyrosine kinase Wee1 inhibits mitotic entry, and its degradation is essential for exit from the G 2 phase of the cell cycle (1). Here, Wee1 inactivates the mitosis-specific cyclin-dependent kinase Cdk1-cyclin B complex during the S and G 2 phases by phosphorylating Cdk1 at tyrosine 15. However, Wee1 activity is opposed by the phosphatase Cdc25, which removes phosphate from tyrosine 15 on Cdk1, thereby activating Cdk1-cyclin B at the G 2 -M transition (2). A major mechanism that tips the balance toward Cdc25 and mitotic entry is Wee1 degradation during G 2 , and as this occurs active Cdc25 and Cdk1-cyclin B form a positive feedback loop, which ensures that mitotic entry is unidirectional (3, 4). Thus, Wee1 degradation is an essential component of the Cdk1 activation circuit.Wee1 degradation has been observed in Saccharomyces cerevisiae, Xenopus egg extracts, and somatic cells (5-11). In Xenopus, the nuclei are required for proper Wee1 degradation, and the completion of DNA replication is required to achieve maximal rates of Wee1 degradation (6). Thus, Wee1 degradation is part of a sensing mechanism that signals the completion of the DNA replication phase, ensuring proper timing of entry into mitosis. For example, when DNA replication stalls, this fail-safe mechanism allows defects to be corrected before cells enter mitosis. Indeed, current theories suggest that many cancer cells have ineffective checkpoint pathways that cause them to divide with incompletely replicated DNA, which leads to genomic instability (12). Consistent with this notion are the findings that wee1 knock-out mice are not viable and that wee1 Ϫ/Ϫ cells undergo mitotic catastrophe (13).Wee1 phosphorylation is required for its degradation and is regulated by the DNA replication checkpoint (6, 10). Here in the presence of the DNA replication checkpoint, Wee1 phosphorylation is negligible, whereas when DNA replication proceeds normally, Wee1 phosphorylation and degradation and mitotic entry are normal.One aspect of Wee1 degradation conserved between embryonic and somatic cell cycles is the requirement for SCF 3 ubiquitin ligases th...

Casein Kinase 1δ-dependent Wee1 Protein Degradation

“…We have previously described similar assays (12,13). N-cyclin B-luciferase, p21 cip1 -luciferase, or p27 kip1 -luciferase assays were performed as described previously (12,13).…”

“…They phosphorylate multiple N-terminal residues to initiate recognition by an SCF ubiquitin ligase containing the F-box protein ␤-TrCP (7,8). Wee1 is turned over in cell extracts isolated from HeLa cells in S/G 2 phase cells (8,12). Furthermore, overexpression of nonphosphorylatable versions of Wee1 limits mitotic entry and arrests cells in G 2 phase (8,12).…”

“…Not surprisingly, the control of Wee1 degradation is an important regulator of mitotic entry (5)(6)(7)(8)(9). Degradation of nuclear Wee1 (5, 10) during G 2 phase of the cell cycle increases the activity of Cdk1, leading to mitotic entry (7,8,11,12). To identify kinases that control Wee1 phosphorylation and subsequent degradation, we screened a 16,000-member, kinase-directed, small molecule library available at Scripps Florida and a reporter of Wee1 turnover, K328M-Wee1-luciferase (13).…”

WEE1 Kinase Targeting Combined with DNA-Damaging Cancer Therapy Catalyzes Mitotic Catastrophe

“…At the onset of mitosis, WEE1 must be downregulated rapidly to activate CDK1. Therefore, phosphorylation of WEE1 by CDK1 and PLK1 at S53 and S123 creates phosphodegrons that signal the ubiquitination of WEE1 by CDC34 and proteasome-dependent degradation by the F-box proteins b-trcp-1 and Tome-1 (11,(78)(79)(80). Furthermore, phosphorylation of WEE1 at S642 by AKT1 creates a 14-3-3q peptide-binding site that ports WEE1 from the nucleus and decreases its level of activity (12).…”

The regulatory network of cell-cycle progression is fundamentally different in plants versus yeast or metazoans

“…For instance, the activation domain plays a crucial role in Wee1-degradation via the ubiquitin proteasome pathway in HeLa cells. 26 The C-terminal parts of Wee1 kinases containing the catalytic segment are highly conserved and the active sites can be clearly identified in all homologs.…”