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...
BackgroundThe protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.Methodology/Principal FindingsUsing chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.Conclusions/SignificanceThese results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.
Transcription factor LSF is essential for cell cycle progression, being required for activating expression of the thymidylate synthase (Tyms) gene at the G1/S transition. We previously established that phosphorylation of LSF in early G1 at Ser-291 and Ser-309 inhibits its transcriptional activity and that dephosphorylation later in G1 is required for its reactivation. Here we reveal the role of prolyl cis-trans isomerase Pin1 in activating LSF, by facilitating dephosphorylation at both Ser-291 and Ser-309. We demonstrate that Pin1 binds LSF both in vitro and in vivo. Using coimmunoprecipitation assays, we identify three SP/TP motifs in LSF (at residues Ser-291, Ser-309, and Thr-329) that are required and sufficient for association with Pin1. Coexpression of Pin1 enhances LSF transactivation potential in reporter assays. The Pin1-dependent enhancement of LSF activity requires residue Thr-329 in LSF, requires both the WW and PPiase domains of Pin1, and correlates with hypophosphorylation of LSF at Ser-291 and Ser-309. These findings support a model in which the binding of Pin1 at the Thr-329 -Pro-330 motif in LSF permits isomerization by Pin1 of the peptide bonds at the nearby phosphorylated SP motifs (Ser-291 and Ser-309) to the trans configuration, thereby facilitating their dephosphorylation.
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