Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

BACKGROUND: Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line. METHODS: Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed. RESULTS: In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours. CONCLUSION: We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy

Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation

Crosstalk between different types of post-translational modifications (PTMs) on the same protein molecule adds specificity and combinatorial logic to signal processing, but has not been characterized on a large-scale basis. Here, we developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation, and these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation