Abstract 1063: The activity of the 26S proteasome is modulated during the different phases of the cell cycle

Abstract

Abstract The 26S proteasome is a multi-catalytic protein complex responsible for the degradation of almost all short-lived and most long-lived proteins. Aberrations of the ubiquitin/proteasome system contribute to many human diseases, including cancer. Many aspects of its structural organization and regulation are still unknown. Among many other functions, the 26S proteasome as the major proteolytic machinery in the cell plays a central role in all phases of the cell cycle. At present, the activity of the 26S proteasome is considered static throughout the cell cycle with protein degradation only depending on their ubiquitination status. We hypothesized that regulation of the activity of the proteasome complex itself could be an additional layer of regulation of cell cycle progression. To test our hypothesis we synchronized two human cell lines, PC-3 prostate cancer cells and MDA-MB-231 breast cancer cells, by treatment with Mimosine and measured 26S proteasome activity in these cells in the presence of the drug and 3, 6, 12 and 24h after restarting the cell cycle. Cells cycle distribution was assessed by Propidium Iodide staining and analyzed by flow cytometry. In parallel, using a proteomics approach we investigated changes of the expression levels and posttranslational modifications of proteasome subunits. Therefore, intact 26S proteasomes from PC3 prostate cancer cell stably expressing a 19S subunit, Rpn11, tagged with 6xHis-TEV-biotin-6xHis, were pulled down from synchronized and untreated (normally cycling) cells. Proteasome subunits and proteasome interacting proteins were separated by 2-dimensional gel electrophoresis (2DE). Differences between synchronized and un-treated cells were analyzed by specific staining for total protein and posttranslational modifications. Differentially expressed or post-translationally modified spots were identified by mass spectrometry. Using this approach, we observed that all three activities of the proteasome were regulated in a similar fashion with down regulation of 26S proteasome activity in cells arrested in the G1-phase of the cell cycle. Treatment of isolated proteasome with Mimosine did not affect its function, excluding that the drug could serve as proteasome inhibitor. After restarting the cell cycle, all three activities sharply increased with maximum activities in cells in the G2/M phase of the cell cycle, which exceeded the 26S proteasome activity of non-synchronized cells about 1.5fold. 2DE and mass spectrometry revealed that proteasome subunit expression was not affected. However, cell cycle synchronization caused specific changes in the phosphorylation pattern of 20S alpha proteasome subunits. In conclusion, we demonstrated that protein degradation and accumulation throughout the cell cycle is not only controlled at the level of proteins involved in cell cycle progression but also by fine-tuning of the activity of the 26S proteasome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1063.