Abstract
The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53. Under normal conditions, p53 is a short-lived protein that is present in cells at a barely detectable level. Upon exposure of cells to various forms of exogenous stress, such as DNA damage, there is a stabilization of p53 which is then responsible for an ensuing cascade of events. To further investigate the effect of p53 activation, we used a MS-based proteomics method to provide an unbiased, quantitative and high-throughput approach for measuring the subcellular distribution of the proteome that is dependent on p53. The spatial proteomics method analyses a whole cell extract created by recombining differentially labeled subcellular fractions derived from cells in which proteins have been mass labeled with heavy isotopes [Boisvert, F.-M., Lam, Y. W., Lamont, D., Lamond, A. I., Mol. Cell. Proteomics 2010, 9, 457–470]. This was used here to measure the relative distribution between cytoplasm, nucleus and nucleolus of around 2000 proteins in HCT116 cells that are either expressing wild-type p53 or null for p53. Spatial proteomics also facilitates a proteome-wide comparison of changes in protein localization in response to a wide range of physiological and experimental perturbations. We used this method to study differences in protein localization in HCT116 cells either with or without p53, and studied the differences in cellular response to DNA damage following treatment of HCT116 cells with etoposide in both p53 wild-type and null genetic backgrounds.
Highlights
The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53
P53 is a short-lived protein that is present in cells at a barely detectable level
Upon exposure of cells to various form of exogenous stress, such as DNA damage, heat shock, hypoxia, etc., there is a stabilization of p53 which is responsible for an ensuing cascade of events, resulting in either cell cycle arrest, or in apoptosis
Summary
The nucleolus is involved in regulating several aspects of stress responses and cell cycle arrest through the tumor suppressor p53. Upon exposure of cells to various form of exogenous stress, such as DNA damage, heat shock, hypoxia, etc., there is a stabilization of p53 which is responsible for an ensuing cascade of events, resulting in either cell cycle arrest, or in apoptosis. Accumulation of p53 in the cell induces the p21-mediated inhibition of cyclin D/cdk and cyclinE/cdk, resulting in cell cycle arrest in G1. Hdm functions as a specific E3 ubiquitin ligase for p53, resulting in a low level of p53 under normal growth conditions due to proteasome-mediated degradation of ubiquitin-conjugated p53. Exposure of cells to various forms of stress such as DNA damage, heat shock and aberrant ribosome biogenesis results in an increase in p53 and cell cycle arrest. The nucleolus acts as a sensor for cellular stress signals through p53 stabilization [6]
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