Abstract
Proteolytic processing at the end of the G(1) phase generates a CUX1 isoform, p110, which functions either as a transcriptional activator or repressor and can accelerate entry into S phase. Here we describe a second proteolytic event that generates an isoform lacking two active repression domains in the COOH terminus. This processing event was inhibited by treatment of cells with synthetic and natural caspase inhibitors. In vitro, several caspases generated a processed isoform that co-migrated with the in vivo generated product. In cells, recombinant CUX1 proteins in which the region of cleavage was deleted or in which Asp residues were mutated to Ala, were not proteolytically processed. Importantly, this processing event was not associated with apoptosis, as assessed by terminal dUTP nick end labeling assay, cytochrome c localization, poly(ADP-ribose) polymerase cleavage, and fluorescence-activated cell sorting. Moreover, processing was observed in S phase but not in early G(1), suggesting that it is regulated through the cell cycle. The functional importance of this processing event was revealed in reporter and cell cycle assays. A recombinant, processed, CUX1 protein was a more potent transcriptional activator of several cell cycle-related genes and was able to accelerate entry into S phase, whereas mutants that could not be processed were inactive in either assay. Conversely, cells treated with the quinoline-Val Asp-2,6-difluorophenoxymethylketone caspase inhibitor proliferated more slowly and exhibited delayed S phase entry following exit from quiescence. Together, our results identify a substrate of caspases in proliferating cells and suggest a mechanism by which caspases can accelerate cell cycle progression.
Highlights
MOP-11590 and by Canadian Cancer Society Grant 014288
The molecular basis for the regulatory effect of p110 CUX1 remains to be defined. We show that another proteolytic processing event results in the removal of the COOH-terminal region of CUX1, which contains two active repression domains
The COOH-terminally cleaved protein was barely visible in early G1 and mid-G1 but was strongly expressed in unsynchronized cells and in S phase using either method of synchronization (Fig. 1B)
Summary
Synchronization of NIH3T3 cells was performed by two methods. Serum Starvation/Stimulation—Post-transfection, cells were maintained in DMEM for 72 h and in DMEM plus 10% FBS for the indicated times. Thymidine Block—Post-transfection, cells were cultured overnight in DMEM plus 10% FBS supplemented with 2 mM thymidine and harvested. Infected Kit225 cells were deprived of IL-2 for 48 h, followed by IL-2 addition for the indicated times. Cell cycle distribution was monitored by fluorescence-activated cell sorting following ethanol fixation and propidium iodide staining [32]. Carboxyfluorescein Diacetate Succinimidyl Ester Staining Cells were stained using the CellTraceTM carboxyfluorescein diacetate succinimidyl ester staining cell proliferation kit and were analyzed by flow cytometry with 488-nm excitation and emission filters appropriate for fluorescein, according to the EXPERIMENTAL PROCEDURES
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