Carboxyl-terminal Proteolytic Processing of CUX1 by a Caspase Enables Transcriptional Activation in Proliferating Cells

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.

CUX1, a haploinsufficient tumour suppressor gene overexpressed in advanced cancers

“…Multiple CUX1 isoforms have been described, two of which are ubiquitously expressed 2,22,[24][25][26][27] (reviewed in REF. 28).…”

“…Using transcription and cellbased assays, a role for p110 CUX1 was shown in many cellular processes, notably in cell cycle progression and cell proliferation 21,22 , strengthening of the spindle assembly checkpoint 19 , establishment of a transcriptional programme that enables efficient DNA damage responses 23 , and cell migration and invasion 13,42 . In addition, from RNA interference (RNAi)-mediated knockdown and genetic inactivation, CUX1 was shown to be required for the resistance to apoptotic signals in pancreatic cancer cells 14 , the repression cytokine genes associated with M1 macrophages 43 , and dendrite branching and spine development in cortical neurons 34 .…”

“…Many transcriptional targets and cellular functions of CUX1 readily suggest mechanisms by which increased p110 or p75 CUX1 expression might promote tumour development and progression, including acceleration of S phase entry 21,22,41,62,63 , stimulation of cell migration and invasion 13,42,[64][65][66] , resistance to apoptosis 14 , and promotion of bipolar mitosis in the presence of supernumerary centrosomes 19 (reviewed in REF. 67).…”

“…Certainly, the idea that CUX1 represses genes that are involved in cell cycle progression runs contrary to the bulk of the evidence showing that CUX1 stimulates expression of histone genes and many genes involved in DNA replication, while repressing expression of the cyclin-dependent kinase inhibitors p21 and p27 (REFS 15,41,60,62,(68)(69)(70)(71)(72)(73)(74)(75). Moreover, in cell-based assays, MEFs from Cux1-knockout mice showed a longer G1 phase and proliferated more slowly than their wild-type counterparts; whereas, in many cell types, ectopic expression of p110 CUX1 accelerated S phase entry and stimulated proliferation 21,22 .…”

“…Cell-based assays have shown transcriptional roles for CUX1 in cell cycle progression 21,22 , DNA damage responses 23 , and resistance to apoptotic signals 14 and other pathways (FIG. 2).…”