CDC25A Phosphatase Is a Target of E2F and Is Required for Efficient E2F-Induced S Phase

Abstract

Functional inactivation of the pRB pathway is a very frequent event in human cancer, resulting in deregulated activity of the E2F transcription factors. To understand the functional role of the E2Fs in cell proliferation, we have developed cell lines expressing E2F-1, E2F-2, and E2F-3 fused to the estrogen receptor ligand binding domain (ER). In this study, we demonstrated that activation of all three E2Fs could relieve the mitogen requirement for entry into S phase in Rat1 fibroblasts and that E2F activity leads to a shortening of the G 0 -G 1 phase of the cell cycle by 6 to 7 h. In contrast to the current assumption that E2F-1 is the only E2F capable of inducing apoptosis, we showed that deregulated E2F-2 and E2F-3 activities also result in apoptosis. Using the ERE2F-expressing cell lines, we demonstrated that several genes containing E2F DNA binding sites are efficiently induced by the E2Fs in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation.

Cdc25A stability is controlled by the ubiquitin-proteasome pathway during cell cycle progression and terminal differentiation

“…These observations are consistent with the reported identi®cation of cdc25A as a myc target gene (Galaktionov et al, 1996). However, the kinetics of expression of cdc25A and c-myc transcripts suggest that cdc25A transcription is not regulated solely by cmyc; consistent with this suggestion is the recent report that E2F also regulates the transcription of cdc25A (Vigo et al, 1999). The observation that cdc25A continued to be expressed in M1myc cells, prompted us to investigate whether cdc25A mediates the c-mycblock in M1 terminal di erentiation.…”

Life and death decisions by E2F-1

“…As eluded to earlier, many genes that are regulated in a cell-specific manner have E2F-binding sites as their promoters, and in some cases E2F-1 has been demonstrated to induce their expression directly. Some of these gene products play a direct regulatory role in the cell cycle, for example, Cdc2, cdc25a and cyclin E. [30][31][32] As well as this, forced expression of E2F-1, as with E2F-2 and E2F-3, in quiescent cells is sufficient to induce entry into DNA synthesis, and each of these E2Fs can function as oncogenes in transforming assays. 33,34 It was amazing, therefore, to find that targeted deletion of the E2F-1 gene in mice resulted in animals that spontaneously developed tumours in a number of tissues.…”

A genetic screen to identify genes that rescue the slow growth phenotype of c-myc null fibroblasts

“…To test this, we infected the c-myc null cells with retroviral vectors expressing either c-Myc (as a positive control), SV40 Tantigen, adenovirus E1A (13S), RasV12, SKP2, E2F1 (which activates the c-Myc target gene CDC25A (Vigo et al, 1999)), E2F2, E2F3, cyclin D1, cyclin D2 or cyclin E. Cell lines were generated by hygromycin selection for 2 weeks and analysed for reversion of the slow growth phenotype. All cell lines were tested by Western analysis for the expression of the introduced cell cycle proteins (Figure 1b).…”