Transcription factors of the E2F family play an important role in the control of cell cycle and proliferation in many different species, including mammals, flies, nematodes, amphibians, and plants. Their activity is regulated by a variety of mechanisms, frequently mediated by proteins binding to individual members or a subgroup of the family.
Herein we describe the identification, cloning, and characterization of a novel protein that interacts with a subset of E2F factors and influences E2F-dependent promoter activity. We isolated this protein as an E2F-interacting factor in a yeast two-hybrid screen using the amino-terminal domain of E2F-1 as the bait. It is strongly phosphorylated and consequently we named it EAPP. The overall phosphorylation of EAPP does not seem to change significantly throughout the cell cycle as judged by in vitro phosphorylation experiments and migration in SDS-PAGE. This does not rule out that the phosphorylation status of individual sites changes in certain stages of the cell thereby influencing the activity of the protein.
The interaction with E2F-1 was confirmed in vivo and in vitro by mammalian two-hybrid and coimmunoprecipitation assays. GST-pulldown assays with in vitro–translated E2Fs showed that EAPP also interacts with E2F-2 and -3, which contain a similar amino-terminal domain, but not with E2F-4. The N-terminal domain of E2F-1, -2, and -3 not only contains the nuclear localization signal but also binding sites for cyclin A (Krek et al., 1994
), transcription factors of the Sp1 family (Karlseder et al., 1996
(Marti et al., 1999
), p53 (Hsieh et al., 2002
), and EBP1 and EBP2 (Jordan et al., 1996
). The interaction with EAPP might interfere with the binding of one or more of these proteins, thereby influencing E2F activity.
Regulated degradation of proteins is essential for the progression of the cell cycle. Without Cdk1 inactivation by cyclin B destruction chromosomes do not decondense and cells do not divide (for reviews see Peters, 1998
). The disappearance of EAPP during mitosis indicates that it might interfere with the completion of the cell cycle and therefore has to be removed in this phase. That it takes 3 h after the release from nocodazole until EAPP completely disappears could be explained by the observation that not all cells seem to arrest exactly at the same stage after nocodazole addition (). A significant fraction of cells might lag behind after the release from nocodazole. Whether EAPP becomes destroyed by ubiquitin-dependent proteolysis like cyclin B remains to be investigated. The quick reappearance suggests that EAPP is needed in G1, presumably, but perhaps not exclusively, to enhance S-phase stimulating E2F activity.
Interestingly, EAPP enhanced the E2F dependent activity of growth stimulated promoters like the thymidine kinase promoter, but inhibited the promoter of the p14ARF
can act as a mediator of E2F-induced apoptosis (Bates et al., 1998
). The observed down-regulation of the p14ARF
promoter by EAPP does not have to be mediated by E2F. This promoter is also activated by transcription factors of the Sp1 family (Parisi et al., 2002
; Berkovich et al., 2003
) and repressed by T-box factors (Lingbeek et al., 2002
) and p53 (Robertson and Jones, 1998
; Stott et al., 1998
). Any of these factors could mediate the repressing effect of EAPP. The inactivation of p53 seems to increase p14ARF
expression (Robertson and Jones, 1998
; Stott et al., 1998
), which can result in p53-independent apoptosis (Hemmati et al., 2002
; Eymin et al., 2003
). If the repression of the p14ARF
promoter by EAPP is p53 independent, overexpression of EAPP might offer a cell with inactivated p53 an escape from p14ARF
-mediated apoptosis. E2F-1 induced expression of p14ARF
also results in binding of this protein to E2F-1 (Eymin et al., 2001
; Mason et al., 2002
), thereby promoting the binding of p45skp2
(Marti et al., 1999
) and subsequently degradation of E2F-1 via proteasome pathways (Martelli et al., 2001
). Overexpression of EAPP could interfere with this negative feedback control of E2F-1 activity. Concordant with this model we found a slight increase of E2F-1 levels in cells transiently overexpressing EAPP (unpublished data). Transcription of the E2F-1 gene is regulated by E2F-binding sites (Neuman et al., 1994
). The elevated E2F-1 level could therefore be the result of EAPP-enhanced E2F activity, or of both, increased E2F-1 promoter activity, and reduced p14ARF
-mediated E2F-1 degradation. Thus, EAPP on the one hand seems to enhance transcription of growth-correlated, E2F-controlled genes like thymidine kinase, resulting in the observed S-phase induction, and on the other hand it inhibits the expression of the tumor suppressor p14ARF
. This implies that EAPP could play a role in malignant transformation. In line with this speculation, compared with diploid fibroblasts, EAPP levels were elevated in almost all investigated transformed human cell lines.
How could EAPP stimulate E2F activity? One possibility would be an increase of the DNA-binding ability of the activating E2Fs. We examined this in electrophoretic mobility shift assays (EMSA) and found neither an increase in DNA binding, nor EAPP as a component of the E2F complexes (unpublished data). This does not rule out that EAPP acts in this way, because weakly interacting proteins are often not detectable in EMSAs and binding of proteins to the naked DNA of an oligonucleotide might differ from binding to DNA organized as nucleosomes in the context of a promoter. Alternative mechanisms would be increased transactivation activity caused by EAPP binding or by EAPP-mediated posttranslational modification of E2F proteins or chromatin rearrangement caused by EAPP bound proteins. E2F-1 activity can be activated or repressed by modifications (for a review see Mundle and Saberwal, 2003
). Although there are no indications that EAPP itself is an enzyme, it might act as a bridging factor for modifying factors. We have found kinase activity in immunoprecipitations of EAPP capable of phosphorylating added GST-E2F-1 (unpublished data). The increase of the S-phase fraction in EAPP-overexpressing cells and the inhibition (or slow down) of S-phase entry in EAPP knockdown cells indicate that EAPP is required for cell cycle progression. Whether the observed S-phase–enhancing activity of EAPP is mediated only by E2F or if it also involves other factors remains to be determined.
EAPP is conserved not only among mammals. Open reading frames corresponding to the EAPP gene can be found in the genomes of many species. RNA interference experiments in Caenorhabditis elegans
suggest that inhibiting the expression of the corresponding gene is embryonic lethal (Kamath et al., 2003
Taken together, EAPP might play an important role in the fine-tuning of both major E2F-1 activities, the regulation of the cell cycle and the induction of apoptosis. By stimulating S-phase entry and at the same time inhibiting p14ARF expression, overexpression of EAPP could contribute to the malignant transformation of a cell.