Characterization of EAPP, a novel E2F binding protein

E2F is a family of heterodimeric transcription factors which regulate the expression of genes, whose products are essential for progression through the mammalian cell cycle. Moreover, it integrates cell-cycle progression with transcription through its cyclical interactions with important growth and cell cycle regulators, such as the retinoblastoma-tumor-suppressor-gene product (pRB), cyclins and cyclin dependent kinases. The activity of the E2F transcription factors is regulated by a multitude of mechanisms, often involving protein-protein interactions. The amino terminal domain of E2F1 plays an important role in the regulation of its activity. It contains a binding site for cyclin A/cdk2 which mediates phosphorylation and subsequent downregulation of the DNA binding activity of the heterodimeric transcription factor complex. The binding of p45(SKP2) results in ubiquitination and rapid degradation of E2F1. The interaction with transcription factors of the Sp1 family seems to be required for the activation of certain promoters. To get more insight into the function of E2F1 we used the yeast two hybrid system to look for novel proteins binding to the amino-terminal domain of E2F1. By screening a human cDNA-library, we identified a hitherto uncharacterized protein. This protein is strongly phosphorylated and consequently we named it EAPP (E2F Associated PhophoProtein). EAPP mRNA is low in resting cells but increases soon after addition of serum. Immunofluorescence experiments showed that EAPP is a nuclear protein like E2F1. Co-expression of EAPP with E2F1 strongly enhanced E2F dependent promoter activity. This indicates the EAPP may play an important role in E2F dependent transcription. Aim of this project is to scrutinize the role of EAPP in E2F dependent transcription. We will study the interaction of EAPP with E2F1 and possibly other E2F proteins, map the binding site within each protein and create specific mutants. The E2F-stimulating activity of EAPP will be investigated by reporter gene assays with E2F dependent and independent promoters and their respective mutants. To study the effect of EAPP expression in resting cells we will create a cell line carrying the EAPP cDNA under the control of an inducible promoter. Finally we will carry out size fractionation of cellular protein complexes and check each fraction for the presence of EAPP and E2F1.

Aim of this project was the functional and structural characterization of EAPP (E2F-associated phosphoprotein) which has been identified in our laboratory as an E2F binding factor and which seemed to enhance E2F-dependent transcription. Transcription factors of the E2F family are important components of the cell cycle- and proliferation controlling machinery. We confirmed the interaction and found out that EAPP interacts with the activating E2Fs, E2F-1, -2, and -3, but not with the primarily repressing E2F-4. To investigate endogenous EAPP we raised murine poly- and monoclonal antibodies. Immunoprecipitation- and GST-pull down experiments with extracts from metabolically labeled tissue culture cells suggested that EAPP interacts with a wide range of cellular proteins. EAPP levels are rather constant throughout most of the cell cycle but immunofluorescence and nocodazole experiments showed that it disappears rapidly with the onset of mitosis. Prolonged serum withdrawal also resulted in a reduction of EAPP levels. Moreover, we could show that EAPP can interact with itself indicating that it exists as a dimer or even as a multimer. EAPP is a nuclear phosphoprotein with a nuclear localization signal (NLS) between amino acids 121-135. Northern analyses showed that the EAPP gene is expressed in a variety of human tissues. Reporter gene assays indicated that EAPP enhances the expression of cell cycle regulated and E2F dependent genes. Surprisingly, the promoter of the tumor suppressor p14ARF that is upregulated by E2F-1 was repressed by co-expressed EAPP. p14ARF seems to induce apoptosis in p53 dependent and independent manners. Repression of p14 ARF by EAPP might help cells with aberrant growth characteristics to escape apoptosis. That means that EAPP could be a proto-oncogene. In line with this hypothesis we have found that most transformed human cells have higher EAPP levels than diploid fibroblasts. Over-expression of EAPP in U2OS cells resulted in a significant increase of cells in S-phase, whereas RNAi-mediated knock down of EAPP reduced the fraction of cells in S- phase. Taken together, these data suggest that EAPP modulates E2F-regulated transcription, stimulates proliferation and may be involved in the malignant transformation of cells.