Epstein±Barr virus nuclear antigen 2 (EBNA2) and the Notch protein both function within the nucleus as transcriptional adaptor proteins. EBNA2 plays a key role during the immortalization of primary B-cells by Epstein±Barr virus (EBV). Notch proteins are involved in lymphomagenesis as well as in multiple cell fate decisions during tissue differentiation and development. Both, EBNA2 and Notch interact with the DNA binding protein RBP-J and thereby gain access to the promoter of their target genes. In order to identify regions within the J recombination signal sequence binding protein (RBP-J), that are relevant for either the Notch or the EBNA2 interaction, we have performed a mutational analysis of RBP-J. A library of RBP-J mutants was screened by a reverse two-hybrid system for alleles that fail to bind to either EBNA2 or Notch. The sequence analysis of these alleles reveals that a limited and particularly distinct number of amino-acid positions are relevant for either interaction only. Given the important role of RBP-J in B-cell immortalization, the EBNA2/RBP-J protein±protein interaction could be a candidate target for therapeutic intervention in EBV related diseases. The Notch pathway is a cellular pathway, that targets the RBP-J protein. The Notch proteins are a family of highly conserved transmembrane receptors. Upon ligand binding, the transmembrane protein is cleaved and an intracellular fragment of Notch translocates into the nucleus and, similarly to EBNA2, binds to RBP-J and activates target genes [23±25]. In mammals, four Notch genes are differentially expressed during development and control processes as diverse as neurogenesis, somite formation and hematopoeisis [4,26,27].As EBNA2/RBP-J signalling is essential for the immortalizing function of EBNA2, the cellular protein RBP-J is a candidate target for therapeutic intervention in EBV associated diseases. On the other hand, we know that four mammalian Notch proteins also interact with RBP-J and we know or expect these interactions to be involved in a broad range of essential biological functions. The most desirable drug should thus interfere with EBNA2/RBP-J signalling, without impairing Notch/RBP-J signalling. A prerequisite for a specific interference with EBNA2/RBP-J signalling is the detailed knowledge of the respective protein±protein interaction.Structural data on the RBP-J protein are not available. The primary sequence of the RBP-J protein has been highly conserved during evolution, but pattern and profile searches so far have only identified one conserved domain, called the IPT/ TIG domain, which lies outside the minimal
TGF- induces apoptosis and inhibits the proliferation of EBV-negative B-lymphoma cell lines. In contrast, EBV-immortalized B cells are resistant to both the proapoptotic and the antiproliferative activities of TGF-. We have generated a lymphoblastoid cell line, in which we can switch on and off the EBV-specific transcriptional program driven by EBNA2. When these cells express the EBNA2-driven phenotype, they are resistant to TGF--mediated growth arrest. We used this cell line to readdress the question of how EBV can overcome the antiproliferative TGF- activity. We show here that EBVdriven cells remain TGF--responsive since TGF- target genes are readily induced. Thus, EBV can overcome TGF--mediated growth arrest without interfering with the core machinery of the TGF- signaling pathway, which links ligand binding to the induction of TGF- target genes.
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