Definition of a major p53 binding site on Ad2E1B58K protein and a possible nuclear localization signal on the Ad12E1B54K protein

Abstract

Previous studies have established that adenovirus 2/5 early region 1B (Ad E1B) 58K protein binds p53 strongly and co-localizes with it to cytoplasmic dense bodies whilst the homologous Ad12E1B54K protein binds only weakly and co-localizes primarily to the nucleus in Ad12E1 transformed cells. We have used these properties of the E1B proteins from di erent viral serotypes to map the p53 binding site on the Ad2/5 protein. A set of chimaeric genes was constructed containing di erent proportions of the Ad12 and Ad2E1B DNA. These, together with Ad12E1A and E1B19K DNA, were transfected into baby rat kidney cells and transformed lines isolated. From an examination of the properties of these Ad12/Ad2E1B fusion proteins in co-immunoprecipitation and subcellular localization experiments it has been concluded that the p53 binding site on Ad2E1B58K protein lies between amino acids 216 and 235 and that the homologous region on Ad12E1B54K protein also binds p53. In addition, a unique nuclear localization signal is located on Ad12E1B54K between residues 228 and 239. We suggest that primary structure di erences in these regions of the Ad2 and Ad12E1B proteins are responsible for the di erent subcellular localizations in AdE1 transformants.

Consequences of disruption of the interaction between p53 and the larger adenovirus early region 1B protein in adenovirus E1 transformed human cells

“…It can be seen from the data presented in Figure 1 that whilst the concentration of complex was reduced in the presence of peptide it was not totally dissociated, as described previously . Similar results were obtained when Ad12 E1 HER cells were treated with peptides 1507 and 1508 (equivalent to the Ad5 and Ad12 sequences [data not shown and Grand et al, 1999]). The consequences for Ad E1-transformed cells of disruption of the complex are examined in the work presented below.…”

“…Whilst the abilities of the two peptides to disrupt both Ad5E1B and Ad12E1B/p53 complexes in vitro were similar (Figure 1 and Grand et al, 1999) di erences were observed in the in vivo experiments described here. Thus, as a rule peptides were more e cient at disrupting the complex in Ad12 transformed cells than in Ad5 (compare Figure 4b, panels [ii] and [iii]).…”

The adenovirus type 5 E1B-55K oncoprotein is a highly active shuttle protein and shuttling is independent of E4orf6, p53 and Mdm2

“…80 K) favour active import rather than passive di usion and further studies are ongoing to dissect the E1B import pathway. Previously, Grand and coworkers (Grand et al, 1999) described a NLS in the Ad12 E1B protein. The di erence in localization (cytoplasmic for the Ad5 E1B-55K versus predominantly nuclear for the Ad12 E1B-54K protein) was explained by the lack of the postulated NLS in the Ad5 E1B-55K protein.…”

Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription

“…Collectively available data suggest that these activities are linked in part to p53 transcription-independent pathways (Lo¨ber et al, 2002;Hobom and Dobbelstein, 2004;Sieber and Dobner, 2007), and to the ability of Ad2/5 E1B-55K products to inactivate p53 and the tumor suppressor protein WT1 by sequestration into a cytoplasmic inclusion body (Maheswaran et al, 1998;Grand et al, 1999;Hutton et al, 2000; typically situated around the microtubule organizing center (MTOC) (Brown et al, 1994). The cytoplasmic restriction imposed upon p53 requires binding of Ad2/5 E1B-55K to p53, but is independent of CRM1-mediated nuclear export of the viral protein in transformed rat cells (Endter et al, 2005).…”