Adenovirus E1B 55K Represses p53 Activation In Vitro

The cellular phosphoprotein p53 acts as a tumor suppressor, and inactivation of this function is the most prevalent alteration found in human and animal tumors (32, 35). p53 acts as a G1 checkpoint control ensuring completion of DNA replication and the integrity of the genome prior to cells entering the S phase. In response to DNA damage, p53 levels are increased and p53 induces genes whose products result in either growth arrest at the G1/S interface or apoptosis (reviewed in reference 32). p53-inducible genes include p21/WAF21/Cip1 (cyclin/cdk inhibitor) (10, 66), cyclin G (45), Wip1 (a PP2A protein phosphatase) (12), Bax1 (apoptosis inducer) (43), insulin-like growth factor binding protein 3 (5), GADD45 (28), and Mdm2 (46). Mdm2 could be a natural biological regulator of p53 through an autoregulatory feedback loop with p53, since p53 activates Mdm2 expression and Mdm2 inhibits p53 activation (64). As a consequence of the inhibition of p53’s activation by Mdm2, induction of growth arrest and apoptosis are also inhibited (28, 46, 64, 69). p53 appears to exert most of its biological effects by acting as a sequence-specific DNA-binding transcription factor (Fig. ​(Fig.1)1) (11) that binds as a tetramer (24) to four tandem, alternatively inverted copies of a 5-bp consensus sequence (7, 9). The transactivation domain maps to the acidic amino-terminal region of the polypeptide (1 to 73 amino acid residues), and it is within this region that p53 has been demonstrated to directly interact with components of the general transcriptional machinery, TATA box-binding protein (TBP), TAFII31, and TFIIH (p62) (41, 42, 58, 60, 65), as well as its negative regulator, Mdm2 (38). FIG. 1 Schematic diagram illustrating the functional domains in Ad E1B 55K (A) and the tumor suppressor p53 (B). (A) Hatched boxes indicate regions in E1B 55K which when mutated result in loss of the described functions. Positions where amino acids have been ... The inactivation of p53 is critical for the replication of DNA tumor viruses, which require cells to enter the S phase, and which, in the case of adenovirus (Ad), would otherwise induce apoptosis (63). The DNA tumor viruses each encode oncoproteins, such as Ad E1B 55K, human papillomavirus E6, and simian virus 40 T antigen (9, 11, 27, 67), which bind to and inhibit the normal biological functions of p53. Binding of HPV E6 to wild-type p53 results in polyubiquination and subsequent degradation of p53 (53). Simian virus 40 T antigen interacts with the DNA binding domain of p53, thus preventing p53 from binding to DNA and regulating gene expression (11). The Ad E1B 55K protein also forms a stable complex with p53 in vitro and in vivo (27, 50) and inhibits p53-mediated transcriptional activation (67). E1B 55K binds to the amino-terminal transactivation domain of p53 (27) (Fig. ​(Fig.1).1). Mutation of p53 at either the proline (amino acid 24) or tryptophan (amino acid 27) residue reduces p53’s affinity for E1B 55K, although the transactivation function remains wild type (38). Previous studies with a panel of E1B 55K linker insertion mutants showed that the ability of E1B 55K to inhibit p53-mediated activation correlated directly with the ability of E1B 55K to transform cells in conjunction with Ad E1A (Fig. ​(Fig.1)1) (68). Furthermore, the interaction between E1B 55K and p53 was necessary but not sufficient for the repression and transformation functions of E1B 55K, since an E1B 55K insertion mutant at position 443 binds to p53 with wild-type affinity but was defective for both repression of p53 transcriptional activation and transformation (see Fig. ​Fig.1)1) (67, 68). These and other studies have indicated that the carboxy-terminal region of E1B 55K and phosphorylation of serine residues 490 and 491 and threonine at 495 within this region are important for the repression functions of E1B 55K (42a, 56, 57, 68). Moreover, these results suggested that E1B 55K has an active role in repression and was not simply preventing p53 from binding to DNA nor sterically blocking the activation domain of p53. Strong evidence suggesting that E1B 55K has a generalized transcriptional repression activity came from transient expression experiments in which a Gal4-55K fusion was shown to repress expression from several target promoters containing Gal4 binding sites. These promoters did not contain any common activator binding sites, implying that E1B 55K does not inhibit a specific activation mechanism, but rather inhibits a general process required for transcription from most promoters (68). Furthermore, the ability of Gal4-55K to repress transcription was independent of p53, because similar results were obtained from experiments performed with p53-minus cell lines (68). These results suggested the model that E1B 55K acts as a general repressor of RNA polymerase II transcription and that the repression function of E1B 55K is directed toward p53-activated promoters by virtue of its binding to DNA-bound p53. E1B 55K could mediate its repressing effect either by acting directly on the general transcriptional machinery (22, 25) or through modification of chromatin (47) (Fig. ​(Fig.11C). Given the central role that aberration of the normal function of p53 has in the development of some cancers, it is important to establish how its activity is regulated and/or inhibited by oncoproteins such as E1B 55K. Furthermore, the mechanism of action by repressors like E1B 55K, which do not simply compete for the DNA binding sites of activating proteins, is not clearly understood for many eukaryotic repressors. Understanding how E1B 55K mediates its effect may yield insights into how other repressors function and how repressors regulate the activity of activators. Here, we report on the physical and biochemical properties of purified, recombinant E1B 55K expressed from a baculovirus vector and demonstrate through in vitro transcription assays that it is indeed a transcriptional repressor.