Regulation of p53-dependent apoptosis, transcriptional repression, and cell transformation by phosphorylation of the 55-kilodalton E1B protein of human adenovirus type 5

Abstract

The adenovirus type 5 55-kDa E1B protein (E1B-55kDa) cooperates with E1A gene products to induce cell transformation. E1A proteins stimulate DNA synthesis and cell proliferation; however, they also cause rapid cell death by p53-dependent and p53-independent apoptosis. It is believed that the role of the E1B-55kDa protein in transformation is to protect against p53-dependent apoptosis by binding to and inactivating p53. It has been shown previously that the 55-kDa polypeptide abrogates p53-mediated transactivation and that mutants defective in p53 binding are unable to cooperate with E1A in transformation. We have previously mapped phosphorylation sites near the carboxy terminus of the E1B-55kDa protein at Ser-490 and Ser-491, which lie within casein kinase II consensus sequences. Conversion of these sites to alanine residues greatly reduced transforming activity, and although the mutant 55-kDa protein was found to interact with p53 at normal levels, it was somewhat defective for suppression of p53 transactivation activity. We now report that a nearby residue, Thr-495, also appears to be phosphorylated. We demonstrate directly that the wild-type 55-kDa protein is able to block E1A-induced p53-dependent apoptosis, whereas cells infected by mutant pm490/1/5A, which contains alanine residues at all three phosphorylation sites, exhibited extensive DNA fragmentation and classic apoptotic cell death. The E1B-55kDa product has been shown to exhibit intrinsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4 DNA-binding domain, even in the absence of p53. Such repression activity was totally absent with mutant pm490/1/5A. These data suggested that inhibition of p53-dependent apoptosis may depend on the transcriptional repression function of the 55-kDa protein, which appears to be regulated be phosphorylation at the carboxy terminus.

Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells

“…Mutant E1B/55K Ϫ (originally pm2019/2250) fails to express the E1B 55-kDa protein, and mutant E1B/19K Ϫ (originally pm1716/2072) does not express the E1B 19-kDa polypeptide (36). Additional E1B 55-kDa protein mutants included pm490/1A and pm490/1/5A, which contain alanine residues in place of the Ser-490, Ser-491, and Thr-495 phosphorylation sites, thus reducing transforming and transcriptional repression activities and the ability to inhibit p53 activity but not affecting complex formation with p53 (58,59), and mutants A262 and R309, which contain insertions next to residues 262 and 309 which block interactions with p53 (28,65). E1A and E1B mutant viruses were propagated on 293 cells.…”

“…Inhibition of E1A-induced accumulation of p53 requires a functional E1B 55-kDa protein. The 55-kDa E1B product possesses intrinsic repression activity which is believed to block the action of the p53 activation domain following complex formation with p53 (58,66). To examine the role of this E1B protein in the inhibition of E1A-induced accumulation of p53, the levels of p53 protein were examined in extracts from E1B mutant-infected cells by Western blotting.…”

“…Studies were also conducted with mutants pm490/1A and pm490/1/5A, which contain Ala residues in place of the Ser-490, Ser-491, and Thr-495 phosphorylation sites of the E1B 55-kDa species. These alterations have been found to diminish cell transformation dependent on the 55-kDa protein, reduce its ability to inhibit p53 activation activity, and eliminate its transcriptional repression activity without affecting the efficiency of binding to p53 or the induction of host cell shutoff (58,59). The precise role of phosphorylation in the regulation of these functions has still not been established.…”

“…The 55-kDa protein binds to and inhibits p53 (28,53,64,66), a cellular transcriptional regulator which induces growth arrest and/or apoptosis following the induction of unscheduled DNA synthesis or DNA damage (reviewed in reference 29). We have shown recently that the 55-kDa protein is able to block p53-dependent apoptosis induced by E1A (58) or by expression of p53 in p53-null Saos-2 human osteosarcoma cells (34). Thus, one role of E1B products in both productive infection and cell transformation appears to be to prevent untimely E1A-induced cell death, thus permitting production of high yields of progeny or survival of transformants.…”

Pleiotropic mechanisms of virus survival and persistence

“…The E1A gene product stabilizes p53 and induces p53-dependent apoptosis. 14,16 In contrast, the adenovirus E3 gene product promotes degradation of Fas, and the adenovirus E1B proteins antagonize p53 function. Viral homologs of Bcl-2, an apoptosis suppressor that binds with Bax, are produced by adenovirus, Epstein-barr virus (EBV, BHRF1 protein), and other viruses.…”