REGULATION OF P53-DEPENDENT APOPTOSIS, TRANSCRIPTIONAL REPRESSION, AND CELL-TRANSFORMATION BY PHOSPHORYLATION OF THE 55-KILODALTON E1B PROTEIN OF HUMAN ADENOVIRUS TYPE-5

The adenovirus type 5 55-kDa E1B protein (E1B-55kDa) cooperates with E 1A gene products to induce cell transformation. EIA proteins stimulate DNA synthesis and cell proliferation; however, they also cause rapid cell death by p53-dependent and p53-independent apoptosis. It is belie ved that the role of the E1B-55kDa protein in transformation is to pro tect against p53-dependent apoptosis by binding to and inactivating p5 3. 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 previousl y mapped phosphorylation sites near the carboxy terminus of the E1B-55 kDa protein at Ser-490 and Ser-491, which lie within casein kinase II consensus sequences. Conversion of these sites to alanine residues gre atly reduced transforming activity, and although the mutant 55-kDa pro tein was found to interact with p53 al normal levels, it was somewhat defective for suppression of p53 transactivation activity. We now repo rt that a nearby residue, Thr-495, also appears to be phosphorylated. We demonstrate directly that the wild-type 55-kDa protein is able to b lock E1A-induced p53-dependent apoptosis, whereas cells infected by mu tant pm490/1/5A, which contains alanine residues at all three phosphor ylation sites, exhibited extensive DNA fragmentation and classic apopt otic cell death. The E1B-55kDa product has been shown to exhibit intri nsic transcriptional repression activity when localized to promoters, such as by fusion with the GAL4 DNA-binding domain, even in the absenc e of p53. Such repression activity was totally absent with mutant pm49 0/1/5A. These data suggested that inhibition of p53-dependent apoptosi s may depend on the transcriptional repression function of the 55-kDa protein, which appears to be regulated be phosphorylation at the carbo xy terminus.