Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins

Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G₀-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo ³²P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G₀-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.