PHOSPHORYLATION WITHIN THE TRANSACTIVATION DOMAIN OF ADENOVIRUS E1A PROTEIN BY MITOGEN-ACTIVATED PROTEIN-KINASE REGULATES EXPRESSION OF EARLY REGION-4

A critical role of the 289-residue (289R) E1A protein of human adenovi rus type 5 during productive infection is to transactivate expression of all early viral transcription. Sequences within and proximal to con served region 3 (CR3) promote expression of these viral genes through interactions with a variety of transcription factors requiring the zin c binding motif in CR3 and in some cases a region at the carboxy-termi nal end of CR3, including residues 183 to 188. It is known that 3',5' cyclic AMP (cAMP) reduces the level of phosphorylation of the 289R E1A protein through the activation of protein phosphatase 2A by the E4orf 4 protein. This study was designed to identify the E1A phosphorylation sites affected by E4orf4 expression and to determine their importance in regulation of E1A activity. We report here that two previously uni dentified sites at Ser-185 and Ser-188 are the targets for decreased p hosphorylation in response to cAMP. At least one of these sites, presu mably Ser-185, is phosphorylated in vitro by purified mitogen-activate d protein kinase (MAPK), and both are hyperphosphorylated in cells whi ch express a constitutively active form of MAPK kinase, Analysis of E1 A-mediated transactivation activity indicated that elevated phosphoryl ation at these sites increased expression of the E4 promoter but not t hat of E3. We have recently shown that one or more E4 products induce cell death due to p53-independent apoptosis, and thus it seems likely that one role of the E4orf4 protein is to limit production of toxic E4 products by limiting expression of the E4 promoter.