Phosphorylation of the Human Cytomegalovirus 86-Kilodalton Immediate-Early Protein IE2

We have investigated the phosphorylation state of the human cytomegalovirus 86-kDa immediate-early (IE) protein IEP86 from transfected and infected cells. We show that multiple domains of IEP86 are phosphorylated by cellular kinases, both in vitro and in vivo. Our data suggest that serum-inducible kinases play a significant role in cell-mediated IE protein phosphorylation and that a member of the mitogen-activated protein (MAP) kinase (MAPK) family, extracellular regulated kinase 2 (ERK2), phosphorylates several domains of IEP86 in vitro. Alanine substitution mutagenesis was performed on specific serines or threonines (T(27), S(144), T(233)/S(234), and T(555)) found in consensus MAP kinase motifs. Analysis of these mutations showed that T(27) and T(233)/S(234) are the major sites for serum-inducible kinases and are the major ERK2 sites in vitro. S(144) appeared to be phosphorylated in a serum-independent manner in vitro. All of the mutations except T(555) eliminated specific phosphorylation in vivo. In transient transfection analyses, IEP86 isoforms containing mutations in S(144) and, especially, T(233)/S(234) displayed increased transcriptional activation relative to the wild type, suggesting that phosphorylation at these sites in wild-type IEP86 may result in reduction of its transcriptional activation ability.