PHOSPHORYLATION OF THE HUMAN CYTOMEGALOVIRUS 86-KILODALTON IMMEDIATE-EARLY PROTEIN IE2

We have investigated the phosphorylation state of the human cytomegalo virus 86-kDa immediate-early (IE) protein IEP86 from transfected and i nfected cells. We show that multiple domains of IEP86 are phosphorylat ed by cellular kinases, both in vitro and in vivo. Our data suggest th at serum-inducible kinases play a significant role in cell-mediated IE protein phosphorylation and that a member of the mitogen-activated pr otein (MAP) kinase (MAPK) family, extracellular regulated kinase 2 (ER K2), phosphorylates several domains of IEP86 in vitro. Alanine substit ution mutagenesis was performed on specific serines or threonines (T-2 7, 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 i n vitro. S-144 appeared to be phosphorylated in a serum-independent ma nner in vitro. All of the mutations except T-555 eliminated specific p hosphorylation in vivo. In transient transfection analyses, IEP86 isof orms containing mutations in S-144 and, especially, T-233/S-234 displa yed increased transcriptional activation relative to the wild type, su ggesting that phosphorylation at these sites in wild-type IEP86 may re sult in reduction of its transcriptional activation ability.