Ny. Harel et Jc. Alwine, PHOSPHORYLATION OF THE HUMAN CYTOMEGALOVIRUS 86-KILODALTON IMMEDIATE-EARLY PROTEIN IE2, Journal of virology, 72(7), 1998, pp. 5481-5492
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.