Identification of constitutive phosphorylation sites on the Epstein-Barr virus ZEBRA protein

ZEBRA, the product of the Epstein-Barr virus gene bzlf1, and a member of the AP-1 subfamily of basic zipper ( bZIP) transcription factors, is necessary and sufficient to disrupt viral latency and to initiate the viral lytic cycle. Two serine residues of ZEBRA, Ser(167) and Ser(173), are substrates for casein kinase 2 ( CK2) and are constitutively phosphorylated in vivo. Phosphorylation of ZEBRA at its CK2 sites is required for proper temporal regulation of viral gene expression. Phosphopeptide analysis indicated that ZEBRA contains additional constitutive phosphorylation sites. Here we employed a co-migration strategy to map these sites in vivo. The cornerstone of this strategy was to correlate the migration of P-32- and S-35- labeled tryptic peptides of ZEBRA. The identity of the peptides was revealed by mutagenesis of methionine and cysteine residues present in each peptide. Phosphorylation sites within the peptide were identified by mutagenesis of serines and threonines. ZEBRA was shown to be phosphorylated at serine and threonine residues, but not tyrosine. Two previously unrecognized phosphorylation sites of ZEBRA were identified in the NH2-terminal region of the transactivation domain: a cluster of weak phosphorylation sites at Ser(6), Thr(7), and Ser(8) and a strong phosphorylation site at Thr(14). Thr(14) was embedded in a MAP kinase consensus sequence and could be phosphorylated in vitro by JNK, despite the absence of a canonical JNK docking site. Thus ZEBRA is now known to be constitutively phosphorylated at three distinct sites.