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, Ser167 and Ser173, 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 32P- and 35S-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 Ser6, Thr7, and Ser8 and a strong phosphorylation site at Thr14. Thr14 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.
El-Guindy
Ayman S
AS
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Paek
So Yeon
SY
Countryman
Jill
J
Miller
George
G
D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D045744:Cell Line, Tumor; D002465:Cell Movement; D003545:Cysteine / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008715:Methionine / Q000737:chemistry; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D014162:Transfection; D014357:Trypsin / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014764:Viral Proteins / Q000737:chemistry / Q000502:physiology*
C063676:BZLF1 protein, Herpesvirus 4, Human; D004268:DNA-Binding Proteins; D010455:Peptides; D015534:Trans-Activators; D014764:Viral Proteins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D008715:Methionine; D048031:JNK Mitogen-Activated Protein Kinases; D014357:Trypsin; D003545:Cysteine