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J. Biol. Chem., Vol. 281, Issue 6, 3085-3095, February 10, 2006

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Identification of Constitutive Phosphorylation Sites on the Epstein-Barr Virus ZEBRA Protein^*

Ayman S. El-Guindy, So Yeon Paek, Jill Countryman, and George Miller^¶1

From the Departments of Molecular Biophysics and Biochemistry, Pediatrics, and ^¶Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut 06520

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.

Received for publication, June 3, 2005 , and in revised form, November 28, 2005.

^* This work was supported by National Institutes of Health Grants CA12055 and CA16038 (to G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Rm. 420 LSOG, 333 Cedar St., P.O. Box 208064, New Haven, CT 06520-8064. Tel.: 203-785-4758; Fax: 203-785-6961; E-mail: George.Miller{at}yale.edu.

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				A. El-Guindy, L. Heston, H.-J. Delecluse, and G. Miller Phosphoacceptor Site S173 in the Regulatory Domain of Epstein-Barr Virus ZEBRA Protein Is Required for Lytic DNA Replication but Not for Activation of Viral Early Genes J. Virol., April 1, 2007; 81(7): 3303 - 3316. [Abstract] [Full Text] [PDF]