Identification of major phosphorylation sites of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP): ability of EBNA-LP to induce latent membrane protein 1 cooperatively with EBNA-2 is regulated by phosphorylation.
Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization of B cells. One of the potential functions of EBNA-LP is a cooperative induction with EBNA-2 of viral and cellular gene expression, including that of the genes for viral latent membrane protein 1 (LMP-1) and cellular cyclin D2. We report here that the phosphorylation of EBNA-LP by cellular kinase(s) is critical to its ability to cooperate with EBNA-2 in up-regulating the expression of LMP-1 in a B-lymphoma cell line. Our conclusion is based on the following observations. (i) Mass-spectrometric analysis of purified EBNA-LP and mutational analyses of EBNA-LP revealed that the serine residue at position 35 in the W2 repeat domain is the major phosphorylation site of EBNA-LP in vivo. (ii) Substitutions of this site in each W2 repeat domain with alanine markedly reduced the ability of the protein to induce LMP-1 expression in combination with EBNA-2 in Akata cells. (iii) Replacement at the major phosphorylation sites with glutamic acids restored the wild-type phenotype. It is well established that this substitution mimics constitutive phosphorylation. These results indicated that the coactivator function of EBNA-LP is regulated by phosphorylation.
Yokoyama
A
A
Department of Tumor Virology, Division of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan.
Tanaka
M
M
Matsuda
G
G
Kato
K
K
Kanamori
M
M
Kawasaki
H
H
Hirano
H
H
Kitabayashi
I
I
Ohki
M
M
Hirai
K
K
Kawaguchi
Y
Y
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001402:B-Lymphocytes; D001665:Binding Sites; D002051:Burkitt Lymphoma; D019556:COS Cells; D002460:Cell Line; D002461:Cell Line, Transformed; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D017353:Gene Deletion; D018698:Glutamic Acid / Q000378:metabolism; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation; D014763:Viral Matrix Proteins / Q000096:biosynthesis*; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C402641:EBNA-2 protein, Human herpesvirus 4; C402640:EBNA-LP protein, Human herpesvirus 4; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D018698:Glutamic Acid; D010770:Phosphotransferases; D000409:Alanine